Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification

From the Department of Molecular and Cellular Biochemistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2012; 287(40):33179-90. DOI: 10.1074/jbc.M111.337063
Source: PubMed


Endochondral ossification is temporally and spatially regulated by several critical transcription factors, including Sox9, Runx2, and Runx3. Although the molecular mechanisms that control the late stages of endochondral ossification (e.g. calcification) are physiologically and pathologically important, these precise regulatory mechanisms remain unclear. Here, we demonstrate that Osterix is an essential transcription factor for endochondral ossification that functions downstream of Runx2. The global and conditional Osterix-deficient mice studied here exhibited a defect of cartilage-matrix ossification and matrix vesicle formation. Importantly, Osterix deficiencies caused the arrest of endochondral ossification at the hypertrophic stage. Microarray analysis revealed that matrix metallopeptidase 13 (MMP13) is an important target of Osterix. We also showed that there exists a physical interaction between Osterix and Runx2 and that these proteins function cooperatively to induce MMP13 during chondrocyte differentiation. Most interestingly, the introduction of MMP13 stimulated the calcification of matrices in Osterix-deficient mouse limb bud cells. Our results demonstrated that Osterix was essential to endochondral ossification and revealed that the physical and functional interaction between Osterix and Runx2 were necessary for the induction of MMP13 during endochondral ossification.

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Available from: Shiho Honma, Nov 09, 2015
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    • "Given that SOX9 was capable of directly binding to a conserved motif in the MMP13 gene; potentially SOX9's effect on lowering MMP13 expression in activated HSCs could be either direct, indirect via Epim or both. Our data are concordant with chondrogenesis during development when SOX9 plays a key role in regulating cell proliferation and the expression of cartilage matrix genes [5]; in the later stages of chondrocyte maturation SOX9 is absent concomitant with an increase in MMP13 [42], [43]. In contrast to our data, retroviral transfer of SOX9 in chick fibroblasts increased EPIM expression in cells cultured as pellets [21]. "
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    ABSTRACT: Background and Aims Liver fibrosis is a major cause of morbidity and mortality. It is characterised by excessive extracellular matrix (ECM) deposition from activated hepatic stellate cells (HSCs). Although potentially reversible, treatment remains limited. Understanding how ECM influences the pathogenesis of the disease may provide insight into novel therapeutic targets for the disease. The extracellular protein Epimorphin (EPIM) has been implicated in tissue repair mechanisms in several tissues, partially, through its ability to manipulate proteases. In this study, we have identified that EPIM modulates the ECM environment produced by activated hepatic stellate cells (HSCs), in part, through down-regulation of pro-fibrotic Sex-determining region Y-box 9 (SOX9). Methods Influence of EPIM on ECM was investigated in cultured primary rat HSCs. Activated HSCs were treated with recombinant EPIM or SOX9 siRNA. Core fibrotic factors were evaluated by immunoblotting, qPCR and chromatin immunoprecipitation (ChIP). Results During HSC activation EPIM became significantly decreased in contrast to pro-fibrotic markers SOX9, Collagen type 1 (COL1), and α- Smooth muscle actin (α-SMA). Treatment of activated HSCs with recombinant EPIM caused a reduction in α-SMA, SOX9, COL1 and Osteopontin (OPN), while increasing expression of the collagenase matrix metalloproteinase 13 (MMP13). Sox9 abrogation in activated HSCs increased EPIM and MMP13 expression. Conclusion These data provide evidence for EPIM and SOX9 functioning by mutual negative feedback to regulate attributes of the quiescent or activated state of HSCs. Further understanding of EPIM's role may lead to opportunities to modulate SOX9 as a therapeutic avenue for liver fibrosis.
    Full-text · Article · Jun 2014 · PLoS ONE
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    • "New in vitro data demonstrated that Sox9 negatively regulates Runx2 by enhancing Bapx1 expression, which leads to the inhibition of terminal chondrocyte differentiation [17]. Osterix, which acts downstream of Runx2 during bone formation, is expressed in chondrocyte progenitors and prehypertrophic chondrocytes in rib, spine, and limb cartilages, suggesting that Osterix may play a critical role during the primary cartilage maturation in combination with Runx2 and Sox9 [6, 7]. "
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    ABSTRACT: The purpose of this study is to investigate the spacial expression pattern and functional significance of three key transcription factors related to bone and cartilage formation, namely, Sox9, Runx2, and Osterix in cartilages during the late development of mouse mandible. Immunohistochemical examinations of Sox9, Runx2, and Osterix were conducted in the mandibular cartilages of the 15 neonatal C57BL/6N mice. In secondary cartilages, both Sox9 and Runx2 were weakly expressed in the polymorphic cell zone, strongly expressed in the flattened cell zone and throughout the entire hypertrophic cell zone. Similarly, both transcriptional factors were weakly expressed in the uncalcified Meckel's cartilage while strongly expressed in the rostral cartilage. Meanwhile, Osterix was at an extremely low level in cells of the flattened cell zone and the upper hypertrophic cell zone in secondary cartilages. Surprisingly, Osterix was intensely expressed in hypertrophic chondrocytes in the center of the uncalcified Meckel's cartilage while moderately expressed in part of hypertrophic chondrocytes in the rostral process. Consequently, it is suggested that Sox9 is a main and unique positive regulator in the hypertrophic differentiation process of mandibular secondary cartilages, in addition to Runx2. Furthermore, Osterix is likely responsible for phenotypic conversion of Meckel's chondrocytes during its degeneration.
    Full-text · Article · May 2013
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