Abstract TP402: The Cerebral Cavernous Malformation Disease Causing Gene Krit1 Participates in Epithelial Barrier Maintenance and Regulation

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Abstract

Background: Cerebral cavernous malformation (CCM) patients develop mulberry-like microvessels in the brain. These lesions can bleed, causing clinical symptoms. Germline mutations of CCM genes have been identified in familial form of CCM, and decreased endothelial CCM protein expression leads to RhoA-Rho kinase (ROCK) dependent actin cytoskeleton reorganization and increased endothelial permeability. It is not known if CCM mutations, which occur in all cells of a familial CCM patient, can impact barrier function of other body cavity lining cells, such as epithelial cells. Understanding how CCM protein function in epithelia may help us identify risks of CCM patients to epithelium-related diseases.

Aim: To determine if and how the CCM gene KRIT1/CCM1 affects epithelial barrier maintenance and regulation.

Methods: KRIT1 knockdown (KD) intestinal epithelial Caco-2 cells were generated by stably transfecting an anti-KRIT1 siRNA expressing plasmid. Epithelial barrier function, measured as transepithelial resistance (TER) and size selective cation permeability was determined by current clamp. Protein expression and localization was determined by western blot and immunofluorescent microscopy, respectively.

Results: KRIT1 KD intestinal epithelial Caco-2 cell monolayers have decreased TER and selectively increased permeability to small cations, along with decreased claudin-1expression. ROCK inhibition (Y-27632) and myosin motor inhibition (blebbistatin) both decreased TER in control Caco-2 monolayers, but not in KRIT1 KD monolayers, indicating KRIT1 is important for myosin regulation of epithelial barrier function. Tumor necrosis factor (TNF), a cytokine that decreases epithelial barrier function through inducing myosin contraction, caused more severe barrier loss in KRIT1 KD monolayers relative to control monolayers, which is blocked by pan-caspase inhibition (zVAD), indicating KRIT1 loss promotes TNF-induced barrier loss through inducing cell death.

Conclusion: KRIT1 regulates epithelial barrier by affecting tight junction protein expression and impacting myosin- and apoptosis- dependent processes. These changes in epithelial function may increase familial CCM patients’ susceptibility to intestinal and other diseases.

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