Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubuleassociated protein 1 light chain 3 b) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvationinduced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/ threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.
Autophagy, an intracellular degradation process, is essential for maintaining cell homeostasis by removing damaged organelles and proteins under various conditions of stress. In cancer, autophagy has conflicting functions. It plays a key role in protecting against cancerous transformation by maintaining genomic stability against genotoxic components, leading to cancerous transformation. It can also promote cancer cell survival by supplying minimal amounts of nutrients during cancer progression. However, the molecular mechanisms underlying how autophagy regulates the epithelial-to-mesenchymal transition (EMT) and cancer metastasis are unknown. Here, we show that starvation-induced autophagy promotes Snail (SNAI1) degradation and inhibits EMT and metastasis in cancer cells. Interestingly, SNAI1 proteins were physically associated and colocalized with LC3 and SQSTM1 in cancer cells. We also found a significant decrease in the levels of EMT and metastatic proteins under starvation conditions. Furthermore, ATG7 knockdown inhibited autophagy-induced SNAI1 degradation in the cytoplasm, which was associated with a decrease in SNAI1 nuclear translocation. Moreover, cancer cell invasion and migration were significantly inhibited by starvation-induced autophagy. These findings suggest that autophagy-dependent SNAI1 degradation could specifically regulate EMT and cancer metastasis during tumorigenesis.
Antioxidant enzymes are decreased in osteoarthritis (OA) patients, implying the role of oxidative stress in osteoarthritis pathogenesis. The aim of this study was to evaluate the cytoprotective effects of delphinidin, a potent antioxidant, in human chondrocytes and the underlying mechanisms. The cytoprotective mechanism induced by delphinidin against oxidative stress (H2O2) in human chondrocytes was investigated. Cell viability and death were evaluated using proapoptotic and antiapoptotic markers such as cleaved caspase-3 (c-caspase-3), cleaved poly(ADP-ribose) polymerase N-acetylcysteine (c-PARP), Bcl-XL, and transcription factors associated with redox and inflammation regulation, including nuclear factor kappa B (NF-κB) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Induction of autophagy was assessed by formation of LC3-II and autophagosome-(LC3 punctate, monodansylcadaverine (MDC) and acridine orange staining) in the presence or absence of an autophagy inhibitor. Treatment with delphinidin itself at concentration below 50 µM for 24 h did not affect viability of chondrocytes. Delphinidin inhibited reactive oxygen species (ROS)-induced apoptosis by significantly decreasing apoptosis markers such as c-caspase-3 and c-PARP while increasing antiapoptotic marker Bcl-XL and antioxidant response NF-κB and Nrf2 pathways. Delphinidin also activated cytoprotective autophagy to protect chondrocytes during oxidative stresses. Activation of autophagy with autophagy inducer rapamycin also inhibited ROS-induced cell death and decreased proapoptotic proteins but increased antiapoptotic protein Bcl-XL, NF-κB, and Nrf2. Delphinidin can protect chondrocytes against H2O2-induced apoptosis via activation of Nrf2 and NF-κB and protective autophagy. Thus, it can inhibit OA with protection of chondrocytes. Delphinidin can protect chondrocytes against H2O2-induced ROS with maintenance of homeostasis and redox. These results suggest that delphinidin could be used to protect chondrocytes against age-related oxidative stress and other oxidative stresses in the treatment of OA. Thus, delphinidin may play a critical role in preventing the development and progression of OA.
Raf kinase inhibitor protein (RKIP) plays a critical role in many signaling pathways as a multi-functional adapter protein. In particular, the loss of RKIP’s function in certain types of cancer cells results in epithelial to mesenchymal transition (EMT) and the promotion of cancer metastasis. In addition, RKIP inhibits autophagy by modulating LC3-lipidation and mTORC1. How the RKIP-dependent inhibition of autophagy is linked to EMT and cancer progression is still under investigation. In this study, we investigated the ways by which RKIP interacts with key gene products in EMT and autophagy during the progression of prostate cancer. We first identified the gene products of interest using the corresponding gene ontology terms. The weighted-gene co-expression network analysis (WGCNA) was applied on a gene expression dataset from three groups of prostate tissues; benign prostate hyperplasia, primary and metastatic cancer. We found two modules of highly co-expressed genes, which were preserved in other independent datasets of prostate cancer tissues. RKIP showed potentially novel interactions with one EMT and seven autophagy gene products (TGFBR1; PIK3C3, PIK3CB, TBC1D25, TBC1D5, TOLLIP, WDR45 and WIPI1). In addition, we identified several upstream transcription modulators that could regulate the expression of these gene products. Finally, we verified some RKIP novel interactions by co-localization using the confocal microscopy analysis in a prostate cancer cell line. To summarize, RKIP interacts with EMT and autophagy as part of the same functional unit in developing prostate cancer.
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