Event Title

Tumor Necrosis Factor Alpha and Inflammation Disrupt the Atypical PKC Rescue Machinery in Intestinal Epithelial Cells

Start Date

10-2-2012 12:00 AM

Description

Objective. In the present work we tested the hypothesis that the cytoskeleton and Hsp70 dependent atypical PKC (aPKC) rescue machinery is sensitive to pro-inflammatory signals. Background. Loss of tight junction (TJ) competence is an important pathophysiological mechanism in inflammatory bowel disease (IBD). Increased TJ permeability facilitates the diffusion of small antigens and bacterial toxins, which in turn can exacerbate and perpetuate inflammatory process. aPKC is one of the key players in polarity organization and TJ assembly in epithelial cells. Recently, we reported that the interaction of aPKC with intermediate filament cytoskeleton and Hsp70 is crucial to prevent activity-induced aPKC degradation. Hsp70 proteins were previously shown to be downregulated under pro-inflammatory signaling. Bearing in mind that a PKC rescue mechanism is dependent on Hsp70, we hypothesized that active aPKC levels may decrease during inflammation, thus becoming an additionalmolecular mechanism for the disruption of epithelial function. Methods.We performed confocal microscopy, metabolic labeling, luciferase refolding and in vitro rephosphorylation assays to study the components of aPKC rescue protein complex in TNF-alpha treated CACO-2 (human colon carcinoma) epithelial cells. Results. We found that aPKC is downregulated by TNF-alpha signaling. Decrease in aPKC levels is mediated via inhibition of Hsp70 chaperoning activity, which subsequently results in failure of the aPKC rescue machinery. Conclusion. We show that aPKC stability is under control of pro-inflammatory signals. Moreover, this study indicates that some potentially important pathophysiological events in IBD may not be detectable by mRNA screens. Grants. Supported by NIH grant R01-DK087359 (to P.J.S.).

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Feb 10th, 12:00 AM

Tumor Necrosis Factor Alpha and Inflammation Disrupt the Atypical PKC Rescue Machinery in Intestinal Epithelial Cells

Objective. In the present work we tested the hypothesis that the cytoskeleton and Hsp70 dependent atypical PKC (aPKC) rescue machinery is sensitive to pro-inflammatory signals. Background. Loss of tight junction (TJ) competence is an important pathophysiological mechanism in inflammatory bowel disease (IBD). Increased TJ permeability facilitates the diffusion of small antigens and bacterial toxins, which in turn can exacerbate and perpetuate inflammatory process. aPKC is one of the key players in polarity organization and TJ assembly in epithelial cells. Recently, we reported that the interaction of aPKC with intermediate filament cytoskeleton and Hsp70 is crucial to prevent activity-induced aPKC degradation. Hsp70 proteins were previously shown to be downregulated under pro-inflammatory signaling. Bearing in mind that a PKC rescue mechanism is dependent on Hsp70, we hypothesized that active aPKC levels may decrease during inflammation, thus becoming an additionalmolecular mechanism for the disruption of epithelial function. Methods.We performed confocal microscopy, metabolic labeling, luciferase refolding and in vitro rephosphorylation assays to study the components of aPKC rescue protein complex in TNF-alpha treated CACO-2 (human colon carcinoma) epithelial cells. Results. We found that aPKC is downregulated by TNF-alpha signaling. Decrease in aPKC levels is mediated via inhibition of Hsp70 chaperoning activity, which subsequently results in failure of the aPKC rescue machinery. Conclusion. We show that aPKC stability is under control of pro-inflammatory signals. Moreover, this study indicates that some potentially important pathophysiological events in IBD may not be detectable by mRNA screens. Grants. Supported by NIH grant R01-DK087359 (to P.J.S.).