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Human and monkey trabecular meshwork accumulate ??B-crystallin in

response to heat shock and oxidative stress

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Human and Monkey Trabecular Meshwork Accumulate aB-Ciystallin in Response to Heat Shock and Oxidative Stress

Ernst R. Tamm,* Paul Russell,^ Douglas H. Johnson,^ andjoram Piatigorsky*

Purpose. Oxidative stress and other forms of injury to trabecular meshwork (TM) cells maycontribute to changes seen with age and primary open-angle glaucoma. This study was de-signed to investigate if TM expresses aB-crystallin, a small heat-shock protein with chaperoneactivity, and whether it might be overexpressed under stress conditions.

Methods. The TM from human and monkey eyes, as well as organ and primary cell culturesderived from these eyes, were investigated for aB-crystallin by immunohistochemistry, two-dimensional gel electrophoresis, Northern and Western blot analysis. The TM cell cultureswere stressed by heat shock (44°C for 15 minutes) or hydrogen peroxide (200 (imo\ for Ihour). Semiquantitation of aB-crystallin messenger RNA (mRNA) or protein was obtainedby densitometry.

Results. In both species, aB-crystallin could be detected in fresh and cultured TM by two-dimensional gel electrophoresis in conjunction with Western blot analysis. Immunohistochem-istry of fresh samples showed that aB-crystallin was expressed predominantly in the cribriformarea. Protein expression was enhanced in 4- to 7-day organ cultures. Primary cultures fromhuman TM cells expressed two sizes (approximately 0.8 and 1.1 kb) of aB-crystallin mRNAin Northern blots. In monkey TM cultures, a 0.8-kb band was observed, which comigratedwith lens aB-crystallin. In both species, heat shock caused a significant increase in aB-crystallinmRNA with a peak after 4 hours. An increase in aB-crystallin mRNA also was observed afteroxidative stress; however, the onset of mRNA induction was slower. After heat shock, but not

after oxidative stress, a transient change in mRNAanalysis showed a 3.4-fold increase in protein 24 houafter 48 hours. No constitutive mRNA expression aiheat shock could be observed in simian virus 40 tra

mobility was observed. Western dot blots after heat shock and a 20-fold increased only a minimal increase 4 hours afternsformed cell lines from human TM.

Conclusions. Overexpression of aB-crystallin might be an important mechanism for TM toprevent cellular damage associated with various stress conditions. Invest Ophthalmol Vis Sci.1996;37:2402-2413.

A he tissues facing the anterior chamber of the eyeare exposed constantly to aqueous humor containingreactive oxygen products generated by light-catalyzedreactions. Among these is hydrogen peroxide, whichis found in human aqueous humor at a mean level of

Prom the * Laboratory of Molecular and Developmental Biology and the ̂ Laboratoryof Mechanisms of Ocular Diseases, National Eye Institute, National Institutes ofHealth, liethesda, Maryland; and the \Depmtmml of Ophthalmology, Mayo Clinic,Rochester, Minnesota./'resented in part at the Annual Meeting of the Association for Research in Visionand Ophthalmology, Fort Lauderdale, Honda, April 1996,Supported by grants from the. Glaucoma Research Foundation (to PR and DHJ), theDeutsche Forsckmigsgemeinschaft (grant Ta 115/8-1 to ERT), and the Ria Freifmuvon Frilsch-Stiflung (to FAT).Submitted for publication March 4, 1996; revised May 21, 1996; accepted July 9,1996.Proprietaiy interest category: N.Reprint requests: Ernst R. Tamm, National Eye Institute, laboratory of Molecularand Developmental Biology, 6 Center Drive, MSC 2730, Building 6/Roovi 204,liethesda, Ml) 20S92.

25 /^mol.1 Hydrogen peroxide in aqueous humor isthought to originate from a light-catalyzed oxidationof ascorbic acid,2 which is enriched in aqueous humorof diurnal mammals at concentrations 10- to 50-foldhigher than in blood plasma.3 In general, hydrogenperoxide and ascorbic acid are reactants in mixedfunction oxidation processes that can generate a num-ber of free radicals.4 Such radicals may cause increasedoxidation in tissues of the anterior eye segment andfinally lead to oxidative damage. In support of thishypothesis, higher than normal levels of hydrogenperoxide are present in the aqueous humor of somepatients with senile cataract.1

Because the bulk of aqueous humor leaves the eyevia the trabecular meshwork, it seems reasonable toassume that normal function of the trabecular mesh-

2402Investigative OphthaCopyright © Associal

nology & Visual Science, November 1996, Vol. 37, No. 12on for Research in Vision and Ophthalmology

Trabecular Meshwork and arB-Crystallin 2403

work requires a number of defenses against oxidativeinsult. Indeed, glutathione that can act as reducingagent and trap free radicals and the hexose mono-phosphate shunt that provides NADPH which is usedin a number of reducing reactions have both beenshown in calf trabecular meshwork.J~7 In addition, thistissue contains rather high amounts of glutathioneperoxidase and catalase to metabolize hydrogen per-oxide as well as superoxide dismutase to detoxify oxy-gen.8 Experimental studies in perfused calf eyes indi-cate that these mechanisms are sufficient to removephysiologic concentrations of hydrogen peroxidefrom the aqueous humor as it passes through the out-flow structures.9 Oxidative stress that exceeds the ca-pacity of the trabecular meshwork for detoxificationcould result in damage of trabecular meshwork cellsand subsequent alteration of aqueous outflow resis-tance. Indeed, anterior chamber perfusion studies inglutathione-depleted enucleated calf eyes showed a33% decrease in facility when hydrogen peroxide wasadded to the perfusion medium.6 In cultured humantrabecular meshwork, a 30-minute exposure to 300//mol hydrogen peroxide resulted in a considerablealteration in cellular morphology.10

In addition to detoxifying enzymes, cells also maysynthesize a specific set of proteins that act as molecu-lar chaperones to prevent oxidative damage. Molecu-lar chaperones stabilize native protein folding andconformations, correct oligomeric assemblies of pro-teins, and protect proteins from denaturation due toheat and other stresses." Many chaperones are heatshock proteins that belong to multigene superfamilieshighly conserved during evolution. Among these isthe small heat shock protein family that in humansis composed of the heat shock protein 27 and twocharacteristic lens proteins, aA-crystallin and aB-crys-tallin, respectively.12 The aA-crystallin is highly lens-preferred in its expression pattern, whereas aB-crys-tallin is expressed constitutively in many nonlenticulartissues.13 In addition, aB-crystallin accumulates in neu-ral tissue in a number of diseases.13 Both crystallinscan act as molecular chaperones and protect againstthermal denaturation of proteins.14 In vitro, aB- butnot aA-crystallin can be induced in a variety of differ-ent cell types by various cellular stresses.15"21 In thepresent investigation, we have extended our studies'3

on aB-crystallin to the trabecular meshwork. We showfor the first time that aB-crystallin is expressed consti-tutively in human and monkey trabecular meshworkand accumulates in trabecular meshwork cells afterheat shock and oxidative stress.

MATERIALS AND METHODS

Twelve pairs of human autopsy eyes (age, 48 to 91years) and 10 pairs of rhesus monkey eyes {Macaco,

mulatto,, age, 2 to 3 years) were investigated. Humaneyes were enucleated within 2 to 6 hours after death.None of the donors showed any abnormalities in thechamber angle. Monkey eyes were enucleated immedi-ately after death from animals that were used in thevaccine testing program of the Center for BiologiesEvaluation and Research of the Food and Drug Ad-ministration. Methods for securing human and animaltissue were humane, included proper consent and ap-proval, and complied with the National Institutes ofHealth Guidelines on the Care and Use of Animals inResearch, the Declaration of Helsinki, and the Associa-tion for Research in Vision and Ophthalmology Reso-lution on the Use of Animals in Research.

Immunohistochemistry

Eight pairs of human eyes and two pairs of monkeyeyes were used for immunohistochemistry. All eyeswere cut equatorially behind the ora serrata, and theanterior segment was dissected in quadrants. Fromeach quadrant, wedge-shaped specimens of 2-mm cir-cumferential width, containing ciliary muscle, scleralspur, and trabecular meshwork, were cut and quick-frozen in isopentane, precooled with liquid nitrogen,without prior chemical fixation. We used frozen sec-tions rather than paraffin sections because of the re-quirements of our aB-crystallin antibodies. All speci-mens from human autopsied eyes were placed in fixa-tive within 6 to 8 hours after death. From all quadrantsof each individual eye, at least one specimen was exam-ined. Meridional and frontal cryostat sections were cutat a thickness of 15 to 20 //m. The sections were placedon slides covered with 0.1% poly-L-lysine, fixed in ace-tone for 10 minutes at —20°C, and preincubated for45 minutes in Blotto's dry milk solution. After preincu-bation, the sections were incubated overnight at roomtemperature with rabbit antibodies specific against aB-crystallin (provided by Joseph Horwitz, Jules Stein In-stitute, UCLA, Los Angeles, CA) at a dilution of 1:100.The polyclonal antibodies (LAP 70), raised against aC-terminal peptide from aB-crystallin, have been de-scribed elsewhere.22'23 After overnight incubation, thesections were washed in Tris-buffered saline: (Tris HC10.05 M, pH 7.5, NaCl 0.15 M (TBS) for 30 minutes(three times each for 10 minutes), reacted with mouseantirabbit immunoglobulin followed by rabbit anti-mouse immunoglobulin (each for 30 minutes, 1:125,Dako, Carpinteria, CA) washed again, and coveredwith an alkaline phosphatase anti-alkaline phospha-tase complex (Dako) diluted 1:50 for 30 minutes. Thesections again were washed in TBS, and alkaline phos-phatase was visualized using new fuchsin as stainingsubstrate. Control sections were incubated with TBSor a preimmune serum replacing the primary anti-body. Additional controls included preabsorption

2404 Investigative Ophthalmology & Visual Science, November 1996, Vol. 37, No. 12

with aB-crystallin (1 mg/100 fA) before immunocyto-chemical incubation.

Cell Cultures

Primary trabecular meshwork cultures were estab-lished from the eyes of two human donors (age, 66and 80 years) and six rhesus monkeys according topreviously published protocols.24 In brief, excised tra-becular meshwork explants were placed in laminin-coated 35-mm Petri culture dishes under sterile glasscoverslips with 2 ml of HAM's F-10 medium supple-mented with 50-U ml"1 penicillin, 50 fig mP1 strepto-mycin, and 20% fetal calf serum (all Gibco Ltd, Pais-ley, Scotland). Coating was performed with lamininfrom basement membrane of Engelbreth-Holm-Swarm sarcoma (Sigma, St. Louis, MO). Laminin wasadded at a concentration of 10 mg/ml. The mediumwas changed every second day, and all cultures wereincubated at 37°C in humidified air enriched with10% carbon dioxide. Confluent cultures formed anendothelium-like monolayer with the typical morpho-logic characteristics of cultured human and monkeytrabecular meshwork cells20'26 and clearly were distin-guishable from contaminating fibroblasts of neigh-boring tissues or ciliary muscle cells.27 Phase-contrastmicrographs of our human and monkey trabecularmeshwork cultures are shown elsewhere.24 Confluentcultures were trypsinized with 0.25% trypsin for a fewminutes, transferred to laminin-coated culture flasksat a split ratio of 1:4, and incubated in F-10 10% fetalcalf serum and antibiotics.

In addition, two simian virus 40 (SV 40) trans-formed human trabecular meshwork cell lines, onefrom a normal (HTM-5) and the other from a glauco-matous donor (HTM-3), were investigated. Both celllines were provided by Dr. Iok-Hou Pang and Dr.Louis DeSantis (Alcon Research Laboratories, FortWorth, TX). The HTM-3 cells have been reported toexpress similar cytoskeletal elements, extracellular ma-trix components, and second messenger systems aspresent in trabecular meshwork tissue and nontrans-formed trabecular meshwork cells.28 The SV 40 trans-formed cell lines were cultured according to pre-viously published protocols.28

Confluent cultures were exposed to a transitoryheat shock by immersion in a 44°C water bath for 15minutes followed by recovery at 37°C. For oxidativestress, confluent cultures were washed three times withserum-free medium and subsequently incubated withserum-free medium containing 200 /umol hydrogenperoxide for 1 hour. After this time, the medium waschanged and regular medium was added. Control cul-tures were incubated for 1 hour in serum-free mediumwithout hydrogen peroxide. Each experiment was per-formed at least three times.

Protein AnalysisTrabecular meshwork from the eyes of two humandonors (age, 44 and 63 years) and two monkeys wereinvestigated. The trabecular meshwork was cut outfrom monkey eyes and from one eye of each humandonor and deep frozen on dry ice, while the anteriorsegment of the contralateral eye of the human donorswas placed in an organ culture system. The culturesystem introduced by Johnson and Tschumper29 wasused without modifications. After 4 and 7 days of or-gan culture, the trabecular meshwork was cut out andfurther processed. Ex situ trabecular meshwork sam-ples and cultured human and monkey trabecularmeshwork cell pellets were solubilized in 9 M ureawith 2% Nonidet P-40 (Sigma Chemical, St. Louis,MO) and 105 ng/jul of trypsinogen. The trypsinogen(24 kDa) was added to all samples as an internal refer-ence standard by which individual proteins in the sam-ples could be compared and analyzed. The trabecularmeshwork was crushed repeatedly with a pipette tip,and all samples were allowed to stand at room temper-ature for a minimum of 5 minutes. Samples were cen-trifuged at 14,000 X g for 10 minutes, and the finalprotein concentration was approximately 2.5 mg/mlin fresh human trabecular meshwork and culturedtrabecular meshwork cells, and 0.77 mg/ml in freshmonkey trabecular meshwork. Protein was estimatedwith a Bradford assay (BioRad Laboratories, Rich-mond, CA). Bovine serum albumin was used as stan-dard. One microliter samples were analyzed by two-dimensional gel eleCtrophoresis as reported pre-viously.30 The first dimension was a pH 3 to 9 gelcontaining 8 M urea and 2% Nonidet P-40 (SigmaChemical). The second dimension was a gradient gelof 8% to 25% acrylamide. Gels were silver stained.Western blots were done with the PhastSystem (Phar-macia, Piscataway, NJ) according to the manufactur-er's protocol. The nitrocellulose paper was blocked(SuperBlock; Pierce, Rockford, IL) and incubatedwith a 1:100 dilution of the same antibody to aB-crys-tallin as used for immunohistochemistry, or with ana-crystallin antibody (dilution 1:100) that binds bothto aA- and aB-crystallin (LAP-69, raised against the N-terminal peptide from aA-crystallin) .22'23 After incuba-tion and washing, the Western blots were developedusing the chemiluminescent Tropix kit (Tropix, Bed-ford, MA). A minimum of two Western blots were usedto confirm the position of the aB-crystallin. Dot blotswere done by spotting 1 p.g of protein on nitrocellu-lose paper, blocking and incubating with a 1:100 dilu-tion of primary antibody to aB-crystallin. These dotblots were developed with a Tropix kit (Tropix).

RNA AnalysisTotal RNA was isolated from cell cultures using RNA-zol (Tel Test, Friendswood, TX), separated on a 2.2-

Trabecular Meshwork and aB-Crystallin 2405

M formaldehyde 1.2% agarose gel, and blotted ontoa Duralon (Stratagene, Lajolla, CA) membrane. Afterthe transfer, the blot was cross-linked using an ultravio-let Stratalinker (Stratagene). Blots were hybridizedwith a 244 base pair Bacillus amyloliquefaciens H(BamHI)-HaemophilusinfluenzaeRd (Hindlll) restric-tion fragment isolated from the human aB-crystallingene ( + 2849 to +3092) containing much of exonIII.31 The probe was labeled with saP-dCTP using anick translation kit (Gibco BRL, Gaithersburg, MD).Prehybridizations were performed at 55°C for 1 hourand hybridizations at 55°C overnight using Hybrisol(Oncor, Gaithersburg, MD) according to the manu-facturer's instruction (including 10% formamide inthe hybridization solution). Membranes were washedtwice for 15 minutes each with 2 X SSC-0.1% sodiumdodecyl sulfate (SDS) at 55°C and twice with 0.2 XSSC-0.1% SDS at room temperature, and autoradio-graphed using a Kodak XAR5 film (Eastman Kodak,Rochester, NY) at -80°C with an intensifying screen(1 to 2 days). Hybridization to an end-labeled oligo-deoxynucleotide complementary to human 28S ribo-somal RNA (rRNA) was performed as described pre-viously311 to monitor the integrity of RNA, the relativeamounts of RNA loaded on the gel, and the efficiencyof transfer to Duralon (Stratagene) membranes. Themessenger RNA (mRNA) size was estimated by refer-ence to the mobility of RNA size markers (Life Tech-nologies, Gaithersburg, MD) stained with methyleneblue.

Intensity of hybridization was determined by scan-ning densitometry using a personal densitometer andImageQuant software (Molecular Dynamics, Sun-nyvale, CA). Autoradiograms were normalized to therelative intensity of the 28S band. In control experi-ments, autoradiograms with different exposure timesand from blots with different dilutions of total RNAwere scanned to check that the measurements werein a linear range and not at saturation.

RESULTS

ImmunohistochemistryIn the eyes of seven human donors, staining for aB-crystallin was found in the cribriform or juxtacanalicu-lar region of Schlemm's canal (Fig. 1A, IB). Bothcribriform cells and endothelial cells of Schlemm'scanal were stained. In addition, some cells in the outerwall of Schlemm's canal showed positive immunoreac-tivity. No staining was observed in the corneoscleralor uveal parts of the meshwork. Frontal sections paral-lel to Schlemm's canal showed that staining for aB-crystallin was distributed equally along the cribriformarea. In the eyes of one human donor, positive immu-nostaining was found in cells of all parts of the trabecu-

lar meshwork as well as in the endothelial cells allaround Schlemm's canal (Fig. 1C). In monkey eyes,staining was confined to the outer regions of the tra-becular meshwork, such as the corneoscleral and crib-riform parts (Fig. ID). In addition, endothelial cellsof Schlemm's canal and of collector channels werestained. In both species, ciliary muscle cells were im-munoreactive, but cells in the scleral spur were not.No staining was seen in control sections, after replace-ment of the primary antibody with preimmune serumor after preabsorption with aB-crystallin (1 mg/100/Ltl) before immunocytochemical incubation (data notshown).Two-Dimensional Gel Electrophoresis andWestern Blot AnalysisSilver stained two-dimensional gels from fresh humantrabecular meshwork resolved a polypeptide with ap-proximately similar molecular mass and pi as bovinelens aB-crystallin (Fig. 2A). This polypeptide was rec-ognized in the two-dimensional Western blots by anti-bodies against aB-crystallin (Fig. 2B). The same anti-body reacted with another protein spot of the samemobility and pi as the phosphorylated form of aB-crystallin (Fig. 2B). Staining with antibodies that reactwith aA- and aB-crystallin gave comparable results andindicated that aA-crystallin was not present or existedat levels below that detectable in this antibody tech-nique. Densitometry showed that the spot for thephosphorylated aB-crystallin had about 10% to 15%of the intensity of that for the nonphosphorylatedform. Similar results were obtained when human tra-becular meshwork cell cultures were investigated (Fig.2C). After several days in organ culture, the aB-crys-tallin spot was enhanced when compared with thatfrom the trabecular meshwork of the fellow eye, whichwas excised and processed shortly after enucleation(Fig. 3). Silver stained two-dimensional gels from freshmonkey trabecular meshwork and trabecular mesh-work cell culture showed a polypeptide with a similarmobility and pi as the unphosphorylated human aB-crystallin (Fig. 2D, 2F). This protein also reacted withantibodies against aB-crystallin in two-dimensionalWestern blots (Fig. 2E). In contrast to human trabecu-lar meshwork, the phosphorylated form of aB-crys-tallin was minimal, and a small spot only could bedetected after exposure of the gel for 2 days (data notshown), compared with a normal exposure time of 1to 2 hours. Based on densitometry, this spot repre-sented less than 1% of the nonphosphorylated one. Incontrast, in monkey lens, phosphorylated aB-crystallincould be detected readily at an amount similar to thatin the human lens (data not shown).Northern Blots, Heat Shock, and OxidativeStress

Northern blots of untreated human trabecular mesh-work cell cultures showed two faint bands (marked by

2406 Investigative Ophthalmology & Visual Science, November 1996, Vol. 37, No. 12

TMTM

* '

Trabecular Meshwork and aB-Crystallin 2407

FIGURE 1. Immunohistochemistry of normal human (A to C) and monkey (D) trabecularmeshwork (D) in situ with antibodies against aB-crystallin. Acetone fixed frozen sections,alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. (A) In most eyes, stain-ing for aB-crystallin is found in the cribriform orjuxtacanalicular region of Schlemm's canal(arrows), but not in the corneoscleral or uveal parts of the meshwork (sagittal section,magnification = X500). (B) Frontal sections parallel to Schlemm's canal show that stainingfor aB-crystallin is equally distributed along the cribriform area (mrmus, magnification =X100). (C) In the eyes of one human donor, positive immunostaining is found in cells ofall parts of the trabecular meshwork as well as in the endothelial cells all around Schlemm'scanal (sagittal section, magnification = X200). (D) In monkey eyes, staining is confined tothe outer regions of the trabecular meshwork, such as the corneoscleral and cribriformparts. In addition, endothelial cells of Schlemm's canal and of collector channels (mrows)are stained. Ciliary muscle cells also are immunoreactive, but not cells in the scleral spur(sagittal section, magnification = X200). TM: trabecular meshwork; S: sclera; AC: anteriorchamber; CM: ciliary muscle; asterisk: Schlemm's canal.

the arrows [Co lane] in Fig. 4) after hybridization withan aB-crystallin-DNA probe, which were approxi-mately 0.8 and 1.1 kb in length. By contrast, in un-treated monkey trabecular meshwork cells, only the0.8-kb band was observed (marked by the arrow [Colane] in Fig. 5), which migrated with monkey lens aB-crystallin mRNA (data not shown). In both human(Fig. 4) and monkey (Fig. 5) cells, a strong accumula-tion in aB-crystallin mRNA could be seen 2 hours afterheat shock. To get a semiquantitative measure, a scanwas done and the intensities of the aB-crystallin bandswere normalized to the 28S-bands. The relative densi-

tometric intensity showed a 24-fold increase in hybrid-ization in human and an 8-fold increase in monkeytrabecular meshwork cells 2 hours after heat shock.The increase in mRNA expression was the highest at4 hours, with a 40-fold increase in human and a 19-fold increase in monkey trabecular meshwork cells,and decreased by 8 and 16 hours (Figs. 4, 5). In hu-mans, the mobility of the induced mRNA band after2 hours heat shock appeared to be between that ofthe two bands seen in control cultures, such as be-tween 0.8 and 1.1 kb. During later stages (4 and 8hours), the mobility of the heat shock-induced mRNA

FIGURE 2. Two-dimensionalgel electrophoresis of un-treated human (A) andmonkey (D) trabecularmeshwork ex situ and hu-man (C) and monkey (F)trabecular meshwork cellcultures. The acidic pole ison the right-handed side ofeach gel. In human samples,2.5 /zg total protein was ana-lyzed, and in monkey sam-ples, 0.7 (j,g was analyzed.The first dimension was a pH3 to 9 gel containing 8 Murea and 2% Nonidet P-40.The second dimension was agradient gel of 8% to 25%acrylamide. Gels were silverstained. Western blots (B,E)were incubated with antibod-ies to aB-crystallin. Arrowson Western blots show posi-tions of aB-crystallin and themodified (phosphorylated)aB (aBm[p])-crystaHin.

k k

B

A A

- 24 -

aB

24 -

2408 Investigative Ophthalmology 8c Visual Science, November 1996, Vol. 37, No. 12

Acidic Basic

A B CFIGURE 3. Two-dimensional gel electrophoresis of control (A, right eye) and 4 d organcultured (B, left eye) trabecular meshwork from a 63-year-old man. In each sample, 2.5 figtotal protein was analyzed. The first dimension was a pH 3 to 9 gel containing 8 M ureaand 2% Nonidet P-40. The second dimension was a gradient gel of 8% to 25% acrylamide.Gels were silver stained. The Western blot (C) is from the organ culture sample and wasincubated with antibodies to aB-crystallin. Arrows (A,B) show positions of aB-crystallin (right)and the phosphorylated aB-crystallin (left).

increased and comigrated with the 0.8-kb species at16 hours (Fig. 4). Similarly, in monkey trabecularmeshwork, the mRNA transcript 2 hours after heatshock had a lower mobility than that of the control,and between 4 and 16 hours, the mobility of heatshock-induced mRNA returned to that of controlmRNA in a stepwise fashion (Fig. 5).

Co 2h 4h 8h 16h

Both in monkey and human Northern blots, theaB-crystallin probe hybridized with an additional highmolecular weight band at approximately 2.6 to 2.9 kb.This band also was inducible after heat shock (Figs.4, 5) and consisted presumably of preprocessed aB-crystallin RNA.

Finally, no band for aB-crystallin mRNA could be

Co 2h 4h 8h 16h

1 . 1 -*•0.8 — m

— 1.77— 1.52— 1.28— 0.78 0.8 •

1.77= 1.52— 1.28

~ 0.78

1 24 40 21 6

— 28S

RDI

FIGURE 4. Northern blot analysis of aB-crystallin messengerRNA {mRNA) in confluent human trabecular meshworkcells under control conditions (Co) and 2 to 16 hours afterheat shock. Loaded on each lane were 20 fj,g of total RNA.The size of molecular markers is given in kilobases. Thearrows mark the position of the 0.8- and 1.1-kb aB-crystallinmRNA species in the control. Relative amounts of RNA thatwere loaded were controlled by reprobing the membranewith an oligonucleotide specific for 28S ribosomal RNA(rRNA). Clearly, some differences were evident that allowedappropriate normalization for the amount of aB-crystallinmRNA. RDI: relative densitometric intensity. RDI (normal-ized to 28S rRNA) of the 2.6- to 2.9-kb bands (presumablypreprocessed aB-crystallin RNA) are as follows: Co: 1; 2 h:88; 4 h: 101; 8 h: 76; 16 h: 198. h = hours.

f • — 28S

1 8 19 6 16 RDI

FIGURE 5. Northern blot analysis of aB-crystallin messengerRNA (mRNA) in confluent monkey trabecular meshworkcells under control conditions (Co) and 2 to 16 hours afterheat shock. Loaded on each lane was 15 fig of total RNA.The size of molecular markers is given in kilobases. Thearrow marks the position of the 0.8-kb aB-crystallin mRNAspecies in the control. Relative amounts of RNA that wereloaded were controlled by reprobing the membrane withan oligonucleotide specific for 28S ribosomal RNA (rRNA).Clearly, some differences were evident that allowed appro-priate normalization for the amount of aB-crystallin mRNA.RDI: relative densitometric intensity. RDI (normalized to28S rRNA) of the 2.6- to 2.9-kb bands (presumably prepro-cessed aB-crystallin are as follows: Co: 1; 2 h: 11; 4 h: 45; 8h: 36; 16 h: 13. h = hours.

Trabecular Meshwork and aB-Crystallin

Lens Co 2h 4h

2370 —

1350 —

t1.771.521.280.78

2409

2h 4h 8h 16h Co

••-0.8

28S —

FIGURES. Northern blot analysis of aB-crystallin messengerRNA (mRNA) in monkey lens (lens) and confluent simianvirus 40 (SV 40) transformed human trabecular meshworkcells under control conditions (Co) and 2 to 4 hours afterheat shock. Loaded were 100 ng of lens total RNA and 20fj,g of total RNA for each meshwork lane. The arrow marksthe lens aB-crystallin position, the arrowhead the weak aB-crystallin mRNA band that was induced after 4-hour heatshock. The size of molecular markers is given in kilobases.Integrity of RNA was confirmed by reprobing the membranewith an oligonucleotide specific for 28S ribosomal RNA.

observed in untreated cultures of SV 40 transformedmeshwork cells. Four hours after heat shock, the aB-crystallin probe hybridized with a weak band that mi-grated slightly slower than that for monkey lens aB-crystallin mRNA (Fig. 6)

Western dot blot analysis showed a 3.4-fold in-crease in protein 24 hours after heat shock in humantrabecular meshwork cell cultures and a 20-fold in-crease after 48 hours in monkey trabecular meshwork

WESTERN DOT-BLOTSFOR aB-CRYSTALLIN

Heat Shock {15min, 44°C)

Human Trabecular MeshworkCo 12h24h

1 1.8 3.4

Monkey Trabecular MeshworkCo 48h

• •1 20

FIGURE 7. Western dot blots of confluent human and mon-key trabecular meshwork cells under control conditions(Co) and 12 to 48 hours after heat shock. For each dot,1 jj,g of protein was spotted on nitrocellulose paper andincubated with an antibody to aB-crystallin. The numbersbelow the spots show the densitometric intensity.

28S —

RDI 1 2 4 16 6

FIGURE 8. Northern blot analysis of aB-crystallin messengerRNA (mRNA) in confluent human trabecular meshworkcells 2 to 16 hours after oxidative stress (1 hour serum-free medium with 200 /j,mol hydrogen peroxide) and undercontrol conditions (Co, 16 hours after 1 hour serum-freemedium). Loaded on each lane was 15 fig of total RNA.The size of molecular markers is given in kilobases. Thearrows mark the position of the 0.8- and 1.1-kb aB-crystallinmRNA species in the control. Relative amounts of RNA thatwere loaded were controlled by reprobing the membranewith an oligonucleotide specific for 28S ribosomal RNA(rRNA). Clearly, some differences were evident that allowedappropriate normalization for the amount of aB-crystallinmRNA. RDI: relative densitometric intensity. RDI (normal-ized to 28S rRNA) of the 2.6- to 2.9-kb bands (presumablypreprocessed aB-crystallin RNA) are as follows: Co: 1; 2 h:1.1; 4 h: 1.8; 8 h: 1.1; 16 h: 2.5. h = hours.

cell cultures (Fig. 7). Thus, the increases in aB-crys-tallin mRNA are followed by a corresponding increasein translation and, consequently, protein.

Both in human (Fig. 8) and monkey (Fig. 9) mesh-work cells, oxidative stress caused an accumulation ofaB-crystallin mRNA. The time course for aB-crystallinmRNA accumulation was slower than that after heatshock. In the human cells, the 0.8- and 1.1-kb bandswere both induced, whereas in the monkey cells, the0.8-kb band increased. Although the relative densito-metric intensity values indicated that the amount ofaB-crystallin mRNA in the control (16 hours after 1hour serum-free medium) was in human cells 6-fold,and in monkey cells 3-fold higher than 2 hours afteroxidative stress (1 hour serum-free medium with 200//mol hydrogen peroxide), the relative densitometricintensity of controls was always lower than that 16hours after oxidative stress (16 in the human cellsand 40 in the monkey cells). As in the heat shockexperiments, there was an induction of the high mo-lecular weight mRNA band hybridizing with the aB-crystallin probe, especially in monkey cells, althoughthis was quite modest in the human cells. In neitherhuman nor in monkey cells was any change in electro-phoretic mobility observed after oxidative stress.

2410 Investigative Ophthalmology & Visual Science, November 1996, Vol. 37, No. 12

2h 4h 8h 16h Co

I0.8 —

1 60 30 44 3

1.771.521.280.78

— 28S

RDIFIGURE 9. Northern blot analysis of aB-crystallin messengerRNA (mRNA) in confluent monkey trabecular meshworkcells 2 to 16 hours after oxidative stress {1 hour serum-free medium with 200 /imol hydrogen peroxide) and undercontrol conditions (Co, 16 hours after 1 hour serum-freemedium). Loaded on each lane was 7 /izg of total RNA. Thesize of molecular markers is given in kilobases. The arrowmarks the position of the 0.8-kb aB-ciystallin mRNA speciesin the control. Relative amounts of RNA that were loadedwere controlled by reprobing the membrane with an oligo-nucleotide specific for 28S ribosomal RNA (rRNA). Clearly,some differences were evident that allowed appropriate nor-malization for the amount of aB-crystallin mRNA. RDI: rela-tive densitometric intensity. RDI (normalized to 28S rRNA)of the 2.6- to 2.9-kb bands presumably preprocessed aB-crystallin RNA) are as follows: 2 h: 1; 4 h: 88; 8 h: 101; 16h: 76; Co: 198. h = hours.

DISCUSSION

Our results show that aB-crystallin is expressed consti-tutively in human and monkey trabecular meshworkin situ and that cultured trabecular meshwork cellsfrom both species express and translate mRNA for aB-crystallin. The aB-crystallin was expressed predomi-nantly in the cribriform or juxtacanalicular area ofthe trabecular meshwork, which is the main site ofoutflow resistance. Only in one eye, aB-crystallin wasfound throughout the entire meshwork, which mightbe because of stress-related events before enucleation.

The aB-crystallin mRNA from monkey trabecularmeshwork cell cultures migrated with monkey lensmRNA at approximately 0.8 kb. In contrast, humantrabecular meshwork cells expressed equal amountsof two distinct mRNAs for aB-crystallin approximately0.8 and 1.1 kb in length. Two sizes of aB-crystallinmRNA transcripts also have been found in mouse andrat tissues by Northern blot analysis.33"33 The shorteraB-crystallin mRNA predominated in lens, heart, skel-etal muscle, and kidney, whereas the longer transcriptwas found mainly in lung and brain. Although thelonger mRNA band in Northern blots might be from

a major transcription start site that has been character-ized at position —474 of the aB-crystallin promoter/1'there also may be other upstream transcription initia-tion sites that contribute to longer aB-crystallin tran-scripts.35'37'38 In addition to bands of 0.8 and 1.1 kb,Northern blots for aB-crystallin showed in both spe-cies an additional band of 2.6 to 2.9 kb, which wasinduced after heat shock and oxidative stress. A bandof comparable size was not shown after similar experi-ments in human glioma cells or rat astrocytes16'39 Themost likely explanation is that this band reflects pre-processed aB-crystallin mRNA. The accumulation ofsuch mRNA might be because of a stress-related delayin mRNA processing. Interruption of mRNA splicinghas been reported to be a generalized response tohigh temperature stress.40 In meshwork cells fromboth species, the time course of aB-crystallin mRNAinduction after heat shock was comparable to thatobserved in rat astrocytes.16 Another similarity to heatshock in astrocytes was that both monkey and humanmRNA showed a transitory decrease in electrophoreticmobility, suggesting an increase in molecular weightafter heat shock. This increase might be because of atransient increase in transcription of mRNA withlonger 5' leading sequences or result from transitoryheat shock-related changes in extent of polyadenyla-tion. Such an increase in the length of the poly(A)tail after heat shock has been observed for the mRNAsof the small heat shock proteins in Drosophild11 andthe plant heat shock protein 21.42 A longer poly (A)tail might result from the addition of more adenylateresidues in the nucleus, from decreased deadenylationor increased readenylation in the cytoplasm, or frominhibition of RNA transport out of the nucleus, whichwould prevent shortening by deadenylation. A longerpoly(A) tail might confer increased stability or trans-latability to the aB-crystallin transcripts, because forsome RNAs, poly(A) tail length is correlated with de-creased mRNA degradation and with increased trans-latory efficiency.43 In contrast to heat shock experi-ments, aB-crystallin mRNA induction in human mesh-work cells after oxidative stress involved both mRNAsizes similarly and was not associated with any changesin electrophoretic mobility in monkey or in humancells. In addition, the time course of induction wasslower than that observed after heat shock. In general,heat shock gene induction is thought to be mediatedby binding of heat shock factor 1 to heat shock ele-ments (inverted NGAAN repeats).44 Indeed, putativeheat shock elements are present in the 5' flankingsequence of the mouse, rat, and human aB-crystallingenes in similar relative positions.3133'36'38 Conversely,oxidative stress and increase in reactive oxygen inter-mediates activates other transcription factors such asNF-KB or AP-1.45 Human and rodent aB-crystallin 5'flanking regions contain an API-like consensus se-

Trabecular Meshwork and aB-Crystallin 2411

quence,31'30'38 and a consensus NF-KB site was reportedfor the rat aB-crystallin gene.'6 Clearly, further studiesare necessary to identify the molecular basis of regula-tory elements that modulate the stress responses ofthe aB-crystallin gene.

The fact that SV 40 transformed cell lines fromhuman meshwork did not show constitutive mRNAexpression for aB-crystallin, and only a weak inductionafter heat is consistent with a dedifferentiation ofthese cells and limits their use in further studies onaB-crystallin expression in trabecular meshwork.

Comparison of the proteins in fresh and culturedtrabecular meshwork displayed by two-dimensional gelelectrophoresis did not show any large differences inaB-crystallin. The small enhancement of aB-crystallinafter several days of organ culture might be becauseof stress-related events while establishing and main-taining the culture system. In contrast to the humanmeshwork (present study), the lens,46 the bovineheart,41' and Alexander's disease brain,'17 phosphory-lated aB-crystallin barely was detectable in fresh orcultured monkey trabecular meshwork. Phosphoryla-tion of a-subunits are catalyzed by a cyclic adenosinemonophosphate (cAMP)-dependent kinase48; in addi-tion, the a-crystallin subunits can autophosphorylateserine residues in an cAMP-independent manner.40

The functional role of aB-crystallin phosphorylationis not clear, but there is evidence that phosphorylationis not important for stress-related aB-crystallin accu-mulation, induction of thermotolerance, or chaper-one activity.lr>'20'50

The aB-crystallin is a molecular chaperone thatsuppresses heat-induced aggregation of lens and non-lenticular proteins.14'51 In vitro studies using trans-fected National Institutes of Health 3T3 cells, gliomacells, or murine L929 fibroblasts show that enhancedexpression of aB-crystallin confers cellular thermore-sistance15'52 and protection against oxidative stress.53

The mechanisms by which aB-crystallin performsthese functions are not clear. One role of aB-crystallinis to interact with cytoskeletal structures of cells. Theassociation of aB-crystallin with various types of inter-mediate filaments in characteristic inclusion bodies ofa variety of human diseases is well documented.i9-a4-55

Under normal, nonstress conditions, aB-crystallin isco-localized with desmin in the Z-bands of cardiac andslow-twitch, high-oxidative skeletal muscle fibers.56'57

In vitro aB-crystallin is able to bind to actin, desmin,and GFAP.56'58'5" It prevents spontaneous aggregationof desmin, low pH-induced aggregation of actin fila-ments, and the in vitro assembly of glial fibrillaryacidic protein and vimentin.50'59 Reduction of aB-crys-tallin in glioma cells by antisense transfection changesthe morphology of these cells and causes a loss ofactin stress fibers.52

There is considerable evidence that cytoskeletal

elements are important for a regular function of thetrabecular meshwork. Ethacrynic acid, a compoundthat in vivo increases outflow facility in human andmonkey eyes, causes in vitro changes of major cytoskel-etal components of human trabecular meshwork cells,including actin, vimentin, and tubulin.00 CytochalasinB, an inhibitor of actin aggregation, increases outflowfacility in monkey eyes, whereas phalloidin, an inhibi-tor of actin filament depolymerization, inhibits thefacility-increasing actions of epinephrine and cytocha-lasin B.01'02 Because actin is oxidized readily in mixed-function oxidation systems,03 aB-crystallin in themeshwork might play a role in protecting damage ofactin and other cytoskeletal elements. Several studieshave reported that the amount of actin in the trabecu-lar meshwork decreases with age and primary open-angle glaucoma.04"06 Interestingly, the ability of lensa-crystallin to protect against heat-induced aggrega-tion is age-dependent,67 raising the possibility that aB-crystallin in the trabecular meshwork might play foraging or glaucomatous processes in this tissue.

Key Words

anterior eye segment, crystallin, Northern blot analysis,small heat shock protein, two-dimensional gel electrophore-

Acknowledgments

The authors thank J. Cogan, Center for Biological Evalua-tions and Research (FDA), for obtaining the monkey eyes,J. Horwitz (Jules Stein Institute, UCLA, Los Angeles, CA)for providing the antibodies for aB-crystallin, and lok-HouPang and Louis DeSantis (Alcon Research Laboratories,Forth Worth, TX) for providing SV 40 transformed mesh-work cells. The authors also thank A. Cvekl, M. Duncan, P.Frederikse, R. Gopal-Srivastava, ]. Haynes 11, M. Kantorow,T. Kays, and B. Norman for their advice and discussion.

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