SCLEROSTIN REGULATION OF WISP1 AND MMPS IN CHONDRCYTES IS DEPENDENT ON THE LRP5/6 BINDING DOMAIN BUT NOT SECRETION FROM THE CELL — The Association Specialists
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SCLEROSTIN REGULATION OF WISP1 AND MMPS IN CHONDRCYTES IS DEPENDENT ON THE LRP5/6 BINDING DOMAIN BUT NOT SECRETION FROM THE CELL (257)

Benjamin Chan 1 , Anthony Ashton 2 , Margaret Smith 1 , Christopher Little 1
  1. Raymond Purves Research Labs, Kolling Institute of Medical Research, University of Sydney, St Leonards, NSW, Australia
  2. Perinatal Medicine, Kolling Institute of Medical Research, University of Sydney, St Leonards, NSW, Australia

Purpose: Sclerostin (SOST) is a secreted protein that inhibits Wnt signaling by binding to LRP5/6. SOST has been reported to be an osteocyte-specific protein, and has recently been demonstrated expression in non-calcified-articular cartilage and protects from IL-1-mediated proteoglycan loss and inhibits metalloproteinase expression and activity in-vitro. The present study aimed to investigate the function of chondrocyte SOST, and explore the molecular mechanism whereby it may have chondroprotective effects.

Methods: Human full-length SOST (WT), non-secretable-SOST (M1), secretable and non-screable-SOST with inactive LRP5/6 binding domain (M2/M3, respecticely) constructs were generated by PCR. SW1353 human chondrosarcoma cells were stably transfected with different constructs or empty vector. SOST expression was determined by Western blotting of cell-lysates and ELISA quantitation of culture media. Cells were cultured ± IL-1 for 24 hours and cell viability (trypan blue exclusion), cell metabolism (MTT assay), MMP1/MMP3/MMP13 gene (qRT-PCR) and protein (Western-blot) expressions were measured.

Results: Transfected cell synthesized SOST, with similar levels in all cells but secreted protein in the media only in WT and M2 cells. No effect of SOST-expressing constructs on cell viability or metabolism. IL-1 significant stimulated Wnt-β-catenin activity as measured by β-catenin nuclear translocation. WT suppressed IL-1 induced β-catenin nuclear translocations and significantly (p<0.05) suppressed MMP-1/-13 mRNA and protein expression. Interestingly, M1 which does not suppressed β-catenin nuclear translocation can also inhibits MMP-1/-13 mRNA and protein expression. In contrast, M2 and M3 had no effect on WISP1 or MMP gene and protein expression in the presence of IL-1.

Conclusion: Our data demonstrate the LRP5/6 binding domain is critical for SOSTs inhibit IL-1 induced Wnt-β-catenin activation and MMP expression. Surprisingly, secretion from cell was not necessary for SOST to inhibit IL-1-stimulated MMP-activity. This suggests a hitherto unrecognized intra-cellular role for the LRP5/6 binding domain of SOST, potentially through cross-talk between BMP and Wnt-pathways.