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

Abstract

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 hours 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 hours 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.

Extracellular matrix networks in bone remodeling

“…Type III collagen mice are embryonic lethal, but analysis of null cells in culture or heterozygous mice suggests that type III collagen promotes differentiation (Volk et al, 2014). The matricellular proteins SPARC (osteonectin) and thrombospondin-2 (Alford et al, 2013) have been implicated in collagen fiber assembly (Alford et al, 2013;Bornstein, 2000;Harris et al, 2011;Rentz et al, 2007;Bradshaw et al, 2003), and also modulate osteoblast lineage progression (Hankenson and Bornstein, 2002;Hankenson et al, 2000;Alford et al, 2009;Delany et al, 2003Delany et al, , 2000. Since collagen-dependent signaling induces osteoblast differentiation, one possibility is that the effects of ECM protein deficiency in these matricellular knock-out mouse models may be explained, in part, by impaired collagen-mediated signaling.…”

Osteoblast-released Matrix Vesicles, Regulation of Activity and Composition by Sulfated and Non-sulfated Glycosaminoglycans

“…FN1 positively stimulates osteoblast differentiation and survival (81,82) and seems to be the nuclei for mineralization cores in the ECM (83). This mineralization seems to be additionally supported by THBS2 (84). The ECM fibrillogenesis may be provided by a higher abundance of POSTN after sHA1 stimulation.…”

Thrombospondin-2 facilitates assembly of a type-I collagen-rich matrix in marrow stromal cells undergoing osteoblastic differentiation

“…As described previously (13), fibrillar collagen levels in acid-soluble and insoluble matrix fractions were determined using a sirius red dye binding method (Sircol soluble collagen assay; Biocolor Ltd, Carrickfergus, Northern Ireland). After 9 or 30 days in osteoblastic culture, conditioned medium plus acid-soluble and insoluble matrix fractions were collected as outlined above under extraction of extracellular matrix .…”

“…In addition to affecting marrow stromal cell proliferation, TSP2 promotes osteoblast lineage progression at the expense of adipogenesis (11, 12). Our published data suggest that TSP2 promotes mineralization in an osteoblast cell line, while also facilitating accumulation or retention of collagen and osteocalcin in the ECM (13). Since MSC-collagen interactions are required for osteoblast lineage progression, TSP2 could promote osteoblast differentiation indirectly by facilitating formation of a collagenous matrix.…”