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.

The effects of treatment with squalestatin 1, a potent inhibitor of squalene synthase, the first committed enzyme of sterol biosynthesis, were examined on cytochrome P450 expression in primary cultured rat hepatocytes and rat liver. Incubation of cultured hepatocytes with squalestatin 1 caused marked accumulations (maximal elevations that were approximately 25-100% of phenobarbital-elicited increases) of CYP2B mRNA and immunoreactive protein but not of CYP1A, CYP3A, or CYP4A. Squalestatin 1 treatment increased CYP2B and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA content in hepatocyte cultures with comparable potencies (ED50 = 5.0 and 18 nM, respectively), and significantly induced CYP2B (mRNA, immunoreactive protein, and pentoxyresorufin O-dealkylase activity) in the livers of treated rats, producing maximal increases at a dose of 25 mg/kg/day that were approximately 32-87% of phenobarbital-induced increases. Squalestatin 1 treatment induced both CYP2B1 and CYP2B2 and activated reporter gene expression in cultured hepatocytes transiently transfected with a plasmid containing approximately 2.4 kb of CYP2B1 gene 5'-flanking region or containing a previously described phenobarbital-responsive region. Coincubation of cultured hepatocytes with 25-hydroxycholesterol suppressed squalestatin 1-mediated CYP2B and 3-hydroxy-3-methylglutaryl coenzyme A mRNA induction with approximately the same potency. Treatment of cultures with SQ-34919, a structurally distinct squalene synthase inhibitor, produced the same selective CYP2B mRNA induction as did squalestatin 1. These results suggest that inhibition of hepatic sterol synthesis activates processes that culminate in increased CYP2B gene transcription.

We examined the effects of TSP2 deficiency on assembly of collagenous extracellular matrix (ECM) by primary marrow-derived mesenchymal stromal cells (MSC) undergoing osteoblast differentiation in culture. After 30 days, wild-type cells had accumulated and mineralized a collagen-rich insoluble matrix, whereas the TSP2-null cultures contained markedly lower amounts of matrix collagen and displayed reduced mineral. Differences in matrix collagen were seen as early as day 9, at which time wild-type cultures contained more total collagen per cell than did TSP2-null cells. Collagen was unevenly distributed amongst different extracellular compartments in the two cell-types. Collagen levels in conditioned medium of wild-type cells were higher than those of TSP2-null cells, but were roughly equivalent in the acid-soluble, newly cross-linked matrixes. Conversely, the mature, cross-linked acid-insoluble matrix layer of wild-type cells contained about twice as much collagen as TSP2-null cell-derived matrix. Western blot analysis of type I collagen in detergent-soluble and insoluble matrix fractions supported the premise that matrix collagen levels were reduced in TSP2-null MSC undergoing osteoblastic differentiation in vitro. Western blot and immunofluorescent analysis suggested that assembly of fibronectin into matrix was not affected by TSP2 deficiency. Instead, western blots of conditioned medium demonstrated a marked reduction in mature, fully processed type I collagen in the absence of TSP2. Our data suggest that, in the context of osteoblast differentiation, TSP2 promotes the assembly of a type I collagen-rich matrix by facilitating pro-collagen processing.

We report two immuoreactive species of thrombospondin-2 (TSP2), sized approximately 200 and 125 kDa, in the long bones of growing, but not skeletally mature, mice. In vitro osteoblasts secrete a 200-kDa species into the culture medium as early as day 3, and it appears in the cell-matrix layer by day 7. A 125-kDa species appears in the cell-matrix layer in parallel with mineralization; it is not detected in cell-conditioned medium. Unilateral tibial fracture induced a time-dependent upregulation of the 200-kDa species at the site of trauma. By contrast, relative levels of the 125-kDa species at the fracture site were lower than in bones from naive control animals. In the contralateral untouched control tibia, the 200-kDa species was rapidly and substantially reduced compared to bone harvested from naive control mice. Levels of the 125-kDa species in the untouched tibia declined gradually with time postfracture. TSP2 gene expression in uninjured control bone decreased modestly by 21 days postfracture. On the day of fracture, the osteoblast differentiation potential of MSCs harvested from uninjured bones decreased compared to those harvested from naive control animals. The presence of two isoforms suggests that TSP2 may undergo posttranscriptional or posttranslational processing in skeletal tissue. Our data also suggest that, in the context of trauma, the two TSP2 isforms are differentially modulated at injured and noninjured skeletal sites in an animal undergoing fracture healing.

Thrombospondin-2 (TSP2) is a matricellular protein component of the bone extracellular matrix. Long bones of adult TSP2-deficient mice have increased endosteal bone thickness due to expansion of the osteoblast progenitor cell pool, and these cells display deficits in osteoblastic potential. Here, we investigated the effects of TSP2 deficiency on whole bone geometric and mechanical properties in growing 6-wk-old male and female wild-type and TSP2-knockout (KO) mice. Microcomputed tomography and mechanical testing were conducted on femora and L2 vertebrae to assess morphology and whole bone mechanical properties. In a second series of experiments, femoral diaphyses were harvested from wild-type and TSP2-KO mice. Detergent-soluble type I collagen content was determined by Western blot of right femora. Total collagen content was determined by hydroxyproline analysis of left femora. In a third series of experiments, cortical bone was dissected from the anterior and posterior aspects of the femoral middiaphysis and imaged by transmission electron microscopy to visualize collagen fibrils. Microcomputed tomography revealed minimal structural effects of TSP2 deficiency. TSP2 deficiency imparted a brittle phenotype on cortical bone. Femoral tissue mineral density was not affected by TSP2 deficiency. Instead, transmission electron microscopy revealed less intensely stained collagen fibrils with altered morphology in the extracellular matrix assembled by osteoblasts on the anterior surface of TSP2-KO femora. Femoral diaphyseal bone displayed comparable amounts of total collagen, but the TSP2-KO bones had higher levels of detergent-extractable type I collagen. Together, our data suggest that TSP2 is required for optimal collagen fibrillogenesis in bone and thereby contributes to normal skeletal tissue quality.