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Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation.

ABSTRACT: The transcription factor AP-2? (activating enhancer-binding protein epsilon) is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2? expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2? mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4) strongly correlated with that of AP-2? suggesting that hypoxia might represent an external regulator of AP-2? expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1), the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2'-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2?, siRNA against HIF1? led to a significantly reduced expression rate of AP-2?. Additionally, we detected a significant upregulation of the AP-2? mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2? gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

SUBMITTER: Niebler S

PROVIDER: S-EPMC4530219 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation.

Niebler Stephan S Angele Peter P Kujat Richard R Bosserhoff Anja K AK

BioMed research international 20150727

The transcription factor AP-2ε (activating enhancer-binding protein epsilon) is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angio ...[more]

PMID: 26273614

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Project description:UnlabelledAmniotic fluid stem (AFS) cells represent a major source of donor cells for cartilage repair. Recently, it became clear that mammalian target of rapamycin (mTOR) inhibition has beneficial effects on cartilage homeostasis, but the effect of mTOR on chondrogenic differentiation is still elusive. Therefore, the objectives of this study were to investigate the effects of mammalian target of rapamycin complex 1 (mTORC1) modulation on the expression of SOX9 and on its downstream targets during chondrogenic differentiation of AFS cells. We performed three-dimensional pellet culturing of AFS cells and of in vitro-expanded, human-derived chondrocytes in the presence of chondrogenic factors. Inhibition of mTORC1 by rapamycin or by small interfering RNA-mediated targeting of raptor (gene name, RPTOR) led to increased AKT activation, upregulation of hypoxia inducible factor (HIF) 2A, and an increase in SOX9, COL2A1, and ACAN abundance. Here we show that HIF2A expression is essential for chondrogenic differentiation and that AKT activity regulates HIF2A amounts. Importantly, engraftment of AFS cells in cell pellets composed of human chondrocytes revealed an advantage of raptor knockdown cells compared with control cells in their ability to express SOX9. Our results demonstrate that mTORC1 inhibition leads to AKT activation and an increase in HIF2A expression. Therefore, we suggest that mTORC1 inhibition is a powerful tool for enhancing chondrogenic differentiation of AFS cells and also of in vitro-expanded adult chondrocytes before transplantation.SignificanceRepair of cartilage defects is still an unresolved issue in regenerative medicine. Results of this study showed that inhibition of the mammalian target of rapamycin complex 1 (mTORC1) pathway, by rapamycin or by small interfering RNA-mediated targeting of raptor (gene name, RPTOR), enhanced amniotic fluid stem cell differentiation toward a chondrocytic phenotype and increased their engrafting efficiency into cartilaginous structures. Moreover, freshly isolated and in vitro passaged human chondrocytes also showed redifferentiation upon mTORC1 inhibition during culturing. Therefore, this study revealed that rapamycin could enable a more efficient clinical use of cell-based therapy approaches to treat articular cartilage defects.

Project description:Tissue engineering strategies often aim to direct tissue formation by mimicking conditions progenitor cells experience within native tissues. For example, to create cartilage in vitro, researchers often aim to replicate the biochemical and mechanical milieu cells experience during cartilage formation in the developing limb bud. This includes stimulating progenitors with TGF-?1/3, culturing under hypoxic conditions, and regulating mechanosensory pathways using biomaterials that control substrate stiffness and/or cell shape. However, as progenitors differentiate down the chondrogenic lineage, the pathways that regulate their responses to mechanotransduction, hypoxia and TGF-? may not act independently, but rather also impact one another, influencing overall cell response. Here, to better understand hypoxia's influence on mechanoregulatory-mediated chondrogenesis, we cultured human marrow stromal/mesenchymal stem cells (hMSC) on soft (0.167?kPa) or stiff (49.6?kPa) polyacrylamide hydrogels in chondrogenic medium containing TGF-?3. We then compared cell morphology, phosphorylated myosin light chain 2 staining, and chondrogenic gene expression under normoxic and hypoxic conditions, in the presence and absence of pharmacological inhibition of cytoskeletal tension. We show that on soft compared to stiff substrates, hypoxia prompts hMSC to adopt more spread morphologies, assemble in compact mesenchymal condensation-like colonies, and upregulate NCAM expression, and that inhibition of cytoskeletal tension negates hypoxia-mediated upregulation of molecular markers of chondrogenesis, including COL2A1 and SOX9. Taken together, our findings support a role for hypoxia in regulating hMSC morphology, cytoskeletal tension and chondrogenesis, and that hypoxia's effects are modulated, at least in part, by mechanosensitive pathways. Our insights into how hypoxia impacts mechanoregulation of chondrogenesis in hMSC may improve strategies to develop tissue engineered cartilage. STATEMENT OF SIGNIFICANCE: Cartilage tissue engineering strategies often aim to drive progenitor cell differentiation by replicating the local environment of the native tissue, including by regulating oxygen concentration and mechanical stiffness. However, the pathways that regulate cellular responses to mechanotransduction and hypoxia may not act independently, but rather also impact one another. Here, we show that on soft, but not stiff surfaces, hypoxia impacts human MSC (hMSC) morphology and colony formation, and inhibition of cytoskeletal tension negates the hypoxia-mediated upregulation of molecular markers of chondrogenesis. These observations suggest that hypoxia's effects during hMSC chondrogenesis are modulated, at least in part, by mechanosensitive pathways, and may impact strategies to develop scaffolds for cartilage tissue engineering, as hypoxia's chondrogenic effects may be enhanced on soft materials.

Project description:BackgroundSkin denervation that develops in patients with diabetes mellitus as a neuropathic manifestation is known as diabetic peripheral neuropathy (DPN). Skin denervation is parallel to neuronal injuries that alter intracellular signaling. To date, the correlation between nerve injury and the activation of intracellular responses to neuropathic manifestations has not been elucidated; specifically, whether activating transcription factor 3 (ATF3) is responsible for neuronal injury and a critical molecule that modulates the activation of intracellular protein kinase C epsilon (p-PKCε) and pain development in DPN is a crucial question.MethodsTo address, ATF3 knockout (atf3 -/- group, C57/B6 genetic background) and wild-type mice (atf3+/+ group) received a single dose of streptozotocin (200 mg/kg) to generate a mouse model of DPN.ResultsBoth atf3+/+ and atf3 -/- mice exhibited hyperglycemia and the same pathology of skin denervation at posttreatment month 2, but only atf3+/+ mice developed thermal hyperalgesia (P<0.001) and mechanical allodynia (P=0.002). The atf3+/+ group, but not the atf3 -/- group, had preferential ATF3 upregulation on p-PKCε(+) neurons with a ratio of 37.7%±6.1% in p-PKCε(+):ATF3(+) neurons (P<0.001). In addition, B-cell lymphoma-extra large (Bcl-XL), an antiapoptotic Bcl2 family protein, exhibited parallel patterns to p-PKCε (ie, Bcl-XL upregulation was reversed in atf3 -/- mice). These two molecules were colocalized and increased by approximately two-fold in the atf3+/+ group compared with the atf3 -/- group (30.0%±3.4% vs 13.7% ± 6.2%, P=0.003). Furthermore, linear analysis results showed that the densities of p-PKCε and Bcl-XL had a reverse linear relationship with the degrees of thermal hyperalgesia and mechanical allodynia.ConclusionCollectively, this report suggested that ATF3 is a critical upstream molecule that modulates p-PKCε and Bcl-XL expression, which consequently mediated the development of neuropathic manifestation in DPN.

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Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation.

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Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation.

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