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Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK-dependent PPARgamma expression and phosphorylation.

ABSTRACT: Mesenchymal stem cells (MSCs) provide us an excellent cellular model to uncover the molecular mechanisms underlying adipogenic differentiation of adult stem cells. PPARgamma had been considered as an important molecular marker of cells undergoing adipogenic differentiation. Here, we demonstrated that expression and phosphorylation of PPARgamma could be found in bone marrow-derived MSCs cultured in expansion medium without any adipogenic additives (dexamethasone, IBMX, insulin or indomethacin). Then, PPARgamma was dephosphorylated in MSCs during the process of adipogenic differentiation. We then found that inhibition of MEK activation by specific inhibitor (PD98059) counteracted the PPARgamma expression and phosphorylation. However, expression and phosphorylation of PPARgamma did not present in MSCs cultured in medium with lower serum concentration. When these MSCs differentiated into adipocytes, no phosphorylation could be detected to accompany the expression of PPARgamma. Moreover, exposure of MSCs to higher concentration of serum induced stronger PPARgamma expression, and subsequently enhanced their adipogenesis. These data suggested that activation of the MEK/ERK signalling pathway by high serum concentration promoted PPARgamma expression and phosphorylation, and subsequently enhanced adipogenic differentiation of MSCs.

SUBMITTER: Wu L

PROVIDER: S-EPMC3823124 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK-dependent PPARgamma expression and phosphorylation.

Wu Ling L Cai Xiaoxiao X Dong Hai H Jing Wei W Huang Yuanding Y Yang Xingmei X Wu Yao Y Lin Yunfeng Y

Journal of cellular and molecular medicine 20090220 4

Mesenchymal stem cells (MSCs) provide us an excellent cellular model to uncover the molecular mechanisms underlying adipogenic differentiation of adult stem cells. PPARgamma had been considered as an important molecular marker of cells undergoing adipogenic differentiation. Here, we demonstrated that expression and phosphorylation of PPARgamma could be found in bone marrow-derived MSCs cultured in expansion medium without any adipogenic additives (dexamethasone, IBMX, insulin or indomethacin). T ...[more]

PMID: 19243475

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Project description:BackgroundMesenchymal stem cells (MSCs) are pluripotent stem cells that can differentiate via osteogenesis and adipogenesis. The mechanism underlying MSC lineage commitment still remains incompletely elucidated. Understanding the regulatory mechanism of MSC differentiation will help researchers induce MSCs toward specific lineages for clinical use. In this research, we intended to figure out the long noncoding RNA (lncRNA) that plays a central role in MSC fate determination and explore its application value in tissue engineering.MethodsThe expression pattern of lncRNAs during MSC osteogenesis/adipogenesis was detected by microarray and qRT-PCR. Lentivirus and siRNAs were constructed to regulate the expression of lncRNA repressor of adipogenesis (ROA). MSC osteogenesis/adipogenesis was evaluated by western blot and alizarin red/oil red staining. An adipokine array was used to select the paracrine/autocrine factor PTX3, followed by RNA interference or recombinant human protein stimulation to confirm its function. The activation of signaling pathways was also detected by western blot, and a small molecule inhibitor, SCH772984, was used to inhibit the activation of the ERK pathway. The interaction between ROA and hnRNP A1 was detected by RNA pull-down and RIP assays. Luciferase reporter and chromatin immunoprecipitation assays were used to confirm the binding of hnRNP A1 to the PTX3 promotor. Additionally, an in vivo adipogenesis experiment was conducted to evaluate the regulatory value of ROA in tissue engineering.ResultsIn this study, we demonstrated that MSC adipogenesis is regulated by lncRNA ROA both in vitro and in vivo. Mechanistically, ROA inhibits MSC adipogenesis by downregulating the expression of the key autocrine/paracrine factor PTX3 and the downstream ERK pathway. This downregulation was achieved through transcription inhibition by impeding hnRNP A1 from binding to the promoter of PTX3.ConclusionsROA negatively regulates MSC adipogenesis through the hnRNP A1-PTX3-ERK axis. ROA may be an effective target for modulating MSCs in tissue engineering.

Project description:Vascular endothelial growth factor (VEGF) is a hypoxia-induced angiogenic protein that exhibits a broad range of neurotrophic and neuroprotective effects in the central nervous system. Given that neurogenesis occurs in close proximity to blood vessels, increasing evidence has suggested that VEGF may constitute an important link between neurogenesis and angiogenesis. Although it is known that VEGF can directly stimulate the proliferation of neuronal progenitors, the underlying signaling pathways responsible in this process are not fully understood. Thus, in the present study, we set out to examine the requirement of two downstream targets of the VEGF/Flk-1 signaling network, the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways, in producing the mitogenic effects of VEGF. Both in vivo and in vitro experiments showed that a single treatment of VEGF activated Erk1/2 and Akt signaling pathways in the adult rat hippocampus and in cultured hippocampal neuronal progenitor cells. This effect was blocked with the VEGF/Flk-1 inhibitor SU5416. Importantly, microinfusion of VEGF into the rat brain also induced pCREB expression in the dentate gyrus and increased the number of BrdU-labeled cells in the dentate subgranular zone. Double immunofluorescence labeling revealed that a large proportion of BrdU-labeled cells expressed activated forms of Flk-1, Erk1/2, and Akt. Interestingly, treatment with the SSRI fluoxetine, which is well known to stimulate neurogenesis and VEGF-signaling, also produced a similar expression pattern of Erk1/2 and Akt in proliferating cells. Finally, pharmacological experiments showed that administration of inhibitors of either MAPK/ERK (U0126) or PI3K (LY294002) blocked VEGF-stimulation of hippocampal cell proliferation in vivo and in vitro. Taken together, our findings demonstrate that the proliferative actions of VEGF require activation of both ERK and Akt signaling cascades and that these intracellular pathways are stimulated almost exclusively in actively proliferating neuronal progenitor cells of the adult hippocampus.

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Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK-dependent PPARgamma expression and phosphorylation.

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Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK-dependent PPARgamma expression and phosphorylation.

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