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.
It has been suggested that free fatty acids (FFA) such as palmitate, which are secreted from enlarged adipocytes in the subcutaneous fat of obese subjects, serve as a link between obesity and altered skin functions. Cyclooxygenease-2 (COX-2) and prostanoids participate in the induction of impaired dermal function. In the current study, we investigated the issue of whether palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB or mitogen-activated protein kinase (MAPK) pathways in human dermal fibroblasts. Palmitate treatment significantly induced COX-2 expression and prostaglandin E2 (PGE2) release in human dermal fibroblasts. In addition, pre-treatment with triacsin C, an inhibitor of acyl-CoA synthetase in de novo ceramide synthesis, was found to reduce palmitate-induced COX-2 expression and PGE2 release in human dermal fibroblast. The findings also show that palmitate-induced COX-2 expression and PGE2 release are mediated by the NF-κB, p38, and extracellular signal-regulated kinase (ERK) MAPK pathways. These findings point to a new mechanism for explaining the link between increased FFAs in obesity and impaired dermal function.
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