Education

Research Interest

The epithelial Tight Junctions form the gut barrier function preventing the diffusion of allergens, toxins and pathogens from the gut lumen into systemic circulation.  Disruption of tight junctions and resulting endotoxemia play crucial role not only in the pathogenesis of most gastrointestinal diseases (IBD, celiac disease, colon cancer, etc) but also in other systemic diseases such as diabetes, alcoholic liver disease, sclerosing cholangitis, etc.  The focus of studies in my laboratory is to understand the molecular organization of tight junctions and its regulation by protein phosphorylation-based cell signaling.  Some of the right junction proteins currently addressed in my laboratory are occludin, claudins, E-cadherin and b-catenin.  Signaling elements such as c-Src, PKC isoforms, PP2A and PTP1B are being studied in relation to phosphorylation of tight junction and adherens junction proteins on tyrosine, serine and threonine residues.  Our recent studies (Suzuki etal 2009; Elias etal 2009) have identified a unique regulatory motif in occludin C-terminal domain.  All cellular and molecular studies are conducted in close relation to understanding the pathogenesis IBD, colon cancer, alcoholic liver disease and cholangitis.

CURRENT TECHNIQUES UTILIZED

Electrophysiology
-Culture of epithelial cells on Transwell inserts and measuring transpithelial
electrical resistance.
    -Measuring macromolecular flux across epithelial monolayers.
Cell biology
    -Analysis of cell morphology by phase contrast microscopy
    -Confocal microscopy to localize specific proteins in epithelial cells.
    -Live cell imaging of fluorescent proteins.
    -FRET and FRAP analysis to analyze protein-protein interactions and protein dynamics in live cells.
Molecular biology
    -Site directed mutagenesis.
    -Construction of expression vectors, transfection and characterization.
    -Cloning transfected cells.
Biochemistry
    -Immunoprecipitation and immunoblot analyses.
    -Analysis of protein phosphorylation
    -Identification of phosphorylation sites by mass spectrometry and point mutations.
Animal studies
    -Breeding and maintenance of transgenic and knockout mice.
    -Ischemia reperfusion-induced tissue injury.
    -Chronic and acute ethanol administration

Research Support

NIH R01 DK055532 (PI: R. K. Rao) ?Intestinal Mucosal Protection by Epidermal Growth Factor? 1998-2013
NIH R01 AA12307 (PI: R. K. Rao) ?Mechanism of Endotoxin Absorption in Alcoholics,? 2000-2010

Selected Publications

1. Suzuki T, Bertha E and Rao RK. PKC is essential for the assembly and maintenance of epithelial tight junctions.  PNAS  In Press 2009.
2. Elias, BC, Suzuki T, Kale G, Giorgianni F, Desiderio DM, Le Shen, Naren AP, Turner JR, Rao RK. Phosphorylation of Y398 and Y402 in Occludin Prevents its Interaction with ZO-1 and Attenuates its Assembly into Tight Junctions (TJ) J. Biol. Chem.  In press 2009.
3. Suzuki T, Seth A, Rao RK.  Role of PLC-induced activation of PKC and PKCI in EGF-mediated protection of tight junctions from acetaldehyde in Caco-2 cell monolayers. J. Biol. Chem. 283:3574-3583, 2008.
4. Rao RK. The mechanisms of oxidative stress-induced disruption of epithelial and endothelial tight junctions.  Fron. Biosci. 13:7219-7226, 2008.
5. Seth A, Yan F, Polk DB, Rao RK. Probiotics Ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC and MAP kinase-dependent mechanism.  Am. J. Physiol. 294:G1016-G1069, 2008.
6. Seth A, Sheth P, Elias B, Rao RK.  PP2A and PP1 interact with occludin and negatively regulate the assembly of tight junctions in Caco-2 cell monolayer.  J. Biol. Chem.
7. Sheth P, Seth A, Atkinson KJ, Gheyi T, Kale G, Giorgianni F, Desiderio DM, Li C, Naren A, Rao RK.  Acetaldehyde dissociates PTP1B-E-Cadherin--Catenin complex in Caco-2 cell monolayer by phosphorylation-dependent mechanism.  Biochem. J. 402:291-300, 2007.
8. Sheth P, Delos Santos N, Seth A, LaRusso NF, Rao RK.  Lipopolysaccharide disrupts tight junctions of cholangiocyte monolayers by c-Src, TLR4 and LBP-dependent mechanism. Am. J. Physiol. 293:G308-318, 2007.
9. Basuroy S, Sheth P, Dandaswamy K, Balasubramanian, Ray RM, Rao RK: Expression of kinase-inactive c-Src delays oxidative stress-induced disassembly and calcium-mediated assembly of tight junction in Caco-2 cell monolayers.  J. Biol. Chem.  278: 11916-11924, 2003.
10. Sheth P, Basuroy S, Li C, Naren AP, Rao RK: Role of phosphatidylinositol 3-kinase in the oxidative stress-induced activation of c-Src and focal adhesion kinase in the cytoskeleton and the disruption of tight junctions in Caco-2 cell monolayer.  J. Biol. Chem.  278:11916-24, 2003.