Alteration of Bone Turnover Markers in Canonical Wingless Pathway in Patients With Ankylosing Spondylitis

Jinxian HUANG1, Guoxiang SONG2, Zhihua YIN3, Zhongchao FU3, Zhizhong YE3

1Department of Rheumatology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China
2Department of Ophthalmology, The Third People’s Hospital of Shenzhen, Shenzhen, China
3Department of Rheumatology, The Fourth People’s Hospital of Shenzhen, Shenzhen, China

Keywords: Ankylosing spondylitis; bone turnover; canonical wingless pathway

Abstract

Objectives: This study aims to determine the levels of bone turnover markers in canonical wingless pathway in patients with ankylosing spondylitis (AS) and the correlation with disease activity indexes.
Patients and methods: We recruited a total of 43 AS patients (34 males, 8 females; mean age 36.8±9.3 years; range 22 to 62 years) and age- and sex-matched 42 healthy controls (32 males, 10 females; mean age 36.1±9.7; range 24 to 59 years). Serum levels of components of canonical wingless pathway including Dickkopf-1, glycogen synthase kinase-3β, β-catenin, alkaline phosphatase, and osteocalcin were detected using enzyme- linked immunosorbent assay method. All patients were assessed in terms of erythrocyte sedimentation rate, C-reactive protein, Bath Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Functional Index, Ankylosing Spondylitis Disease Activity Score, and the modified Stoke’s Ankylosing Spondylitis Spine Score. Pearson’s correlation test was used to analyze the correlation between serum bone turnover markers and clinical assessment indexes.
Results: No significant difference was observed between AS patients and healthy controls for the levels of glycogen synthase kinase-3β, β-catenin, alkaline phosphatase, and osteocalcin, respectively (p>0.05). The level of Dickkopf-1 was significantly higher in AS patients (1914.5±407.8 pg/mL) than in healthy controls (1729.1±352.9 pg/mL) (p<0.05). There was no correlation between high Dickkopf-1 level and any of the clinical parameters contributing to inflammation or bone formation. However, the correlation between osteocalcin and disease duration was significant in AS patients (r=0.323, p=0.034).
Conclusion: Alteration of bone turnover markers in canonical wingless pathway was observed in AS. This might partially explain the complicated mechanism of bone formation in the disease.

Introduction

Ankylosing spondylitis (AS) is a chronic inflammatory disorder mainly affecting the spine and sacroiliac joints, characterized by new bone formation that progressively leads to ankylosis and functional disability. Syndesmophytosis are pathological hallmarks of AS, which results from new bone formation induced by chronic inflammation and further structural damage.

The mechanisms that lead to bony fusion in AS are yet to be fully defined. Aberrant regulation of the wingless (Wnt) pathway has been suggested as a key element in the pathogenesis of AS.(1) There has been evidence for the involvement of this pathway in AS bone formation in vitro studies. As Wnt signaling inhibitors, the expression of Dickkopf (DKK)-1 and sclerostin were reduced in the spine of proteoglycan-induced spondylitis mice(2) and blockade of DKK-1 induces fusion of sacroiliac joints in tumor necrosis factor (TNF) transgenic mice,(3) implicating the Wnt pathway as a likely mediator of the mechanism by which inflammation induces bony ankylosis in spondyloarthritis.

Dickkopf-1 is a bone-specific inhibitor of the canonical Wnt pathway; if DKK-1 is not able to bind to low-density lipoprotein receptor-related protein 5/6,(4) Wnt proteins might interact with the receptors, resulting in dephosphorylation of glycogen synthase kinase-3β (GSK-3β) and activation of β-catenin. Beta-catenin is subsequently translocated into the nucleus to form a transcriptionally active β-catenin T-cell factor/lymphoid enhancer factor deoxyribonucleic acid-binding complex that regulates the Wnt target gene. Canonical Wnt signaling promotes osteogenesis by directly stimulating Runx2 gene expression. Runx2 activates osteocalcin (OC), which is an osteoblast-specific gene expressed by differentiated osteoblasts.(5,6)

In theory, the activation of Wnt pathway would be facilitated by decreased expression of DKK-1, while the serum expression of DKK-1 in AS has been controversial.(7-18) To the best of our knowledge, other components of Wnt pathway have not yet been studied.

We hypothesize that alteration of the components of canonical Wnt signaling, such as down regulation of negative regulators, might contribute to the activation of the pathway and effector gene expression and finally lead to the bone formation in AS. Therefore, in this study, we aimed to determine the levels of bone turnover markers in canonical Wnt pathway in patients with AS and the correlation with disease activity indexes.

Patients and Methods

We consecutively enrolled 43 AS patients (34 males, 8 females; mean age 36.8±9.3 years; range 22 to 62 years) and age and sex similar 42 healthy controls (32 males, 10 females; mean age 36.1±9.7; range 24 to 59 years). All patients were diagnosed with AS according to the modified New York criteria.(19) Bath Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Functional Index, and Ankylosing Spondylitis Disease Activity Scores(20,21) were assessed for all patients. All patients underwent an X-ray of the cervical and lumbar spine to calculate the modified Stoke’s Ankylosing Spondylitis Spine Score.(22) The study was approved by the local bioethics committee and all participants signed informed consents. The study was conducted in accordance with the principles of the Declaration of Helsinki.

Samples were centrifuged immediately and serum was separated and stored at -80 °C. Markers involving in the Wnt signaling pathway including DKK-1, GSK-3β, β-catenin, alkaline phosphatase (ALP) and OC were measured by using commercially available enzyme-linked immunosorbent assay tests (DKK-1, GSK-3β and β-catenin: EIAab Science Co. Ltd., Wuhan, China; OC: Immunodiagnostic Systems Ltd.). Erythrocyte sedimentation rate and C-reactive protein as well as human leukocyte antigen-B27 were detected for all AS patients.

Statistical analysis

Data for continuous variables are presented as mean ± standard deviation. The Kolmogorov Smirnov test was used to test the normality of distribution of continuous variables. Independent t-test was used to test for the differences in continuous variables between two groups. Pearson’s coefficient of correlation was used for bivariate correlations between continuous variables. P value <0.05 was considered statistically significant in all the above tests. Statistical analysis was performed using SPSS for Windows version 13.0 software (SPSS Inc., Chicago, IL, USA).

Results

Human leukocyte antigen-B27 positivity was 88.37%. Among the 43 AS patients, six had peripheral arthritis involvement including four males and two females. Mean disease duration was 8.9±7.6 years. Erythrocyte sedimentation rate and C-reactive protein were 22.3±20.6 mm/h and 12.8±13.6 mg/L, respectively. Mean Bath Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Functional Index and Ankylosing Spondylitis Disease Activity Score were 4.0±1.9, 43.0±21.5 and 2.0±1.0, respectively. Mean modified Stoke’s Ankylosing Spondylitis Spine Score was 13.9±16.0.

The levels of DKK-1 was significantly higher in AS patients (1914.5±407.8 pg/mL) than in healthy controls (1729.1±352.9 pg/mL) (p<0.05). No significant difference was observed between AS patients and healthy controls for the levels of GSK-3β, β-catenin, ALP and OC, respectively (p>0.05) (Table 1).

Table 1. Levels of bone turnover markers in canonical wingless signaling in ankylosing spondy- litis patients and healthy controls

Among the bone turnover markers in canonical Wnt pathway, no correlation was observed between the two markers in AS patients. Nonetheless, the correlation between several markers (DKK-1 and GSK-3β: p=0.050; DKK-1 and β-catenin: p=0.085; β-catenin and OC: p=0.070) almost reached significance (Table 2).
Table 2. Correlations between bone turnover markers in canonical wingless pathway in ankylosing spondylitis patients

Likewise, there was no correlation between high DKK-1 level and any of the clinical parameters contributing to inflammation or bone formation. However, the correlation between OC and disease duration was significant in AS patients (r=0.323, p=0.034). We also noticed that the correlation between ALP and Bath Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Functional Index, and Ankylosing Spondylitis Disease Activity Score nearly achieved significance with p values of 0.055, 0.072, 0.062, respectively (Table 3).
Table 3. Correlations between bone turnover markers and clinical parameters in ankylosing spondylitis patients

Discussion

Our study demonstrated the expression of bone turnover markers in the downstream of canonical Wnt pathway. As for the widely investigated negative regulator of canonical Wnt pathway, DKK-1 upregulation was replicated in our study. Some previous studies supported the downregulation of DKK-1 and the probable activation of Wnt signaling, while others showed no significant difference between AS patients and controls, or even higher expression of DKK-1 in AS patients than in controls(7,18) (supplemental data 1). There are at least three explanations for this contradictory situation. First, the dysfunction of DKK-1 was more in cellular level, instead of that in serum. DKK-1 binding to low-density lipoprotein receptor-related protein-6 has been reported to be reduced in AS.(23) And the functional capacity of DKK-1 from the sera of patients with AS to directly antagonize Wnt signaling was proved in a previous study.(15) Second, there might be a compensatory effect for DKK-1 in the pathway during AS bone formation, as suggested by a recent study. AS patients with no syndesmophyte formation show significantly higher functional DKK-1 levels, suggesting that blunted Wnt signaling suppresses new bone formation and consequently syndesmophyte growth and spinal ankylosis.(18) Third, other negative regulator in the signaling pathway might also contribute. A low serum level of sclerostin in the setting of AS is linked to increased structural damage, emphasizing the role of sclerostin in the suppression of bone formation.(24,25)

Figure 1. Supplemental data 1. Dickkopf-1 expression in ankylosing spondylitis patients in published studies

Except for DKK-1, ALP and OC, the other markers including GSK-3β and β-catenin were to date the first to be evaluated in serum samples of AS patients. No significance was identified for these two markers between patients and controls, supporting future detection in cellular level to prove the dysfunction of this pathway.

As well known bone turnover markers, OC(26-38) (supplemental data 2) and ALP(29,32,36,39-41) (supplemental data 3) have been frequently assessed in AS in former studies; however, to our knowledge, they have not yet been evaluated in parallel with other markers in the Wnt signaling pathway. OC expression was similar between AS patients and controls. OC was the effector gene of the canonical Wnt pathway, but it might also be downstream factors of other signaling pathway. This complicated situation will interrupt the real expression that link to the canonical Wnt pathway. The result of ALP in our study was consistent with the former ones. ALP had a higher trend in AS, although it did not always reach significance. Increased serum ALP levels were associated with high disease activity, low bone mineral density, and higher structural damage scores in spondyloarthritis patients,(42) which was also suggestive of activation of the signaling pathway.

Figure 2. Supplemental data 2. Osteocalcin levels in ankylosing spondylitis and controls
Figure 3. Supplemental data 3. Alkaline phosphatase levels in ankylosing spondylitis and controls

In our study, when we investigated the correlation between components of the canonical Wnt pathway, high level of DKK-1 was not correlated with GSK-3β and β-catenin and also with disease activity indexes as well as pathologic indexes, suggesting that besides DKK-1, there must be other regulators contributing in the complicated mechanism of bone formation in AS.

Persistent systemic inflammation was associated with radiographic progression.(43) The key treatment for AS was evaluated to be the blockade of bridging signal between inflammation and bone formation. Interestingly, we did not discover any correlation between bone turnover markers in canonical Wnt pathway and disease inflammation and radiographic indexes in AS patients in our study. Thus, further investigations are required to show whether Wnt signaling would trigger inflammation causing new bone formation in AS.

A limitation of this study was that the influence of treatment on bone turnover markers in Wnt signaling was not included. Some studies reported decreased DKK-1 level after TNF inhibitor treatment, which might theoretically lead to activation of Wnt pathway and eventually cause bone formation. According to the previous clinical trials,(44-48) the structural progression in AS seems to be independent of TNF, despite the fact that TNF is responsible for the signs and symptoms due to inflammation in this disease. The paradoxical effects of TNF inhibitors on radiographic progression suggested that the inflammation and bone formation might be two independent processes in AS disease progress. The bone formation process was not retarded after anti-TNF treatment, implicating that other inflammation factors which may also trigger bone formation might exist synchronously. Explanation of this phenomenon needs further investigation.

In conclusion, the alteration of bone turnover markers in canonical Wnt pathway was observed in AS patients, while the results were not completely consistent with our hypothesis. The mechanism of canonical Wnt pathway in bone formation in AS might be explained by molecular dysfunction of these bone turnover markers and may not be reflected merely in serum level.

Acknowledgments
We thank Professor Yubin Deng from translational medicine research center of the First Affiliated Hospital of Sun Yat-sen University for assistance in study design and preparation of the manuscript.

Conflict of Interest

The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Financial Disclosure

This research was supported by National Natural Sci ence Foundation of China (81301529), Natural Science Foundation of Guangdong Province (S2013040012296), Medical Scientific Research Foundation of Guangdong P rovince (A2015395), Science and Technology Planning Project of Guangdong Province (2014A020212617), Science and Technology Planning Project of Shenzhen (JCYJ20150331142757389) and Public Welfare Scientific Research Project of Futian District Shenzhen (FTWS201308).

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