dickkopf-1 level is lower in patients with ankylosing spondylitis than in healthy people and is not...
TRANSCRIPT
Rheumatol Int (2012) 32:2523–2527
DOI 10.1007/s00296-011-1981-0ORIGINAL ARTICLE
Dickkopf-1 level is lower in patients with ankylosing spondylitis than in healthy people and is not inXuenced by anti-tumor necrosis factor therapy
Seong-Ryul Kwon · Mie-Jin Lim · Chang-Hee Suh · Shin-Goo Park · Yeon-Sik Hong · Bo-Young Yoon · Hyoun-Ah Kim · Hyo-Jin Choi · Won Park
Received: 9 August 2010 / Accepted: 3 July 2011 / Published online: 16 July 2011© Springer-Verlag 2011
Abstract (1) To compare the serum levels of Dickkopf-1(DKK-1) and bone biomarkers in patients with ankylosingspondylitis (AS) and healthy controls. (2) To examine theeVects of anti-tumor necrosis factor-� (TNF-�) therapy for3 months on bone biomarkers in patients with AS. Wemeasured the levels of DKK-1, osteocalcin, osteoproteg-erin, and C-terminal telopeptide of type I collagen (CTX-1)in patients with AS and in healthy controls at baselineand 3 months after initiating anti-TNF-� therapy in ASpatients. Bath Ankylosing Spondylitis Disease Activity
Index (BASDAI) scores were also measured before andafter anti-TNF-� therapy in AS patients. Serum levels ofDKK-1 were signiWcantly lower in the AS patients than inthe controls (P < 0.0001). Osteocalcin and osteoprotegerinlevels were signiWcantly higher in the AS patients than inthe controls (P < 0.0001). Serum levels of DKK-1 were notchanged after the 3-month anti-TNF-� therapy. Osteocalcinlevel increased (P < 0.0001), osteoprotegerin level andBASDAI scores decreased (P = 0.025 and P < 0.0001,respectively) signiWcantly after the 3-months anti-TNF-�therapy. Serum DKK-1 level was lower in patients with ASthan in healthy controls and did not change after 3 monthsof anti-TNF-� therapy in the AS patients despite themarked improvement in BASDAI scores. These Wndingssuggest the low serum DKK-1 level is related to the patho-genesis of new bone formation in AS, which is resistant toTNF-� blocking therapy.
Keywords Dickkopf-1 · Ankylosing spondylitis · TNF blocker · Osteocalcin · Osteoprotegerin · CTX-1 · BASDAI
Introduction
Ankylosing spondylitis (AS) is a chronic inXammatory dis-ease characterized by inXammation of the spine and canlead to bone erosion, new bone formation, and ankylosis ofthe spine. Tumor necrosis factor-� (TNF-�) plays an impor-tant role in the inXammatory response observed in patientswith AS. Treatment with TNF-� blocking agents (etaner-cept, adalimumab, and inXiximab) has been shown to besafe and eVective for reducing the signs and symptomsassociated with AS [1–3] and can signiWcantly improve thehealth-related quality of life in AS patients [4]. However,this treatment has little or no eVect on structural remodeling.
S.-R. Kwon · M.-J. Lim · W. Park (&)Rheumatism Center, Inha University Hospital, 3rd street Sinheung-dong, Jung-gu, Incheon 400-711, Republic of Koreae-mail: [email protected]
C.-H. Suh · H.-A. KimDepartment of Rheumatology, Ajou University School of Medicine, Suwon, Republic of Korea
S.-G. ParkDepartment of Occupation and Environmental Medicine, Inha University Hospital, Incheon, Republic of Korea
Y.-S. HongDepartment of Rheumatology, St. Mary’s Hospital, Incheon, Republic of Korea
B.-Y. YoonDepartment of Rheumatology, Inje University Ilsan Paik Hospital, Goyang, Republic of Korea
H.-J. ChoiDepartment of Rheumatology, Gachon School of Medicine, Gachon University of Medicine and Science, Incheon, Republic of Korea
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Recent studies have reported that up to 2 years of treatmentwith TNF-� blockers does not inhibit the progression of ASas shown in radiological assessment [5, 6]. The molecularaspects of structural remodeling in patients with AS requirefurther study.
AS is characterized by the fusion of joints and interverte-bral spaces. Fusion of the joints is caused by increasedendochondral ossiWcation, which allows bone formationand bridges the joint space. When inXammation occurs inthe joint space, this inXammation leads to joint damage.After the initial destructive changes occur, the jointsrespond by forming osteophytes, which are bony apposi-tions originating from the juxta-articular periosteal lining[7].
To explain these phenomena, Diarra et al. showed thatDickkopf-1 (DKK-1) plays a key role in the remodeling ofjoints [8]. They hypothesized that the joint pathologyaVects the regulators of bone formation that drive eitherbone resorption or formation of new bone in the diseasedjoint. Proteins synthesized by the group of wingless (Wnt)genes are key mediators of osteoblastogenesis and modu-late the formation of the skeleton during the developmentof the embryo [9]. Several members of the Wnt proteinfamily bind to a receptor complex (comprising LPR5/6 andfrizzled receptors) on the plasma membrane of mesenchy-mal cells, and this binding signals osteoblast diVerentiationby engaging the intracellular protein �-catenin [10, 11].Wnt signaling is modulated by several diVerent families ofsecreted negative regulators. Among these, DKK is a fam-ily of cysteine-rich proteins comprising at least four diVer-ent forms (DKK-1, DKK-2, DKK-3, and DKK-4). The beststudied of these is DKK-1, which functions as a naturalinhibitor of Wnt signaling [12, 13]. Binding of DKK-1 tothe LPR5/6 receptor and a cell surface co-receptor, Kre-men-1/2, promotes internalization of the receptor complexand dampens the Wnt signal. Deletion of a single allele ofDKK-1 increases bone mass in mice [14]. Aberrant expres-sion of DKK-1 in myeloma cells was recently shown to beassociated with increased bone erosion in human multiplemyeloma [15].
Inhibition of DKK-1 reverses the bone-destructive pat-tern in a mouse model of rheumatoid arthritis to the bone-forming pattern of osteoarthritis [8]. Diarra et al. suggestedthat DKK-1 is a key regulator of joint remodeling. Theyshowed that the serum DKK-1 level was diVerent inpatients with AS and rheumatoid arthritis, and in healthycontrols, but the number of enrolled subjects is not clearfrom their report.
We compared the DKK-1 levels and markers associ-ated with bone turnover in patients with AS and healthycontrols, and we examined the eVects of anti-TNF-� ther-apy for 3 months on these bone biomarkers in patientswith AS.
Patients and methods
Patients
Fifty-six individuals diagnosed with AS according to themodiWed New York criteria [16] with active disease thatwas refractory to non-steroidal anti-inXammatory drugs(NSAIDs) and disease-modifying anti-rheumatic drugtreatment and 40 healthy volunteer controls were enrolledfrom multiple centers. Patients were allowed to receive sul-fasalazine, methotrexate, or NSAIDs only at stable dosesduring the study. The type of TNF-� blocking therapy wasdetermined mainly by patient choice after each patientreceived medical information from the rheumatologist.
Patients with AS received etanercept at a dosage of25 mg twice weekly or adalimumab 40 mg every otherweek by subcutaneous administration during the 12-weeksstudy or 5 mg/kg inXiximab infusion at weeks 0, 2, 6, and14. Our study was approved by the local ethics committee,and patients signed written informed consent forms accord-ing to the Declaration of Helsinki before participating.
Clinical outcomes and laboratory methods
Serum samples were obtained at baseline and after12 weeks of etanercept or adalimumab or 14 weeks ofinXiximab therapy in patients with AS.
Erythrocyte sedimentation rate (ESR) and C-reactiveprotein (CRP) levels were measured after the patientsarrived at each center. Serum samples used for the measure-ment of the levels of DKK-1, osteocalcin, osteoprotegerin,and C-terminal telopeptide of type I collagen (CTX-1) werefrozen quickly and stored at ¡80°C until analyzed.
Serum levels of DKK-1, CTX-1, osteocalcin, and osteo-protegerin were measured using commercially availableenzyme-linked immunosorbent assay kits: DKK-1, AssayDesigns (Ann Arbor, MI, USA); CTX-1, Immunodiagnos-tic Systems Ltd. (Boldon, UK); osteocalcin, Quidel (Hano-ver, Germany); and osteoprotegerin, Biomedica (Vienna,Austria). The Bath Ankylosing Spondylitis Disease Activ-ity Index (BASDAI) [17] was also measured.
Statistical analysis
Each value is described using the mean § SD. Statisticalanalysis was performed using the SPSS software packagefor Windows v17.0. DiVerences in the levels of DKK-1 andbiomarkers between baseline and after TNF-� blockingtherapy in patients with AS were analyzed using a pairedt test. DiVerences in levels of DKK-1 and biomarkersbetween patients with AS and controls were analyzed usingan independent samples t test. DiVerences were consideredsigniWcant when P values were less than 0.05.
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Results
Characteristics of the AS patients and controls
The patients with AS included 47 men and 9 women (age35.1 § 9.6 years) and the controls 31 men and 9 women(age 43.5 § 8.6 years). The age and sex ratio did not diVersigniWcantly between the patients with AS and the controls.Among the 56 patients with AS, 20 had axial involvementonly and 36 had axial and peripheral involvement. Twelvehad a history of uveitis. Six patients received etanercept, 22patients inXiximab, and 21 patients adalimumab. Thedemographic characteristics of the AS patients are summa-rized in Table 1.
DKK-1 and bone biochemical markers in patients with AS and controls
Serum DKK-1 levels were signiWcantly lower in thepatients with AS than in the controls (P < 0.0001). Meanserum CTX-1 levels did not diVer signiWcantly between theAS patients and controls. Serum osteocalcin and osteopro-
tegerin levels were signiWcantly higher in patients with ASthan in the controls (P < 0.0001 for both; Table 2).
Clinical response following TNF-� blocking therapy
Both the clinical and laboratory measures of disease activ-ity improved after 3 months of TNF-� blocking therapy.BASDAI scores improved signiWcantly after TNF-� block-ing therapy (P < 0.0001). CRP levels and ESR decreasedsigniWcantly after TNF-� blocking therapy (P < 0.001 forboth; Table 3).
DKK-1 and bone biochemical markers following 3 months of TNF-� blocking therapy in patients with AS
Serum DKK-1 and CTX-1 levels did not change after3 months of TNF-� blocking therapy. However, serumosteocalcin levels increased signiWcantly after the TNF-�blocking therapy (P < 0.0001). Serum osteoprotegerin lev-els decreased after the TNF-� blocking therapy (P = 0.025;
Table 1 Baseline demographic characteristics
Mean § SD. DMARDs, disease-modifying antirheumatic drugs; BMD,bone densitometry; CRP, C-reactive protein; ESR, erythrocyte sedi-mentation rate; BASDAI, the bath ankylosing spondylitis disease activ-ity index
Characteristics Ankylosing spondylitis Controls
Number 56 40
Age, year 34.6 § 9.0 43.5 § 8.6
Female/male 9:47 9:31
Years after diagnosis 6.9 § 4.8
Schober test, cm 2.9 § 1.8
Chest expansion, cm 3.0 § 1.5
Occiput to wall, cm 1.7 § 3.7
Peripheral arthritis, no. (%) 36 (64.3)
Uveitis, no. (%) 12 (21.4)
BMD (T score)
Lumbar spine total ¡1.0 § 1.6
Femur total ¡0.7 § 1.1
Femur neck ¡0.7 § 1.1
DMARDs use
Sulfasalazine, no. (%) 55 (98.2)
Methotrexate, no. (%) 11 (19.6)
HLA B27, no. (%) 55 (98.2)
Serum CRP, mg/dl 1.7 § 2.0
ESR, mm/h 26.0 § 23.9
BASDAI 7.7 § 1.9
Table 2 DKK-1 and bone biochemical markers in patients with ASand in controls
DKK-1, Dickkopf-1; CTX-1, C-terminal telopeptide of type I collagen;OPG, osteoprotegerin
* Independent sample t test
AS Controls P value*
n Mean § SD n Mean § SD
DKK-1, pg/ml 49 12,321 § 6,136 39 20,811 § 5,671 <0.0001
CTX-1, ng/ml 51 0.41 § 0.28 39 0.35 § 0.20 0.194
Osteocalcin, ng/ml
54 14.5 § 5.6 39 8.9 § 3.4 <0.0001
OPG, pmol/L 55 3.5 § 1.1 40 2.0 § 1.0 <0.0001
Table 3 EVects of TNF-� blocker therapy for 3 months on biochemi-cal and clinical measures in patients with AS
DKK-1, Dickkopf-1; CTX-1, C-terminal telopeptide of type I collagen;OPG, osteoprotegerin; BASDAI, bath ankylosing spondylitis diseaseactivity index; ESR, erythrocyte sedimentation rate; CRP, C-reactiveproteina Mean § SD9 Paired t test
n Baselinea 3mo latera P value9
DKK-1, pg/ml 49 12,321 § 6,136 11,726 § 5,961 0.459
CTX-1, ng/ml 54 0.41 § 0.28 0.47 § 0.36 0.173
Osteocalcin, ng/ml
56 14.5 § 5.6 17.6 § 4.5 <0.0001
OPG, pmol/l 56 3.5 § 1.1 3.1 § 1.2 0.025
BASDAI 56 7.7 § 1.9 3.8 § 1.7 <0.0001
ESR, mm/h 56 26.0 § 23.9 7.0 § 7.1 <0.0001
CRP, mg/dl 56 1.7 § 2.0 0.1 § 0.2 <0.0001
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Table 3). The bone biomarkers (DKK-1, CTX-1, osteocal-cin, and osteoprotegerin) levels did not diVer between thesubgroups treated with diVerent kinds of TNF-� blockers.
Discussion
We measured DKK-1 levels in patients with AS and foundthat this level was signiWcantly lower in patients than inmatched healthy controls. In patients with AS, after the initialdestructive changes by inXammation of the joint, blockadeof DKK-1 relieves Wnt signaling from DKK-1-mediatedsuppression and induces bone formation, as mirrored by thegrowth of osteophytes [8]. Canonical Wnt proteins induceosteoprotegerin expression, which blocks the receptor acti-vator of nuclear factor kappa b (NF-�B) ligand-mediatedbone resorption [18]. Consistent with the model of Diarraet al., we would expect the serum DKK-1 level in patientswith AS to be suppressed but the levels of osteocalcin andosteoprotegerin to increase during osteophyte formation.This is consistent with our results (Table 2), which presentstrong evidence to support the conclusions of the studyreported by Diarra et al. [8].
Blockade of DKK-1 leads to a profound inhibition ofosteoclast formation and bone resorption in joints, suggest-ing the existence of cross talk between the bone anabolicWnt and the bone catabolic receptor activator of NF-�B(RANK) ligand pathways [8]. At the molecular level,osteoprotegerin, a key modulator of the RANK–RANKligand system, appears to be regulated by the Wnt systemand vice versa; this is also supported by another recentstudy of Glass et al. [18]. The study of Glass et al. providesindirect evidence that the low DKK-1 level in patients withAS allows the osteoprotegerin level to be higher than in thecontrols. This dual action identiWes DKK-1 as a key regula-tor of pathological joint remodeling, which is a function ofthe interplay between anabolic and catabolic pathways.
Serum DKK-1 levels did not change, and the osteocalcinlevels increased after 3 months of TNF-� blocking therapyin AS patients. These results suggest that DKK-1 plays amajor role and TNF-� minor role in the new bone formationin the pathophysiology of AS, although the latter is a keyproinXammatory cytokine that is involved in the patho-physiology of AS. If TNF-� played a major role in the path-ophysiology of AS, serum DKK-1 levels should haveincreased after 3 months of TNF-� blocking therapy. How-ever, DKK-1 levels were unchanged, supporting a majorrole for DKK-1 in the structural damage associated withAS. Uderhardt et al. [19] studied human TNF-transgenicmice and found that joint ankylosis did not form spontane-ously, suggesting that TNF is not the driving factor for jointfusion. TNF blockade did not induce joint fusion, suggest-ing that blocking TNF does not lead to suYcient Wnt acti-
vation, which is necessary for the induction of bonyproliferation.
Increases in serum osteocalcin level after TNF-� block-ing therapy have been reported in AS patients [20–22].TNF-� is a key proinXammatory cytokine in AS, but it isalso a potent inhibitor of bone formation. Therefore, TNF-�might inhibit whereas Wnt signaling accelerates bone for-mation. After treatment with a TNF-� blocking agent, boneformation would not be inhibited by TNF-�, and the serumosteocalcin level would increase. Surprisingly, the serumosteoprotegerin level decreased after TNF-� blocking ther-apy. However, osteoprotegerin is a decoy receptor ofRANK ligand, and RANK ligand expression mightdecrease after TNF-� blocking therapy. Osteoprotegerinmight be inXuenced more by RANK ligand than by Wntsignaling. Therefore, it is possible that the serum osteopro-tegerin level decreases after TNF-� blocking therapymainly in response to the decrease in RANK ligand.
How can we explain the fusion of the sacroiliac jointwithout DKK-1? In an animal study, arthritis in TNF-trans-genic mice was caused by mesenchymal overexpression ofTNF, and inXammatory lesions were conWned to the ana-tomical sites with synovial tissue such as diarthrodial jointsand the sacroiliac joint [19]. By contrast, regions withoutsynovial tissue, such as the intervertebral spaces, werespared from inXammatory lesions. Thus, the TNF- trans-genic mice could be regarded as an excellent model ofbilateral erosive sacroiliitis, but not as a model of spond-yloarthropathy per se, because the pathological changesalong the intervertebral spaces including syndesmophyteformation were absent. This contrasts with the HLA B27-transgenic rat and the proteoglycan immunization modelsof BALB/c mice [23, 24], where both develop spinalfusion, and with the model of male DBA mice, whichshowed ankylosis of peripheral joints without a precedingerosive phase of the disease [25]. Therefore, HLA B27might be another factor in the pathophysiology of jointfusion and osteophyte formation in ankylosing spondylitis.Whereas ankylosis of the intervertebral spaces and periphe-ral joints was similar in these models, sacroiliac joint anky-losis was not reported in any of these models, although itconstitutes a major part of the disease process in humanspondyloarthropathy.
In conclusion, our data show lower serum DKK-1 levelsin patients with AS than in healthy controls and that theserum DKK-1 levels in patients with AS did not changeafter anti-TNF-� therapy despite the dramatic improvementin BASDAI scores. These Wndings suggest that low serumDKK-1 is involved in the pathogenesis of new bone forma-tion in AS, which is also known to be resistant to the TNF-�blocking therapy. Another strategic therapy is needed inaddition to the current treatment method to prevent thestructural changes associated with AS.
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Acknowledgments This study is supported by INHA UNIVERSITYResearch Grant (INHA 37476-01).
ConXict of interest The authors have declared no conXict of interest.
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