Thrombospondin-2 regulates matrix mineralization in MC3T3-E1 pre-osteoblasts

The matricellular protein thrombospondin-2 (TSP2) has context-dependent effects on osteoblast lineage proliferation and differentiation. Mice lacking TSP2 display increased endocortical bone thickness, which is associated with increased marrow stromal cell (MSC) number and in vitro proliferation. TSP2-null MSC also exhibit delayed osteoblastogenesis and enhanced adipogenesis compared to cells harvested from wild type mice. The goal of the present work was to more precisely characterize the contribution that TSP2 makes to the maturation of osteoblast-derived extracellular matrix (ECM) using a highly characterized pre-osteoblast cell line. Specifically, we asked whether TSP2 influences mineralization indirectly through its known effects on proliferation, or whether TSP2 directly promotes osteoblast differentiation. To pursue these questions, we used RNA-interference (RNAi) to inhibit TSP2 gene expression in MC3T3-E1 pre-osteoblasts. Introduction of siRNA oligonucleotides resulted in reduced TSP2 mRNA expression as early as 24 h post-transfection, and TSP2 mRNA levels remained low for 10 days. Similarly, TSP2 protein levels in both conditioned medium and the cell-matrix layer were reduced at 24 h post-transfection and remained reduced for 7 days. At day 21, mineralization was significantly reduced in cells transfected with TSP2 siRNA when compared to cells treated with scrambled siRNA. This decrease in mineralization occurred without a concomitant change in cell number. Twenty-four hours after transfection, runx2 gene expression was transiently enhanced in TSP2 siRNA-treated cultures. Between 6 and 14 days post-transfection, runx2, osterix, alkaline phosphatase, type I collagen, osteocalcin and bone sialoprotein all displayed moderate increases in gene expression with TSP2 RNAi. As well, soluble osteocalcin levels were markedly higher in the conditioned medium of cells treated with TSP2 siRNA than in control siRNA-treated cells. Increased soluble osteocalcin occurred without a concomitant change in the levels of osteocalcin in the cell-ECM layer. TSP2 reduction also elicited a transient change in the distribution of collagen between the acid soluble cell-ECM protein fraction and the insoluble matrix. Together, our data suggest that TSP2 may promote mineralization, by facilitating proper organization of the osteoblast-derived ECM.