Poster Presentation 6th Australian Health and Medical Research Congress 2012

Matrilin-3 interacts with chondrogenic differentiation growth factor TGF-b1 during spontaneous differentiation of ATDC5 cells  (#463)

Fiona H Zhou 1 2 , ZhengKe Wang 1 , Qian Chen 1
  1. Department of Orthopaedics Research, Rhode Island Hospital/ Brown University, Providence, Rhode Island, USA
  2. Department of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia, Australia

The absence of extracellular matrix ECM protein, matrilin (MATN)3 in mice leads to increased hypertrophy of the developing growth plate, higher bone density and early onset of osteoarthritis (OA), indicating that MATN3, a component of cartilage ECM, is critical for regulating chondrogenesis. In addition, transient expression of MATN3 by synovial fibroblasts enhanced chondrogenesis induced by TGF-β1.. In order to further elucidate the involvement of MATN3 in chondrogenesis, we stably transfected teratoma-derived cell line ATDC5 with wild type MATN3 plasmid and then we examined the effect of MATN3 on cell proliferation using MTT assay, Col2a and Aggrecan mRNA expression using Real-time PCR, and accumulation of proteoglycan using Alcian blue staining in the absence and presence of TGF-β1 (10ng/ml). We found that both MATN3 transfected and TGF-β1 treated wild-type ATDC5 cells increased cell proliferation, up regulated expression of chondrogenic markers and enhanced accumulation of alcian blue on day 12 of culture in DMEM:F12 without ITS induction. Interestingly however, we found that the addition of TGF-β1 to MATN3-ATDC5 cells did not induce an additive effect on cell proliferation and spontaneous chondrogenesis.  These results suggest that there may be a possible interaction between MATN3 and TGF-β1 to control their chondrogenic effects on ATDC5. Therefore, we further analyzed the direct biochemical binding interaction of MATN3 with TGF-β1 using solid phase ELISA based assay and surface plasmon resonance (SPR) analysis using BIAcore T100. From solid phase assay, the saturation of TGF-β1 binding to MATN3 indicates that MATN-3 and TGF-β1 have a strong biochemical interaction. Furthermore, high binding affinities of 11nM and 128nM were determined from SPR analysis using BIAevalution software. This indicates that there are two TGF-β1 binding sites on MATN3. The findings suggest MATN3 not only interact with structural proteins of ECM to maintain integrity of cartilage tissue but may also function in regulating cellular changes of chondrocytes by directly binding to growth factor TGF-β1.