The role of the cytoskeleton in articular cartilage chondrocyte homeostasis

Abstract

Introduction The aetiology of osteoarthritis (OA) is unknown although abnormal loading of the joint is a contributory factor. We have demonstrated previously that a cyclic, compressive load (0.5 MPa, 1Hz) applied to immature articular cartilage induces a significant increase in the expression and activation of MMP‐2 and MMP‐9 (Blain et al. 2001). Using differential RNA display, we identified a mechanically regulated gene –thymosin β4 (Blain et al. 2002). The primary function of thymosin β4 is in the sequestration of filamentous actin (F‐actin). Therefore, we hypothesize that the mechanical induction of matrix degradation, i.e. the up‐regulation of MMP gene expression, is initiated via the actin cytoskeleton, whether directly or indirectly remains to be elucidated. Thus, the objective of this study was to determine whether the actin cytoskeleton, in addition to the tubulin and vimentin cytoskeletal networks are involved in the signalling pathways involved in chondrocyte MMP regulation.Materials and methods Primary chondrocytes, isolated from 7‐day‐old bovine calves, were seeded at a density of 1 × 106 cells/ml, and individual cytoskeletal elements were disrupted with 10 µm cytochalasin‐D (for F‐actin), 10 µm colchicine (for tubulin) or 5 mm acrylamide (for vimentin) for 1–7 days. Amounts of sulphated glycosaminoglycan (sGAG) were determined using the DMMB assay, and total collagen content was assessed using the hydroxyproline assay. MMP activity was measured using gelatin substrate zymography and the amounts of their inhibitors, the TIMPs, assessed by reverse zymography.Results We have demonstrated that disruption of the cytoskeletal elements can affect cartilage chondrocyte homeostasis. There was a significant decrease in sGAG release for all three cytoskeletal disruption treatments when compared to untreated controls (P < 0.01). There was a reduction in total collagen released from the cells which was significant after 7 days in actin‐disrupted (P = 0.012) and vimentin‐disrupted cells (P = 0.05). No collagen was detected in tubulin‐disrupted cells at any time point, which may be due to a decrease in cell viability. Interestingly, at days 3 and 7, both tubulin and vimentin disruption abrogated synthesis of pro‐MMP‐2 and significantly reduced the amount of MMP‐2 activation (P < 0.01). Actin disruption significantly enhanced both synthesis and activation of MMP‐2 (P < 0.02). In comparison, TIMP‐1 expression was also abrogated in cells without a functioning tubulin or vimentin network, whereas in actin‐disrupted cells, there was a reduction in TIMP‐1 compared to untreated controls (P < 0.001).Discussion Clearly disruption of the cytoskeletal networks can affect cartilage chondrocyte homeostasis. Thymosin β4 has been shown to induce MMP activity in our cell culture system which may be directly attributable to F‐actin depolymerization. Addition of cytochalasin‐D to chondrocytes revealed an increase in MMP‐2 synthesis/activation and reduced TIMP‐1 expression implicating the actin cytoskeleton in this process, whether directly or indirectly remains to be determined. We are currently using antibodies that recognize key signalling intermediates, i.e. FAK125, p38 kinase and ERK 1/2 to assess the involvement of these molecules in events proceeding cytoskeletal disruption and prior to the mediation of MMP expression. We are also starting to investigate the mechanisms involved in abrogation of MMP synthesis after tubulin and vimentin disruption in chondrocytes. Elucidation of the role that the three cytoskeletal elements play in cartilage homeostasis will enable us to fully appreciate their functions in cartilage tissue turnover and dysregulation in disease.This work is supported by the EU 5th Framework and ARC (Grant No. D0600).