Two Molecular Weight Species of Thrombospondin-2 Are Present in Bone and Differentially Modulated in Fractured and Nonfractured Tibiae in a Murine Model of Bone Healing

Department of Orthopaedic Surgery, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA.
Calcified Tissue International (Impact Factor: 3.27). 02/2012; 90(5):420-8. DOI: 10.1007/s00223-012-9580-y
Source: PubMed


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.

Download full-text


Available from: Andrea I Alford, Oct 24, 2014
  • Source
    • "Bones were trimmed to remove the distal and proximal epiphyses and flushed with PBS to remove bone marrow. The remaining cortical bone was homogenized with a rotor-stator homogenizer (Omni) in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl pH 8, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.1% SDS) supplemented with 5 mM Na3VO4, 10 mM NaPPi, 100 mM NaF, 500 μM PMSF, and 5 mg/ml eComplete Mini protease inhibitor tablet (Roche) [33]. Western analyses was also used to examinee lysates from SAOS-2 or UMR-106 cells collected after the indicated treatments. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Bone continually adapts to meet changing physical and biological demands. Osteoblasts, osteoclasts, and osteocytes cooperate to integrate these physical and biochemical cues to maintain bone homeostasis. Although TGFβ acts on all three of these cell types to maintain bone homeostasis, the extent to which it participates in the adaptation of bone to mechanical load is unknown. Here, we investigated the role of the TGFβ pathway in load-induced bone formation and the regulation of Sclerostin, a mechanosensitive antagonist of bone anabolism. We found that mechanical load rapidly represses the net activity of the TGFβ pathway in osteocytes, resulting in reduced phosphorylation and activity of key downstream effectors, Smad2 and Smad3. Loss of TGFβ sensitivity compromises the anabolic response of bone to mechanical load, demonstrating that the mechanosensitive regulation of TGFβ signaling is essential for load-induced bone formation. Furthermore, sensitivity to TGFβ is required for the mechanosensitive regulation of Sclerostin, which is induced by TGFβ in a Smad3-dependent manner. Together, our results show that physical cues maintain bone homeostasis through the TGFβ pathway to regulate Sclerostin expression and the deposition of new bone.
    Full-text · Article · Jan 2013 · PLoS ONE
  • [Show abstract] [Hide abstract]
    ABSTRACT: Abstract 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.
    No preview · Article · Jun 2013 · Connective tissue research
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Increasing evidence indicates that the secretome of mesenchymal stem cells (MSCs) has therapeutic potential for the treatment of various diseases, including cartilage disorders. However, the paracrine mechanisms underlying cartilage repair by MSCs are poorly understood. Here, we show that human umbilical cord blood-derived MSCs (hUCB-MSCs) promoted differentiation of chondroprogenitor cells by paracrine action. This paracrine effect of hUCB-MSCs on chondroprogenitor cells was increased by treatment with synovial fluid (SF) obtained from osteoarthritis (OA) patients, but was decreased by SF of fracture patients, compared to that of an untreated group. To identify paracrine factors underlying the chondrogenic effect of hUCB-MSCs, the secretomes of hUCB-MSCs stimulated by OA SF or fracture SF were analyzed using a biotin label-based antibody array. Among the proteins increased in response to these 2 kinds of SF, thrombospondin-2 (TSP-2) was specifically increased in only OA SF-treated hUCB-MSCs. In order to determine the role of TSP-2, exogenous TSP-2 was added to a micromass culture of chondroprogenitor cells. We found that TSP-2 had chondrogenic effects on chondroprogenitor cells via PKCα, ERK, p38/MAPK, and Notch signaling pathways. Knock-down of TSP-2 expression on hUCB-MSCs using small interfering RNA (siRNA) abolished the chondrogenic effects of hUCB-MSCs on chondroprogenitor cells. In parallel with in vitro analysis, the cartilage regenerating effect of hUCB-MSCs and TSP-2 was also demonstrated using a rabbit full-thickness osteochondral-defect model. Our findings suggested that hUCB-MSCs can stimulate the differentiation of locally presented endogenous chondroprogenitor cells by TSP-2, which finally leads to cartilage regeneration.
    Full-text · Article · Oct 2013 · Stem Cells
Show more