Progress 09/01/99 to 06/30/04
Outputs
The long-term goal of this proposal is to understand the mechanism of canalicular bile formation and cholestasis. 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. Cyclic AMP stimulates bile formation by translocating solute transporters to the plasma membrane and reverses acute cholestasis associated transporter dislocation. The goal of this project was to define the signaling pathway involved in short-term regulation of sinusoidal Na+/TC cotransport. More specifically, we proposed to define the role of the PI3K signaling pathway in cAMP-mediated translocation of Ntcp. In addition, we proposed studied to define the role of protein phosphatases 2A and 2B in cAMP-induced Ntcp dephosphorylation and translocation. Studies were conducted in isolated rat hepatocytes and in HuH7 cells stably transfected with Ntcp. The role of
a signaling pathway was investigated using specific inhibitors of key enzymes in hepatocytes, and using wildtype or dominant negative form of key enzymes in HuH7 cells. Conclusions from these studies are as follows: 1. Cyclic AMP mediates Ntcp translocation by activating the PI3K/PKB and PI3K/PKC-zeta signaling pathway. 2. There is a cross talk between PKB and PKC-zeta involving a requirement for PKC-zeta in the activation of PKB. 3. Cyclic AMP-mediated dephosphorylation of Ntcp is dependent on its ability to increase cytosolic [Ca 2+]. Cyclic AMP, by increasing cytosolic [Ca2+], activates PP2B, which in turn dephosphorylates Ntcp. Although the effect of cAMP is not directly mediated via PP2A, PP2A is necessary for cAMP to increase cytosolic [Ca2+] and hence to activate PP2B. 4. Cyclic AMP-induced Ntcp translocation is dependent on intact microfilaments. 5. Cyclic AMP activates p38 MAPK, but cAMP-stimulated TC uptake is not mediated via p38 MAPK. 6. Cell swelling does not activate
ERK1/2 MAPK in hepatocytes.
Impacts
Functional abnormalities associated with various liver diseases lead to the accumulation of toxic products (such as bilirubin in jaundice and bile acids) in blood and premature death of liver cells. One of the major determinants is the inability of the liver to properly regulate bile acid transport from blood to bile. Studies are conducted to better understand the mechanisms regulating bile acid transport. The overall goal is to determine regulatory pathways that are derailed and then find ways to reestablish normal regulation. These studies should provide a basis for therapeutic interventions needed to ameliorate secondary problems associated with various chronic and acute liver diseases.
Publications
- Mukhopadhdayay, S., Ananthanarayanan, M., Stieger, B., Meier, P.J., Suchy, F.J., Anwer, M.S.: Sodium taurocholate cotransporting polypeptide is a serine, threonine phosphoprotein and is dephosphorylated by cyclic AMP in rat hepatocytes. Hepatology 28:1629-1636, 1998.
- Mukhopadhdayay, S., Webster, C.R.L., Anwer, M.S.: Role of protein phosphatases in cyclic AMP-mediated stimulation of hepatic Na+/taurocholate cotransport. J. Biol. Chem. 273:30039-30045, 1998.
- Wesbter, C.R.L., Anwer, M.S.: Cyclic AMP mediated protection against bile acid induced apoptosis in cultured rat hepatocytes. Hepatology 27:1324-1331, 1998.
- Webster, C.R.L. Anwer, M.S.: Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp. Am. J. Physiol. 277:G1165-G1172, 1999.
- Webster, C.R.L., Blanch, C.J., Phillips, J., Anwer, M.S.: Cell swelling-induced translocation of rat liver Ntcp is mediated via the PI3K signaling pathway. J. Biol. Chem. 275:29754-29760, 2000.
- Webster, C.R.L., Anwer, M.S.: Phosphoinositide-3-kinase, but not mitogen-activated protein kinase, pathway is involved in hepatocyte growth factor-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes. Hepatology 33:608-615, 2001.
- Webster, C.R.L., Blanch, C., Anwer, M.S.: Role of protein phosphatase 2B in cyclic AMP induced dephosphorylation and translocation of Ntcp. Am. J. Physiol. 283:G44-G50, 2002.
- Webster, C.R.L., Srinivasulu, U., Ananthanarayanan, M., Suchy, F.J., Anwer, M.S.: Protein kinase B/Akt mediates cAMP- and cell swelling-stimulated Na+/taurocholate cotransport and Ntcp translocation. J. Biol. Chem. 277:28578-28583, 2002.
- Webster, C.R.L., Usechak, P., Anwer, M.S.: Cyclic AMP inhibits bile acid induced apoptosis by blocking caspase activation and cytochrome C release. Am. J. Physiol. 283:G727-G736, 2002.
- Webster, C.R.L., Boria, P., Usechak, P., Anwer, M.S.: S-Adenosylmethionine and cAMP confer differential cytoprotection against bile acid-induced apoptosis in canine renal tubular cells and primary rat hepatocytes. Vet. Therap. 3:474-484, 2002.
- McConkey, M., Gillin, H. Webster, C.R.L., Anwer, M.S.: Role of PKC-zeat in cAMP mediated TC uptake and Ntcp translocation in hepatocytes. J. Biol. Chem. 279: 20882-20888, 2004.
- Cullen, K., McCool, J., Anwer, M.S., Webster, C.R.L.: Activation of cAMP-guanine exchange factor (cAMP-GEF) confers protein kinase A independent protection from hepatocyte apoptosis. Am. J. Physiol. 287: G334-G343, 2004.
- Anwer, M. S., Webster, C.R.L.: Signal transduction in bile formation and cholestasis. In "Molecular Pathogenesi of Cholestasis" (M. Trauner & P. Jansen, Eds), Landes Bioscience, Georgetown, TX, 2004, pp112-125, (also 2002 Eurekah.com).
- Anwer, M.S.: Cellular regulation of hepatic bile acid transport in health and cholestasis. Hepatology 39:581-590, 2004.
- Anwer, M.S.: Mechanism of bile formation and cholestasis. In "Liver Biology in Health and Disease" (E. Bittar, Ed), Elsevier, Amsterdam, 2004 (in press).
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