The Regulation Of Adipogenic Differentiation Of Mesenchymal Stem Cells By NCoR

BackgroundBone marrow mesenchymal stem cells have the potential of multi-directional differentiation, they can differentiate into osteoblast, adipocyte and chondrocyte. When observing the proliferation and osteogenesis differentiation of BMSCs, some studies found that under the proper condition, BMSCs could divide and proliferate for 38+4 times in vitro, and were arranged swirl with the original spindle. After frozen storage and recovery, these cells could be still subcultured for more than 20 generations and had the property of stem cells. BMSCs not only exists in the bone marrow, but also exists outside of bone tissue. When stimulated by the appropriate signal, BMSCs can drift away the bone tissue, and reach the target organ, this biological behavior is called "homing". The homing ability of BMSCs is closely related to cell cycle, and the recognition and engraftment can be mediated by the interactions of complex molecules in the bone marrow. So BMSCs showed a good prospect in clinical application.Adipose tissue regulate energy balance of our body, and maintain the stability of the metabolism and body temperature by adjusting the metabolism of fat and sugar. It also can serve as an important barrier to cushion the impact of mechanical force and protect our organs. Now obesity and metabolic disease caused by obesity has become the top threat to human health on the world scale, and they are related to adipose differentiation.Differentiation of BMSCs to adipocytes involves a comprehensive network including transcription factors responsible for expression of key proteins that induce mature adipocyte formation. The process of adipogenesis also involves changes in cell morphology, induction of insulin sensitivity and changes in secretory capacity of cells. In mammalian proliferator-activated receptor y (PPARy) and CCAAT/enhancer binding protein a(C/EBPα) are the main regulators of adipogenesis. PPARγ is induced during differentiation of preadipocytes to adipocytes and is essential for thisprocess. Without it, precursor cells are unable to differentiate into mature adipocytes. Furthermore, PPARy is capable of promoting adipogenesis in C/EBPa-deficient cells. However, C/EBPa is not able to promote adipogenesis in PPARy-deficient cells, demonstrating that PPARy is the master regulator of adipogenesis. Although cells deficient in C/EBPa are capable of differentiating into adipocytes, this differentiation is defective in that they accumulate less lipid droplets and do not induce expression of PPARy, demonstrating that cross-regulation between C/EBPa and PPARy is important for maintenance of differentiated state. And PPARy and C/EBPa were activated by transcription factors, co-activators and co-repressors together, histone acetyltransferase and histone deacetylase play an important role in this process.NCoR is a number of the wide family of co-regulator molecules, Repression is mediated by recruiting multiple histone deacetylase enzymes such as HDAC1, HDAC7, HDAC4, HDAC3 and Sirtl. The relative importance of each of these enzymes has yet to be fully established; however, it has been clearly demonstrated that HDAC3 recruitment to the complex is essential for repression by the thyroid hormone receptor. Tremendous progress has been made in understanding the mechanisms, regulation and functions of NCoR and its interacting proteins in transcription, and involvement in diverse pathways that regulate metabolism, inflammation and oncogenesis. While the literature has supported the concept that deregulated corepressor function facilitates cancer development by disrupting the homeostatic balance between corepressors and coactivators (or other regulatory proteins) in the regulation of chromatin structure, transcription, DNA repair, inflammation and other important biological processes. Corepressors may have different mechanisms and play either pro-or anti-tumorigenic roles in different types of cancers and leukemias. Jin Z reported that HDAC9 inhibits PPARy activity in synergy with silencing mediator of retinoic acid and thyroid hormone receptors (SMRT)/NCoR corepressors and identify HDAC9 as a novel, important and physiologically relevant modulator of bone remodeling and skeletal homeostasis. Chun guo found that GPS2 can mediate a novel corepressor repression pathway that allows NCoR to directly repress active PPARy-mediated transcription, which is important for the optimal corepressor function of NCoR for PPARy. Ping L found that NCoR deletion leads to adipogenesis, reduced inflammation, and enhanced systemic insulin sensitivity in NCoR knock-out mice. So what is the effect of NCoR on the differentiation of BMSCs to adipocytes.PurposeBone marrow mesenchymal stem cells (BMSCs) are considered a promising cell source for tissue engineering. Methods to advance the proliferation and anti-apoptotic capacity of mesenchymal stem cells (MSCs) are required to improve the efficiency of MSC-based therapy. In this study, we aimed to observe the effect of nuclear receptor corepressor (NCoR) on BMSC proliferation, as well as the relationship between NCoR and insulin. And we want to investigate the effects of NCoR on adipogenic differentiation of MSCs isolated from the rats.Methods1. According to the NCoR sequence from Gen Bank (EU006039.1), we designed three sequences of NCoR siRNA and pCMV-NCoR. For transient transfection, cells were cultured with serum-free medium for 2 days and then transfected with construct for 2 days using HiPerFect (Qiagen) according to the manufacturer’s protocols. The efficiency of NCoR siRNA and pCMV-NCoR were determined by Western blot analysis, and NCoR expression was detected using Western blot assay. Gene expression of NCoR was analyzed by real-time PCR.2. Effect of NCoR on the proliferation of BMSCs. In brief, isolated and identified rat MSCs were seeded in 96-well plates at a density of 1.0x106/mL in medium. When cells attained 65% confluence, cells were transfected with NCoR siRNA and pCMV-NCoR. After incubation for 1,2,3,4, and 5 days in medium, cell viability was evaluated by methyl thiazolyl tetrazolium (MTT) assay and cell growth curve assays.3. Relationship between NCoR and insulin on the proliferation of BMSCs. We added different concentrations of insulin (0,5,15, and 45 mmol/L) to the medium for interfering with MSC growth. We added insulin to the medium for every group, and cell viability was evaluated by MTT and the cell counter after incubation for 2 days.4. Effect of NCoR on adipocyte viability. Cells cultured in adipocytic medium attaining 65% confluency were treated with NCoR siRNA or pCMV-NCoR. At 0,3,5, 7, and 14 d following NCoR inhibition or over-expression, MTT activity in each well was used to determine the viability of MSC-derived adipocytes.5.Effect of NCoR on adipogenic differentiation of rat MSCs. Cells cultured in adipocytic medium for 2 d were treated with NCoR siRNA or pCMV-NCoR for 2 d, adipogenic differentiation of rat MSCs was investigated. Oil Red O staining was used to determine the quantity of lipid droplets in cells. The expressions of three marker genes of differentiation including PPARy, C/EBPa, and LPL were determined by Western blot or real-time RT-PCR.Results1. BMSCs were treated with NCoR siRNA before cells were counted with the cell counter. NCoR siRNA was shown to decrease BMSC proliferation after 2 days, pCMV-NCoR can increase BMSCs proliferation.2. NCoR siRNA inhibits the effect of insulin on BMSC proliferation.BMSCs were treated with insulin at various concentrations (0,5,15, and 45 mmol/L) before the rate of cell proliferation was examined via MTT assay. Insulin at 15 mmol/L was shown to remarkably enhance BMSC proliferation, where as insulin at 45 mmol/L had no effect on BMSC proliferation. However, when the gene NCoR was knocked down from BMSC, the effect of various concentrations of insulin on cell proliferation was inhibited. Over-expression of NCoR make the effect of various concentrations of insulin on cell proliferation was inhibited.3. NCoR inhibited cell viability of rat MSCs.Cells receiving NCoR siRNA treatment showed the higher proliferation responses in comparison to the control and non-targeting NCoR siRNA control (P<0.05). However, over-expression of NCoR resulted in the down-regulation of cell viability significantly at indicated times. It indicated that NCoR had the ability to inhibit cell viability of rat MSCs cultured in adipocytic medium.4. NCoR down-regulated adipogenic differentiation of rat MSCsThe results demonstrated that, compared to the control and non-target siRNA control, the quantity of lipid droplets in cells was increased significantly in NCoR siRNA treatment. An increase in OD value further proved that NCoR siRNA enhanced adipogenic differentiation of rat MSCs. Additionally, the expressions of three marker genes of differentiation including PPARy, C/EBPa, and LPL were determined by Western blot or real-time RT-PCR. The results indicated that the absence of NCoR increased protein expression of PPARy including PPARyl and PPARy2 and C/EBPa significantly (P<0.01). Compared to the control, mRNA expression of LPL was also increased in NCoR siRNA-treated cells. Over-expression of NCoR in differentiated MSCs for 2 d could reduce lipid droplet accumulation in cells, compared to the control and non-target siRNA control, and inhibit OD value and mRNA expression of PPARyl, PPARy2, C/EBPa, and LPL. Taken together, NCoR could negatively regulate adipogenic differentiation of rat MSCs via down-regulating the expression of PPARγ and C/EBPa.ConclusionBMSC proliferation is critical for MSC-based therapy or cell trials. Our results showed that NCoR siRNA could inhibit BMSC growth and proliferation, and cell morphology did not change.Insulin could promote BMSC growth and proliferation, and insulin at 15 mmol/L produced the best effect. When various concentrations of insulin were added to the medium for incubating BMSCs with knocked out NCoR, cell proliferation was inhibited. Thus, NCoR inhibited the function of insulin in cell proliferation, and PI3K signaling may play an important role in the relationship between NCoR and insulin. the present study demonstrated that the differentiation of rat MSCs to adipocytes is negatively regulated by NCoR through the influence on differential genes PPARy and C/EBPa activity. Thus, our results provided insight into the possible function of NCoR, suggesting that it might be physiologically important in the regulation of adipose accumulation and the maintenance of the mass.