The long-term goal of this proposal is to understand the mechanism of. Our present understanding of the pathogenesis of cholestasis is based on studies to define the physiological regulation of transporters involved in bile formation and their deregulation in cholestasis. During the last granting period we have defined the role of aPKC , Rab4 and phosphorylation in Ntcp translocation and started defining the role of nPKC , nPKC and p38 MAPK in Mrp2 translocation. The present proposal extends these studies to further define the role of nPKC and nPKC in Ntcp/NTCP and Mrp2/MRP2 translocation/retrieval. One of the key goals is to define the mechanism involved in opposing effects of these kinases in hepatocytes. The following hypotheses are proposed: 1) nPKC -pThr505 mediates translocation of Ntcp/NTCP by cAMP and translocation of Mrp2/MRP2 by cAMP and TUDC to the plasma membrane, 2) nPKC mediates TLC-induced retrieval of Mrp2/MRP2 from the plasma membrane by phosphorylating MARCKS and/or Mrp2/MRP2, and cAMP and TUDC reverse this effect by inhibiting TLC-induced nPKC activation. Proposed studies will be conducted in perfused livers and hepatocytes from rat livers, primary human hepatocytes and hepatic cell lines. In addition, studies will be conducted in perfused livers and hepatocytes isolated from nPKC -/- and nPKC -/- mice to further confirm the role of these kinases. Role of these kinases will be evaluated by manipulating their activity and expression using chemical inhibitors, wild type-, constitutively active- and dominant negative-plasmids and Si- and/or ShRNA. Immunofluorescence and co-immunoprecipitation studies will be used to determine colocalization of desired proteins in subcellular organelles and with other proteins. Collectively, proposed studies should further define the role of nPKC in Ntcp/NTCP and Mrp2/MRP2 translocation, the role of nPKC in Mrp2/MRP2 retrieval, and mechanism of reversal of TLC-induced cholestasis by cAMP and TUDC. Since a kinase may produce beneficial or toxic effect in an isoform specific manner, a better understanding of isoform specific effects should allow for better therapeutic agents that could avoid potential liver toxicity.

Public Health Relevance

The long-term goal of this proposal is to understand the mechanism of canalicular bile formation and cholestasis. The goal of the current proposal is to further define the role of intracellular signaling mechanisms involved in vectorial transport of solutes from blood to bile under physiological and pathological (cholestasis) conditions. More specifically, this proposal will attempt to define the role of protein kinase C isoforms in bile formation and cholestasis. By defining the isoforms that are involved in the cholestatic effect, we should be able to design therapeutic agents that selectively reverse cholestatic effect without affecting beneficial effects.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK090010-04
Application #
8489290
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Sherker, Averell H
Project Start
2010-09-30
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$462,279
Indirect Cost
$182,110
Name
Tufts University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
Anwer, M Sawkat; Stieger, Bruno (2014) Sodium-dependent bile salt transporters of the SLC10A transporter family: more than solute transporters. Pflugers Arch 466:77-89
Anwer, M Sawkat (2014) Role of protein kinase C isoforms in bile formation and cholestasis. Hepatology 60:1090-7
Park, Se Won; Schonhoff, Christopher M; Webster, Cynthia R L et al. (2014) Rab11, but not Rab4, facilitates cyclic AMP- and tauroursodeoxycholate-induced MRP2 translocation to the plasma membrane. Am J Physiol Gastrointest Liver Physiol 307:G863-70
Schonhoff, Christopher M; Webster, Cynthia R L; Anwer, M Sawkat (2013) Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase C? in HUH-NTCP Cells. Hepatology 58:284-92
Ramasamy, Umadevi; Anwer, M Sawkat; Schonhoff, Christopher M (2013) Cysteine 96 of Ntcp is responsible for NO-mediated inhibition of taurocholate uptake. Am J Physiol Gastrointest Liver Physiol 305:G513-9