BAG-1 DIFFERENTIALLY REGULATES INTERMEDIATE FILAMENT-BASED HSP70 CHAPERONING OFAPKC IN INTESTINAL CELLS UNDER PRO-INFLAMMATORY SIGNALING

Objective. Our goal was to identify the molecule(s) that block activity of Hsp70 chaperone in intestinal epithelium during inflammation. Background. Atypical protein kinase C (aPKC) plays an essential role in the establishment of epithelial polarity. aPKC acquires inactive conformation after normal activity and can be rescued from ubiquitinylation and degradation by Hsp-70- and intermediate filament-dependent mechanism. We have shown previously that aPKC was strongly downregulated by TNF-alpha –mediated signaling in intestinal epithelial cells and also in vivo during intestinal inflammation. Furthermore we have demonstrated that decrease of aPKC levels under pro-inflammatory conditions was mediated through inhibition of Hsp70 chaperoning activity, resulting in failure of the aPKC rescue machinery. Methods. We conducted a transcriptome PCR screen detecting chaperones and co-chaperones and compared mRNAs from control and TNF-alpha treated Caco-2 cells (human colon carcinoma). Subsequent validation experiments allowed us to focus on BAG-1, a multifunctional protein that assists Hsp70 in nucleotide exchange but also blocks its activity at higher concentrations. Results. We found that BAG-1 isoform BAG-1M was upregulated up to 3 fold in Caco-2 cells following stimulation with TNF-alpha. In addition, BAG-1M levels increased up to 6 fold in mouse enterocytes after treatment with dextran sodium sulfate (DSS) to induce colitis. Overexpression of BAG-1M in Caco-2 cells decreased levels of phosphorylated aPKCs, similar to TNF-alpha stimulation. In contrast, knockdown of BAG-1 abolished the TNF-alpha- induced decrease of phosphorylated aPKC. Conclusion. We conclude that BAG-1M mediates inhibition of Hsp70 chaperoning activity during epithelial inflammatory response. Grants. The Nova Southeastern University Health Professions Research Grant to Dr. Mashukova.