A novel mechanism for H₂O₂-induced autophagic cell death in GSH-depleted RAW 264.7 cells, a murine macrophage cell line, is proposed. Under GSH-depleted conditions, H₂O₂-induced autophagic cell, characterized by an increased LC3-II/I ratio, a decreased level of p62 and the formation of autophagic vacuoles, was inhibited by bafilomycin A1 and by Atg5 siRNA transfection, whereas the cell death was not inhibited by zVAD-fmk, by PI3K inhibitors or by Beclin 1 siRNA transfection. In addition, H₂O₂ treatment reduced the activity of mTOR and promoted the ubiquitination and degradation of Rheb, a key upstream activator of mTOR. Furthermore, proteasome inhibition with MG132 restored the expression of Rheb and increased mTOR activity, resulting in an increased viability of H₂O₂-treated cells. Collectively, these findings demonstrate that H₂O₂ induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells.
a b s t r a c tDifferentiation of preadipocytes into adipocytes is controlled by various transcription factors. Recently, the pro-adipogenic function of XBP1, a transcription factor upregulated by endoplasmic reticulum stress, has been reported. In this study, we demonstrated that XBP1 suppresses the expression of Wnt10b, an anti-adipogenic Wnt, during the differentiation of 3T3-L1 preadipocytes. The expression pattern of XBP1 was reciprocal to that of Wnt10b during the early stage of adipogenesis. The intracellular protein levels of b-catenin were negatively regulated by XBP1. Direct binding of XBP1 to the Wnt10b promoter and the subsequent decrease of the b-catenin signalling pathway represent a novel adipogenic differentiation mechanism.
X‐box binding protein 1 (XBP1), a transcription factor of the unfolded protein response, plays various roles in many biological processes. We examined its pro‐adipogenic activity and target genes during adipogenic differentiation in wild‐type and genetically modified 3T3‐L1 cells. Signalling pathways that contribute to Xbp1 mRNA splicing, and the correlation of the transcriptionally active XBP1 isoform (XBP1s) level with body mass index and the level of peroxisome proliferator‐activated receptor γ2 (PPARγ2) in human adipose tissues were also examined. The mRNA and nuclear protein expression levels of XBP1s increased immediately following hormonal induction of adipogenesis, reaching a peak at 6 h. Results from cDNA microarray and gene expression analyses using genetically modified cells indicated that PPARγ2 was a principal target of XBP1s. The XBP1s‐specific binding motif, which is distinct from the CCAAT/enhancer‐binding protein α binding site, was identified in the PPARγ2 promoter by site‐directed mutagenesis. Fetal bovine serum, insulin, 3‐isobutyl‐1‐methylxanthine and dexamethasone contributed independently to Xbp1 mRNA splicing. In human subcutaneous adipose tissues, the levels of both Xbp1s and Pparγ2 mRNA increased proportionally with body mass index, and there was a significant positive correlation between the two genes. These data suggest for the first time that positive regulation of PPARγ2 is a principal mechanism of XBP1s‐mediated adipogenesis in 3T3‐L1 cells.
Wnt10b, an endogenous inhibitor of adipogenesis, maintains preadipocytes in an undifferentiated state by suppressing adipogenic transcription factors. We have previously demonstrated that Wnt10b transcription during adipogenesis is negatively regulated by X-box-binding protein 1 (XBP1), an important transcription factor of the unfolded protein response. In this report, we demonstrate that XBP1s can directly induce the transcription of microRNA-148a, which in turn mediates the silencing of Wnt10b mRNA during adipogenic differentiation of 3T3-L1 cells. Stability of Wnt10b mRNA was found to be significantly increased by knockdown of XBP1s. Using computational algorithms, a set of microRNAs was predicted to bind Wnt10b mRNA, of which microRNA-148a was selected as a potential target for XBP1s. Our results revealed that microRNA-148a could bind to the 3′UTR of Wnt10b mRNA. Its ectopic expression significantly suppressed both Wnt10b expression and β-catenin activity. When we altered the expression of XBP1 in 3T3-L1 cells, microRNA-148a levels changed accordingly. A potential XBP1 response element was found in the promoter region of microRNA-148a, and XBP1s directly bound to this response element as shown by point mutation analysis and chromatin immunoprecipitation assay. In addition, a microRNA-148a mimic significantly restored adipogenic potential in XBP1-deficient 3T3-L1 cells. These findings provide the first evidence that XBP1s can regulate Wnt10b by a post-transcriptional mechanism through directly inducing microRNA-148a.
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