Foxc2 Promotes Thermogenesis Of Adipocytes Via Inhibiting Endoplasmic Reticulum Stress And Autophagy

Forkhead box C2(Foxc2) gene is a member of the forkhead/winged helix transcription factor family and is able to express in many tissues. It is vital for individual development and lipid metabolism. But, whether Foxc2 affects thermogenesis of adipocytes via ER stress and autophagy is still unknown.In this experiment, to clear whether Foxc2 affects on thermogenesis of adipocytes via ER stress and autophagy or not, we forced expression of Foxc2 and knock-down Foxc2 gene in 3T3-L1 adipocytes. We treat adipocytes with tunicamycin to establish the ER stress models.Tunicamycin and Earle’s Balanced Salt Solution(EBSS)are used to establish autophagy models.we detect the change of ER stress and autophagy related signaling pathway.To further verify the influence of Foxc2 on thermogenesis of adipocytes via ER stress and autophagy,4-BPA(the inhibitors of ER stress) and 3-MA(the inhibitors of autophagy) are used in this experiment. Results are as follows:1. Foxc2 alleviated ER stress induced by tunicamycin in adipocytes.Establishing ER stress model induced by tunicamycin in adipocytes, the markers of ER stress Chop, Grp78 were elevated significantly over time( p<0.05), overexpression of Foxc2 significantly decreased the expression of ER stress factors, interference of Foxc2 significantly increased the expression of ER stress factors(p<0.05); overexpression of Foxc2 significantly decreased the phosphorylation level of ERK1/2, interference of Foxc2 significantly increased the phosphorylation level of ERK1/2(p<0.05); after that we used U0126 to inhibit ERK1/2signaling pathway, the results showed that overexpression of Foxc2 also significantly decreased phosphorylation level of ERK1/2; The results are opposite, when in Foxc2 knockdown group( p<0.05). It indicated that Foxc2 alleviated ER stress induced by tunicamycin in adipocytes though ERK1/2 signalling pathway.2. Foxc2 inhibited autophagy induced by ER stress in adipocytes. Establishing autophagy model induced by ER stress with tunicamycin in adipocytes. The markers of autophagy Atg7, Beclin1 and the proportion of LC3II/LC3 I were significantly elevated over time( p<0.05), and the cargo marker of autophagy p62 is significantly decreased(p<0.05).When treating cells with tunicamycin for 8h, the markers of ER stress were alsoelevated significantly( p<0.05), MDC staining showed that the numbers of autophagic vacuole were significantly increased(p<0.05); After transfection with three types of carriers and induction of autophagy with tunicamycin in adipocytes, the results showed that overexpressing Foxc2 supressed the markers of ER stress Chop, Grp78(p<0.05), it also inhibited the markers of autophagy Atg7、Beclin1 as well as the proportion of LC3II/LC3 I,the results were opposite in interference of Foxc2 group(p<0.05); overexpression of Foxc2 dramaticlly decreased the phosphorylation of AMPK and increased the phosphorylation of m TORC1,the results were opposite in interference of Foxc2 group(p<0.05). Then we used compound C to inhibit AMPK and rapamynin to inhibit mTORC1 signaling pathways respectively at indicated time, it turned out that overexpression of Foxc2 also dramaticlly lowered the phosphorylation of AMPK and elevated the phosphorylation of mTORC1,The results were opposite in interference of Foxc2 group(p<0.05); These datas showed that Foxc2 inhibited autophagy induced by tunicamycin though AMPK and mTORC1 signaling pathways.3. Foxc2 inhibited autophagy induced by EBSS in adipocytes. To further prove the effect of Foxc2 on autophagy induced by different inducers, we used EBSS to induce autophagy,when overexpressing Foxc2, the expression of markers of autophagy Atg7, Beclin1 and the proportion of LC3II/LC3 I were significantly lowered(p<0.05),and the marker cargo of autophagy P62 was elevated(p<0.05), the result were opposite in interference of Foxc2 group.At the same time, the MDC staining showed that overexpression of Foxc2 inhibited the product of autophagic vacuoles induced with EBSS( p<0.01). To further explain the influence of Foxc2 on autophagy, we detected the change of phosphorylation level of AMPK and mTORC1 respectively, the resulted showed that whatever overexpression or interference of Foxc2, the phosphorylation level of AMPK was not varied, but the phosphorylation of m TORC1 was increased noteworhily(p<0.05)in overexpression Foxc2 group, the result were opposite in interference of Foxc2 group(p<0.05); Then we used compound C to inhibit AMPK and rapamynin to inhibit mTORC1 signaling pathways respectively, it proved that the phosphorylation level of AMPK was also not varied in overexpression or interference of Foxc2 group( p>0.05). the phosphorylation of m TORC1 was significantly increased in overexpressing Foxc2 group, the phosphorylation of m TORC1 was significantly decreased in interferencing Foxc2(p<0.05); Foxc2 inhibited autophagy induced by EBSS in adipocytes via mTORC1 signaling pathway.4. Foxc2 promoted thermogenesis of adipocytes via inhibiting ER stress and autophagy.the mRNA expression of Foxc2 and thermogenic genes in adipocytes were inhibited when ER stress or Autophagy happened, the results were opposite when ER stress or autophagy wasinhibited( p<0.05 or p<0.01); Foxc2 can further promoted the protein and mRNA expression of thermogenic genes though partly inhibition of ER sress or autophagy in adipocytes(p<0.05 or p<0.01).In conclusion, in this study, after using 4-PBA to inhibit ER stress, Foxc2 could further inhibit ER stress. Foxc2 inhibits ER stress in adipocytes via ERK1/2 signaling pathway. In other experiments, Foxc2 inhibits autophagy induced by tunicamycin in adipocytes via AMPK and m TORC1 signaling pathway, in autophagy model induced by EBSS, Foxc2 inhibited autophagy induced by EBSS via mTORC1 signaling pathway.besides, and we also explored the effect of Foxc2 on the expression of thermogenic genes in adipocytes via mediated ER stress or autophagy.