Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Robins, Jared C.; Akeno, Nagako; Mukherjee, Aditi; Dalal, Ravi R.; Aronow, Bruce J.; Koopman, Peter; Clemens, Thomas L.

doi:10.1016/j.bone.2005.04.040

Cited by 277 publications

(238 citation statements)

References 60 publications

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“…These data indicate that hypoxia promoted chondrocytic commitment of the pluripotent C3H10T1/2 mesenchymal cells and inhibited osteoblastic differentiation. Our results are in accordance with previous reports, which describe that hypoxia enhances Col2a1 and Aggrecan gene expression in C3H10T1/2 cells (66). To further investigate the role of oxygen tension in chondrocytic differentiation, we used a mouse embryo forelimb explant culture system.…”

Section: Discussionsupporting

confidence: 91%

“…However, very interestingly, our results indicate that hypoxia-mediated chondrocytic differentiation did not involve the up-regulation of Sox9 gene expression, its phosphorylation, or its transcriptional activity, suggesting that hypoxia-induced Col2a1 induction was independent of Sox9. However, in the previous report, it was described that Sox9 gene expression was up-regulated by hypoxia in association with transactivation of the Sox9 promoter in ST2 stromal cells (66). Although the difference in the results might depend on the difference in cell type, further confirmation about the Sox9 promoter may be necessary.…”

Section: Discussionmentioning

confidence: 81%

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Oxygen Tension Regulates Chondrocyte Differentiation and Function during Endochondral Ossification

Hirao

Tamai

Tsumaki

et al. 2006

Journal of Biological Chemistry

168

129

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Cartilage functions at a lower oxygen tension than most other tissues. To determine the role of oxygen tension in chondrocyte differentiation and function, we investigated the influence of oxygen tension in the pluripotent mesenchymal cell line C3H10T1/2 and 14.5E mice embryo forelimb organ culture. 10T1/2 cells and embryo forelimbs were cultured under normoxia (20% O 2 ) or hypoxia (5% O 2 ) in the presence of recombinant human bone morphogenetic protein 2. To elucidate the mechanism by which oxygen tension influences chondrocyte differentiation, the Smad pathway was examined using Smad6 overexpression adenovirus and Smad6 transgenic mice embryo forelimbs. The p38 MAPK pathway was examined using dominant-negative MKK3 and FR167653, a specific p38 MAPK inhibitor. The transcriptional activities of Sox9 and Runx2 were also investigated. Hypoxia promoted bone morphogenetic protein 2-induced glycosaminoglycan production and suppressed alkaline phosphatase activity and mineralization of C3H10T1/2. Thus, hypoxia promoted chondrocytic commitment rather than osteoblastic differentiation. In the mice embryo forelimb organ culture, hypoxia increased cartilaginous matrix synthesis. These effects were primarily mediated by p38 MAPK activation, independent of Sox9. Hypoxia inhibited Col10a1 (type X collagen ␣1) expression via downregulation of Runx2 activity by Smad suppression and histone deacetylase 4 activation. In conclusion, hypoxia promotes chondrocytic differentiation and cartilage matrix synthesis and suppresses terminal chondrocyte differentiation. These hypoxiainduced phenomena may act on chondrocytes to enhance and preserve their phenotype and function during chondrocyte differentiation and endochondral ossification.A number of pathophysiological findings suggest that a correlation exists between hypoxia and chondrogenesis. For example, articular cartilage is an avascular tissue that functions at an oxygen tension that is lower than that of most other tissues. Articular cartilage derives both its nutrition and oxygen supply by diffusion from the synovial fluid and the subchondral bone. It has been estimated that articular chondrocytes in the deepest layers may have access to no more than 1-6% O 2 (1-6). Furthermore, although the majority of mammalian cells derive their energy by using oxygen for mitochondrial oxidative phosphorylation (7), few mitochondria are present in articular chondrocytes (8). Carbohydrate breakdown in articular cartilage is dominated by the conversion of glucose to lactate via the Embden-Meyerhof-Parnas pathway (9 -11) that consumes no O 2 . Similarly, during the endochondral ossification processes that occur in the growth plate, chondromodulin-1, an endogenous inhibitor of neovascularization, is highly expressed by chondrocytes. Of note, most of the growth plate is avascular (12). Recently, in an in vivo experiment, it was found that hypoxia-inducible factor 1, which appears to be one of the major regulators of the hypoxic response, is essential for chondrocyte growth arrest and survival (1...

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 81%

Oxygen Tension Regulates Chondrocyte Differentiation and Function during Endochondral Ossification

Hirao

Tamai

Tsumaki

et al. 2006

Journal of Biological Chemistry

168

129

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“…This study is the first to provide direct evidence that hypoxia promotes the articular chondrocyte phenotype through SOX9 and, furthermore, that this is mediated specifically by HIF-2␣. Robins et al previously suggested that HIF could be involved in SOX9 up-regulation by hypoxia in mouse stromal cells (8). Those investigators identified HREs (sequences known to bind HIF proteins) in the mouse Sox9 promoter.…”

Section: Discussionmentioning

confidence: 99%

“…Domm and colleagues also showed a positive effect of hypoxia on Col␣1(II) levels in bovine chondrocytes (7). Hypoxia has been shown to promote chondrogenic differentiation of mesenchymal stem cells, and it was recently shown that the mouse Sox9 promoter is activated by hypoxia in mouse mesenchymal cells (8).…”

mentioning

confidence: 96%

Hypoxia‐inducible factor 2α is essential for hypoxic induction of the human articular chondrocyte phenotype

Lafont¹,

Talma²,

Murphy³

2007

Arthritis & Rheumatism

134

155

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Objective. To uncover the mechanism by which hypoxia enhances cartilage matrix synthesis by human articular chondrocytes.Methods. The hypoxic response was investigated by exposing normal (nonarthritic) human articular chondrocyte cultures to 20% oxygen and 1% oxygen. Induction of the differentiated phenotype was confirmed at the gene and protein levels. In its first reported application in human articular chondrocytes, the RNA interference method was used to directly investigate the role of specific transcription factors in this process. Small interfering RNA directed against hypoxiainducible factor 1␣ (HIF-1␣), HIF-2␣, and SOX9 were delivered by lipid-based transfection of primary and passaged human articular chondrocytes. The effect of each knockdown on hypoxic induction of the chondrocyte phenotype was assessed.Results. Hypoxia enhanced matrix synthesis and SOX9 expression of human articular chondrocytes at both the gene and protein levels. Although HIF-1␣ knockdown had no effect, depletion of HIF-2␣ abolished this hypoxic induction. Thus, we provide the first evidence that HIF-2␣, but not HIF-1␣, is essential for hypoxic induction of the human articular chondrocyte phenotype. In addition, depletion of SOX9 prevented hypoxic induction of matrix genes, indicating that the latter are not direct HIF targets but are up-regulated by hypoxia via SOX9.Conclusion. Based on our data, we propose a novel mechanism whereby hypoxia promotes cartilage matrix synthesis specifically through HIF-2␣-mediated SOX9 induction of key cartilage genes. These findings have potential application for the development of cartilage repair therapies.Being avascular, cartilage has a low oxygen concentration (1,2). Chondrocytes, the unique resident cells, are therefore adapted to these hypoxic conditions, e.g., by having lower levels of oxidative phosphorylation (3) and enhanced anaerobic glycolysis (4). Furthermore, recent studies suggest that hypoxia triggers essential positive signals for the chondrocyte phenotype beyond such survival responses. Indeed, we previously reported that reduced oxygen tension increases levels of the cartilage matrix genes COL2A1 and aggrecan and the cartilage transcription factor SOX9 in cultured articular chondrocytes (5,6). Domm and colleagues also showed a positive effect of hypoxia on Col␣1(II) levels in bovine chondrocytes (7). Hypoxia has been shown to promote chondrogenic differentiation of mesenchymal stem cells, and it was recently shown that the mouse Sox9 promoter is activated by hypoxia in mouse mesenchymal cells (8).In various cell types, hypoxic effects have been shown to be mediated by hypoxia-inducible factor (HIF) transcription factors. These proteins belong to the Per-ARNT-Sim (PAS) subfamily of basic helix-loop-helix (bHLH) transcription factors and consist of an ␣ subunit and a ␤ subunit (9). Under conditions of normoxia, HIF-1␣ is hydroxylated on specific proline residues, ubiquitinated through interaction with the von HippelLindau tumor suppressor protein, pVHL, and subsequently degraded b...

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“…This expression of SOX9 is abolished by deletion of hypoxia responsive element sites in the SOX9 promoter (6). SOX9 has also been described as a specific marker and maintenance factor for multipotential progenitors during pancreas organogenesis.…”

Section: Introductionmentioning

confidence: 95%

Hypoxia-Independent Gene Expression Mediated by SOX9 Promotes Aggressive Pancreatic Tumor Biology

Čamaj

Jäckel

Krebs

et al. 2014

Molecular Cancer Research

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Pancreatic cancer aggressiveness is characterized by its high capacity for local invasion, ability to promote angiogenesis, and potential to metastasize. Hypoxia is known to represent a crucial step in the development of aggressive malignant features of many human cancers. However, micrometastatic tumors are not typically subjected to hypoxic events during early stages of dissemination; therefore, it is unclear how these tumors are able to maintain their aggressive phenotype. Thus, the identification of regulators of hypoxia-related genes in aggressive/ metastatic tumors represents a fundamental step for the design of future therapies to treat pancreatic cancer. To this end, transcriptomic profiles were compared between the nonmetastatic pancreatic cancer cell line FG (LMET) and its angiogenic/metastatic derivate L3.6pl (HMET) under normoxic or hypoxic conditions. Cluster analysis revealed a number of transcripts that were induced by hypoxia in nonmetastatic cancer cells. Strikingly, this cluster was determined to be constitutively activated under normoxia in the metastatic cancer cells and could not be further induced by hypoxia. A subset of these transcripts were regulated by the transcription factor SOX9 in the aggressivemetastatic cells, but driven by hypoxia-inducible factor-1a (HIF-1a) in the parental nonmetastatic cell line. Moreover, these transcripts were enriched in cancer-related networks including: WNT, CXCR4, retinoic acid, and (FAK) focal adhesion kinase, gene PTK2 signaling pathways. In functional assays, inhibition of SOX9 expression in HMET cells led to increased apoptosis and reduced migration in vitro and a significant reduction in primary tumor growth, angiogenesis, and metastasis following orthotopic tumor cell injection. At the molecular level, the control of SOX9 expression was associated with changes in the methylation status of the SOX9 promoter. Finally, SOX9 upregulation was verified in a series of tumor specimens of patients with pancreatic carcinoma.

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Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Cited by 277 publications

References 60 publications

Oxygen Tension Regulates Chondrocyte Differentiation and Function during Endochondral Ossification

Oxygen Tension Regulates Chondrocyte Differentiation and Function during Endochondral Ossification

Hypoxia‐inducible factor 2α is essential for hypoxic induction of the human articular chondrocyte phenotype

Hypoxia-Independent Gene Expression Mediated by SOX9 Promotes Aggressive Pancreatic Tumor Biology

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