| Bone marrow mesenchymal stem cells?BMSCs? are a population of pluripotent cells within the bone marrow. BMSCs are able to differentiate toward adipocyte and myocyte, which affects the proportion of fat and muscle in the meat tissue of domestic animal. Therefore, studies of p BMSCs proliferation and adipogenesis have important implications for regulating fat deposition in different body parts, and improving meat quality. Ca2+ is a universal and impotant second messenger, which has been shown to play a pivotal role in controlling cell proliferation and adipogenic differentiation. However, the effects of extracelluar calcium([Ca2+]o) on the proliferation and adipogenic differentiation of porcine BMSCs?p BMSCs?, as well as the possible mechanisms underlying these process remain unclear. Firstly, we investigated the effects of [Ca2+]o on the expressions of plasma membrane calcium channel, calcium-sensing receptor?Ca SR? during the adipogenesis of p BMSCs. we applied the Real-time quantitative PCR to detected the m RNA expression patterns of calcium channel, voltage-dependent alpha-2/delta subunit 1?CACNA2D1?, calcium release activated calcium channel modulator1?Orai1?, transient receptor potential vanilloid receptor1?TRPV1?, Ca SR at different times of adipocyte differentiation in p BMSCs. Secondly, to investigated the role of [Ca2+]o in p BMSCs proliferation and the underlying mechanisms. We applied the methods of CCK-8, flow cytometer, Real-time quantitative PCR and Western blot to determined the effects of [Ca2+]o and/or nifedipine?an antagonist of VGCC?, NPS2143?an inhibitor of Ca SR?, U0126?an inhibitor of ERK kinase? on the proliferation of viable cells, cell cycle distribution, intracellular calcium([Ca2+]i) levels, as well as the expression of proliferative genes, Ca SR, and ERK. Finally, we explored the effects of [Ca2+]o on p BMSCs adipogenesis, glucose uptake and the involved signaling pathways. We firstly applied the Oil red staining and intracellular triglycerides?TG? assay to assess the effects of [Ca2+]o and/or nifedipine, NPS2143, BAPTA-AM(a chelator of [Ca2+]i), KN-93?an inhibitor of Ca MKII?, Wortmannin?an inhibitor of PI3K? on the lipid accumulation of p BMSCs after 10 days adipocyte differentiation. Meanwhile, the effects of [Ca2+]o and/or these inhibitors on the protein expressions of adipogenic genes?PPAR?, CEBP?, a P2?, Ca MKII, PI3K/Akt, Fox O1, AS160 were detected by Western blot. In addition, the role of [Ca2+]o and/or these inhibitors on GLUT4 expression and translocation, glucose consumption and uptake during the adipogenesis of p BMSCs were detected by the methods of Western blot, 2-NBDG?a fluorescent glucose analogue? uptake assay kit, and glucose consumption assay kit.The results were showed as follows:1. The m RNA expression of CACNA2D1, Orai1, TRPV1, Ca SR were obviously enhanced by 4 m M [Ca2+]o after p BMSCs adipogenesis for 5 days, suggesting that [Ca2+]o and the related calcium channels, Ca SR may contribute to the adipogenesis of p BMSCs.2. [Ca2+]o significantly stimulated p BMSCs proliferation in a dose-dependent manner, and promoted Ca SR m RNA and protein expression during the p BMSCs proliferation. Meanwhile, 4 m M [Ca2+]o was able to increase [Ca2+]i levels and to promote cell cycle progression?increased S phase and inhibited G0/G1 phase?. In addition, 4 m M [Ca2+]o elevated pro-proliferative genes?cyclin A2/D1/D3/E2, PCNA? and inhibited p21 m RNA and/or protein expression, as well as enhenced the phosphorylation of ERK. Moreover, the inhibition of Ca SR or ERK resulted in the elimination of the above-mentioned effects of [Ca2+]o. However, the inhibition of VGCC have no effect on [Ca2+]o-sitimulated p BMSCs proliferation. These results suggested that the enhanced proliferation of p BMSCs induced by extracellular calcium is associated with the activation of Ca SR and ERK signaling pathway.3. [Ca2+]o?4 m M? significantly elevated intracellular TG contents and [Ca2+]i levels, enhenced the protein expression of PPAR?, C/EBP?, a P2 during the adipogenesis of p BMSCs. While the inhibition of VGCC or Ca SR reversed [Ca2+]o-stimulated [Ca2+]i levels in p BMSCs. In addition, 4 m M [Ca2+]o significantly improved the phosphorylation of Ca MKII, PI3K/Akt, Fox O1 during the adipogenesis of p BMSCs. However, the activation of above-mentioned signaling pathways induced by [Ca2+]o were reversed by nifedipine?1 ?M?, NPS2143?0.1 ?M?, BAPTA-AM?0.1 ?M?, KN-93?1 ?M?, Wortmannin?50 n M?. These results indicated that increase of [Ca2+]i levels through VGCC and Ca SR, and the activation of Ca MKII-PI3K/Akt-Fox O1 signalling pathways were contributed to [Ca2+]o-sitimulated p BMSCs adipogenesis.4. [Ca2+]o?4 m M? significantly promoted GLUT4 expression and translocation, glucose consumption and uptake. Meanwhile, [Ca2+]o markedly increased the phosphorylation of Ca MKII and PI3K/Akt, AS160 during the adipogenesis of p BMSCs, which were reversed by nifedipine, BAPTA-AM, KN-93, Wortmannin. These results suggested that [Ca2+]o-sitimulated GLUT4 translocation, and glucose uptake during the adipogenesis of p BMSCs were resulted from the increase of [Ca2+]i levels via VGCC, and the activiation of Ca MKII-PI3K/Akt-AS160 signalling pathways.In conclusion, our data revealed that [Ca2+]o promoted the expression of plasma membrane calcium channels and Ca SR during the adipogenesis of p BMSCs. In addition, the enhanced proliferation of p BMSCs induced by extracellular calcium was associated with the activation of Ca SR and ERK signaling pathway. Furthermore, the stimulation of adipogenesis and glucose uptake by extracellular Ca2+ is associated with increase of [Ca2+]i levels through VGCC or Ca SR activition, activation of Ca MKII and enhancement of insulin-mediated PI3K/Akt-Fox O1/AS160 signaling pathways in p BMSCs. The data revealed the effects and mechanisms of [Ca2+]o on p BMSCs proliferation and adipogenic differentiation and provided the scientific basis for the regulation of fat deposition in animal body and the improvement of meat quality. |
PDF Full Text Request