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HIF-1A and C/EBPs transcriptionally regulate adipogenic differentiation of bone marrow-derived MSCs in hypoxia.

ABSTRACT: Bone marrow-derived mesenchymal stem cells (BMSCs, also known as bone marrow-derived mesenchymal stromal cells) are known to be a component of the tumor microenvironment. BMSCs are multipotent stromal cells that can differentiate into a variety of cell types, including osteocytes, chondrocytes, adipocytes, epithelial cells and endothelial cells. Stem cells found in niches or transplanted into injured tissues constantly encounter hypoxic stress. Areas with very low to no oxygen pressure exist in solid tumors. The differentiation capacity of BMSCs under hypoxic conditions remains controversial.In this study, a hypoxic workstation, set at an oxygen concentration of 0.2% was used to mimic the hypoxic microenvironment of cancer in vivo. Oil red O staining and alkaline phosphatase staining were used to examine the adipogenic or osteogenic differentiation, respectively, of BMSCs. Real-time PCR was performed to explore the expression of adipocyte- or osteocyte-specific genes. An RT2 Profiler™ PCR Array was used to screen a panel of 84 genes associated with human adipogenesis in BMSCs under normal and hypoxic conditions. A dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) were applied to analyze promoter activity to evaluate the possible regulatory mechanism of adipocyte-specific gene expression.We found that this extreme hypoxia impaired osteogenic differentiation as indicated by the attenuation of alkaline phosphatase (ALP) activity and the reduced expression of osteogenic markers osteocalcin and osteopontin. Moreover, extreme hypoxia enhanced adipogenic differentiation, as indicated by the accumulation of lipid droplets and the expression of the adipocyte-specific genes leptin, LPL, CFD, PGAR and HIG2. In the extreme hypoxic conditions (0.2% oxygen), the overexpression of CCAAT enhancer-binding proteins (C/EBPs), especially C/EBP?, and HIF-1A upregulated the promoter activities of adipocyte-specific genes such as leptin, CFD, HIG2, LPL, PGAR. In the present study, peroxisome proliferator-activated receptor-gamma (PPAR?) exerted a negative effect on the differentiation of BMSCs into adipocytes.In view of these findings, extreme hypoxia induced the adipogenic differentiation of BMSCs through HIF-1A and C/EBPs. These findings might provide clues regarding the roles of BMSCs in the cancer microenvironment.

SUBMITTER: Jiang C

PROVIDER: S-EPMC4559195 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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HIF-1A and C/EBPs transcriptionally regulate adipogenic differentiation of bone marrow-derived MSCs in hypoxia.

Jiang Chen C Sun Jun J Dai Yafei Y Cao Pengfei P Zhang Liyang L Peng Shuping S Zhou Yanhong Y Li Guiyuan G Tang Jingqun J Xiang Juanjuan J

Stem cell research & therapy 20150312

<h4>Introduction</h4>Bone marrow-derived mesenchymal stem cells (BMSCs, also known as bone marrow-derived mesenchymal stromal cells) are known to be a component of the tumor microenvironment. BMSCs are multipotent stromal cells that can differentiate into a variety of cell types, including osteocytes, chondrocytes, adipocytes, epithelial cells and endothelial cells. Stem cells found in niches or transplanted into injured tissues constantly encounter hypoxic stress. Areas with very low to no oxyg ...[more]

PMID: 25889814

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Project description:BackgroundMesenchymal stem cells (MSCs) selectively differentiate into adipocytes or osteoblasts, and several molecules control the fate determination of MSCs. Understanding these key checkpoints greatly contributes to the ability to induce specific MSC differentiation for clinical applications. In this study, we aimed to explore whether TNF receptor-associated factor 4 (TRAF4) affects MSC adipogenic differentiation, which we previously reported that could positively regulated the osteogenic differentiation.MethodsWestern blotting and Real-time Polymerase Chain Reaction were used to detected the expression pattern of TRAF4 during adipogenic differentiation. Lentivirus was constructed to regulate TRAF4 expression, and oil red O staining and Western blotting were used to assess its role in adipogenesis, which was confirmed in vivo by implanting an MSC-matrigel mixture into nude mice. Western blotting was used to detect the activated signaling pathways, and a specific inhibitor and agonist were used to clear the roles of the key signaling pathways. Additionaly, Co-Immunoprecipitation was conducted to find that Pyruvate kinase isozyme type M2 (PKM2) interacts with TRAF4, and to further explore their binding and functional domains. Finally, an RNA-binding protein immunoprecipitation assay and Western blotting were used to detect whether N6-methyladenosine mediates the decreased TRAF4 expression during adipogenic differentiation.FindingsThe results demonstrated that TRAF4 negatively regulates MSC adipogenesis in vitro and in vivo. Mechanistically, we revealed that TRAF4 binds to PKM2 to activate the kinase activity of PKM2, which subsequently activates β-catenin signaling and then inhibits adipogenesis. Furthermore, TRAF4 downregulation during adipogenesis is regulated by ALKBH5-mediated N6-methyladenosine RNA demethylation.InterpretationTRAF4 negatively regulates the adipogenesis of MSCs by activating PKM2 kinase activity, which may act as a checkpoint to fine-tune the balance of adipo-osteogenic differentiation, and suggests that TRAF4 may be a novel target of MSCs in clinical use and may also illuminate the underlying mechanisms of bone metabolic diseases.FundingThis study was supported by the National Natural Science Foundation of China (81871750 and 81971518) and the Science and Technology Project of Guangdong Province (2019B02023600 and 2017A020215070).

Project description:BackgroundTransfer RNA-derived small RNAs (tsRNAs) are a recently discovered form of non-coding RNA capable of regulating myriad physiological processes. The role of tsRNAs in hMSC adipogenic differentiation, however, remains incompletely understood. The purpose of this study was to identify the novel tsRNA-16902 as a regulator of hMSC adipogenic differentiation.MethodsIn this study, we conducted transcriptomic sequencing of hMSCs after inducing their adipogenic differentiation, and we were thereby able to clarify the molecular mechanism underlying the role of tsRNA-16902 in this context via a series of molecular biology methods.ResultsWhen we knocked down tsRNA-16902 expression, this impaired hMSC adipogenic differentiation and associated marker gene expression. Bioinformatics analyses further revealed tsRNA-16902 to target retinoic acid receptor γ (RARγ). Luciferase reporter assays also confirmed the ability of tsRNA-16902 to bind to the RARγ 3'-untranslated region. Consistent with this, RARγ overexpression led to impaired hMSC adipogenesis. Further analyses revealed that Smad2/3 phosphorylation was increased in cells that either overexpressed RARγ or in which tsRNA-16902 had been knocked down. We also assessed the adipogenic differentiation of hMSCs in which tsRNA-16902 was knocked down and at the same time a Smad2/3 inhibitor was added to disrupt Smad2/3 phosphorylation. The adipogenic differentiation of hMSCs in which tsRNA-16902 was knocked down was further enhanced upon the addition of a Smad2/3 signaling inhibitor relative to tsRNA-16902 knockdown alone.ConclusionsThrough a comprehensive profiling analysis of tsRNAs that were differentially expressed in the context of hMSC adipogenic differentiation, we were able to identify tsRNA-16902 as a previously uncharacterized regulator of adipogenesis. tsRNA-16902 is able to regulate hMSC adipogenic differentiation by targeting RARγ via the Smad2/3 signaling pathway. Together, our results may thus highlight novel strategies of value for treating obesity.

Dataset Information

HIF-1A and C/EBPs transcriptionally regulate adipogenic differentiation of bone marrow-derived MSCs in hypoxia.

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HIF-1A and C/EBPs transcriptionally regulate adipogenic differentiation of bone marrow-derived MSCs in hypoxia.

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