differential expression of proteoglycan epitopes by ovine intervertebral disc cells in calcium...

British Connective Tissue Society Meeting, Cardiff,30–31 March 1998

Molecular Interaction and Matrix Assembly

The Spring meeting of the BCTS was held at UniversityHall, Cardiff University on 30–31st March, and had as itstheme molecular interaction and matrix assembly. About170 delegates, from all around the UK, registered for themeeting. In order to promote and increase internationalcollaborations and interactions, speakers were invitedfrom laboratories in the USA, Germany, Belgium, andAustralia, and, also from the biomedical industries in theUK. The meeting was organised by Drs Alvin Kwan(Connective Tissue Biology Laboratories, Cardiff ) andMalcolm Davies (Institute of Nephrology, Cardiff ) andfinancial support for international speakers was obtainedfrom the Wellcome Trust, Arthritis Research Campaign,Roche Ltd and Pfizer Ltd. Additional donations werereceived from Hybaid Ltd., R & D Systems, Calbiochem-Novabiochem (UK) Ltd., Carl Zeiss Ltd. and NovexInternational.

The scientific programme was divided into foursessions, (1) extracellular matrix assembly and remodel-ling, (2) cell signalling, (3) collagen metabolism and (4)matrix organisation and turnover. The first sessionexamined the assembly of basement membrane andthe analyses of plasmin and metalloproteinase systemsin matrix remodelling. Dale Abrahamson (University ofAlabama at Birmingham, USA) described the processesleading to the assembly of the glomerular basementmembrane. Lieve Moons (University of Leuven, Belgium)described the use of Apolipoprotein E deficient mice asan experimental model to investigate the roles of tissueplasminogen activator (tPA) and urokinase-type plas-minogen activator (uPA) in the activation of plasmin andmatrix metalloproteinase proteolysis in the remodelling ofthe arterial intimal elastic lamina.

The second session was devoted to the discussion ofthe roles of the cell-surface heparan sulphate proteo-glycans – the syndecans. John Couchman (University ofAlabama at Birmingham, USA) described the use ofdeletion constructs of Syndecan-2 core protein in studiesto investigate the functional roles of this heparan sul-phate proteoglycan in regulating matrix assembly. GuidoDavid (University of Leuven, Belgium) continued with thetheme of syndecans and described that the highly con-served FYA C-terminal sequence of the syndecans bindsto a novel protein syntenin which contains a tandemrepeat of PDZ-domains.

The third session explored the molecular compositionof cartilage collagen fibrils and the molecular pathologyof collagens. Peter Bruckner (University of Munster,Germany) presented the concept that cartilage collagenfibrils are biological alloys i.e. heterotypic fibrils com-posed of collagen types II, IX and XI. John Bateman(University of Melbourne, Australia) presented a briefreview of genetic diseases associated with collagengene mutations.

The fourth session was devoted to the discussion ofmatrix organisation. Neera Borkakoti (Roche, UK) gavea detailed review of studies on several members of thezinc metalloproteinases (MMPs) family. Mats Paulsson(University of Cologne, Germany) described thestructure of matrilins 1, 2 and 3 – a new family ofextracellular matrix proteins with von Willebrand FactorA-like domains. Dick Heinegard (University of Lund,Sweden) described changes in the articular cartilageextracellular matrix synthesis at early stage of cartilagedegeneration.

In addition there were two sessions designed to allowyoung investigators to present their work. These presen-tations were chosen on the basis of the relevance oftopics to the main theme of this meeting. Jo Lewthwaite(Eastman Dental Institute, London) presented data onthe induction of pig articular cartilage breakdown inexplant cultures by chaperonin 60 (cpn60). Mark Bond(Bristol Heart Institute, UK) presented data that showedthat the transcription factor NF-kB is essential for upregu-lating MMP-9 expression in rabbit dermal fibroblasts andsmooth muscle cells. Ian Clark (University of EastAnglia, UK) described the pathway of human TIMP-1gene activation by all-trans retinoic acid (ATRA) andbFGF. Alison Reith (University of Manchester, UK)described a newly developed three-dimensional mixedaggregates of dermal fibroblasts and keratinocytes inculture, used as a model for the dermal-epidermal junc-tion of skin. Maddy Parsons (University College London,UK) presented data showing that mechanical loadingenhanced the effect of FCS, TGFb and thrombin on theincrease in collagen synthesis and deposition. MichaelBriggs (University of Manchester, UK) described thescreening for candidate genes in 7 families with Multipleepiphyseal dysplasia (MED). Anne Vaughan Thomas(Cardiff University, UK) presented findings on age-related changes in the articular cartilage matrix. Neil

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Smyth (University of Cologne, Germany) described thepreparation of LAMC1 knock-out mice. Carl Flannery(Cardiff University, UK) described the detection of twochondrocyte hyaluronidases in human articular cartilage,explant cultures and cultured chondrocytes by RT-PCR.

Alvin Kwan

The role of lysosomal enzymes, in particularcathepsin B, in cartilage proteoglycanbreakdown within a bovine model

C. ADCOCKS*, A. FRAZER*, V. EVERTS†AND D.J. BUTTLE**Division of Biochemical and Musculoskeletal Medicine,

Section of Human Metabolism and Clinical Biochemistry,

University of Sheffield Medical School, Sheffield, UK and

†Laboratory of Cell Biology, University of Amsterdam,

Academic Medical Centre, Amsterdam, The Netherlands

Introduction

The aim of this investigation was to determine the role oflysosomal enzymes, in particular cathepsin B, in cartil-age proteoglycan degradation, and to ascertain whetherthe action is intra- or extracellular. Four methodologicalapproaches will be discussed, (i) immunolocalization ofcathepsin B in bovine nasal chondrocytes, (ii) competi-tion between lysosomal enzymes and free mannose-6-phosphate for the mannose-6-phosphate receptor duringtransport to the lysosome (iii) utilization of lysosomo-tropic agents that elevate the lysosomal pH and (iv)utilization of a cathepsin B fluorescent substrate, Z-R-R-NHMec in conjunction with a specific inactivator ofcathepsin B, CA074 to determine levels of cathepsin B inculture media.

Methods

Bovine nasal septum or metacarpopharangeal jointexplants were maintained in serum-free DMEM for theappropriate culture period with various concentrations ofmannose-6-phosphate, NH4Cl, bafilomycin or folimycin.Proteoglycan degradation was assessed using thedimethylmethylene blue assay.

Immunolocalization was performed on bovine nasalchondrocytes cultured in the presence of TNFa, IL-1a orretinoic acid (Ret). The antibodies used were affinitypurified anti-cathepsin B IgG and as a negative controlanti-chymopapain IgG.

Results

In the presence of NH4Cl, cartilage proteoglycan degra-dation was significantly increased in a dose-dependentmanner. However the lysosomotropic agents bafilomycinand folimycin did not produce a marked alteration in therate of proteolgycan degradation.

The use of the fluorescent substrate Z-R-R-NHMecalso showed that in the presence of NH4Cl there is anincrease in cathepsin B levels in the culture media.

In the presence of mannose-6-phosphate (10 mM),there was a significant and dose-dependent reductionin proteoglycan degradation.

The immunolocalization of cathepsin B in bovine nasalchondrocytes demonstrated that in the presence ofIL-1a, TNFa and Ret. for a 5 day culture period, cathep-sin B can be detected intracellularly. A punctate stainingpattern was observed, perhaps showing cathepsin Bwithin the lysosomes. The staining appeared moreintense for the cells treated with Ret.

Discussion

These results suggest that an alteration of the localisa-tion and trafficking of cathepsin B can modulate rates ofproteoglycan breakdown in ways which require furtherstudy.

Matrix metalloproteinase levels are elevated ininflamed areas of the colitic bowel

M.D. BAUGH* G.S. EVANS*,A.P. HOLLANDER† D.R. DAVIES§,M.J. PERRY§, S. CROSS‡, A.J. LOBOAND C.J. TAYLOR*Division of Child Health, †Human Metabolism and Clinical

Biochemistry in the Division of Biochemical and Musculoskeletal

Medicine and ‡Division of Pathology, University of Sheffield and

‡Department of Gastroenterology & Liver Unit,

Royal Hallamshire Hospital, Sheffield, UK

Introduction

MMP’s are a family of Znþþ containing neutral endo-peptidases that degrade extracellular matrix moleculessuch as collagen, fibronectin and proteoglycans. Recentevidence suggests that proteolytic enzymes may con-tribute to tissue damage in inflammatory bowel disease(IBD). Increases in collagen degradation have beenfound in patients with active Crohns’ disease (Kjeldsenet al. 1995). Loss of GAGs (Murch et al. 1993), and

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expression of MMP-3 have also been demonstrated inareas of mucosal loss in ulcerative colitis (UC) (Bailey etal. 1994).

Materials and methods

Endoscopic biopsies (n ¼ 24) were taken with ethicalapproval from the rectum and colon of adults with UC andchildren with indeterminate colitis. From each site twobiopsies were removed for enzyme analysis and one forhistological grading. On the basis of endoscopic criteria,biopsies were taken from inflammed tissue and non-involved areas in each patient. In addition biopsieswere removed from control patients (n ¼ 9) with non-inflammatory bowel complications. The majority of IBDpatients were receiving immunosuppressive therapy.Biopsy samples were snap frozen and stored at ¹708C.Each sample was homogenised and assayed for pro-tein concentration. The biopsies were investigated byzymography for gelatinolytic, caseinolytic and elastase-like enzymes, and using antibodies to MMP-1, 2, 3 and 9(Binding Site, U.K.) by Western blotting. Digital images ofthe zymographs were analysed using Kodak 1D imageanalysis software and measurement were made in termsof the total proteolysis per lane. Comparisons of zymo-graphic activity in paired samples were made usingWilcoxon matched pairs signed ranks. A numericalscoring was used to grade the histological features ofeach biopsy and the relationship between the histologicalgrade and zymographic activity was analysed usingSpearman’s rank coefficient of correlation.

Results

A significant increase (P ¼ 0.005) in gelatinolytic activitywas found in areas of inflammed vs non-involvedmucosa in patients with IBD and this increase correlatedwith the histological score of inflammation (rs ¼ 0.37,P < 0.0005). There was also a significant increase in thegelatinolytic activity in inflammed (P < 0.001) and noninvolved (P < 0.01) regions of bowel in IBD patients vsthe control group. The majority of bands on the gelatinzymograms were inhibited by 10 mM EDTA, a metallo-protease inhibitor, and comigrated with MMP-2 fromisolated intestinal fibroblasts and MMP-9 from neutro-phils. Western immunoblotting confirmed that MMP-9was present in 10/21 inflamed, but only 1/21 non-involved, and 0/9 control biopsies. Pro and activeMMP-1 was detected in all biopsies from IBD patientsbut only the proform was found in control biopsies.Stromelysin was not detected by either method.

Conclusions

An increase in MMPs in IBD has been demonstrated.These observations suggest that connective tissue cellsand neutrophils may be the major source of matrixdegrading enzymes in the colitic bowel, with MMP-2 &9 being amongst the most abundant.

Acknowledgments

This work was supported by The Children’s Appeal,Sheffield, the Nuffield Foundation and a University ofSheffield bursary.

References

BAILEY, C.J., HEMBRY, R.M., ALEXANDER, A., IRVING, M.H., GRANT,M.E. & SHUTTLEWORTH, C.A. (1994) Distribution of the matrixmetalloproteinases stromelysin, gelatinases A and B, andcollagenase in Crohn’s disease and normal intestine. J. Clin.Path. 47, 113–116.

KJELDSEN, J., SCHAFFALITZKYDE MUCKADELL, O.B. & JUNKER, P.(1995) Seromarkers of collagen I and III metabolism inactive Crohn’s disease. Relation to disease activity andresponse to therapy. Gut 37, 805–810.

MURCH, S.H., MACDONALD, T.T., WALKER-SMITH, J.A., LEVIN, M.,LIONETTI, P. & KLEIN, N.J. (1993) Disruption of sulphated glyco-saminoglycans in intestinal inflammation. Lancet 341, 711–714.

p38 MAPK negatively regulates and p42/44MAPK positively regulates expression of thehuman tissue inhibitor of metalloproteinases-1(TIMP-1) gene by ATRA in combination withbFGF

H.F. BIGG*, R.M. MCLEOD*,T.E. CAWSTON‡ AND I.M. CLARK†*School of Biological Sciences, University of East Anglia,

Norwich, UK, †University of British Columbia, Department of

Oral, Medical and Surgical Sciences, Vancouver, Canada and

‡Department of Rheumatology, University of Newcastle,

Newcastle, UK

Introduction

The matrix metalloproteinases (MMPs) are a family ofneutral enzymes implicated in turnover and degradationof the extracellular matrix. The active forms of all of theMMPs are inhibited by a family of specific inhibitors,the tissue inhibitors of metalloproteinases (TIMPs)(Denhardt et al. 1993). The balance between MMPsand TIMPs is therefore critical in matrix homeostasisand any alteration in this balance which favours MMPactivity may lead to uncontrolled matrix destruction. Theexpression of TIMP-1 is regulated by a number of

British Connective Tissue Society Meeting A21


different growth factors and cytokines, and the inductionof this gene may be a therapeutic target in degradativediseases such as arthritis. We have previously describedthe synergistic induction of the human TIMP-1 gene by acombination of all-trans retinoic acid (ATRA) and basicfibroblast growth factor (bFGF) (Bigg & Cawston 1995),here we explore the pathways by which this takes place.


Primary human skin fibroblasts were grown in monolayerculture in MEM þ 10% FCS. Cells were transferred intomedium containing 1% acid-treated FCS for 48 hoursprior to addition of test reagents for up to 72 hours;ATRA was used at 10¹5 or 10¹6 M, bFGF was usedat 10–100 ng/ml. TIMP-1 and MMP-1 were assayed inculture medium by ELISA. Expression and activation ofMAP kinases was assessed on Western blot usingspecific antibodies (New England Biolabs).

Results

The addition of ATRA in combination with bFGF resultsin a synergistic induction of both TIMP-1 protein andmRNA, whilst ATRA suppressed bFGF-induced MMP-1expression. The induction of mRNA is maximal atbetween 24 and 48 hours, whilst protein levels aremaximal at 72 hours. A short incubation (10 minutes to12 hours) with bFGF alone followed by ATRA alone gavea synergistic induction of TIMP-1 protein similar to thatseen with both agents together. Longer incubations withbFGF were not as effective and eventually ineffective.Treatment of cells with ATRA first followed by bFGF wasineffective. Control TIMP-1 mRNA was stable in thepresence of the transcriptional inhibitor DRB for 48hours; no differences were seen with bFGF, ATRA, orboth reagents. The induction of TIMP-1 mRNA by ATRAand bFGF was greatly diminished by cycloheximide. Atyrosine kinase inhibitor genistein, and a MEK1 inhibitor(PD98059) blocked the induction of TIMP-1 by ATRAand bFGF. No effect was seen in response to the proteinkinase C inhibitor bisindolylmaleimide or the proteinkinase A inhibitor H-89. Specific p38 MAP kinase inhibi-tors (SB203580 and SB202190) both further enhancedthe synergistic induction of TIMP-1 by ATRA and bFGF.Western blot analysis has demonstrated activation of thep38 and p42/44 MAP kinases from 5 minutes to 1 hourafter addition of ATRA and bFGF.

Discussion

We have described the potent, synergistic induction ofTIMP-1 gene expression in response to ATRA in

combination with bFGF. It appears that bFGF inducesa transient event which potentiates the effect of ATRA.New protein synthesis is required for the induction ofTIMP-1 by these agents and we are currently investi-gating the expression of retinoic acid receptors. Theinduction of TIMP-1 described is independent of PKCand PKA, but dependent on tyrosine kinase activity. Aninhibitor of the p42/44 MAP kinase pathway, blocksTIMP-1 induction, whilst inhibition of the p38 MAPkinase pathway potentiates the response. Furthercharacterisation of the role of these signalling pathwaysis underway.

Acknowledgements

This work was funded and supported by the ArthritisResearch Campaign, UK

References

BIGG, H.F. & CAWSTON, T.E. (1995) All-trans-retinoic acid inter-acts synergistically with basic fibroblast growth factor andepidermal growth factor to stimulate the production of tissueinhibitor of metalloproteinases from fibroblasts. Arch. Biochem.Biophys. 319, 74–83.

DENHARDT, D.T., FENG, B. & EDWARDS, D.R. et al. (1993) Tissueinhibitor of metalloproteinases (TIMP, aka EPA): structure,control of expression and biological functions. Pharmac Ther59, 329–341.

The regulation of metalloproteinaseexpression by cytokines and growth factors:The role of NF- kB and AP-1 transcriptionfactors

M. BOND, A.H. BAKER AND A. NEWBYBristol Heart Institute, Bristol University, Bristol, UK

Introduction

The matrix metalloproteinase (MMPs) enzymes havebeen widely implicated in matrix remodelling during anumber of pathological processes such as rheumatoidarthritis and atherosclerosis. We have recently demon-strated a synergistic interaction between interleukin-1a

(IL-1a) and platelet-derived growth factor (PDGF) thatenhances MMP-9 expression in rabbit vascular smoothmuscle cells (SMC) (Fabunmi et al. 1996). Analysis ofthe MMP-9 promoter has identified essential AP-1 andPEA-3 elements and an additional upstream NF-kB site(Sato & Seiki 1993). This study seeks to determinewhether growth factor and cytokine synergy is acommon mechanism regulating MMP-9 expression and



seeks to clarify the roles played by the NF-kB and AP-1transcription factors.

Methods

MMP expression was analysed by western blotting,zymography and northern blotting. NF-kB and AP-1activation was analysed by immunocytochemistry andelectromobility shift assay. The role of NF-kB in MMPexpression was determined by adenovirus mediatedoverexpression of IkBa (Wrighton et al. 1996).

Results

MMP-9 secretion from rabbit dermal fibroblasts andsmooth muscle cells was not stimulated by PDGF ofbFGF alone, only slightly by TNF-a or IL-1a andsynergistically upregulated by combinations of growthfactors and cytokines. NF-kB nuclear translocation andDNA binding activity was strongly induced by cytokinebut not growth factor stimulation, with no potentiationof this response by combinations of these agonists.However, AP-1 DNA binding activity increased additivelyin response to cytokine and growth factor stimulation.Inhibition of NF-kB activity by overexpression of IkBa

resulted in a complete inhibition of MMP-9 secretion.

Discussion

These experiments demonstrate the synergistic regu-lation of MMP-9 secretion by cytokine and growthfactor combinations, implying that enhanced basementmembrane and matrix turnover results were bothfactors are present together. NF-kB activity is essentialbut insufficient for full upregulation of MMP-9 expression.Taken together, the data presented here imply thatsynergistic regulation of MMP-9 expression is achievedby enhanced AP-1 DNA binding in concert with cytokineinduced NF-kB activity.

References

FABUNMI, R.P., BAKER, A.H., MURRAY, E.J., BOOTH, R.F.G. &NEWBY, A.C. (1996) Divergent regulation by growth factorsand cytokines of 95 kDa and 72 kDa gelatinases and tissueinhibitors or metalloproteinases-1, -2, and -3 in rabbit aorticsmooth muscle cells. Biochem. J. 315, 335–342.

SATO, H. & SEIKI, M. (1993) Regulatory mechanism of 92 kDatype IV collagenase gene expression which is associated withinvasiveness of tumor cells. Oncogene 8, 395–405.

WRIGHTON, C.J., HOFER-WARBINEK, R., EYTNER, R., MOLL, T., BACH,F.H. & DE MARTIN, R. (1996) Inhibition of endothelial cellactivation by adenovirus-mediated expression of I kappa Balpha, an inhibitor of the transcription factor NF-kappa B. J.Exp. Med. 483, 1013–1022.

Immunohistochemical localization of TGF b inequine tendons. A study in age-relatedchanges in TGF b expression

E.R. CAUVIN*, R.K. SMITH*, S.A. MAY*AND M.W.J. FERGUSON†*FAEMS, Royal Veterinary College, University of London,

North Mymms and †School of Biological Sciences,

University of Manchester, Manchester, UK

Introduction

Tendinitis is common in equine athletes and is frustratingto treat. Yet physiology of tendons remains poorly under-stood. Growth factors play a major role in the quality ofrepair in skin (Shah et al. 1995). Their modulation mayoffer great potential in the management of tendinitis. Theaim of this study was to determine the normal TGFb

profile in tendons depending on age and location.


Samples were collected post-mortem from the commondigital extensor and superficial digital flexor tendons inthe metacarpal (ST) and metacarpo-phalangealregions (SC), snap frozen and sectioned on a cryo-tome. Immunohistochemistry was performed using anti-TGFb1, b2 and b3 antibodies (AutogenBioclear UK Ltd)and Avidin-Biotin/DAB stain (Vector Laboratories, UK).

Results

There were significant differences in the pattern andintensity of staining, depending on age and location.TGFb1 was only seen in vessel walls and fibroblasts inendotenon septa. In the flexor tendon, the intensity ofstaining increased from birth to 2 years, decreased from4 to 9 and increased in older horses. The staining wasmore intense in the ST than in the SC, where thecompressed, dorsal part stained weakly. The extensortendon showed a weak stain at birth, moderate later inlife. TGFb2 was found in septa and vessel walls, but alsoin the cytoplasm of tenocytes within fibre bundles at birthand 2 years. The intensity of the stain reduced with age.The compressed, dorsal aspect of the SC stained stronglyfrom birth to 2 years, while accumulating pressure-resist-ing proteogycans. A similar distribution was seen withTGFb3 but the intensity of the stain was strong at birthand faded more rapidly with age than TGFb2.

Discussion

Unlike in bovine tendons (Robbins et al. 1997), no TGFb



isoforms were seen within the matrix. The strong peri-and intracellular stain for TGFb3 in the newbornsdiffered from the pattern observed in skin and wouldcorrelate with a potential for better quality repair and lessinflammation in foal tendons (Whitby & Ferguson 1991).TGFb1 and 2 have similar effects in tissues (Shah et al.1995). Differences between their staining patterns mayrelate to phenotypical differences between tenocytesand non-differentiated fibroblasts. This study indicatesthat the growth factor environment varies with age andthis may relate to the gradual production of normal matrixin weight-bearing tendons while the animal grows andthe apparent lack of anabolism in the adult. TGFb1appears not to be involved in the development andmaturation within tendon fibres, although it may beimportant in processes within the septa. The peri- andintracellular distribution of TGFb2 and 3 in tenocytessuggests that these growth factors may be involved innormal homeostatic mechanisms in tendon fibres.

Acknowledgements

The authors wish to thank Drs Joe Bird and MalcolmGaynes, Miss Kim Francis, Miss Linda Williams and MrsCaroline Ruaut for their help and advice.

References

ROBBINS, J.R., EVANKO, S.P. & VOGEL, K.G. (1997) Mechanicalloading and TGFb regulate proteoglycan synthesis in tendon.Archives of Biochemistry and Biophysics 342, 203–211.

SHAH, M., FOREMAN, D.M. & FERGUSON, M.W.J. (1995) Neutralisa-tion of TGFb1 and TGFb2 or exogenous addition of TGFb3 tocutaneous rat wounds reduces scarring. J. Cell Science 108,985–1002.

WHITBY, D.J. & FERGUSON, M.W.J. (1991) Immunohistochemicallocalisation of growth factors in fetal wound healing.Developmental Biology 147, 207–215.

Isolation and partial characterisation ofrecombinant human TIMP-1 from ChineseHamster Ovary (CHO) cell transfectants

J.K.G. CREAN, P. UPTON, P. HIRCOCK,AND I. COLLINSR þ D Systems Europe Ltd., Abingdon, Oxford, UK

Introduction

The inhibitors of metalloproteinases comprise a family ofproteins that includes TIMP-1, TIMP-2, TIMP-3 and

TIMP-4. TIMP-1 is a small (Mr 28 kD), N-glycosylatedprotein sharing sequence homology with other TIMP’sand between species. It inhibits active forms of MMPs byforming strong, non-covalent complexes in 1 : 1 molarratios. TIMP-1 has been demonstrated to bind morerapidly to MMP-3 than other TIMP’s and is a morepotent inhibitor of MMP-1 than TIMP-2. We describe asimple and reproducible method for the purification andpartial characterisation of recombinant human TIMP-1from transfected CHO cells.


CHO cells expressing human TIMP-1 were grown inhollow fibre bioreactor culture with DMEM as intra-capillary media and CHO-S-SFM II with penicillin/streptomycin as the extracapillary media. The condi-tioned media was then passed over a gelatin matrix(Prosep-gelatin, Bioprocessing) to remove any endo-genous gelatinases and buffered by mixing conditionedmedia (50% v/v), 100 mM MES, pH 6 (25% v/v), 3 M NaCl(1.5% v/v) and water (23.5% v/v) together. The adjustedconditioned media was then loaded onto a cationexchange column (Poros HS 20, Perceptive Bio-systems), washed with 120 mM NaCl, 25 mM MES,pH 6, and eluted with a 150 to 900 mM NaCl gradient.TIMP-1 containing fractions were identified by reversezymography (Oliver et al. 1991), pooled, concentratedand buffer exchanged (Pd 10, Pharmacia) into 10 mM

HEPES, pH 9. This solution was then loaded onto aanion exchange column (Poros HQ 20, PerceptiveBiosystems), washed with 25 mM HEPES, pH 8.5 andeluted with a 0 to 300 mM NaCl gradient. TIMP-1 con-taining fractions were identified by reverse zymography,pooled, concentrated and buffer exchanged into PBS,pH 7.5. These pooled fractions were loaded onto acolumn of Concanavalin A (ConA Type III-ASCL, Sigma)equilibrated with PBS, pH 7.5, with 1 mM MnCl2 andwashed with the equilibration buffer (Ward et al. 1991).The column was then eluted with PBS, pH 7.5, containing1 M mannose. Again, TIMP-1 containing fractions wereidentified by reverse zymography, pooled, concentratedand buffer exchanged into PBS, pH 7.5. Purified proteinwas examined by SDS-PAGE and Western blot usingTIMP-1 specific antibodies. Metalloproteinase inhibitoryactivity was confirmed by the inhibition of MMP-2 asdemonstrated by gelatin zymography.

Results and discussion

The combination of strong cation and anion exchangechromatography, combined with the use of a gelatin



guard column and a Con A column, facilitated theisolation of human TIMP-1 from CHO cell transfectants.The purified protein migrated with an apparent mole-cular weight of 28 kD in SDS-PAGE and in Western blotsusing a specific anti-TIMP-1 antibody. In the absence ofsuitable antibodies to affinity purify rhTIMP-1 the simple,reproducible, multi-step procedure outlined producedvirtually pure TIMP-1 from TIMP-1 CHO cell trans-fectants. The systematic use of a gelatin guardcolumn allows the removal of endogenous hamsterMMP-2 and MMP-9 while the fact that TIMP-1 is glyco-sylated can be exploited using Con A to eliminatecontaminating hamster TIMP-2.

References

OLIVER, G.W., LEFERSON, J.D., STETLER-STEVENSON, W.G. &KLEINER, D.E. (1997) Quantitative reverse zymography:Analysis of picogram amounts of metalloproteinase inhibitorsusing Gelatinase A and B reverse zymograms. Anal.Biochem. 244, 161–166.

WARD, R.V., HEMBRY, R.M., REYNOLDS, J.J. & MURPHY, G. (1991)The purification of tissue inhibitor of metalloproteinases-2from its 72 kDa progelatinase complex. Biochem. J. 278,179–187.

Transcriptional regulation of the human tissueinhibitor of metalloprotinases 1 (TIMP-1) geneinvolves elements throughout intron 1

G. DEAN AND I.M. CLARKSchool of Biological Sciences, University of East Anglia,

University Plain, Norwich, UK

Introduction

The matrix metalloprotinases (MMPs) are a family ofenzymes involved in the turnover and degradation ofextracellular matrix. The active forms of all the MMPs areinhibited by a family of specific inhibitors, the tissueinhibitors of metalloprotinases (TIMPs). Inhibition ofMMP activity by TIMPs represents an important controlmechanism. Aberrant matrix turnover is involved in anumber of pathologies including arthritis, tumour inva-sion and metastasis, and liver fibrosis (Greenwald &Golub 1994); therefore an understanding of TIMP generegulation maybe critical in developing novel therapiesfor these diseases. Initial studies have suggested thatintron-1 of the human TIMP-1 gene contains elementsinvolved in transcription regulation (Clark et al. 1997).Sequences around the boundary of exon-1/intron-1 ofhuman TIMP-1 have been shown to be involved in basal

and inducable (all trans retinoic acid and phorbol ester)transcription (Clark et al. 1997). Studies with the murineTIMP-1 gene in transgenic animals have demonstratedthat intron-1 is essential for the correct developmentalexpression of murine TIMP-1 (Flenniken & Williams,1990). We describe the deletion mapping of intron-1 ofthe human TIMP-1 gene, showing that at least twopositive and two negative elements are present in intron-1.

Material and methods

Deletions were made from the 30 to the 50 of a constructcontaining – 1718/þ988 of the human TIMP-1 gene(numbered from the transcriptional start), deleting throughintron-1 to the end of exon-1. Deletions were performedusing ExoIII/S1 nuclease (Pharmacia Biotech., nesteddeletion kit) and Bal31 (New England Biolabs). Deletionconstructs were subcloned into a CAT reporter plasmid(pBLCAT3) (Luckow & Schutz 1987). The CAT constructswere transiently transfected into primary human skinfibroblasts using the FuGene 6 transfection reagent(Boehringer Mannheim). Cells were harvested, andCAT protein levels assayed using ELISA (BoehringerMannheim).

Results

Deletion constructs were made containing intron-1sequences ranging from þ96 to þ988. When basallevel transcription was assessed, several distinct regionsof positive and negative control within intron-1 wereidentified. Deletion from þ748 to þ684 removes astrong negative element giving high levels of basaltranscription. Between þ684 and þ561, there is apositive element driving transcription only in the absenceof the negative element between þ748 and þ684; furtherdeletion of this positive element reduced basal trans-cription to zero, revealing a further negative elementbetween þ561 and þ190, after which another positiveelement gives a high level of basal transcription.

Discussion

The data from an extensive range of deletion mutants,suggests that there are at least four distinct controlregions within intron-1 of the human TIMP-1 gene; twopositive and two negative elements. These negativeelements are dominant in basal transcription, whichcorrelates with work with transgenic animals where thepresence of the whole intron suppresses expression.The role of the intronic elements in induction of thegene is unknown. Further characterisation of intron-1 isrequired to determine precisely where these positive and



negative elements are located and which transcriptionfactors are binding to them.

Acknowledgements

All work funded by the Arthritis Research Campaign, UK.

References

CLARK, I.M., ROWAN, A.D., EDWARDS, D.R., BECH-HANSEN, T., MANN,D.A., BAHR, M.J. & CAWSTON, T.E. (1997) Transcriptionalactivity of the human tissue inhibitor of metalloproteinases 1(TIMP-1) gene in fibroblasts involves elements in the promo-ter, exon 1 and intron 1. Biochem. J. 324, 611–617.

FLENNIKEN, A.M. & WILLIAMS, B.R.G. (1990) Developmentalexpression of the endogenous TIMP gene and a TIMP-lacZfusion gene in transgenic mice. Genes Dev. 4, 1094–1106.

GREENWALD, R.A. & GOLUB, L.M. (eds.) (1994) Low dose doxy-cycline inhibits pyridinoline excretion in selected patients withrheumatoid arthritis. Ann. N.Y. Acad. Sci. 732, 419–421.

LUCKOW, B. & SCHUTZ, G. (1987) CAT constructions with multipleunique restriction sites for the functional analysis of eukaryoticpromoters and regulatory elements. Nucl. Acid Res. 15(13),5490.

The effect of mechanical strain on thephosphorylation of CD44 in articularfibrocartilage cells

G.P. DOWTHWAITE†, C.R. FLANNERY*,C.W. ARCHER* AND A.A. PITSILLIDES†The Royal Veterinary College, London and *School of

Molecular and Medical Biosciences, Cardiff University,

Cardiff, UK

Introduction

The interaction between hyaluronan (HA) and its cellsurface binding proteins (HABPs) is believed to play animportant role in the development of diarthrodial joints(Edwards et al. 1994; Pitsillides et al. 1995). Indeed,disruption of HA : HABP interactions prevents formationof functional joint cavities (Dowthwaite et al. 1998). It isalso well established that immobilization of embryosprevents formation of functional joint cavities and thejoint fusions evident in such animals are associated withdecreases in HA synthetic capability and HABP expres-sion (Pitsillides 1997). Whilst these results providephenomenological evidence for the removal of move-ment-induced stimuli in mediating HA : HABP inter-actions they do not directly address the role ofmechanical stimuli in mediating HA synthesis andHA : HABP interactions in developing joints. In addition,it is known that the actin cytoskeleton and the actin

capping protein moesin, may play a role in controllingHA : HABP interactions during joint development and thatthe phosphorylation of specific amino acid residues inCD44, and possibly moesin, may facilitate changes incell : matrix interactions during development and matrixmetabolism (Dowthwaite et al. 1998; Knudson et al.1997). Therefore, the aim of this study was to assessthe effect of mechanical stimuli applied to cells isolatedfrom developing chick articular surfaces on HA synthesisand HABP expression and to examine changes in aminoacid phosphorylation of CD44 and moesin in strainedcells.

Methods

Chick articular fibrocartilage cells isolated from St 42tibio-tarsi by collagenase digestion were grown to con-fluence on plastic strips in DMEM containing 5% chickserum. Confluent cells were serum deprived for 16 hoursthen subjected to uniaxial 4 point bending at 3800 microstrain for 10 minutes at 1 Hz (n ¼ 15 strips). Controlsconsisted of cells subjected to perturbation of the culturemedia alone (flow) and unperturbed (static) cells. Atvarious time points up to 24 hrs, medium samples wereassayed for HA concentration using an ELISA plate-based assay. At 24 hours, cells were immunolabelledwith antibodies to CD44 (Ab 30189), reacted forUDPGD activity (an indicator of HA synthetic capability,4) and labelled with bHA to assess HA bindingcapacity. Alkaline phosphatase end-products for CD44and bHA were measured semi-quantitatively andUDPGD reaction products were measured quantita-tively using microdensitometry. To assess changes inamino acid phosphorylation, strained and static sam-ples at 24 h (n ¼ 5) were extracted in TBS (pH 7.5)containing 0.5% Triton X100. Detergent insolublepellets were run on 10% SDS-PAGE, electrotransferredto nitrocellulose and probed with antibodies to CD44(Ab H1), moesin, phospho-serine, phospho-tyrosineand phospho-threonine.

Results

Significant increases in media HA concentration wereapparent 10 minutes (400%, P ¼ 0.001), 6 hours (337%,P ¼ 0.001) and 24 hours (531%, P ¼ 0.001) after treat-ment in strained cells compared with controls. In addi-tion, UDPGD activity was significantly increased instrained samples at 24 hours compared with the controls(153%, P ¼ 0.001 for flow and 495%, P ¼ 0.0001 forstatic). At 24 hours, strained cells showed increasedlabelling for CD44 relative to flow (32% increase,



P ¼ 0.03) and static (69% increase, P ¼ 0.01) controls.Consistent with these results, binding of bHA to hyalur-onidase pretreated cells (total HA binding sites) wassignificantly increased in strained cells compared withflow (162% increase, P ¼ 0.01) and static (140%increase, P ¼ 0.01) cells. Using detergent insolubleextracts, two distinct bands were visible in controlsamples probed with anti-CD44 antibody (Ab H1) withan apparent molecular mass of 80–85 kD. With strain,the larger of these 2 bands had disappeared. Alsoapparent at 24 hours in static cells was a distinct bandof approximately 70 kD which labelled with antibody tothe actin capping protein moesin, with strain this bandwas decreased in intensity. Labelling with antibodies tophosphorylated amino acid residues revealed the CD44band and 70 kD moesin band to be p.ser, p.tyr and p.thrpositive in controls. The single band identified withanti-CD44 antibody in strained samples only labelledwith anti-p.thr antibody. No bands corresponding tomoesin were detectable in strained samples probed forphosphorylated amino-acid residues.

Discussion

These experiments indicate that embryonic articularfibrocartilage cells respond directly to mechanical stimuliby increasing CD44 expression, bHA binding sites andHA release into the media and that these changes inmatrix synthesis and cell : matrix interactions are asso-ciated with changes in phosphorylation of amino acidresidues in CD44 and moesin. Thus, the application ofstrain in vitro induces a number of HA related charac-teristics which resemble those evident at the develop-ing articular surfaces in ovo (Pitsillides et al. 1995;Dowthwaite et al. 1998). These results, in addition tothose indicating immobilisation-induced changes in HAsynthesis and CD44 expression in developing joints(Pitsillides et al. 1997) and HA oligosaccharide-inducedfailure to cavitate (Dowthwaite et al. 1998) endorse theconcept that HA : HABP interactions play a pivotal role inthe mechanism of diarthrodial joint morphogenesis andthat this mechanism is mediated by mechanicallyinduced stimuli. In addition, changes in the phosphoryla-tion of amino acid residues in CD44 and moesin areassociated with increases in CD44 expression, bHAbinding and increased HA synthetic capability as is alsowitnessed in articular chondrocytes after matrix depletion(Knudson et al. 1997).

Acknowledgements

This work was funded by the Arthritis and RheumatismCampaign.

References

DOWTHWAITE, G.P., EDWARDS, J.C. & PITSILLIDES, A.A. (1998) Anessential role for the interaction between hyaluronan andhyaluronan binding proteins during joint development. J.Histochem. Cytochem. 465, 1–11.

EDWARDS, J.C., WILKINSON, L.S., JONES, H.M., SOOTHILL, P., HEN-

DERSON, K.J., WORRALL J.G. & PITSILLIDES A.A. (1994) Theformation of human synovial joint cavities: a possible role forhyaluronan and CD44 in altered interzone cohesion. J. Anat.185, 355.

KNUDSON, W. ET AL. (1997) Trans. ORS 22, 34.PITSILLIDES, A.A., ARCHER, C.W., PREHM, P., BAYLISS, M.T. &

EDWARDS, J.C. (1995) Alterations in hyaluronan synthesisduring developing joint cavitation. J. Histochem. Cytochem.43, 263.

PITSILLIDES, A.A. (1997) Biology of the Synovial Joint (inpress).

Identification of chondrocyte hyaluronidasesand their differential expression in freshlyisolated and cultured human articular cartilageand chondrocytes

C.R. FLANNERY, C.B. LITTLE,C.E. HUGHES AND B. CATERSONConnective Tissue Biology Laboratories, Cardiff University, UK

Introduction

The load-bearing capacity of articular cartilage is facili-tated by the presence of high concentrations of hydro-philic sulphated-glycosaminoglycans (S-GAGs) whichare attached to the core protein of aggrecan. Main-tenance of high S-GAG concentrations is achieved viathe binding of the N-terminal G1 domain of aggrecanmonomers to hyaluronan (HA), resulting in the formationof large multimolecular aggregates. Studies conductedusing model cartilage explant culture systems havedemonstrated that both HA and aggrecan are activelymetabolized. Independent reports have demonstratedthat under steady state conditions, aggrecan and HAare catabolized in a coordinated fashion (Morales &Hascall 1988; Ng et al. 1992). In these studies, aggrecanlost from the extracellular matrix was released intoculture media, while HA appeared to be internalizedand presumably degraded by the chondrocytes (a pro-cess consistent with CD44-mediated endocytosis; Ref.Fosang et al. 1991). Conversely, under conditions ofelevated matrix catabolism (e.g. treatment with IL-1)aggrecan fragments and HA are detected in culturemedium (Hua et al. 1993), thereby implicating the poten-tial for the presence of extracellular hyaluronidase(s), anenzyme activity which has not previously been detectedin cartilage. In order to identify putative chondrocytehyaluronidases, we adopted a gene homology cloning



approach and performed RT-PCR reactions on chondro-cyte mRNAs using degenerate primers based on aminoacid sequences which are identical in two vertebrate(human) hyaluronidases, the plasma hyaluronidaseHyal-1 (Frost et al. 1997) and the sperm-associatedhyaluronidase PH-20 (Gmachl et al. 1993). Two formsof chondrocyte hyaluronidase were identified, and theexpression of mRNAs for these ‘isozymes’ in freshlyisolated and cultured human articular cartilage andchondrocytes was performed using primers specific foreach gene product.

Methods

Articular cartilage was obtained from the knee jointsof patients undergoing arthroplasty for osteoarthritis.Portions of intact cartilage were taken for direct RNAextraction or maintained as explant cultures in DMEMprior to RNA extraction. In addition, chondrocytesliberated by pronase/collagenase digestion were grownas monolayer cultures in DMEM prior to RNA extraction.Individual explant or monolayer cultures were treated for3 days with DMEM 6 20 mg/ml IL-1a or 1 mM all-transretinoic acid. Total RNA was extracted from mono-layer cultures by direct addition of Tri-Reagent (MRC,Cincinnati, OH, USA). For fresh tissue and explantcultures, samples were snap-frozen in liquid nitrogenand pulverized prior to addition of Tri-Reagent andRNA isolation. Homology cloning RT-PCR was per-formed using primers based on cDNA sequencesencoding amino acid sequences AVIDWE, ALYPSI andFPDCYN (identical for Hyal-1 and PH-20). Gene-specificRT-PCR was performed using cDNA sequencesspecific to the two hyaluronidase ‘isozymes’ identifiedby homology cloning RT-PCR.


Homology cloning to RT-PCR of chondrocyte mono-layer RNA resulted in the generation of six distinct PCRproducts (,180 bp to ,405 bp). Each PCR product wasisolated and sequenced. A homology BLAST search ofthe GenBank database revealed that three of thesequences obtained had significant homology tohuman cDNAs with the following accession numbers:U96078 (plasma hyaluronidase Hyal-1; Frost et al.1997), U03056 (candidate tumor suppressor LUCA-1;unpublished), AJ000099 (lysosomal hyaluronidaseHyal-2; unpublished), U90577 (candidate tumor sup-pressor LUCA-2; unpublished) and AC002455 (cosmidclone LUCA-13; unpublishedobservation). Thesequencesfor LUCA-1 and -2 are identical to those for plasma Hyal-1and lysosomal Hyal-2, respectively, and are contained

within thecosmidcloneLUCA-13(29 314 bpfromchromo-some 3p21.3) with LUCA-2 occurring 50 to LUCA-1.Sequence alignment of lysosomal Hyal-2/LUCA-2 withplasma Hyal-1/LUCA-1 revealed ,26% amino acid iden-tity, with highest homology occurring at and around thepeptide sequences used for primer design.

Gene-specific RT-PCR was then performed usingprimers selective for Hyal-1/LUCA-1 and Hyal-2/LUCA-2. Hyal-1 mRNA was present in all monolayercultures, but was not detected in fresh tissue or culturedexplants. Hyal-2 mRNA, however, was present in freshtissue and cultured explants, as well as in monolayercultures. Thus, the present results indicate that theexpression of Hyal-1 by chondrocytes cultured inmonolayer is an artifact of these culture conditions, andthat in intact articular cartilage, this hyaluronidase activ-ity is not present. Expression of lysosomal Hyal-2 inintact cartilage is consistent with intracellular hyaluroni-dase activity following internalization of HA and mayrepresent the only ‘true’ chondrocyte hyaluronidase.

Acknowledgements

This work was funded by the Arthritis ResearchCampaign.

References

FOSANG, A.J., TYLER, J.A. & HARDINGHAM, T.E. (1991) Effect ofinterleukin-1 and insulin like growth factor-1 on the release ofproteoglycan components and hyaluronan from pig articularcartilage in explant culture. Matrix 11, 17–24.

FROST, G.I., CSOKA, T.B., WONG, T. & STERN, R. (1997) Purifica-tion, cloning, and expression of human plasma hyaluronidase.Biochem. Biophys. Res. Comm. 236, 10–15.

GMACHL, M., SAGAN, S., KETTER, S. & KREIL, G. (1993) The humansperm protein PH-20 has hyaluronidase activity. FEBS Lett.336, 545–548.

HUA, Q., KNUDSON, C.B. & KNUDSON, W. (1993) Internalization ofhyaluronan by chondrocytes occurs via receptor-mediatedendocytosis. J. Cell Science 106, 365–375.

MORALES, T.I. & HASCALL, V.C. (1988) Correlated metabolism ofproteoglycans and hyaluronic acid in bovine cartilage organcultures. J. Biol. Chem. 263, 3632–3638.

NG, C.K., HANDLEY, C.J., PRESTON, B.N. & ROBINSON, H.C. (1992)The extracellular processing and catabolism of hyaluronan incultured adult articular cartilage explants. Arch. Biochem.Biophys. 298, 70–79.

Structural requirements for fibromodulinbinding to collagen and the control of type Icollagen fibrillogenesis

B. FONT, D. EICHENBERGER,D. GOLDSCHMIDT, M.-M. BOUTILLONAND D.J.S. HULMES



Institut de Biologie et Chimie des Proteines, CNRS UPR 412,

Lyon, France

Introduction

Fibromodulin belongs to the family of small, leucine-richproteoglycans which have been reported to interact withcollagens and to inhibit type I collagen fibrillogenesis.Decorin and fibromodulin exhibit a noticeable degree ofsequence similarity. However, as previously reported(Font et al. 1996) the domains of these moleculesimplicated in the interactions with type XII and type XIVcollagens are different, these being the dermatan sul-phate/chondroitin sulphate chain for decorin and the coreprotein for fibromodulin. At the present time the fibro-modulin domains implicated in the interactions withfibrillar collagens remain unknown. In experimentsreported here, we have sought to identify the structuralrequirements for fibromodulin interaction with collagenand for the control of type I collagen fibrillogenesis.

Results

Circular dichroism spectra and fibrillogenesis inhibitionstudies show that fibromodulin structure and its collagenfibrillogenesis control function are strictly dependent onthe presence of intact disulphide bridge(s). In addition,we show that the binding of fibromodulin (or fibromodulinderived fragments) to type I collagen is not necessarilycorrelated with fibrillogenesis inhibition. In order to iso-late fibromodulin domains, the native proteoglycan wassubmitted to mild proteolysis. We have isolated an a-chymotrypsin resistant fragment which contains the bulkof the N-terminal and central region of the moleculeincluding the leucine-rich repeats 4 and 6 reported fordecorin to be implied in type I collagen binding. Thisfragment does not bind to type I collagen. Using enzymeswith different specificities, a number of large fragments offibromodulin were obtained, suggesting a compactstructure for this molecule which is relatively resistantto proteolysis. None of these N-glycosylated fragmentswere able to bind to type I collagen in co-sedimentationexperiments.

Discussion

Taken together these results suggest that fibromodulin-type I collagen interactions leading to fibrillogenesisinhibition require more than one binding domain. Oneof these domains could be the C-terminal end of themolecule containing the disulphide loop which is absentin the chymotrypsin resistant fragment.

Acknowledgements

This work was funded by the CNRS and by the Fondationpour la Recherche Medicale.

Reference

FONT, B., EICHENBERGER, D., ROSENBERG, L.M. & VAN DER REST, M.(1996) Characterization of the interactions of type XII collagenwith two small proteoglycans from fetal bovine tendon, decorinand fibromodulin. Matrix Biology 15, 341–348.

Purification and functional studies ofpregnancy associated plasma protein A

G. GREENACRE*, A. FRAZER† ANDR.A.D. BUNNING**Department of Biomedical Sciences, Sheffield Hallam

University, Sheffield and †Section of Human Metabolism and

Clinical Biochemistry, University of Sheffield Medical School,

Sheffield, UK

Introduction

Increasing levels of pregnancy associated plasmaprotein A (PAPP-A) are produced with advancingpregnancy, a major source being the placenta (Lin etal. 1974; Bonno et al. 1994). These increasing levelsand reports of possible proteinase inhibitory andimmunomodulatory activities (Sinosich & Saunders,1987) suggest that it may act as an antiarthriticagent, involved in the amelioration of rheumatoidarthritis observed during pregnancy (Persellin 1978).We have developed a purification scheme for PAPP-Afrom late pregnancy plasma and have studied itseffects on prostaglandin E (PGE) and interleukin 6(IL-6) production by MG-63 osteosarcoma cells inorder to determine whether it is potentially immuno-suppressive and/or antiinflammatory. We have alsostudied binding of PAPP-A to human cartilage sincethis may facilitate any protective effects of PAPP-A,such as proteinase inhibition, on cartilage.


PAPP-A was purified from late term plasma using thefollowing purification protocol: ammonium sulphateprecipitation, gel filtration on Ultrogel AcA 34, ionexchange chromatography on DEAE Trisacryl usingFast Protein Liquid Chromatography and finally negativeaffinity chromatography on Sepharose 2B to which totalhuman serum antibody has been bound.



Confluent MG-63 cells were incubated for 24 hoursin the presence or absence of IL-1b (10 U/ml) and/orPAPP-A (0.5 ug/ml–50 ug/ml). The supernatants werethen assayed for PGE and IL-6 levels. PGE was mea-sured by radioimmunoassay (Richardson et al. 1985)and IL-6 using a commercial ELISA kit from LifescreenLimited. Binding of PAPP-A to human cartilage wasstudied using unfixed cryostat sections of human femoralhead cartilage. Bound PAPP-A was determined usingestablished immunohistochemical techniques.

Results

The purified PAPP-A produced a major band of ,200 kDon SDS-PAGE under reducing conditions and one bandon western blotting.

Levels of PGE were increased when the MG-63 cellswere stimulated with IL-1. PAPP-A had no effect on IL-1stimulated PGE levels and PAPP-A alone did notstimulate PGE production. IL-1 also stimulated IL-6production by the MG-63 cells. PAPP-A had no effecton IL-1 stimulated IL-6 production, but caused a slightstimulation of basal IL-6 production. Immunohistochemi-cal studies showed intense staining of the cartilagematrix particularly around the edges of the cartilage.

Discussion

PAPP-A has no effect on IL-1 stimulated PGE and IL-6production by MG-63 cells even at levels (50 ug/ml)found in blood at term, suggesting it is not immuno-suppressive or antiinflammatory. It does however bindto cartilage. The significance of this may become cleareronce other functions of PAPP-A, such as is proteinaseinhibitory activity, are fully elucidated.

Acknowledgements

This work was funded by the Arthritis and RheumatismCouncil, UK.

References

BONNO, M., OXVIG, C., KEPHART, G.M., WAGNER, J.M., KRISTENSEN,T., SOTTRUP-JENSEN, L. & GLEICH, G.J. (1994) Localization ofpregnancy associated plasma protein A and colocalization ofpregnancy associated plasma protein A messenger ribo-nucleic acid and eosinophil granule major basic proteinmessenger ribonucleic acid in placenta. Lab. Invest. 71,560–566.

LIN, T.M., HALBERT, S.P., KEIFER, D., SPELLACY, W.N. & GALL, S.(1974) Characterisation of human pregnancy associatedplasma proteins. Am. J. Obstet. Gynaecol. 118, 223.

PERSELLIN, R.H. (1978) The effect of pregnancy on rheumatoid

arthritis. Bull. Rheum. Dis. 7, 747–748.RICHARDSON, H.J., ELFORD, P.R., SHARRARD, R.M., MEATS, J.E. &

RUSSELL, R.G.G. (1985) Modulation of connective tissuemetabolism by partially purified human interleukin 1. CellImmunol. 90, 41–51.

SINOSICH, M.J. & SAUNDERS, D.M. (1987) Potential role of preg-nancy associated plasma protein A in human reproduction. J.Reprod. Immunol. 10, 55–65.

Modulation of gene expression in articularchondrocytes by oxygen partial pressure

M.J. GRIMSHAW AND R.M. MASONDivision of Biomedical Sciences, Imperial College School of

Medicine at Charing Cross Hospital, London, UK

Introduction

There is an oxygen gradient across articular cartilageranging from pO2 ,50 mm Hg at the surface to possiblyless than 10 mm Hg in the deep zone (1). Articularchondrocytes have been shown to modulate theirresponse to growth factors within this range (2). Sincethe oxygen gradient will be perturbed in OA and RAcartilage, specific chondrocyte genes are likely to beup- or down-regulated in these disorders. We are identi-fying genes which are modulated by oxygen partialpressure by culturing chondrocytes in alginate underdifferent oxygen tensions in the physiological range(0–10% pO2) and under normoxia (20% pO2).


Chondrocytes were isolated from bovine articular carti-lage of the metacarpalphalangeal joint. Cells were cul-tured in alginate beads at a density of 7.5 × 106 cells/mlalginate in DMEM medium supplemented with 10%FCS and 25 mg/ml ascorbic acid. Oxygen tensions ofthe cultures were 0, 5, 10 and 20% O2 with 5% CO2

and the balance N2. The cells were recovered from thealginate and the total RNA extracted. RNA arbitrarilyprimed polymerase chain reaction (RAP-PCR) wasperformed on the total RNA incorporating 32P-dCTPduring PCR. The products were separated by electro-phoresis on sequencing gels and the bands compared.Differentially expressed bands were amplified, TAcloned and sequenced. Differential expression was con-firmed by semi-quantitative RT-PCR by comparison withhousekeeping genes.

Results

Chondrocytes were cultured for 2 and 7 days underdifferent oxygen tensions. By semi-quantitative methods



it was found that total RNA yield is markedly reducedafter 7 days under anoxia compared to normoxia. RNAyield under hypoxia (5 and 10% Oxygen) is unaffected.Approximately 4000 RAP-PCR products were analysed.32 were found to be putatively differentially expressed.21 were up-regulated in hypoxia or anoxia whilst 9 weredown-regulated. 2 were ‘aberrantly’ up-regulated in 5or 10% oxygen, but not in 0 or 20%. These putativedifferentially expressed genes have been identified bycloning and sequencing. Genes found to be up-regulatedin hypoxia include those coding for TIMP-1 and Inter-leukin-8 receptor b. Genes found to be up-regulated innormoxia include those coding for collagen V, collagenXII and T-cell Early Activation Protein.

Discussion

<1% of genes expressed by chondrocytes are modulatedby oxygen tension although the chondrocyte apparentlydown-regulates total RNA synthesis under anoxic con-ditions. Modulation of gene expression occurs within thephysiological range of pO2 as well as between hypoxiaand normoxia. Genes modulated by oxygen includethose coding for both structural and regulatory proteins.Particularly important is the finding that TIMP-1 isexpressed under hypoxia but not under normoxia, thepO2 under which most previous laboratory investigationshave taken place.

Acknowledgements

This work was supported by an Arthritis and RheumatismCouncil post-graduate studentship (GMJ) and by TheSpecial Trustees Of Charing Cross Hospital.

References

SILVER, I.A. (1975) Measurement of pH and ionic composition ofpericellular sites. Phil. Trans. Roy. Soc. Lond., Series B, 271,261–272.

YSART, G.E. & MASON, R.M. (1994) Responses of articularcartilage explant cultures to different oxygen tensions. Bio-chim. Biophys. Acta. 1221, 15–20.

Alginate beads stabilise chondrocytephenotype despite abnormal collagenassembly

K.E. GREGORY*, M.M. MARSDEN*,J. ANDERSON-MACKENZIE*J.B.L. BARD†, S.P. ROBINS‡ ANDD.J.S. HULMES*§

Departments of *Biochemistry and †Anatomy, University of

Edinburgh, Edinburgh, UK, ‡The Rowett Research Institute,

Aberdeen, UK and §Institut de Biologie et Chimie des Proteines,

UPR412-CNRS, Lyon, France

Introduction

Alginate bead cultures are commonly used to studychondrocoyte differentiation in vitro. While proteoglycanmetabolism in this system has been studied in detail,comparatively few studies have been carried out onthe structure and composition of the collagenouscomponents of the matrix.


The collagens produced by chick embryo chondro-cytes cultured in alginate beads were investigatedboth biochemically (SDS-PAGE, immunoblotting andcolumn chromatography) and morphologically (electronmicroscopy).

Results

The cartilage phenotype was maintained for at least 14days, as indicated by the production of the cartilagespecific collagens II, IX and XI. The distributions ofcollagens among the three different compartments(cells and their associated matrix (CM), further removedmatrix (FRM) and culture medium) were different. Whilethe ratio of collagens II/IX/XI in the combined CM/FRMwas similar to that in vivo, there were large amounts ofcollagen IX (mainly in proteoglycan form) in the culturemedium. Inhibition of lysyl oxidase activity by b-amino-propionitrile led to an overall decrease in collagenproduction. In contrast to the biochemical observations,collagen morphology in the extracellular matrix ofalginate cultures was not in the form of the expected64 nm banded fibrils, but instead it consisted of a thinfilamentous network together with segment-long-spacing(SLS)-like aggregates. This abnormal morphology wasfound to be a result of alginate disrupting normalassembly.

Discussion

These results thus confirm that the alginate environmentallows chondrocytes in culture to maintain their pheno-type, while normal fibrillogenesis is disrupted. Thus themorphology of the collagenous matrix has little effect onthe differentiated phenotype of chondrocytes.



Acknowledgements

This work was funded by the BBSRC (studentship toKEG) and by the Arthritis and Rheumatism Council(project grant to DJSH and SPR).

Quantification of rare mRNA’s derived fromdifferential display

J.F. HUGGETT AND D.J. MASONSchool of Molecular and Medical Biosciences,

University of Wales College Cardiff, Cardiff, UK

Introduction

Osteocytes form a network of intercommunicatingcells throughout cortical bone that are known to respondto mechanical loading in vivo. Genes regulated bymechanical loading in these cells are likely to be involvedin the osteogenic response and are therefore potentialtargets for therapeutic manipulation of bone mass.Previously we have used differential display (DRD), onosteocyte RNA, to identify genes regulated bymechanical loading in vivo (Mason et al. 1997). SinceDRD is a PCR based technique it is often impossible toconfirm gene regulation in small in vivo samples usingstandard techniques. One transcript found to beregulated by mechanical loading is a Naþ dependentglutamate transporter (GLAST) previously reported tobe responsible for termination of excitatory signals in theCNS. The role of GLAST in osteocytes is not clear, so weare currently using a novel method of quantitativeRT-PCR to investigate its role in bone cell signalling.


We have used a combined thermocycler and fluorometer(Lightcycler, Idaho Technology) that allows calculation ofrelative amounts of specific mRNA’s by real time PCR.Thermocycling is achieved by regulated airflow andsamples are amplified in glass capillary tubes allowingvery fast reaction times. The reaction mix contains afluorescent dye (Sybr Green) that binds dsDNA and theLight cycler detects fluorescence of each sample afterevery PCR cycle. By comparing PCR product concentra-tions within the logarithmic phase of the reaction, specificmRNA levels in each sample may be estimated.

b-actin and GLAST PCR products were each clonedinto pGEM-T (Promega) and transformed into competentE. coli (JM109, Promega). Plasmid DNA was extractedfrom the transformants and its concentration and puritydetermined by absorbance ratios at wavelengths of 230,

260 and 280. A dilution series generated from each clonewas amplified in the Light cycler using published primers(b-actin, Araki 1993; GLAST, Mason et al. 1997). StartingDNA concentration was calculated from the PCRproduct fluorescence and found to be a good estimateof the actual DNA concentration. Levels of mRNAexpression were compared in rat bone and brain, andhuman osteosarcoma cells (HOS). Total RNA wasextracted using Ultraspec II (Biogenesis) and reversetranscribed after priming with oligo dT (Promega).cDNA’s from each sample were then amplified withb-actin and GLAST primers and the concentration ofeach product determined by comparison with theappropriate dilution series. Sample cDNA concentrationswere normalised to the housekeeping gene b-actin andrelative amounts of GLAST cDNA calculated.

Results

The starting copy numbers estimated by comparison ofPCR product concentrations in the logarithmic phase ofthe reactions are shown in Table 1.

Discussion

These data show that GLAST mRNA is 100× moreabundant in brain than in bone or HOS after normali-sation to the housekeeping gene b-actin. So far we haveaccurately quantified a starting copy number of 27 000and believe that further optimisation using mRNA con-structs and template-specific probes will increase thissensitivity. We conclude that this technology is ideallysuited to quantifying specific mRNA’s in very small bonesamples derived from in vivo experiments.

References

ARAKI, N. (1993) Rapid and sensitive method for quantitation ofbone gla protein mRNA using competitive PCR. J. BoneMiner. Res. 8, 313–322.

MASON, D.J., SUVA, L.J., GENEVER, P.G. ET AL. (1997) Mechanicallyregulated expression of a neural glutamate transporter inbone: A role for excitatory amino acids as osteotropicagents. Bone 20(3), 199–205.



Table 1

b-actin copy GLAST copy GLAST copy numberSample number number normalized to b-actin

Brain 1.16 × 109 3.05 × 107 4.5 × 107

Bone 1.71 × 109 2.54 × 105 2.5 × 105

HOS 4.53 × 108 1.57 × 104 5.9 × 105

Human cathepsin K cleaves native type I and IIcollagens at the N-terminal end of the triplehelix

W. KAFIENAH*, D. BROMME†,D.J. BUTTLE*, L.J. CROUCHER* ANDA.P. HOLLANDER**Section of Human Metabolism and Clinical Biochemistry in the

Division of Biochemical and Musculoskeletal Medicine,

University of Sheffield Medical School, Sheffield, UK and

†Department of Human Genetics, Mount Sinai School of

Medicine, New York, USA

Introduction

Cathepsin K is highly expressed in osteoclasts (Drake etal. 1996) with some reports demonstrating expression inhypertrophic chondrocytes and synoviocytes from RAand OA patients. It has potent collagenolytic activityagainst type I collagen even at pH values as high as6.5 (Bromme et al. 1996). The aim of this study was todetermine if it also cleaves type II collagen in the triplehelix and to identify the helical cleavage site(s) in types Iand II collagens.


Soluble human and bovine type II collagen, and rat type Icollagen were incubated with cathepsin K at 258C for 6 hbefore the reaction was stopped with L-trans-epoxysuc-cinyl-leucylamido(4-guanidino)-butane. Reaction mix-tures were analysed by SDS-PAGE. Anti-peptideantibodies specific to an N-terminal helical region(AH12L3), a C-terminal helical region (AH9L2), or acentral helical region (CB11B) of a1(II) chain, were usedto probe for fragments using western blotting. N-terminalsequencing was performed for some fragments.

Results

Analysis by SDS-PAGE of the collagen digests showedthat optimal activity of cathepsin K against native type IIcollagen was between pH 5.0 and 5.5 and againstdenatured collagen between pH 4.0 and 7.0. Theenzyme cleaved telopeptides as well as the a1(II)chains, generating multiple fragments in the range of90–120 kD. The collagenolytic activity was not due to acontaminating metalloenzyme or serine proteinase as itwas not inhibited by 1,10-phenanthroline, EDTA, or3,4-dichloroisocoumarin. Western blotting using anti-peptide antibodies to different regions of the a1(II)chain suggested that cathepsin K cleaved native a1(II)

chains in the N-terminal region of the helical domainrather than at the well-defined collagenase cleavage site.This was confirmed by N-terminal sequencing of one ofthe fragments, revealing cleavage at a Gly-Lys bond, 58residues from the N-terminus of the helical domain.Using a similar approach, cathepsin K was found tocleave native type I collagen close to the N-terminus ofits triple helix.

Discussion

These results indicate that cathepsin K could play a rolein the turnover of cartilage type II collagen, as well astype I collagen.

Acknowledgment

This research was funded by a grant from the UK Arthritisand Rheumatism Council.

References

BROMME, D., OKAMOTO, K., WANG, B.B. & BIROC, S. (1996) Humancathepsin O2, a matrix protein-degrading cysteine proteaseexpressed in osteoclasts. Functional expression of humancathepsin O2 in Spodoptera frugiperda and characterizationof the enzyme. J. Biol. Chem. 271, 2126–2132.

DRAKE, F.H., DODDS, R.A. & JAMES, I.E. ET AL. (1996) Cathepsin K,but not cathepsins B, L, or S, is abundantly expressed inhuman osteoclasts. J. Biol. Chem. 271, 12511–12516.

Identification of an a2b1-binding sequence incollagen I

C.G. KNIGHT, L.F. MORTON, D.J. ONLEY,A.R. PEACHEY, R.W. FARNDALE ANDM.J. BARNESBiochemistryDepartment,CambridgeUniversity,Cambridge,UK

Introduction

Integrin a2b1 is an important collagen receptor that playsa crucial role in collagen-platelet interaction. Severalconformation-dependent a2b1-binding sites have beendetected in the collagen I molecule (Morton et al. 1994).One such site occurs in the fragment a1(I)CB3 (Mortonet al. 1994; Staatz et al. 1991), and on the basis ofinhibitory activity of short, linear (non-helical) peptides,this site has been ascribed to the sequence DGEA,residues 435–438 of the a1(I) chain (Staatz et al.1991). We have sought to define a2b1-bindingsequences in a1(I)CB3 by synthesizing the fragment asseven overlapping triple-helical peptides and examining



their ability to support a2b1-mediated cell adhesion andbinding of isolated a2b1.


Peptides were synthesized by standard Fmoc chemistryand characterized by mass spectrometry. Each sequencewas synthesized with additional GPP* (P* ¼ Hyp) tripletsat each end, to ensure triple-helical stability, which wasassessed by polarimetry. a2b1 was isolated from humanplatelets by affinity chromatography on collagen-Sephar-ose. Binding to immobilized peptides was assessed usingbiotinylated receptor. Integrin-mediated cell attachmentto peptides was measured as before, using anti-a2 mAb6F1 to block (Morton et al. 1997).

Results

Only two peptides, peptides 5 and 6, bound a2b1,binding being at least as good as to collagen. Onlythese two peptides supported HT 1080 cell adhesion.This adhesion was fully blocked by 6F1. Plateletadhesion occurred to all peptides, part of which wasMg2þ-dependent. However, only peptides 5 and 6exhibited cation-dependent adhesion which could beblocked by 6F1. A peptide containing the overlapsequence between 5 and 6 also bound a2b1 andsupported a2b1-mediated cell adhesion, indicating thelocation of an a2b1-binding sequence in the overlap, a15-mer sequence, GFP*GERGVEGPP*GPA, residues502–516 of the a1(I) chain. We were unable to findevidence of an a2b1-binding site in peptide 2, thetriple-helical peptide containing the DGEA sequence.

Discussion

The sequence described here as an a2b1-recognitionsite in collagen I is at a locus close to one we partiallyidentified in collagen III as an a2b1-binding sequence(Morton et al. 1997) but we cannot as yet say if there isexact equivalence. The sequence does not contain anaspartyl residue, as normally required for integrin recog-nition, but we believe the glutamyl residues present maybe essential as we proposed for the sequence in collagenIII (Morton et al. 1997).

Acknowledgements

This work was supported by the British Heart Foundationand the Medical Research Council of which MJB, LFMand ARP are external staff members. The authors aregrateful to Dr. Barry Coller for mAb 6F1, and to Dr. PeterSmethurst and Ms. Anthea Messent for gifts of a2b1.

References

MORTON, L.F., PEACHEY, A.R., ZIJENAH, L.S., GOODALL, A.H.,HUMPHRIES, M.J. & BARNES, M.J. (1994) Conformation-depen-dent platelet adhesion to collagen involving integrin alpha 2beta 1-mediated and other mechanisms: multiple alpha 2 beta1-recognition sites in collagen type I. Biochem. J. 299, 791–797.

MORTON, L.F., PEACHEY, A.R., KNIGHT, C.G., FARNDALE, R.W. &BARNES, M.J. (1997) The platelet reactivity of synthetic pep-tides based on the collagen III fragment alpha1(III)CB4.Evidence for an integrin alpha2beta1 recognition site involvingresidues 522-528 of the alpha1(III) collagen chain. J. Biol.Chem. 272, 11044–11048.

STAATZ, W.D., FOK, K.F. & ZUTTER, M.M., ADAMS, S.P.,RODRIGUEZ, B.A. & SANTORO, S.A. (1991) Identification of atetrapeptide recognition sequence for the alpha 2 beta 1integrin in collagen. J. Biol. Chem. 266, 7363–7367.

Roles played by stromelysin-1, neutrophilcollagenase and collagenase-3 inchondrocyte-mediated cartilage breakdown

L. DIDEM KOZACI, C.C. BROWN,C. ADCOCKS, A.P. HOLLANDER ANDD.J. BUTTLESection of Human Metabolism and Clinical Biochemistry,

Division of Biochemical and Musculoskeletal Medicine,

University of Sheffield Medical School, Sheffield, UK

Introduction

The aim of this study was to determine the roles ofstromelysin-1, neutrophil collagenase and collagenase-3 in chondrocyte-mediated cartilage proteoglycan andtype II collagen breakdown in cartilage.


Bovine nasal cartilage explants were cultured in serumfree DMEM with or without interleukin 1a (rhIL1a),tumour necrosis factor-a (TNF-a) and retinoic acid(Ret), in the absence or presence of low molecularweight synthetic MMP inhibitors BB-94 (batimastat) andBB-3437. Proteoglycan and collagen type II release weredetermined by colourimetric assay and immunoassay,respectively. MMP activity in conditioned culture mediawas determined by using a quenched fluorescentsubstrate assay which detects all MMPs.

Results

Batimastat, a broad spectrum MMP inhibitor produced apotent inhibitory effect on both proteoglycan and typeII collagen loss. BB-3437, a selective inhibitor ofstromelysin, neutrophil collagenase and collagenase-3,



showed a weak but dose-dependent inhibition on IL1stimulated type II collagen breakdown. It had no effect onloss of proteoglycan.

Discussion

It is unlikely that stromelysin-1, neutrophil collagenaseand collagenase-3 contribute to chondrocyte-mediatedproteoglycan breakdown. However, partial inhibition ofIL1-stimulated type II collagen degradation by BB-3437suggests a minor role for one or more of theseenzymes.

References

BUTTLE, D.J., HANDLEY, C.J., ILIC, M.Z., SAKLATVALA, J., MURATA, M.& BARRETT, A.J. (1993) Inhibition of cartilage proteoglycanrelease by a specific inactivator of cathepsin B and an inhibitorof matrix metalloproteinases. Evidence for two convergingpathways of chondrocyte mediated proteoglycan degradation.Arthritis. Rheum. 36, 1709–1717.

KOZACI, L.D., BUTTLE, D.J. & HOLLANDER, A.P. (1997) Degradationof type II collagen, but not proteoglycan, correlates withmatrix metalloproteinase activity in cartilage explant cultures.Arthritis Rheum. 40, 164–174.

Structure of five chondroitin sulphate linkageregion octasaccharides and their digestion bychondroitinase ABC Endolyase

R.M. LAUDER*, T.N. HUCKERBY† ANDI.A. NIEDUSZYNSKI**Department of Biological Sciences and †The Polymer Centre,

University of Lancaster, Lancaster, UK

Introduction

The enzyme chondroitin sulphate ABC lyase (E.C.4.2.2.4) has recently been shown, in most commercialpreparations, to be two enzymes with endolyase andexolyase activities respectively (Hamai et al. 1997). Their

activity has been shown to differ, the endolyase beingunable to cleave a tetrasaccharide into disaccharides,while the exolyase can. The authors proposed thatchondroitin sulphate ABC endolyase could be used toisolate linkage region oligosaccharides retaining at leasta tetrasaccharide attached to the core linkage region.

We report the isolation of five octasaccharides frombovine articular cartilage aggrecan. They vary in theirsulphation pattern and have the following generalstructure:

DUAb(1-3)GalNAc[4/6S]b(1-4)Glcb(1-3)GalNAc[6S]-b(1-4)Glcb(1-3)Galb(1-3)Galb(1-4)Xyl-ol

Methods

Following the isolation of aggrecan by standard methods,CS chains were depolymerised by chondroitin sulphateABC endolyase and the protein core by papain. Thelinkage regions were released by b-elimination andpurified by ion exchange chromatography.

Their structures were determined by HPAE chromato-graphy of the oligosaccharides released followingdigestion by chondroitinase ACII which releases bothdisaccharides and chondroitinase ABC endolyase whichreleased only the non-reducing terminal disaccharide.


This is the first study to isolate CS linkage regionoligosaccharides of this length, allowing an examinationof the sulphation pattern of the tetrasaccharides adjacentto the linkage region. The majority have 6-sulphation ofboth GalNAc residues, however a significant portion(37%) have one or more GalNAc residue unsulphated.Recent work suggests that, on average, only 5% of thetotal GalNAc residues are unsulphated, suggesting apreferential location of such residues close to the linkageregion. This is confirmed by differences in abundance ofoligosaccharides II and III. Oligosaccharide II, with an



Table 1. Occurrence of disaccharides following digestion by chondroitinase enzymes

Disaccharides observed following digestion by

ACII ABC Endolyase

00 04 06 26 00 04 06 26 Deduced linkage type ID %

þ ¹ ¹ ¹ þ ¹ ¹ ¹ DUAb(1-3)GalNAc0Sb(1-4)Glcb(1-3)GalNAc0S-Link I 11þ ¹ þ ¹ ¹ ¹ þ ¹ DUAb(1-3)GalNAc6Sb(1-4)Glcb(1-3)GalNAc0S-Link II 21þ ¹ þ ¹ þ ¹ ¹ ¹ DUAb(1-3)GalNAc0Sb(1-4)Glcb(1-3)GalNAc6S-Link III 5¹ ¹ þ ¹ ¹ ¹ þ ¹ DUAb(1-3)GalNAc6Sb(1-4)Glcb(1-3)GalNAc6S-Link IV 60¹ þ þ ¹ ¹ þ ¹ ¹ DUAb(1-3)GalNAc4Sb(1-4)Glcb(1-3)GalNAc6S-Link V 4

unsulphated GalNAc closest to the linkage region issignificantly more abundant than III, which has thisresidue sulphated and the other unsulphated.

We confirm that chondroitin sulphate ABC endolyasecannot cleave an isolated tetrasaccharide, however it willcleave the GalNAcb(1-4)Glcb(1-3) bond closest to thenon-reducing terminal in these oligosaccharides. Thecleavage yields a disaccharide and hexasaccharidelinkage region, and proceeds irrespective of the sulpha-tion status of the GalNAc residues. No other bond in theoctasaccharide linkage region is susceptible to digestionby this enzyme.

Acknowledgements

The authors thank the Arthritis and Rheumatism Council,UK for support (Grant number N0511).

References

HAMAI, A., HASHIMOTO, N., MOCHIZUKI, H., KATO, F., MAKIGUCHI, Y.,HORIE, K. & SUZUKI, S. (1997) Two distinct chondroitin sulfateABC lyases – an endoeliminase yielding tetrasaccharides andan exoeliminase preferentially acting on oligosaccharides.J. Biol. Chem. 272, 9123–9130.

Breakdown of articular cartilage bychaperonin 60

J.C. LEWTHWAITE*, J. BIRD‡,A. MILLER†, M.T. BAYLISS‡ ANDB. HENDERSON**Eastman Dental Institute, University College London,

‡The Royal Veterinary College, University of London and

†Imperial College, London, UK

Introduction

Chaperonin 60 (cpn60) is a member of a family ofintracellular protein-folding proteins. A number of mole-cular chaperones, including cpn60, have recently beenshown to have activities unrelated to their protein foldingfunction. This includes, the ability to stimulate synthesisof pro-inflammatory cytokines from various cells. Studiesfrom our laboratory have demonstrated the ability ofchaperonins to induce bone resorption in vitro this maybe mediated by recruitment of osteoclasts (Kirby et al.1995; Meghji et al. 1997). Here we have studied theeffects of cpn60 on articular cartilage.

Methods

Pig articular cartilage explants were cultured for fourdays in the presence of purified (LPS-low) cpn60 from

E. coli (also termed groEL). Cartilage proteoglycandegradation was assessed by the release of glycos-aminoglycans (GAGs) into the medium and the loss ofGAGs from the tissue. Cartilage proteoglycan synthesiswas assessed by the incorporation of [35S]sulphate intonewly synthesised proteoglycans during the last 6 hoursof culture.

Results

cpn60 was shown to induce the dose-dependentbreakdown of pig articular cartilage explants by bothstimulating cartilage proteoglycan degradation andinhibiting cartilage proteoglycan synthesis following a 4day culture period.

Discussion

These results suggest that bacterial cpn60 may playa role in the pathology of infective arthritis. Studiesare currently underway to investigate the effects ofmammalian chaperonins on cartilage and bone.

Acknowledgements

This work was supported by grants from the Sir JulesThorn Charitable Trust and Home of Rest for Horses.

References

KIRBY, A.C., MEGHJI, S., NAIR, S.P. ET AL. (1995) The potent bone-resorbing mediator of Actinobacillus actinomycetemcomitans



�16

12

8

4

0Control 1 10 100 1000 IL-1 retA

cpn60 ng/ml

35S

O4 i

nco

rpo

rati

on

nm

ol/

hr/

µg D

NA

* *

**

**

**

***

Figure 1. Inhibition of cartilage proteoglycan synthesisinduced by cpn60 (B) in comparison to IL-1b (10 ng/ml d)and retinoic acid (1 mM B).Control data (A) are expressed asmean 6 standard error. Statistical significance was assessedusing Students t-test where *P < 0.05, **P < 0.01,***P < 0.001.

is homologous to the molecular chapterone GroEL. J. Clin.Invest. 96, 1185–1194.

MEGHJI, S., WHITE, P., NAIR, S. ET AL. (1997) Mycobacteriumtuberculosis chaperonin 10 stimulates bone resorption: apotential contributory factor in Pott’s disease. J. Exp. Med.186(8), 1241–1246.

Normal and accelerated aggrecan turnoverin vitro does not involve MMP cleavage atN341–F342

C.B. LITTLE*, C.R. FLANNERY*,C.E. HUGHES*, C. DENT† ANDB. CATERSON**Connective Tissue Biology Laboratories and †College of

Medicine, University of Wales, Cardiff, UK

Introduction

Articular cartilage contains a high concentration of thelarge aggregating proteoglycan aggrecan. The C-terminal region of aggrecan is heavily substituted withhydrophilic glycosaminoglycan (GAG) chains which con-tribute to the mechanical properties of articular cartilage.Loss of GAG-bearing aggrecan fragments from articularcartilage is one of the central pathophysiological eventsin rheumatic diseases and is believed to occur throughproteolytic cleavage of the core protein in the inter-globular domain (IGD) between the two N-terminalglobular regions (Sandy et al. 1992). Sequence analysisof aggrecan fragments isolated from synovial fluidindicate that the predominant cleavage occurs betweenE373 and A374 by an as yet unidentified enzyme(s)‘aggrecanase’ (Sandy et al. 1992). However, aggrecanfragments terminating with the amino acid sequence. . DIPEN have been isolated from human articularcartilage (Flannery et al. 1992; Sztrolovics et al.1997; Lark et al. 1997), and a small fragment withthe N-terminus FFGVG . . has been found in humansynovial fluid (Fosang et al. 1996). These fragmentscan be generated by matrix metalloproteinase (MMP)cleavage of the IGD between N341 and F342. Whetherthis MMP cleavage of the aggrecan IGD occurs as aprimary or secondary event remains unclear. In thepresent study we have investigated generation of aggre-canase and MMP derived aggrecan fragments in vitroand the expression of MMP-3 and -13 in bovine, porcineand human articular cartilage.

Methods

Articular cartilage explants (10–20 mg wet weight) frombovine and porcine metacarpophalangeal joints and

human knees were cultured for 72 h in DMEM containing10% FCS. Tissue was then washed in serum free DMEMand cultured in triplicate for 96 hours in serum freeDMEM 6 10¹6 M RA or 10 ng/ml IL-1. At the terminationof culture, two explants were used for extraction ofmatrix associated aggrecan fragments. Proteoglycans inextracts and their associated media were ethanol pre-cipitated, dissolved in Tris acetate and the GAG contentdetermined using DMMB. Samples were deglycosylatedwith Chondroitinase ABC and Keratanase I and II andseparated by 4–12% gradient SDS-PAGE (20 mgGAG/lane). Western blotting was performed withmonoclonal antibodies as follows: BC-13 and BC-3recognising the aggrecanase generated C and Ntermini . . . ITEGE and ARGSV . . . respectively; BC-4and BC-14 recognising the MMP generated C and Ntermini . . . DIPEN and FFGVG . . . respectively; and2-B-6 recognising chondroitinase generated C4S epi-tope. The third explant from each experiment was usedfor total RNA extraction and RT-PCR using primers forGAPDH, MMP-3 and MMP-13.

Results

In all three species, RA and IL-1 increased GAG releasefrom cartilage explants over the 4 days of culture whencompared with controls. Bovine and porcine cartilageshowed a greater response to these catabolic agentsthan human cartilage. The release of GAG was highest inIL-1 treated cultures of bovine and porcine cartilage. Inhuman cartilage RA treatment resulted in highest GAGrelease. In all cultures, multiple C4S containing (2-B-6positive) aggrecan fragments (>200 kD–70 kD) werereleased into the medium however the banding patternin IL-1 treated cultures was different from the control andRA samples. In all species the medium from RA and IL-1,but not control cultures contained numerous BC-3positive bands (190 kD–60 kD). In contrast, no BC-14reactive bands were evident in medium from anycultures. The tissue extracts from RA and IL-1 treatedbovine and porcine cultures contained a BC-13immunoreactive band (70 kD) but no BC-4 reactivematerial. In contrast, the extracts of control, RA andIL-1 human cartilage contained both BC-13 and BC-4reactive bands. There was no difference in the intensityof BC-4 reactivity between control, RA and IL-1 treatedcultures while BC-13 staining intensity was increased inRA treated cultures. MMP-13 was expressed in allcartilages examined (although weakly in human tissue)with no apparent differences between the control andstimulated cultures. MMP-3 expression was upregulatedby RA and IL-1 in bovine cartilage and only by IL-1 in



porcine tissue. In human cartilage, expression of MMP-3was similar under all three culture conditions.

Discussion

This is the first report which has simultaneously evalu-ated the generation of aggrecan fragments by bothaggrecanase and MMPs and further, correlated thiswith the expression of two MMPs shown in vitro tocleave the aggrecan IGD. This work has clearly shownthat despite the expression of MMPs by the chondro-cytes, MMP cleavage at the N341 and F342 of aggrecanwas not involved in normal or accelerated turnover invitro, either as a primary or secondary event, in eitherbovine or porcine cartilage. Whether MMP cleavageoccurs at sites not recognised by the BC-4 and BC-14is unclear, however digestion of bovine and porcinearticular cartilage with recombinant MMP-1 and -3resulted in the release of BC-14 reactive fragments.The BC-4 reactive fragments in human cartilage extractsare indicative of MMP related cleavage of the IGD.However, the fact that no increase in BC-4 reactivefragments was evident despite the RA and IL-1 stimu-lated GAG release, coupled with the lack of BC-14reactive fragments in the medium, strongly suggestedthat cleavage at the MMP site was not associated withaggrecan turnover during the course of the culture. Anage related increase in . . VDIPEN epitope has beenobserved in normal human cartilage (Lark et al. 1992;Sztrolovics et al. 1997) and one may postulate in light ofthe results of the present study, that the MMP(s) respon-sible for the aggrecan cleavage in these previous studieswere not of chondrocytic origin.

References

FLANNERY, C.R., LARK, M.W. & SANDY, J.D. (1992) Identification ofa stromelysin cleavage site within the interglobular domain ofhuman aggrecan. Evidence for proteolysis at this site in vivo inhuman articular cartilage. J. Biol. Chem. 267, 1008–1014.

FOSANG, A.J., LAST, K. & MACIEWICZ, R.A. (1996) Aggrecan isdegraded by matrix metalloproteinases in human arthritis.Evidence that matrix metalloproteinase and aggrecanaseactivities can be independent. J. Clin. Invest. 98, 2292–2299.

LARK, M.W., BAYNE, E.K. & FLANAGAN, J. ET AL. (1997) Aggrecandegradation in human cartilage. Evidence for both matrixmetalloproteinase and aggrecanase activity in normal osteo-arthritic, and rheumatoid joints. J. Clin. Invest. 100, 93–106.

SANDY, J.D., FLANNERY, C.R., NEAME, P.J. & LOHMANDER, L.S.(1992) The structure of aggrecan fragments in human syno-vial fluid. Evidence for the involvement in osteoarthritis of anovel proteinase which cleaves the Glu 373-Ala 374 bond ofthe interglobular domain. J. Clin. Invest. 89, 1512–1516.

SZTROLOVICS, R., ALINI, M., ROUGHLEY, P.J. & MORT, J.S. (1997)Aggrecan degradation in human intervertebral disc and articu-lar cartilage. Biochem. J. 326, 235–241.

Differential expression of proteoglycanepitopes by ovine intervertebral disc cells incalcium alginate microspheres

J. MELROSE, S. SMITH, P. GHOSH ANDT.K.F. TAYLORRaymond Purves Bone and Joint Research Laboratories,

The University of Sydney, The Royal North Shore Hospital,

St. Leonards, NSW, Australia

Introduction

The culture of intervertebral disc (IVD) cells has beenhampered by the lack of a suitable culture system whichmaintains cellular viability and phenotypic expression invitro. This study describes a culture system which pro-vides these requirements. We have previously used thealginate bead culture system (Melrose et al. 1997a, b) toculture IVD cells and have identified catabolised frag-ments of aggrecan in media samples by Affinity (Melroseet al. 1995) and Western blotting (Melrose et al. 1997a, b).Elevated levels of decorin and biglycan were alsoidentified in IVD cells cultured from degenerate ovineIVDs (Melrose et al. 1997b). The bead incorporatedproteoglycans (PGs) were not assessed in these earlierstudies, this has been rectified by the present study.


Ovine IVDs were dissected into annulus fibrosus (AF),transitional zone (TZ) and nucleus pulposus (NP) and thecells released by enzymatic digestion using collagenase/pronase/DNAase (Melrose et al. 1997a, b). The disc cellswere established in alginate bead culture at a density of1–3 million/ml and cultured in Hams-F12 : DMEM (1 : 1)containing 10% foetal calf serum. Beads were collectedon days 2, 5 and 10 and their DNA contents determined.Beads were also examined immunohistochemically toshow PG expression patterns using a panel of mono-clonal antibodies to specific PG epitopes. Disc cellviability in culture was assessed using a colorometricformazan cell viability dye (MTS) and also with theformaldehyde fixable semi-permanent fluorescent dyescell tracker green (5-chloromethyl fluorescein diacetate)and ethidium homodimer-1 to differentiate live and deadcells respectively.

Results

Disc cells from the AF, NP and TZ all grew successfully inthe alginate bead culture system. Cells from the AF andTZ proliferated faster than NP cells however NP cellssynthesised more PG per cell than the AF or TZ cells. On



day 10 some AF cells had actually proliferated out of thebeads and established themselves as monolayers whichhad a characteristic fibroblastic morphology, these cellssynthesised type I collagen almost exclusively, virtuallyno type II collagen was detected. PGs substituted withkeratan sulphate and chondroitin sulphate (CS) werestrongly expressed in all disc cell cultures. The 7-D-4epitope, an atypical CS isomer present in embryoniclimb bud, osteoarthritic and hypertrophic growth platecartilage PGs (Visco et al. 1993) was strongly expressedby NP cells in culture.

Discussion

The variable expression pattern of PG epitopes anddifferential proliferative rates of disc cells we observedin alginate bead cultures is consistent with disc cellheterogeneity which has been demonstrated in vivo.

Acknowledgements

This study was funded by the National Health and MedicalResearch Council of Australia. The Mab 7-D-4 was a kindgift from Prof. Bruce Caterson, Dept of Molecular andMedical Biosciences, University of Wales, Cardiff.

References

MELROSE, J., NUMATA, Y. & GHOSH, P. (1995) Biotinylatedhyaluronan: a versatile and highly sensitive probe capableof detecting nanogram levels of hyaluronan binding proteins(hyaladherins) on electroblots by a novel affinity detectionprocedure. Electrophoresis, 17, 205–212.

MELROSE, J., TAYLOR, T.K.F., GHOSH, P., LATHAM, J. & MOORE, R.(1997a) Topographical variation in the catabolism of aggrecanin an ovine annular lesion model of experimental discdegeneration. J. Spinal Dis. 10, 55–67.

MELROSE, J., GHOSH, P., TAYLOR, T.K.F., VERNON-ROBERTS, B.,LATHAM, J. & MOORE, R. (1997b) The elevated synthesis ofdecorin and biglycan by annular fibrochondrocytes in an ovineannular lesion model of experimental disc degeneration.Eur. Spine. J. (in press).

VISCO, D.M., JOHNSTONE, B., HILL, M.A., JOLLY, G.A. & CATERSON,B. (1993) Immunohistochemical analysis of 3-B-3(-) and7-D-4 epitope expression in canine osteoarthritis. ArthritisRheum. 36, 1718–1725.

Endothelial presentation of IL-8 to neutrophils:evidence for chemokine transcytosis

J. MIDDLETON†, S. NEIL*, J. WINTLE*AND A. ROT**Novartis Research Institute, Vienna, Austria and †Leopold

Muller Arthritis Research Centre, Orthopaedic Hospital,

Oswestry, UK

Introduction

Interleukin-8 (IL-8) is implicated in inducing neutrophilemigration at sites of inflammation. It is proposed that inthe process of transmigration the chemokine is pre-sented to the circulating neutrophils immobilised on theendothelium, probably bound to cell surface proteogly-can or receptor (Rot et al. 1996). However there is littleexperimental evidence to support this proposal. There-fore we examined role of endothelial IL-8 binding sites inleucocyte emigration using immuno-electron microscopyand a chemokine-induced leucocyte emigration model(Middleton et al. 1997).


Human recombinant IL-8 was injected into rabbit skin invivo and samples taken at 0, 0.5, 1, 2 and 4 hours. Tissuewas fixed (2% paraformaldehyde, 0.05% glutaralde-hyde), dehydrated and embedded in Lowicryl HM20resin using a progressive lowering of temperature tech-nique in a Leica AFS system. Ultra-thin sections weretaken and immuno-labelled with an anti-IL-8 monoclonalantibody followed by second antibody/gold particles.

Results

At the zero time point, high extravascular levels of IL-8were detected with little or no localisation within venules.At 1 hour after IL-8 administration the endothelium ofvenules was activated, showing numerous protrusions ofthe luminal plasma membrane. Plasmalemmal vesicles,which lined both luminal and abluminal endothelialsurfaces, increased in number suggesting increasedtranscellular transport. Larger electron-lucent vesiclesincreased in abundance as did the number of micro-filaments. At 1 hour IL-8 localised at the luminal surface,and was particularly concentrated on protrusions thatextended out into the luminal space. At this time pointneutrophils interacted with the endothelium and hadbegun to migrate but the strongest neutrophil recruitmentoccurred at 2 and 4 hours. The initial points of contactbetween the neutrophil and the endothelium appeared tobe the endothelial protrusions where the majority of thechemokine was located. Immunoreactive IL-8 waspresent within endothelial cells localising in both luminaland abluminal plasmalemmal vesicles, and non-vesicleassociated label occurred in the cytoplasm. In additionpericyte surface membranes labelled for IL-8. Chemo-kine was also apparent in the extracellular matrix. Thisoccurred in the wall of the venule, including the basallamina, as well as in the collagen fibre bundles of the



dermis. In vehicle-injected control samples no immuno-reactive IL-8 was detected.

When a truncated form of IL-8, IL-8(1-63) which lacksthe c terminus, was injected only background labellingwas present within endothelial cells and at the luminalsurface.

Discussion

These results suggest that injected chemokine is inter-nalised by the plasmalemmal vesicles at the abluminalsurface of the endothelium, then transported trans-cellularly and released from luminal plasmalemmalvesicles on to the cell surface. Here it occurs bound toendothelial presentation molecules which are preferen-tially located on membrane protrusions where IL-8 wouldbe more accessible to the neutrophils. The truncationexperiment indicates that the c terminus of IL-8 isrequired for transcytosis and presentation. Since this isthe domain that binds to heparan sulphate, a role for theglycosaminoglycen is implicated.

References

MIDDLETON, J., NEIL, S., WINTLE, J. ET AL. (1997) Transcytosis andsurface presentation of IL-8 by venular endothelial cells. Cell91, 385–395.

ROT, A., HUB, E., MIDDLETON, J. ET AL. (1996) Some aspects of IL-8pathophysiology. III: Chemokine interaction with endothelialcells. J. Leukocyte Biol. 59, 39–44.

Identification of the TIMP-2 binding site on thehemopexin domain of human gelatinase A bysite-directed mutagenesis

C.M. OVERALL*, A.E. KING*, D.K. SAM*,A.D. ONG† T.T.Y. LAU*, U.M. WALLON*,Y.A. DECLERCK† AND J. ATHERSTONE**Faculty of Dentistry and the Department of Biochemistry and

Molecular Biology, Faculty of Medicine, University of British

Columbia, Vancouver, Canada and †Division of Hematology-

Oncology, Childrens Hospital, Los Angeles and University of

Southern California, Los Angeles, USA

Introduction

Cell membrane activation of the matrix metalloproteinasegelatinase A requires trimolecular association involvingthe anionic carboxyl-terminal peptide of the tissueinhibitor of metalloproteinases-2 (TIMP-2) and mem-brane-type matrix metalloproteinase. Of potential func-tional importance in this interaction, distinct cationic

clusters occur on hemopexin blades III and IV of thefour bladed b-propellor structure of the gelatinase A Cdomain. These are not present in gelatinase B whichdoes not bind TIMP-2.


Twelve site-directed single, double and triple mutationsof the unique cationic clusters in the human gelatinase Ahemopexin carboxyl domain were made to locate theTIMP-2 binding site. The recombinant wild-type andmutant domains were expressed in E. coli according toWallon & Overall (1997).


The mutation Lys617Ala increased the apparent Kd ofTIMP-2 binding by an order of magnitude from3.0 × 10¹8

M to 1.1 × 10¹7M. The importance of Lys617

was further shown by the mutation Lys610Thr/Lys617Alawhich rendered any TIMP-2 interaction undetectable. Animportant contributory, but nonessential role in TIMP-2binding for the triple lysine cluster at positions 566–568was found. Although Lys566/568Ala had little appar-ent effect, no TIMP-2 binding by the triple mutantLys566/568/617Ala was detectable. The mutationLys566/568Ala did not globally destabilize the domainsince essentially full TIMP-2 binding was retainedwhen combined with Lys575Ala, which alone also didnot interfere with TIMP-2 binding. The double alaninemutation of Arg561 and Lys558, which lie either side ofLYs575 in the tertiary structure, resulted in near com-plete loss of TIMP-2 binding. Mutation of the nearby



Lys547 to alanine also significantly reduced TIMP-2affinity whereas the mutations Lys549Ala andLys550Ala, either alone or in the triple mutant Lys547/549/550Ala showed only slight effects on TIMP-2 bind-ing. Thus, these analyses localize the cationic TIMP-2binding site to the junction of b-blades III and IV on theperipheral rim of the gelatinase A hemopexin domain andreveal the differential importance of the basic residuesforming crucial salt bridges for binding TIMP-2.

Acknowledgements

Supported by the National Cancer Institute of Canada.

Reference

WALLON, U.M. & OVERALL, C.M. (1997) The COOH-terminalhemopexin-like domain of human gelatinase A (MMP-2)requires Ca2þ for fibronectin and heparin binding: bindingproperties of recombinant gelatinase A C-domain to extra-cellular matrix and basement membrane components. J. Biol.Chem. 272, 7473–7481.

Retinoic acid-induced type II collagendegradation does not correlate with matrixmetalloproteinase activity in cartilage explantcultures

J.S. PRICE*, WANG-WEIGAND†L.D. KOZACI* AND A.P. HOLLANDER**University of Sheffield Medical School, Sheffield, UK and

†Procter & Gamble Pharmaceuticals, Mason, OH, USA

Introduction

We have previously demonstrated that degradation ofbovine nasal cartilage induced by interleukin-1a (IL-1a)correlates with a large increase in release of matrixmetalloproteinases (MMPs) into the culture medium(Kozaci et al. 1997) We have now examined the role ofMMPs in retinoic acid (Ret) induced degradation of type IIcollagen in cartilage.


Bovine nasal cartilage explants were cultured with 1 mMRet. Proteoglycan and type II collagen released into themedium were measured by colourimetric assay andimmunoassay respectively. MMP activity in the mediumwas determined using a quenched fluorescent substrateassay, while specific collagenases were identified bywestern immunoblotting. In some cases the effects oflow molecular weight synthetic MMP inhibitors andserum on collagen degradation were studied.

Results

Ret promoted maximal breakdown of type II collagenafter 4 or 5 weeks in culture and this degradation wascoincident with a small increase in MMP activity in theculture medium. In western immunoblots of culturemedia from Ret cultures, pro-interstitial collagenaseand active collagenase-3 were sometimes detected,but not in all experiments. A potent, broad spectruminhibitor of MMPs and a weak MMP inhibitor were bothonly weak inhibitors of Ret-induced collagen degrada-tion. When foetal calf serum was included in cartilagecultures, MMP activity in the culture medium wasreduced to low levels, however there was no inhibitionof Ret-induced collagen degradation.

Discussion

We conclude that Ret induces degradation of type IIcollagen in cartilage explants by a mechanism which,unlike IL-1a-stimulated catabolism (Kozaci et al. 1997),is mainly independent of those MMPs that can bedetected in the culture medium.

References

KOZACI, L.D., BUTTLE, D.J. & HOLLANDER, A.P. (1997). Degradationof type II collagen, but not proteoglycan, correlates withmatrix metalloproteinase activity in cartilage explant cultures.Arthritis Rheum. 40, 164–174.

The macromolecular composition of bovinevitreous

A. REARDON, J. SHEEHAN, D. MCLEODAND P. BISHOPWellcome Trust Centre for Cell-Matrix Research,

University of Manchester, Manchester, UK

Introduction

The human vitreous gel is an extremely dilute, trans-parent extracellular matrix. It is known to containheterotypic collagen fibrils (which are essential for gelformation) and hyaluronan. In addition, it contains anumber of poorly defined structural and glycoproteins/proteoglycans. The aim of this work is to characterizethese potentially important macromolecules.

Methods

Bovine vitreous gel was solubilised in 4M GuHCl and the



extract fractionated on a Superose 12 HR gel filtrationcolumn before and after pretreatment with hyaluronanlyase. In-line measurements of refractive index and lightscattering were performed during the chromatographyand fractions collected for biochemical analyses. Specificcomponents were identified by Western blotting. Appro-priate fractions were subjected to trypsin digestion andtryptic peptides purified by reverse phase HPLC. Theamino acid sequence of purified peptides was obtainedby automated Edman degradation.

Results

We have identified a pool of large glycoproteins/proteoglycans (high Mr pool) which elute at and aroundthe void volume of the Superose 12 column. This high Mr

pool has been estimated to contain 22% of the macro-molecules in vitreous. Several components of this poolhave been identified by Western blotting, including thelarge proteoglycan versican. Another has been shown tocarry keratan sulphate chains, although the presence ofaggrecan could not be demonstrated. Partial amino acidsequence data has been obtained which suggests thepresence of fibulin-2. In addition, several sequenceshave been obtained which show no homology to proteinsin databases.

Discussion

Bovine vitreous contains a previously unidentified pool oflarge glycoproteins/proteoglycans, many of which are sofar unidentified. Current work is aimed at obtainingmore amino acid sequence data as a means ofidentifying components within this pool, while apparentlynovel sequences will be used to construct degenerateoligonucleotide probes for use in gene cloning andsequencing studies.

Studies of type II collagen synthesis in articularchondrocyte pellet cultures using a novelassay for the amino-propeptide

N. REBUCK AND A.P. HOLLANDERHuman Metabolism and Clinical Biochemistry in the Division of

Biochemical and Musculoskeletal Medicine, University of

Sheffield Medical School, Sheffield, UK

Introduction

Type II collagen is the major type of collagen in cartilage.The amino- and carboxy-propeptides of fibrillar collagensare considered to be good markers of collagen synthesis

as they are rapidly cleaved from the collagen moleculewhen it has been released into the extracellular matrix.Assays for the carboxy-propeptide of type II collagenhave been in use for several years (Hinek et al. 1987)whilst none are available for the amino-propeptide. Inthis study, we use an inhibition ELISA to assay theamino-propeptide of type II collagen in bovine articularchondrocyte pellet cultures to determine whether theamino-propeptide is retained in the extracellular matrix,as has been shown for the caboxy-propeptide, or if it isimmediately released.


A polyclonal antibody (AH15N1) was raised to a syn-thetic peptide sequence present in the amino-propeptideof type II collagen. The antibody was affinity-purified andin western immunoblot experiments was found torecognise both the truncated (NPIIB) and alternativelyspliced (NPIIA) forms of the amino-propeptide in bovinevitreous extract. Antibody AH15N1 was used to set up aninhibition ELISA for the quantitation of total amino-propeptide and characterised using a bovine chondro-cyte pellet culture system (Xu et al. 1996). The amino-propeptide of type II collagen was measured in theconditioned culture medium and in Guanidinium extractsof the chondrocyte pellets (following removal of theGuanidinium using a Tris-Sephadex column).

Results

We are able to detect type II procollagen both in theconditioned medium and extracellular matrix of bovinearticular chondrocyte pellet cultures. We determined thatup to 97% of the amino-propeptide was immediatelyreleased into the medium when the cultures weresupplemented with 10% foetal calf serum and that upto 80% was released when supplemented with insulin-transferrin.

Discussion

We have demonstrated, using a bovine chondrocytepellet culture system, that the amino-propeptide oftype II collagen is immediately released in to mediumand is not retained within the extracellular matrix. Thisstudy suggests that levels of the amino-propeptide willaccurately reflect of type II collagen synthesis.

Acknowledgement

This work was supported by the Arthritis & RheumatismCouncil (grant H0546).



References

HINEK, A., REINER, A. & POOLE, A.R. (1987) The calcification ofcartilage matrix in chondrocyte culture: studies of the C-propeptide of type II collagen (chondrocalcin). J. Cell Biol.104, 1435–1441.

XU, C., OYAJOBI, B.O., FRAZER, A., KOZACI, L.D., RUSSELL, R.G.G. &HOLLANDER, A.P. (1996) Effects of growth factors and inter-leukin-1alpha on proteoglycan and type II collagen turnoverin bovine nasal and articular chondrocyte pellet cultures.Endocrinology 137, 3557–3565.

Three-dimensional aggregates of humandermal fibroblasts and keratinocytes act as anin vitro model for the dermal-epidermaljunction of skin

A. REITH, C. JONES, C. KIELTY ANDA. SHUTTLEWORTHSchool of Biological Sciences, University of Manchester,

Manchester, UK

Introduction

Normal skin comprises an upper epidermal layer and abasal dermal layer which are connected by a base-ment membrane, frequently referred to as the dermal-epidermal junction (DEJ). The DEJ is responsible forproviding structural stability and maintaining the integrityof skin. Abnormalities in the DEJ can lead to blisteringand more serious phenotypes.

The DEJ is conventionally studied in vitro usingskin equivalent models comprised of fibroblast con-tracted collagen gels with an upper layer of keratino-cytes. Such systems are technically difficult toperform and form a weak DEJ. We have developedan in vitro model for studying the formation of theDEJ which overcomes these problems using three-dimensional mixed aggregates of dermal fibroblastsand keratinocytes.

Material and methods

Primary cultures of epidermal keratinocytes and dermalfibroblasts were isolated from human skin obtained frombreast reduction patients. The HaCaT cell line was usedas a second source of human keratinocytes. Three-dimensional aggregates of dermal fibroblasts, HaCaTcells and primary keratinocytes were prepared byculturing cells on an agar overlay. In the absence of aplastic substratum the cells aggregate to form cohesivespherical structures.


Examination by electron microscopy showed cell aggre-gates formed from HaCaT cells have a well definedapical brush border and glycocalyx at the outer edge ofthe aggregate indicative of cell polarity. The cells linkedby tight cell junctions showed the presence of a cyto-keratin cytoskeleton while desmosomes were observedbetween adjacent keratinocytes. A basement membranewas not detected in these aggregates. In contrast,aggregates of dermal fibroblasts formed a cohesive struc-ture. They showed tight cell packing with no evidence of abasement membrane. Collagen VI was detected in abun-dance within these aggregates. Aggregates formed froma mixture of HaCaT keratinocytes and dermal fibroblastsproduced a cohesive structure which had a randomdistribution of each cell type. A cytokeratin cytoskeletonwas observed within the keratinocytes after three days inculture. A well developed basement membrane wasobserved at the basolateral surface of the keratinocyteswhich lay adjacent to fibroblasts within the aggregatestructure. Hemidesmosomes were detected along thebasement membrane. Unlike the fibroblast aggregates,mixed cell aggregates did not appear to contain collagenVI, however fibrillar structures were observed which mayrepresent fibrillar collagens or fibrillin microfibrils.

Mixed cell aggregates of keratinocytes and fibroblastsprovide a good in vitro model for studying the formation,regulation, composition and organisation of the base-ment membrane formed between these two different celltypes. This technique has the advantage over conven-tional skin equivalent models, that it is quick, easy tohandle, the cells maintain their natural size, polarity, anddifferentiation.

Acknowledgements

This study was supported by the BBSRC.

Aggrecan degradation in humansupraspinatus tendon

G.P. RILEY*, R.L. HARRALL*,B.L. HAZLEMAN* AND R.A. MACIEWICZ†*Rheumatology Research Unit, Addenbrooke’s Hospital,

Cambridge and †Cardiovascular, Metabolism and

Musculoskeletal Research, Zeneca Pharmaceuticals, Alderly

Park, Cheshire, UK

Introduction

A large proteoglycan similar or identical to aggrecanis relatively abundant in the supraspinatus tendon,



compared to flexor tendons which contain predominantlythe small proteoglycan, decorin (Berenson et al. 1996).The major role of aggrecan in cartilage is to resistcompressive forces. In the supraspinatus tendon, whicharticulates around the head of humerus and may besubject to ‘impingement’, aggrecan may have a similarrole. We hypothesized that degradation of aggrecanmight be associated with tendon matrix degeneration(which is common in the supraspinatus tendon) andmay have similarities with osteoarthritic disease of carti-lage, involving changes in proteoglycan synthesis andturnover, leading to structural damage that weakens thetendon, predisposing to rupture. In osteoarthritic cartilage,aggrecan is enzymatically degraded primarily at a singlesite between amino acid residues Glu373-Ala374, althoughthe putative ‘aggrecanase’ enzyme has not been charac-terised (Fosang et al. 1996). In this study, we investigatedwhether there is any aggrecanase-mediated degradationof aggrecan in the degenerate supraspinatus tendon,using an antibody to the carboxyl neo-epitope of aggre-can generated by the action of aggrecanase.


A rabbit polyclonal antisera was raised to the peptideKLH-GCPLPRNITEGE. The antibody, designated RAM3-2, was characterised by immunoblotting, and showedno reaction to uncleaved bovine or human aggrecan, andno reaction with extracts of cartilage, or media fromcontrol incubated bovine nasal cartilage. There wasstrong reaction with media from IL1 stimulated cartilage,with a signal at the expected molecular weight of approxi-mately 70 kD. Specimens of human tendon collectedfrom cadavers or during surgery were fixed in 4% para-formaldehyde, frozen in TissueTek, then cryo-sectionedfor immunoperoxidase staining with RAM 3-2, using theperoxidase anti-peroxidase technique (Dako). Controlswere conducted with the pre-immune sera. Explants oftendon were cultured in DMEM 65 ng/ml IL1-b; themedia was collected at 48 hours, run on 4–15%SDS-PAGE and immunoblotted with RAM 3-2 usingchemiluminescent detection (ECL Amersham).

Results

The NITEGE neo-epitope was detected in 10/16 (63%)ruptured supraspinatus tendons. Positive staining wassimultaneously detected in controls (IL1 stimulatedhuman cartilage) but not in unstimulated cartilage, andno staining was detected with the pre-immune sera. Theintensity and distribution of staining was variable. Stain-ing was generally restricted to discrete foci, often clumpsof cells between fibre bundles and sometimes associated

with blood vessels, although some specimens showedmoderate diffuse matrix staining. Immunostaining wasalso detected in 10/12 cadaver supraspinatus tendons,1/3 biceps brachii tendons and 1/3 subscapularistendons, although in histologically normal tendons,staining was restricted to streaks between fibre bundles.Immunoblotting of culture media from a degeneratetendon maintained in explant culture for 48 hoursshowed a single band of approx. 70 kD in both control(un-stimulated) and IL1 stimulated culture (more intensein the latter), co-migrating with the positive control fromIL1 stimulated bovine nasal cartilage.

Discussion

We present evidence that aggrecan in the humansupraspinatus tendon is degraded by an enzyme simi-lar or identical to aggrecanase, and that this enyzmemay have some involvement in supraspinatus tendonpathology. Although the NITEGE neo-epitope was alsoexpressed in non-ruptured cadaver tendons, its expres-sion within the fibre bundles was generally associatedwith moderate to severe matrix degeneration, whichis common in supraspinatus tendons over 40 years andthought to precede tendon rupture. Further studies arerequired to characterise the enzyme in tendon.

Acknowledgements

This study was supported by the Cambridge ArthritisResearch Endeavour (CARE).

References

BERENSON, M.C., BLEVINS, F.T., PLAAS, A.H.K. & VOGEL, K.G.(1996) Proteoglycans of human rotator cuff tendons.J. Orthop. Res. 14, 518–525.

FOSANG, A.J., LAST, K. & MACIEWICZ, R.A. (1996) Aggrecan isdegraded by matrix metalloproteinases in human arthritis.Evidence that matrix metalloproteinase and aggrecanaseactivities can be independent. J. Clin. Invest. 98, 2292–2299.

Absence of the laminin g1 subunit causes earlyembryonic lethality by inhibition of basementmembrane formation

N. SMYTH†‡, H.S. VATANSEVER*,M. MEYER‡, C. FRIE†, M. PAULSSON†AND D. EDGAR**Department of Human Anatomy and Cell Biology,

University of Liverpool, Liverpool, UK, †Institute for

Biochemistry, University of Cologne, Koln, Germany and

‡Department of Neurochemistry, Max-Planck-Institute of

Psychiatry, Matrinsried, Germany



We have targeted by homologous recombination theLAMC1 genes coding for the laminin g1 subunit inmouse embryonic stem cells. Mice heterozygous forthe mutation had a normal phenotype and were fertile,whereas homozygous nul-mutant embryos did notsurvive beyond day 5.5 post-coitum. These embryoslacked basement membranes and although the blasto-cysts had expanded, primitive endoderm cells remainedin the inner cell mass and did not migrate to form theparietal yolk sac. Cultured nul-mutant ES cells appearednormal, but the embryoid bodies they produced ondifferentiation also lacked basement membranes,having disorganised extracellular deposits of the base-ment membrane proteins collagen IV and perlecan.Secretion of the linking protein nidogen and a trun-cated laminin a1 subunit did occur, but these werenot deposited in the extracellular matrix. Theseresults show that the laminin g1 subunit is necessaryfor laminin assembly and that laminin is in turn essentialfor the organisation of other basement membranecomponents. Surprisingly, during early embryogenesisbasement membranes are first required for endodermalcell differentiation, a functional trophectodermal epithe-lium being able to develop in their absence.

An in vitro investigation into early chondrocytereactions to cartilage wounding

S.R. TEW*, A.P.L. KWAN*,B.M. THOMSON† AND C.W. ARCHER**Anatomy Unit, School of Molecular and Medical Biosciences,

University of Wales, Cardiff and †Smith and Nephew GRC,

York, UK

Introduction

It has long been known that articular cartilage lesionsthat do not penetrate the subchondral bone do notheal. This is due to the limited response of the chondro-cytes at the lesion edge. Classically, examination of thesides of chondral defects has revealed a band of celldeath along the lesion edge (Fisher, 1922), with an areaof cellular proliferation adjacent to it (Mankin, 1962,Calandruccio & Gilmer, 1962). Detailed informationabout what is happening to these cells is scarce. Wehave used an in vitro cartilage explant culture model toinvestigate the processes occurring after cartilage injury.

Methods

Cartilage explants (approx. 1 cm × 2 cm) was excised

from both mature and immature bovine metacarpo- andmetatarsal- phalangeal joint and wounded using a1.7 mm trephine to create a plug of cartilage that waseither removed or replaced immediately. Some cultureshad 1% agarose set into the wound shortly afterwounding. The explants were cultured in DMEM/HAMS-F12 þ 10% Fetal Calf Serum for 1, 2, 5 and 10days before fixation in 10% formal saline. 24 hoursbefore fixation 20 mCi/ml 3H thymidine was added tothe culture media of each explant. Each sample wasdehydrated, cleared in xylene and embedded in paraffinwax. 7 mm sections were cut and mounted on slides formicroscopic analysis.

Results

Sections were examined using TUNEL (Tdt mediateddUTP Nick End Labelling) as a test for programmedcell death. Results were similar for both mature andimmature tissue. At day 1 a band of positive cells couldbe seen at the wound edge and grew by day 2 until it wasapproximately 200mm wide. The band remained at thiswidth and was still visible at days 5 and 10. Interestinglyin samples where the excised plug had been replaced orwhere agarose had been used to fill the lesion, thenumbers of positive cells and the width of the band wasmarkedly reduced suggesting a possible role for diffusiondependent factor in this process. Incorporation of 3Hthymidine into the cell’s nuclei was visualized by auto-radiography. At days 1 and 2 occasional positive cellscould be seen along the lesion in the immature andmature samples. By day 5 incorporation of the labelwas elevated in tissues of both ages and at day 10 anumber of positive cells were still visible.

Conclusions

It seems likely that cell death at the edge of cartilagewounds play a major role in the failure of repair tissuesto fully integrate with existing healthy tissue. Treat-ments that could reduce or even halt this degenerationwould contribute greatly to a successful cartilage repairtreatment.

References

CALANDRUCCIO, R.A. & GILMER, A. (1962) J. Bone Jt. Surg. 44A,431–455.

FISHER, A.G.T. (1922) A contribution to the pathology andetiology of osteo-arthritis: with observations upon the prin-ciples underlying its surgical treatment. Brit. J. Surg. 10, 52.

MANKIN, H.J. (1962) J. Bone Jt. Surg. 44A, 688–698.



Cartilage breakdown by synovium-derivedproteolytic enzymes

M. VANKEMMELBEKE*,S. WANG-WEIGAND† AND D.J. BUTTLE**Section of Human Metabolism and Clinical Biochemistry,

Division of Biomedical and Musculoskeletal Medicine, Sheffield

University Medical School, Sheffield, UK and †Procter &

Gamble Pharmaceuticals, Mason, Ohio, USA

Introduction

It is widely believed that the destruction of articularcartilage in rheumatoid arthritis is mediated, at least inpart, by degradative enzymes secreted by cells of theinflammatory synovial tissue. This can involve the activa-tion of chondrocyte-derived proproteinases by activatingenzymes originating from the synovium, as well as adirect attack by synovium-derived enzymes. Anotherpossible mechanism is the release of soluble factorsfrom synovial cells which stimulate the chondrocytes todegrade their own matrix (Saklatvala 1986; Saklatvalaet al. 1984). We have developed an in vitro system withwhich to monitor the effects of synovial tissue oncartilage breakdown, utilizing cocultures of synovialtissue and cartilage obtained from the bovine meta-carpophalangeal joint (Fell & Jubb 1977).


Both living and dead bovine articular cartilage have beencultured in the presence or absence of serum alongsideminced bovine synovial tissue. The release of proteo-glycan in the medium was quantitated by use ofdimethylmethylene blue; the loss of collagen was deter-mined by a competitive elisa (Hollander et al. 1994).Histochemical analysis has been used to define the sitesof proteoglycan and type II collagen loss.

Results

Both living and dead cartilage lost from 50% to 60% oftheir glycosaminoglycan content by day 7 of culture withliving synovial tissue in the presence of serum. Proteo-glycan breakdown was less effective in the absence ofserum, this effect being more pronounced for livingcartilage. Furthermore cocultures of synovial tissue incontact with articular cartilage had a more degradativeeffect on the proteoglycan degradation than thoseseparated. This difference was more pronounced withliving cartilage. Cocultures in the presence of synovial

tissue which had not been minced, were less efficient inproteoglycan breakdown than those with minced tissue.

Discussion

That cocultures in the presence of serum were moreeffective in proteoglycan breakdown is intriguing sinceserum is known to contain many proteinase inhibitors; itmay suggest that the synovial cells need serum in orderto synthesize the factors responsible for the proteoglycanbreakdown. Further investigations are in progress. Thisin vitro model, once fully characterized, should provideus with a more detailed insight into the role of proteolyticenzymes from synovial tissue in cartilage breakdown.

References

FELL, H.B. & JUBB, R.W. (1977) The effect of synovial tissue onthe breakdown of articular cartilage in organ culture. ArthritisRheum. 20, 1359–1371.

HOLLANDER, A.P., HEATHFIELD, T.F., WEBBER, C., IWATA, Y., BOURNE,R., RORABECK, C. & POOLE, A.R. (1994) Increased damage totype II collagen in osteoarthritic articular cartilage detected bya new immunoassay. J. Clin. Invest. 93, 1722–1732.

SAKLATVALA, J. (1986) Tumour necrosis factor alpha stimulatesresorption and inhibits synthesis of proteoglycan in cartilage.Nature 322, 547–549.

SAKLATVALA, J., PILSWORTH, L.M.C., SARSFIELD, S.J., GAVRILOVIC, J.& HEATH, J.K. (1984) Pig catabolin is a form of interleukin 1.Cartilage and bone resorb, fibroblasts make prostaglandinand collagenase, and thymocyte proliferation is augmented inresponse to one protein. Biochem. J. 224, 461–466.

The effects of exercise on equine meniscalcartilage

J. WARDALE, N. HILLS*, R. D’AURIA ANDV. DUANCEConnective Tissue Biology Laboratories, School of Molecular

and Medical Biosciences, Cardiff University, Cardiff and

†Institute of Medical Genetics, University of Wales College of

Medicine, Cardiff, UK

Introduction

Changes in the biochemistry of the human meniscushave been observed with increasing age and also withthe onset of osteoarthritis. This implies that changes inthe extracellular matrix of meniscal cartilage maypredispose an animal to joint degeneration. The aim ofthis study is to establish whether changes can bedetected in the biochemistry of the meniscal cartilagefollowing 4.4 months and 18 months of exercise whencompared to non-exercised controls.



Methods

Lateral and medial menisci were removed from controland exercised animals and analysed for water content,total collagen, proteoglycan, collagen type, metallo-proteinases (MMPs) and their inhibitors (TIMPs).Sections of tissue were taken across the centre of themeniscal cartilages and freeze-dried to provide a valuefor the water content. These samples were used forsubsequent analysis. Total collagen content wasmeasured by hydrolysis of tissue samples followed byassay for hydroxyproline. Collagen type was establishedby digestion of the tissue with cyanogen bromide,separating the peptides by SDS polyacrylamide gelelectrophoresis and western blotting using specificanti-collagen antibodies. Accurate quantification ofindividual collagen types could then be made by scan-ning laser densitometry of the resultant blots using amethod developed in our laboratory. Proteoglycanlevels were measured by estimation of the sulphatedglycosaminoglycans using the dimethylmethylene blueassay and metalloproteinases (MMPs) and their inhibi-tors (TIMPs) estimated by zymography and reversezymography respectively.

Results

The results are expressed as a comparison between allthe menisci from the exercised group versus the controlgroup and also as a comparison between the exercisedlateral menisci versus the control lateral menisci andthe exercised medial menisci versus the control medialmenisci.

Short term exercise (4 months). A significant decrease inproteoglycan content was observed in the meniscalcartilage of the exercised animals. There is also adecrease in the water content of these menisci althoughthe result is less significant. Interestingly the mostsignificant change in proteoglycan occurs in the lateralcompartment rather than the medial compartment whereit is thought that most joint degeneration occurs. Con-versely, levels of MMP-2 showed a slight increase in theexercised samples and this was as a result of changesonly in the medial menisci. There is a possible decreasein the total collagen content of the medial compartmentsof the exercised meniscal cartilages but more experi-mental work is necessary to confirm the results as sig-nificant. The major collagen type, type I, was unchangedby exercise but type II collagen which makes up approxi-mately 10% of the total collagen was decreased overallby exercise and more specifically in the lateral compart-ment although the results are not yet significant.

Long-term study (18 months). The significant changesseen in the proteoglycan content of the short term studywere not detectable in the long term study. Total collagenhad increased slightly in the exercised group, particularlyin the medial compartment. There was no change inlevels of type I collagen but both types II and III collagenswere reduced in the exercised group. A slight rise in proand active MMP-2 levels was seen in the exercisedgroup with a concomitant drop in levels of TIMPS 1–3.MMP-9 levels were low or undetectable, but from thosesamples that had measurable levels, it appeared to bereduced by long term exercise.

Discussion

It is interesting to note that the changes seen in theanimals exercised in the short-term study are similar tothose that we observe in the human with the onset ofosteoarthritis ie. a decrease in proteoglycan and collagenlevels and an increase in collagenolytic enzymes. How-ever, longer-term exercise appears to allow the tissue torecover the balance of matrix turnover although changesin the proportions of the collagens may lead to amechanically altered tissue. Due to the low samplenumber in these experiments further studies will benecessary in order to improve the statistical significanceof these results before any firm conclusions can bedrawn.

Effects of transforming growth factor-beta 1 onthe assembly of matrix by bovine fibroblastsgrown in a 3-dimensional matrix

J. WARDALE*, N. HILLS†, S. GILBERT*,R. YOUNG* AND V. DUANCE**Connective Tissue Biology Laboratories, School of Molecular

and Medical Biosciences, Cardiff University, Cardiff and


Medicine, Cardiff, UK

Introduction

The basis of connective tissue repair is reliant on theability of cells to produce and assemble a functionalmatrix. Chondrocytes cultured in 3-dimensional matricessuch as alginate or agarose appear to retain theirphenotype as measured by the collagen and proteo-glycan species exported into the culture medium. How-ever, our recent studies have shown that passagedchondrocytes and fibroblasts do not assemble a matrix



if grown in agarose or alginate even after appreciableculture periods in standard tissue culture media such asDMEM with 10% foetal calf serum. The purpose of thisstudy is to establish whether these types of cells can bealtered in such a way as to produce a matrix similar tothat synthesised in vivo. Initial studies have focused onthe effects of growth factors on bovine fibroblasts andhuman meniscal cells with respect to their collagen andcollagenolytic enzyme production.

Methods

Bovine fibroblasts were isolated from cornea, skin andtendon and cultured. Cells were proliferated on plasticand frozen down in sufficient numbers to allow their useat an early passage number (3–4). The three fibroblasttypes were seeded in agarose, allowed to grow for 2 ormore days and then grown in, 5% heat-inactivated, acid-treated (HIAT) FCS or 5% (HIAT) FCS þ 2.5 ng/mlTGF-beta 1. Cultures were allowed to grow on forapproximately 2 weeks and media samples removedevery 3–4 days and replaced with fresh media. Aliquotswere taken and mixed with an equal volume of SDS gelsample buffer and run on Laemmli gels containing0.5 mg/ml gelatin for zymography 2.25 mg/ml gelatinand 7.5% conditioned media for reverse zymography.The gels were washed with 2.5% Triton X-100 to removeSDS and incubated overnight at 378C in 50 mM TrispH 7.8, 50 mM CaCl2, 0.5 M NaCl, containing 0.5 mMAPMA to activate MMPs. Gels were then stained withCoomassie Blue and the MMPs and TIMPS quantified bydensitometry. The remaining media was precipitated withammonium sulphate and the resulting pellet extractedwith 1 mg/ml pepsin in 0.1 M acetic acid overnight andthen freeze-dried before SDS-PAGE and western blot-ting for type I collagen. Agarose plugs were removed atthe end of the experiment for study by electron micro-scopy and the remainder of the cells extracted forwestern blotting for type I collagen as above.

Results

ProMMP-2 was slightly upregulated by TGF-beta 1 inboth cornea and tendon fibroblast cultures (,25%) butwas downregulated by approximately 10% in skin fibro-blasts. Active MMP-2 expression was erratic in cornealand tendon cultures but was downregulated (,50%) inskin cultures. A huge increase in MMP-9 was seen inboth cornea (4000%) and tendon (>1000%) fibroblastsbut skin fibroblasts were again unaffected. TIMP-1 wasupregulated by approximately 70% in cornea and tendonfibroblasts with skin fibroblasts unaffected. TIMP-2 was

upregulated in tendon and skin cultures (,25%) but waslittle changed in corneal cultures.

Type I collagen secretion into the media was slightlyincreased in corneal and tendon fibroblasts but therewere no effects on the skin cultures. Analysis of thecollagen associated with the cell pellet showed that asmall proportion of the total collagen produced by thecornea and tendon cultures was associated with the cellsand this was dramatically increased by the addition ofTGF-beta. However, there was no evidence of any type Icollagen associated with the cellular fraction from theskin fibroblasts.

Examination of the cell cultures by transmissionelectron microscopy showed that fibroblasts grown inagarose or alginate matrices made no discernable matrixin the presence of serum alone. However, the addition ofTGF-beta 1 into the media caused a collagenous matrixto be deposited around the corneal and tendon cells butnot in the skin cultures.

Discussion

We have demonstrated that TGF-beta 1 is necessaryfor fibroblasts to commence assembly of a collagenousmatrix in a 3-dimensional in vitro system. It is not clearfrom these early studies what component(s) are beingaffected by the growth factor in order to allow thisphenotypic change but the profound changes causedby TGF-beta 1 to some of the collagenolytic enzymesinvolved in matrix turnover suggests a possible role forthese enzymes. The fact that skin cells are still unable todeposit a collagenous matrix at their cell surface andtheir levels of enzymes are unaffected by the growthfactor lends weight to this theory.

Alterations in metalloproteinase and TIMPexpression following cyclical compressiveloading of bovine fibroblasts

J. WARDALE*, N. HILLS†, S. GILBERT*,P. PURSLOW‡ AND V. DUANCE**Connective Tissue Biology Laboratories, School of Molecular

and Medical Biosciences, Cardiff University, Cardiff,


Medicine, Cardiff, UK and ‡Department of Dairy and Food

Science, The Royal Veterinary and Agricultural University,

Frederiksberg, Denmark

Introduction

One of the fundamental questions in connective tissueresearch is how a cell perceives the forces acting on it and



whether the surrounding matrix influences those signals.The aim of this project is to compare the responses ofcells derived from different tissues that are normallysubjected to different mechanical loads to an abnormalcompressive load in vitro. A number of parameters werestudied and the results obtained for metalloproteinaseand TIMP expression are reported here.

Methods

Bovine fibroblasts were isolated from cornea, skin andtendon and cultured. Cells were proliferated on plasticand frozen down in sufficient numbers to allow their useat an early passage number (3–4). The three fibroblasttypes were seeded in agarose, allowed to grow for 2 ormore days and then cyclically loaded by uniaxial, uncon-fined, cyclical compression (0.1 kPa, 4 Hz) for 6 hours at37 degrees. Identical control cells were left unloaded.Cultures were allowed to grow on for approximately 2weeks and media samples removed every 2–3 days.Aliquots were taken and mixed with an equal volume ofSDS gel sample buffer and run on Laemmli gels contain-ing 0.5 mg/ml gelatin for zymography or 2.25 mg/mlgelatin and 7.5% conditioned media for reverse zymo-graphy. The gels were washed with 2.5% Triton X-100 toremove SDS and incubated overnight at 378C in 50 mMTris pH 7.8, 50 mM CaCl2, 0.5 M NaCl, containing 0.5 mMAPMA to activate MMPs. Gels were then stained withCoomassie Blue and MMPs and TIMPs quantified byscanning densitometry.

Results

Bovine skin, tendon and corneal fibroblasts exhibiteddifferent responses in vitro with respect to their productionof MMPs and TIMPS.

Prior to loading, all lines produce MMP-2, MMP-9,TIMP-1 and TIMP-2 (Skin q Tendon > Corneal) whenplated on plastic or in agarose. The differences betweenthe cell types were larger on plastic and the overalllevels produced on plastic are much higher than thoseproduced in agarose.

Following loading, skin fibroblasts respond by a slightdownregulation of both MMP-2, MMP-9 and TIMP-2 butare generally unaffected by the loading regime.

Tendon fibroblasts show an initial downregulation ofMMP-2 but this eventually recovers to unloaded levels.MMP-9 is also initially downregulated but recovers tolevels approximately 50% higher than those of theunloaded controls. TIMP-1 is slightly upregulated andTIMP-2 is markedly downregulated.

Corneal fibroblasts show a similar initial downregula-tion of both MMP-2 and MMP-9 to that seen in tendon

cells. However, both enzymes recover to finish sub-stantially higher than the unloaded control. The patternof TIMP expression is similar to that seen in tendon cells.

Discussion

The loading regime used in this study is considered to beless than the cells are normally subjected to in vivo but itis enough to elicit a measurable response from the cells.Each cell type, when cultured identically, respondeduniquely to the applied load indicating that they retaindistinct inherent phenotypic features. As the cells failedto make a matrix in agarose, it must be assumed that thecells are perceiving the applied load either by deforma-tion of the cell or by fluid flow at the cell surface. Studiesare now underway to build up an external matrix aroundthe cells to establish whether this will modulate theeffects of the load.

Immunoelectron microscopy of matrixmolecules in the guinea pig knee duringearly stages of osteoarthritis

R.D. YOUNG, A. VAUGHAN-THOMAS ANDV.C. DUANCEConnective Tissue Biology Laboratories, School of Molecular &

Medical Biosciences, University of Wales, Cardiff, UK

Introduction

Previous studies have described the spontaneousarthritis which occurs with high incidence in the kneejoints of male Dunkin Hartley strain guinea pigs (Bendele& Hulman 1988). The pathological course follows eventsthought to be similar to those seen in human knees,including the appearance of changes in subchondralbone subjacent to the sites of insertion of the cruciateligaments (Dieppe et al. 1993). Sclerosis of subchondralbone and the appearance of bone ‘cysts’ were presentbefore the degeneration of articular cartilage whichhowever still progressed to full-thickness erosion by asearly as twelve months of age (Watson et al. 1994). Inpreliminary immunofluorescence studies we identifiedearlier changes in the posterior cruciate ligaments ofmale guinea pigs (Young et al. 1997). Deposition oftype II collagen was detected at sites within the centralligament body quite separate from the fibrocartilaginousligament attachment sites. The purpose of this work wasto examine the ultrastructure of joint tissue components,cartilage and cruciate ligament at these early stagesin the development of osteoarthritic signs to furtherunderstand the changes in tissue organisation.




Samples of cartilage from the medial tibial plateau andposterior and anterior cruciate ligament were takenfrom guinea pigs of 600 and 1100 gm weight. Thesewere processed for immunoelectron microscopy usingmethods we have previously shown to facilitate preser-vation of both ultrastructure and tissue epitope reactivitywith antibodies. 1.5 mm diameter tissue discs preparedwith a trephine were sandwiched between aluminiumplanchettes and frozen at 2100 bar pressure in a Baltechigh pressure freezer. After storage in liquid nitrogen,the discs were freeze-substituted at ¹908C withoutfixatives in methanol or acetone over 4 A molecular sievefor 18 and 120 h respectively, before infiltration withLowicryl HM20 resin and UV polymerisation at ¹508C.Ultrathin sections from these blocks were exposed toprimary monoclonal antibodies to chondroitin-6-sulphate,keratan sulphate, type II and type IX collagen, or PBS orantibodies to cytoskeletal components as controls. Matrixmolecules were detected with secondary antibodiesconjugated to 5 or 10 nm colloidal gold.

Results

Excellent preservation of ultrastructure was achievedby these methods. Further improvements in cellularpreservation and retention of the cell-matrix interfacewas achieved in samples exposed to controlled rewarm-ing at 18C/h prior to freeze-substitution in acetone. Thisprobably relates to devitrification and crystallisation ofwater during rapid rewarming plus the more aggressiveremoval of ice in methanol substitution media. Antibodyto keratan sulphate labelled throughout the cartilagematrix, as did chondroitin-6-sulphate antibody, but withincreased signal in the immediate pericellular matrix.Type II collagen was detected throughout the cartilage

matrix. In cruciate ligament of the 1100 gm animal, type IIcollagen was detected in the fibrocartilage zone, wherethe ligament attached to tibia and at discrete sites withinmid ligament. The latter appeared as discrete fociassociated with the main collagenous component of theligament.

Discussion

Immunolocalisation of tissue components in tibialcartilage reflected the known composition of this tissuefrom biochemical analyses, without obvious differencesbetween animals of different age/body weight. Type IIcollagen localisation in cruciate ligament confirmed ourearlier studies by immunofluorescence light microscopyand showed that type II collagen appears within theconstituent fibrils of the ligament body which are con-sidered to be primarily type I collgaen. Type II: type Iheterotypic fibrils have not previously been described.Type II copolymerisation with type I collagens in ligamentmay significantly alter the biomechanical properties ofthe tissue, perhaps contributing to the sequellae ofosteoarthritis previously identified in bone and articularcartilage.

References

BENDELE, A.M. & HULMAN, J.F. (1988) Spontaneous cartilagedegeneration in guinea pigs. Arthritis & Rheum. 31, 561–565.

DIEPPE, P., CUSHNAGHAN, J., YOUNG, P. & KIRWAN, J. (1993)Prediction of the progression of joint space narrowing inosteoarthritis of the knee by bone scintigraphy. Ann.Rheum. Dis. 52, 557–563.

WATSON, P.J., HALL, L.D., CARPENTER, T.A. & TYLER, J.A. (1994)J. Rheum. 33 (suppl. 1), 186.

YOUNG, R.D., VAUGHAN-THOMAS, A., WARDALE, R.J., MASON, D.J. &DUANCE, V.C. (1997) Orthop. Trans. 22, 500.



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