Opening of tight junctions is involved in the pathogenesis of inflammatory bowel disease (IBD). Many studies have focused on the role of Tyr-kinases in the removal of tight junction components. However, no one has yet studied the role of atypical Protein Kinase C (aPKC), the evolutionarily conserved organizer of tight junctions, in inflammation. Our preliminary data shows a steep decrease of aPKC in intestinal epithelia exposed to pro- inflammatory signals. We have also shown that this effect is post-trancriptional and related to an Hsp-70- intermediate filament- dependent mechansim of rescue of aPKC. We hypothesize that post-translational mechanisms that recruit and maintain atypical PKC at the apical domain are impaired by inflammatory signals, resulting in increased tight junction permeability in the intestinal epithelium. We propose to: (1) Determine the molecular mechanisms by which Hsp70 chaperones rescue misfolded aPKC otherwise targeted for degradation. A combination of tissue culture of human intestinal cells, subcellular fractionation, and an in vitro reconstitution assay developed in our laboratory will be used to analyze the components of the aPKC rescue mechanisms. (2) Characterize molecular mechanisms that can antagonize loss of aPKC active conformation or degradation. Specific knock-down of Thr/Ser phosphatase catalytic subunits and expression of keratin intermediate filaments in human cells that normally lack them will be used to assess possible mechanisms to protect aPKC from losing its active conformation or to enhance the rescue mechanism. These observations will be validated in transgenic mice overexpressing keratin intermediate filaments. (3) Identify pro-inflammatory signals that downregulate aPKC and their targets in the degradation and rescue machinery that maintains aPKC physiologic levels. We will screen for pro-inflammatory cytokines that decrease aPKC levels, aside of TNF-, and analyze the step of the activation and degradation pathways of aPKC that are affected by pro-inflammatory signaling. Again, the results will be validated in an animal model

Public Health Relevance

IBD is a chronic invalidating disease that affects around 3 million mericans. Increased permeability across the epithelial intestinal barrier is not the cause, but it is widely accepted as an important factor to perpetuate the inflammation. The studies in this project are intended to understand the molecular basis of a novel, as yet non studied mechanism to organize and maintain the "tightness" (tight junction competence) of the epithelial barrier.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK087359-04
Application #
8400421
Study Section
Special Emphasis Panel (ZRG1-DKUS-G (08))
Program Officer
Grey, Michael J
Project Start
2010-04-01
Project End
2013-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
4
Fiscal Year
2013
Total Cost
$303,300
Indirect Cost
$105,065
Name
University of Miami School of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
Kravtsov, Dmitri; Mashukova, Anastasia; Forteza, Radia et al. (2014) Myosin 5b loss of function leads to defects in polarized signaling: implication for microvillus inclusion disease pathogenesis and treatment. Am J Physiol Gastrointest Liver Physiol 307:G992-G1001
Mashukova, Anastasia; Forteza, Radia; Wald, Flavia A et al. (2012) PDK1 in apical signaling endosomes participates in the rescue of the polarity complex atypical PKC by intermediate filaments in intestinal epithelia. Mol Biol Cell 23:1664-74
Mashukova, Anastasia; Wald, Flavia A; Salas, Pedro J (2011) Tumor necrosis factor alpha and inflammation disrupt the polarity complex in intestinal epithelial cells by a posttranslational mechanism. Mol Cell Biol 31:756-65