Rheumatol Int (2013) 33:1313–1319 DOI 10.1007/s00296-012-2548-4

ORIGINAL ARTICLE

Phosphorylation of osteopontin in osteoarthritis degenerative cartilage and its effect on matrix metalloprotease 13 Mai Xu • Lu Zhang • Lei Zhao • Shuguang Gao Rui Han • Dazhi Su • Guanghua Lei

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Received: 28 March 2012 / Accepted: 21 October 2012 / Published online: 7 November 2012 Ó Springer-Verlag Berlin Heidelberg 2012

Abstract The purpose of this study is to observe the differences of osteopontin (OPN) phosphorylation in osteoarthritis (OA) cartilage and normal cartilage, and evaluate the possible correlations between the OPN phosphorylation and MMP-13 expression. Degenerative cartilage (n = 29) and normal cartilage (n = 10) were identified by hematoxylineosin, safranin-O staining and modified Mankin score. The phosphorylation level of OPN in OA cartilage and normal cartilage was detected by immunoprecipitation. Chondrocytes were treated with phospho-OPN, OPN or buffer. Quantitative reverse transcription polymerase chain reaction (qPCR) and ELISA were used to assess the expression of MMP-13 in different treatments. The OD values of phosphorylation of OPN in normal cartilage and OA cartilage were 137.89 ± 10.59 and 153.52 ± 8.80, respectively,

(P = 0.000). Chondrocytes treated with OPN showed a higher MMP-13 expression at gene and protein level compared with control group. Chondrocytes treated with phospho-OPN showed the highest MMP-13 expression in gene and protein. In conclusion, our results revealed a higher phosphorylation level of OPN in OA cartilage than in normal cartilage. We found OPN leads to elevated expression of MMP-13 (both at gene level and protein level), and phospho-OPN had a more obvious upregulation effect on MMP-13 expression than nonphospho-OPN. Further studies are needed to reveal the mechanism of OPN phosphorylation on cartilage degeneration.

M. Xu  S. Gao  G. Lei (&) Department of Orthopaedics, Xiangya Hospital, Central South University, No. 87 Xiangya Road, 410008 Changsha, Hunan, China e-mail: [email protected]

Introduction

L. Zhang Center for Molecular Medicine, Xiangya Hospital, Central South University, No. 87 Xiangya Road, 410008 Changsha, Hunan, China L. Zhao Institutes of Biology and Medical Sciences, Suzhou University, No. 199 Renai Road, 215123 Suzhou, Jiangsu, China R. Han Department of Orthopaedics, Qingdao Haici Hospital, No. 4 Renmin Road, 266000 Qingdao, Shandong, China D. Su Department of Orthopaedics, Zhuzhou No.1 Hospital, No.23 Station Road, 412000 Zhuzhou, Hunan, China

Keywords Osteopontin  Osteoarthritis  Phosphorylation  Matrix metalloproteinase 13

Osteoarthritis (OA) is a complex degenerative joint disease that characterized by recurrent arthralgia and progressive disfunction. However, current treatments only address symptoms without mentioning the underlying causes [1]. As a result, further investigation and understanding of OA pathology is needed and important. The most conspicuous biochemical change in OA cartilage is the loss of proteoglycan and collagen type II. Matrix metalloprotease (MMP)-13 is considered as a wellcharacterized key player in cartilage biology and OA pathology due to its capacity to degrade collagens type II and a wide range of other matrix components [2–4]. The gene and protein expression of MMP-13 increases with severity of cartilage degeneration [5, 6]. These observations support the concept that MMP-13 reflects an intrinsic process of cartilage degradation in OA [6].

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Numerous factors contribute to the overall degradation of cartilage observed in OA, either directly or indirectly by modulating anabolic and catabolic factors [7, 8]. For the past decades, OPN, as an extracellular matrix cytokine, has become one of the hot spots in the research of OA pathogenesis. Previous studies have shown a close relationship between OPN and OA [9–13]. OPN in plasma [11], synovial fluid [10, 11] and cartilage [10, 13] is related to progressive joint damage in OA. Thus, osteopontin may serve as a biochemical marker for determining disease severity. The wild-type OPN is heavily post-translational modified (PTM) with phosphorylation, glycosylation and tyrosine sulfation before it works. This nature of the PTMs decorating OPN has momentous effects on the structure and biological properties of the protein [14, 15]. Phosphorylation of OPN can interfere in OPN binding with integrin [16, 17]. Consequently, phospho-OPN should have an impact on function of integrin which can induce MMP13 expression via MAPKs signaling pathways [18], a major signal-transducing pathway of cartilage catabolism in OA [19]. The purpose of this study is to observe the differences of OPN phosphorylation in OA cartilage and normal cartilage, and evaluate the possible correlations with the OPN phosphorylation and MMP-13 expression, which may serve as a useful tool to mark the degeneration process of cartilage and to further elucidate the pathways involved in the progression of OA.

Materials and methods Cartilage acquisition The study was approved by the institutional review board and ethics committee of Xiangya Hospital affiliated to Central South University, which conformed with the regulations of medical ethics. All patients who donated the knee had signed the informed consent. Normal human knee cartilage (n = 10) was obtained from traumatic low-limb amputee (knee above amputation), and osteoarthritic decayed cartilage (n = 29) was obtained from knee arthroplastic patients. Cartilage degeneration assessment Cartilage specimens were fixed overnight in 2 % paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). After dehydration in alcohol, samples were embedded in paraffin and cut into 3 lm prepared for staining with hematoxylineosin (HE) and safranin-O. The sections were assessed by 2 blinded observers for degenerative changes using a modified Mankin grading system [20].

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Protein extraction from tissue and immunoprecipitation Approximately 100 g (wet weight) of cartilage tissue was washed with phosphate-buffered saline (PBS) solution containing proteinase inhibitors and then ground with a mortar and pestle under liquid nitrogen. The resulting powder was treated with 4.0 M guanidine–HCl to remove nonmineral-associated proteins and then treated with 0.5 M EDTA. EDTA extract was concentrated by ultrafiltration, subjected to buffer exchange. The soluble lysate protein concentration was determined with bicinchoninic acid reagent (Shanghai Biological Technology, Shanghai, China). The lysate was pretreated by incubation at 4 °C for 1 h with protein A-agarose beads coated with anti-OPN (Santa Cruz Biotechnology, Santa Cruz, CA, USA), then separated from the beads followed by centrifugation and transferred to fresh tubes. The immunoprecipitate was resolved with 10 % SDS–polyacrylamide gels and transferred onto PVDF membranes (Thermo Scientific, Rockford, IL, USA). The membranes were blocked using blocking buffer containing 50 lg/l ovalbumin in TTBS and then incubated with phosphotyrosine antibodies (1:500), followed by anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (Amersham Pharmacia, Piscataway, NJ, USA). Membranes were washed three times with cold Triton-free lysis buffer followed by elution of proteins with Laemmli buffer (0.5 M Tris–HCl, 2 % sodium dodecyl sulfate) and shaking. Immunoprecipitated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (4–20 % gradient, NOVEX; San Diego, CA, USA). Gels were dried and exposed to Kodak Biomax films. Phospho-OPN preparation Recombinant human osteopontin (rhOPN) was not phosphorylated in any significant degree. To get a phosphorylated exogenous OPN, MAP kinase was used. Recombinant human osteopontin (rhOPN) (R&D Systems, Minneapolis, MN, USA) was diluted with PBS to 0.1 lg/ml. The diluented OPN (5 ll) was phosphorylated in the presence of 10 mM ATP (10 ll) and mitogen-activated protein kinases (MAPKs) (1 ll) in 5 ll 10 9 kinase buffer in a total volume of 50 ll at 30 °C for 30 min and terminated by extra 10 ll of 1 % SDS/100 mM EDTA. All reagents were purchased from Cell Signaling Technology, Beverly, MA, USA. Cell isolation and culture conditions Samples were minced into pieces of \1 mm3, followed by sequential digestion at 37 °C with 0.15 % collagenase II

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(Invitrogen, Carlsbad, CA, USA) for 5–6 h with stirring every 20 min after 2 h. Chondrocytes were isolated after centrifugation and cultured in DMEM-F12 containing 10 % fetal bovine serum (FBS) and antibiotics for 5–7 days before use. Medium was changed to serum-free antibiotics-free DMEM-F12 with 4 ng/ml of (a) phosphoOPN (P-OPN), (b) rhOPN (OPN) or (c) kinase buffer (KB) 6 h before each experiment. RNA isolation and quantitative reverse transcription polymerase chain reaction assay Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). In brief, chondrocytes were harvested and washed with cold phosphate-buffered saline (PBS) and lysed directly in a 3.5-diameter dish by adding 1 ml of TRIzol reagent. After passing several times through a pipette, the homogenized samples were incubated for 5 min at room temperature; 0.2 ml of chloroform was added to the lysate to extract RNA. The samples were centrifuged at 10,0009g for 15 min at 4 °C, and the upper aqueous phase was transferred into a fresh tube and mixed with 0.5 ml of isopropyl alcohol. Samples were incubated at room temperature for 10 min and then centrifuged under 10,0009g for 10 min at 4 °C. After removing the supernatant, the RNA pellet was washed by adding 75 % ethanol. The mixture was centrifuged under 7,5009g for 5 min at 4 °C before air-dry. Quantitative RT-PCR (qPCR) was performed on the Applied Biosystems 7,500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) using the sense primer: 50 -CTTAGAGGTGACTGGCAA AC-30 and antisense primer: 50 -GCCCATCAAATGGGTA GAA G-30 . Each condition was tested in duplicate and datasets represent a minimum of four independent experiments. Data were normalized using mmp-13 levels in the KB treated samples. Protein extraction from cells and ELISA Cell lysates were prepared using modified cell lysis RIPA buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 0.25 % deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM glycerol phosphate, and 1 mM Na3VO4), with 2 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). Cold RIPA buffer was added to chondrocytes in 60-mm plate. The chondrocytes were scraped off the plate immediately. After concentration, the extract was transferred to a microcentrifuge tube. The soluble lysate protein concentration was determined by enzyme-linked immunosorbent assay using MMP-13 ELISA kit (Boster, Wuhan, China) as described in product booklet.

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Statistical analysis All statistical analyses were performed using Sigmaplot 12.0 software. The normality test (Shapiro–Wilk) was first performed, and P \ 0.05 was considered abnormal distribution. For normal distributed data, the mean difference and the associated standard deviation were calculated via the Student’s t test; for abnormal distributed data, Mann– Whitney rank sum test was used, and the median value and associated 25th percentile and 75th percentile were obtained. A P value \0.05 was considered significant differences.

Results Cartilage degeneration assessment Normal cartilage tissues were obtained from 10 amputees and degenerated cartilage tissues from 29 OA patients. Representative changes of cartilage in HE staining and safranin-O staining were listed in Fig. 1 (a––HE staining; b––Safranin-O staining). The mean modified Mankin score was significantly higher in OA cartilage (9.74 ± 1.23) than in the normal cartilage (0.60 ± 0.52) with the P value of 0.000 (Fig. 1c). Phosphorylation of OPN in normal cartilage and degenerated cartilage Immunoprecipitation method was used to detect the phosphorylation level of OPN. Both normal cartilage and degenerated cartilage exhibited phosphorylation of the OPN. And the OD values of OA cartilage and normal cartilage were 153.52 ± 8.80 and 137.89 ± 10.59, respectively. The differences were considered statistically significant with P value of 0.000. This indicated that the wild-type OPN in cartilage was secreted after phosphorylation. Furthermore, OA cartilage showed higher levels of OPN phosphorylation than the normal one (Fig. 1d). Effect of OPN and its phosphorylation on MMP-13 Figure 2a shows designed different treatments to chondrocytes. To investigate whether OPN phosphorylation induced MMP-13 RNA activation, qPCR was performed to detect RNA of MMP-13. As expected, all of the three groups of chondrocytes showed expression of MMP-13 gene, but the quantification of MMP-13 using RNA from the P-OPN treated chondrocytes showed the highest expression of this gene compared with samples in OPN treated chondrocytes and KB treated group. Moreover, OPN treated chondrocytes showed enhanced MMP-13

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upregulation of MMP-13 expression detected in chondrocytes growing with P-OPN suggested the MMP-13 activation was not only relying on the quantity of OPN but also the post-translational modification—phosphorylation. Therefore, we concluded that OPN upregulated MMP-13 expression at both gene level and protein level by phosphorylation along with the amount.

Discussion

Fig. 1 Representative changes at cartilage in hematoxylin-eosin staining, safranin-O staining, modified Mankin score and expression of phospho-OPN. The left column represented OA degenerative cartilage, and the right column represented normal cartilage. a Hematoxylin-eosin staining (9100 magnification) showed an irregular exfoliative surface of cartilage in OA degenerative cartilage, with chondrocytes clustered in disorganized cartilage lacuna; normal cartilage showed a much more regular surface of cartilage, with chondrocytes distributed neatly and evenly in the cartilage lacuna. b Safranin-o staining (9100 magnification) OA degenerative cartilage was deep stained or stained loss; staining of normal cartilage was natural and tinged. c Comparison of modified Mankin score (mean ± SD) between OA cartilage and normal cartilage. Bars represented the mean modified Mankin score of each group. Errors represented standard deviation. An asterisk capping on bars indicated a significant difference (P value \0.05). d Immunoprecipitation assay showed the expression of phospho-OPN in OA cartilage and normal cartilage. The bands representing expression changes in phosphoOPN were normalized with levels of b-actin. The P-OPN band was located between 37 kDa and 50 kDa

RNA expression (less than twofold) compared with control group. Figure 2b shows the relative expression of MMP-13 mRNA in three treatments of chondrocytes. To discover the effect of OPN phosphorylation on MMP-13, ELISA was employed to detect MMP-13 expression in all three groups of chondrocytes. The highest MMP-13 levels appeared in chondrocytes growing with P-OPN, and OPN treated chondrocytes in the middle (Fig. 2c). Upregulation of MMP-13 expression detected in chondrocytes growing with OPN (whether phosphorylated or not) suggested OPN activated MMP-13 expression, but twofold to threefold

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Currently, many diseases have been found to be associated with increased OPN expression [21–26]. However, besides the amount of protein, the regulatory roles of OPN in normal and pathological states are highly dependent on the phosphorylation status of the protein [15]. Functionally, phosphorylation has been found to regulate adhesion and/or migration of OPN [27–30]. Therefore, differences in phosphorylation of OPN contribute to the complexity of OPN-receptor binding and downstream signaling [16, 27]. The role of OPN in OA is controversial. Previous researches [9–13, 31] were mostly focused on the amount of OPN expression and found elevated OPN level associating with the disease severity. These studies are consistent with OPN exerting a destructive role of OA. In contrast, there is a report that appears consistent with a protective role against OA. Matsui et al. [32] reported OPN deficiency exacerbated OA, indicating OPN a intrinsic critical regulator of cartilage degradation via its effects on MMP-13 expression and proteoglycan loss. With respect to OPN, it is evident that in its phosphorylation state OPN is important to its role in rheumatoid arthritis [33]; thus, we wonder whether phosphorylated modification is required in OPN playing a role in OA. We compared tyrosine phosphorylation of OPN from OA cartilage and normal cartilage and found out that the level of OPN phosphorylation in OA cartilage was statistically higher than it in normal cartilage. To our knowledge, it is the first time OPN phosphorylation being related to OA. With the higher level of phosphorylation of OPN involved in the pathologic process of OA, we hypothesize that there is a cause and effect between differences in phospho-OPN and degeneration of cartilage. MMP-13 is a potential marker for OA. Previously, it was shown that elevated expression of OPN can upregulate the expression of MMP-13 during the early phase of tendon tissue remodeling [34]. Our study suggests a similar trend. Exogenous OPN (whether phosphorylated or not) can stimulate chondrocytes to increase MMP-13 synthesis. Meanwhile, this effect can be greatly enhanced by OPN phosphorylation. However, as the native human osteopotin has 36 potential phosphoric sites and is highly tissue- and cell-specific in the pattern of phosphorylation [35], we can

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Fig. 2 Phospho-OPN and OPN increased expression of MMP-13 in chondrocytes. a Chondrocytes were cultured and treated in three different reagents 6 h before experiment: the purple culture dish represented the chondrocytes treated with phospho-OPN, the gray culture dish represented the chondrocytes treated with rhOPN, the blue culture dish represented the chondrocytes treated with kinase buffer. b qPCR for MMP-13 expression of chondrocytes treated 6 h with P-OPN, OPN and KB. For comparative purposes, mRNA levels in KB treated cells were normalized to 1. Bars represented fold changes of MMP-13 mRNA after normalization with levels of KB

treated chondrocytes. Samples from two independent experiments were measured. Results were given as median value (25th percentile, 75th percentile). An asterisk capping on bars indicated a significant difference (P value \ 0.05). c ELISA for MMP-13 expression of chondrocytes treated 6 h with P-OPN, OPN and KB. Bars represented absolute concentration of MMP-13 protein in ng/ml. Errors represented standard deviation. Samples from two independent experiments were measured. An asterisk capping on bars indicated a significant difference (P value \ 0.05)

hardly tell whether or not the occurrence of OA is due to phosphorylation of specific sites. Some experiment design maybe further improved in our research. First, we used a conventional method (OPN antibody ? phosphotyrosine antibodies) to detect the phosphorylated level of OPN during the immunoprecipitation instead of using specific OPN phosphorylation antibody. This may lead to a poor specificity. In our study, we obtained a distinct band and qualitatively corresponded with OPN. We considered this band represents the phosphorylated OPN. Secondly, MAPKs signal pathway can upregulate the expression of MMP-13 [36], we used a combination of SDS and EDTA to terminate the process of phosphorylation, hoping to eliminate the influence of MAP kinase on MMP-13. Otherwise, the potential phosphokinase of OPN includes

mammary gland casein kinase (MGCK) and casein kinase II (CKII) [35], both of which are from the CK family. Using MAPKs as the phosphokinase of OPN in our study may result in mis-phosphorylation of the non-OA-specific phosphorylation sites. Further investigations of phosphorylation on specific sites are necessary to get detailed insight into the possible mechanism of OPN in OA patients. In conclusion, our results revealed a higher phosphorylation level of OPN in OA cartilage than in normal cartilage. We found OPN lead to elevated expression of MMP13 (both at gene level and protein level), and phospho-OPN had a more obvious upregulation on MMP-13 expression than nonphospho-OPN. Additional studies are needed to reveal the mechanism of OPN phosphorylation on cartilage degeneration.

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Acknowledgments We thank the reviewers and the Rheumatology International (Clinical and Experimental Investigations) manuscript editorial staffs for their helpful comments that have helped to considerably improve this paper. Mai Xu has received Sports Medicine Research Fund of Central South University. Shuguang Gao is currently receiving the Young Teacher’s boosting project of the Fundamental Research Funds for the Central Universities in Central South University, National Natural Science Foundation of China (81201420). Guanghua Lei has received the National 863 project of China (2011AA030101), National Natural Science Foundation of China (81272034), the Provincial Science Foundation of Hunan, the freedom explore Program of Central South University (2012QNZT103), Distinguished Young Scientists fund of Central South university, the foundation of development and reform commission of Hunan province and National Clinical Key Department Construction Projects of China. For the remaining authors none were declared. Conflict of interest of interest.

The authors declare that they have no conflict

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