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A5.5 SMOC2 modulates chondrogenesis by interfering with WNT and BMP signalling
  1. Frédéric Cailotto,
  2. Frank P. Luyten,
  3. Rik J. Lories
  1. Laboratory of Tissue Homeostasis and Disease, Skeletal Biology and Engineering Research Center, Department of Development & Regeneration, KU Leuven, Belgium.

Abstract

Background and Objectives A proteomic analysis of cartilage revealed an increase in SPARC-related modular calcium binding protein-2 (SMOC2) in patients with osteoarthritis (OA). SMOC2 was originally isolated from a chondrogenic extract of articular cartilage together with GDF5 and FRZB, proteins associated with OA. We investigated SMOC2 in chondrocyte differentiation.

Methods Three independent stable clonal colonies of the ATDC5 chondrogenic cell line, control (empty vector/GIPZ) and SMOC2 overexpressing (SMOC2+) or knocked-down cells (SMOC2-), were used. Clones were cultured as high density micromasses (2x105 cells/10µl). Chondrogenesis was induced by culturing cells for 14 days (D) in DMEM/F12 + 5%FBS + ITS [insulin (10µg/ml), transferrin (10µg/ml) and sodium selenite (30nM)]. On D14, cells were switched to mineralization medium (αMEM+5%FBS+ITS). mRNA expression of markers (Aggrecan (Agg), type II (Col2a1) and X (Col10a1)) collagens was assessed by quantitative RT-PCR. Likely, we assessed mRNA expression of Wnts (-3a, -4, -5a, -5b, -11) and BMPs (-2, -4, -6, -7). Quantification of Alcian Blue, Safranin O, Sirius red and Alizarin red staining were used to evaluate proteoglycans, collagens and mineralised content respectively. Western blot was used to study BMP and Wnts pathways.

Results In SMOC2 + cells, during the early (D1-7) and late proliferation phase (D7-14), Col2a1 and Agg mRNA increased less than controls. Safranin O and Alcian blue staining were also less upregulated in SMOC2 + . This was consistent with lower activation of Smad 1/5/8 in SMOC2 + . Col10a1 mRNA increased as expected from D14 onwards, but significantly less in SMOC2 + compared to controls. SMOC2 overexpression affected Wnt mRNA levels. During mineralization (D14-21), Col10a1 mRNA was strongly increased, but to a lesser extent in SMOC2 + , and alizarin red was much stronger in controls. The SMOC2- cells exhibited opposite features as SMOC2 + . In the D1-D14 phases, Collagen and Proteoglycan content was higher than in controls, alike Col2a1 mRNA level. In the D14-21 phase, these differences were maintained, and Col10a1 mRNA level was higher in SMOC2-, and a trend was found for a higher mineralization content in SMOC2-. No difference was detected in cell viability for both SMOC2 + and SMOC2- compared to their respective controls.

Conclusions SMOC2 modulates chondrogenesis by affecting BMP-Smad and Wnt signalling. These data suggest that SMOC2 can play a modulating role in OA.

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