HSF1-mediated BAG3 Expression Attenuates Apoptosis in 4-Hydroxynonenal-treated Colon Cancer Cells via Stabilization of Anti-apoptotic Bcl-2 Proteins

Abstract

4-Hydroxynonenal (HNE) is a pro-apoptotic electrophile generated during the spontaneous decomposition of oxidized lipids. We have previously shown that HNE activates the transcription factor, heat shock factor 1 (HSF1), and promotes cytoprotective heat shock gene expression and that silencing HSF1 sensitizes the colon cancer cell line RKO to HNE-induced apoptosis. Here we report a reduction in the anti-apoptotic proteins Bcl-X L , Mcl-1, and Bcl-2 in HSF1-silenced RKO cells, and we examine the underlying mechanism. To investigate the regulation of the Bcl-2 family by HSF1, microarray analysis of gene expression was performed. We observed that the Hsp70 co-chaperone, BAG3 (Bcl-2-associated athanogene domain 3), is strongly induced by HNE in control but not in HSF1-silenced colon cancer cells. Silencing BAG3 expression with small interfering RNA caused a dramatic reduction in Bcl-X L , Mcl-1, and Bcl-2 protein levels in colon cancer cells and increased apoptosis, similar to the effect of silencing HSF1. Also, immunoprecipitation experiments indicate specific interactions between BAG3, Hsp70, and the Bcl-2 family member, Bcl-X L . Overall, our data reveal that BAG3 is HSF1-inducible and has a unique role facilitating cancer cell survival during pro-apoptotic stress by stabilizing the level of Bcl-2 family proteins.The indiscriminate oxidation of biological membranes is increasingly recognized as a contributing factor in a variety of neurodegenerative and inflammatory diseases, including Alzheimer and Parkinson diseases, atherosclerosis, diabetes, alcoholic liver disease, and cancer (1-5). An established route for lipid oxidation involves hydrogen atom abstraction from unsaturated fatty acids by reactive oxidants, followed by reaction with oxygen to form lipid hydroperoxides (6). Various electrophiles, including the ␣,␤-unsaturated aldehyde 4-hydroxynonenal (HNE), 2 are generated from the nonenzymatic decomposition of lipid hydroperoxides (7). HNE is a diffusible lipid species and is capable of reacting with nucleophiles throughout the cell, such as glutathione, nucleic acids, and proteins (8).HNE promotes apoptosis in mammalian cells at low to mid-micromolar concentrations (9 -11). In response to HNE, cells also activate cytoprotective pathways that abrogate programmed cell death. Microarray analysis of gene expression highlights the variety of protective measures activated in response to HNE, including DNA damage, antioxidant, heat shock, and ER stress pathways (12). The heat shock response, mediated by the transcription factor HSF1, is strongly activated in HNE-treated cells. We previously reported that HNE elicits the nuclear translocation of HSF1 and promotes Hsp40 (DNAJB1) and Hsp72 (HspA1A) expression (13). Using siRNA to silence HSF1, we found that cells deficient in heat shock gene expression are dramatically sensitized to the pro-apoptotic effects of HNE. Investigating the mechanism of cell death in HSF1-silenced cells, we discovered that Bcl-X L protein levels are significantly reduced, thereby predisposi...

IKKγ protein is a target of BAG3 regulatory activity in human tumor growth

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