Constitutive expression of thrombospondin 1 in MC3T3‐E1 osteoblastic cells inhibits mineralization

Ueno, Akihiko; Miwa, Yoshiro; Miyoshi, Keiko; Horiguchi, Taigo; Inoue, Hideo; Ruspita, Intan; Abe, Kaori; Yamashita, Kikuji; Hayashi, Eiji; Noma, Takafumi

doi:10.1002/jcp.20735

Cited by 37 publications

(24 citation statements)

References 64 publications

(70 reference statements)

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“…Our observations are the first to directly implicate TSP2 in the regulation of mineralization of the osteoblast-derived ECM. This is in marked contrast to Ueno and colleagues [17] who demonstrated that TSP1, which shares significant sequence homology with TSP2, inhibits mineralization in MC3T3-E1 cells.…”

Section: Discussioncontrasting

confidence: 92%

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

Alford¹,

Terkhorn²,

Reddy³

et al. 2010

Bone

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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.

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Section: Discussioncontrasting

confidence: 92%

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

Alford¹,

Terkhorn²,

Reddy³

et al. 2010

Bone

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“…These regulatory effects indicate that PC may inhibit OA meniscal cell differentiation and pathological calcification in part by modulating the FGF signaling pathway. Consistently, studies have demonstrated that constitutive expression of thrombospondin 1 (THBS1), a protein involved in FGF signaling pathway, inhibited biomineralization and that THBS1 gene therapy suppressed the progression of arthritis in a rat model of OA [47, 48]. Another study demonstrated that the absence of signaling through FGF receptor 3 (FGFR3) leads to premature cartilage degeneration and early arthritis [49].…”

Section: Discussionmentioning

confidence: 97%

Biological Activities of Phosphocitrate: A Potential Meniscal Protective Agent

Sun

Roberts

Mauerhan

et al. 2013

BioMed Research International

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Phosphocitrate (PC) inhibited meniscal calcification and the development of calcium crystal-associated osteoarthritis (OA) in Hartley guinea pigs. However, the mechanisms remain elusive. This study sought to examine the biological activities of PC in the absence of calcium crystals and test the hypothesis that PC is potentially a meniscal protective agent. We found that PC downregulated the expression of many genes classified in cell proliferation, ossification, prostaglandin metabolic process, and wound healing, including bloom syndrome RecQ helicase-like, cell division cycle 7 homolog, cell division cycle 25 homolog C, ankylosis progressive homolog, prostaglandin-endoperoxide synthases-1/cyclooxygenase-1, and plasminogen activator urokinase receptor. In contrast, PC stimulated the expression of many genes classified in fibroblast growth factor receptor signaling pathway, collagen fibril organization, and extracellular structure organization, including fibroblast growth factor 7, collagen type I, alpha 1, and collagen type XI, alpha 1. Consistent with its effect on the expression of genes classified in cell proliferation, collagen fibril organization, and ossification, PC inhibited the proliferation of OA meniscal cells and meniscal cell-mediated calcification while stimulating the production of collagens. These findings indicate that PC is potentially a meniscal-protective agent and a disease-modifying drug for arthritis associated with severe meniscal degeneration.

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“…Thbs4 is known to be expressed in osteogenic tissues (51,52), but its physiological function in bone has not been established. However, the observations that ablation of the related Thsbs3 results in accelerated endochondrial ossification (53) and overexpression of Thbs1 inhibits mineralization in osteoblast cultures (54), suggest that increased Tbhs4 in Hyp osteoblasts may play a role in the intrinsic mineralization defect. We found that carbonic anhydrase 12 (Car12) and 3 (Car3) as well as the sodium-dependent citrate transporter (Slc13a5), were decreased in Hyp bone.…”

Section: Discussionmentioning

confidence: 98%

Novel Regulators of Fgf23 Expression and Mineralization in Hyp Bone

Liu

Tang

Fang

et al. 2009

Molecular Endocrinology

112

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We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts associated with abnormal Fgf23 production and mineralization in Hyp mice. We found evidence that elevation of Fgf23 expression in osteocytes is associated with increments in Fgf1, Fgf7, and Egr2 and decrements in Sost, an inhibitor in the Wnt-signaling pathway, were observed in Hyp bone. beta-Catenin levels were increased in Hyp cortical bone, and TOPflash luciferase reporter assay showed increased transcriptional activity in Hyp-derived osteoblasts, consistent with Wnt activation. Moreover, activation of Fgf and Wnt-signaling stimulated Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the extracellular matrix protein Dmp1. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced posttranslational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regard to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH-altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone.

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Constitutive expression of thrombospondin 1 in MC3T3‐E1 osteoblastic cells inhibits mineralization

Cited by 37 publications

References 64 publications

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

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

Biological Activities of Phosphocitrate: A Potential Meniscal Protective Agent

Novel Regulators of Fgf23 Expression and Mineralization in Hyp Bone

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