Renal

Principal Investigator(s):	Bradley M. Denker, M.D.
Group Member(s):	Jason Guan, PhD Deguang Zhu, PhD Jennifer Hunt, MD Ernesto Sabath, MD

Role of Heterotrimeric G Proteins in Regulating Epithelial Cell Junctions in Health and Disease

My laboratory is focused upon heterotrimeric G proteins and how unique protein-protein interactions regulate the function of renal tubular epithelial cells and podocytes. Our approach has been to develop in vitro assays, cell culture models and transgenic animals to study the function of G proteins in three major kidney diseases (1) acute (ischemia/reperfusion) and chronic (unilateral ureteral obstruction) renal injury (2) glomerular diseases of the podocyte slit and (3) Polycystic Kidney Disease.  In each of these areas there is strong evidence for a fundamental role of G protein regulation.   We have identified novel interactions of Ga12 with the tight junction (TJ) protein, ZO-1 and elucidated the mechanisms regulating barrier function in established epithelial and during TJ assembly.  The role of Ga12 activation on recovery of TJs in renal injury induced by hypoxia, ischemia/reperfusion and fibrosis models are being investigated in tissue culture and in animal models.  In addition, transgenic mice with proximal tubular expression of constitutively active Ga12 (QLa12) have been established for these and other studies.  The glomerular epithelial cell (podocyte) is a specialized polarized cell that forms multiple foot processes along the glomerular capillary basement membrane.  The “junction” between foot processes (slit diaphragm) shares many features with traditional epithelial cell junctions and is essential for normal kidney function.  Loss of podocyte slit diaphragms results in proteinuria and often leads to progressive renal failure. The function of Ga12 and other G proteins regulating the podocyte slit diaphragm is being investigated with podocyte specific expression of QLa12 in transgenic mice and in tissue culture models.  We recently identified Ga12 binding and stimulation of the serine/threonine phosphatase, PP2A and mapped the interacting domains.  To explore functional consequences of this interaction, we characterized apoptosis in epithelial cells and identified novel stimulation of apoptosis by Ga12 that was PP2A dependent.  These signaling pathways have been elucidated and result in loss of Bcl-2 and up regulation of IkB-a.  Apoptosis is dysregulated in Polycystic Kidney Disease and both Ga12 and PP2A interact with PC1, the large transmembrane protein mutated in the disease.  Current studies are focused on how PC1 may regulate Ga12 function and alter fundamental cell behavior such as apoptosis, proliferation and cell migration.  Through efforts to identify and understand specific protein interactions, fundamental insights have been gained into specialized processes in epithelia. These efforts should lead to novel therapeutic approaches and broaden opportunities for treating injured epithelia from a variety of etiologies.

SELECTED PUBLICATIONS

Meyer, T., Schwesinger, C., Ye, J. Denker, B.M. and Nigam, S.K. Reassembly of the Tight Junction after Oxidative Stress Depends Upon a Tyrosine Kinase. J. Biol. Chem. 2001; 276:22048-22055

Saha, C., Nigam, S.K. and Denker, B.M.  Expanding Role of G Proteins in Tight Junction Regulation; Gs Stimulates TJ Assembly. Biochem. Biophys. Res. Comm. 2001; 285:250-256

Redd, K.J. Oberdick, J., McCoy, J., Denker, B.M. and Luo, Y.  Association and Colocalization of G Protein ao Subunit and Purkinje Cell Protein 2 (Pcp2) in Mammalian Cerebellum. J. Neurosci, Res. 2002; 70:631-637

Meyer, T.N., Schwesinger, C., Denker, B.M. ZO-1 is a Scaffolding Protein for Signaling Molecules; Ga12 Directly Binds to the SH3 Domain and Regulates Paracellular Permeability in Epithelial Cells. J. Biol. Chem. 2002;277:24855-24858

Meyer, T.N., Schwesinger, C., Denker, B.M. G12 Regulates Paracellular Epithelial Cell Permeability Through Src Tyrosine Kinases. Am. J. Physiol. Cell Physiol. 2003; 285:C1281-1293

Yuasa, T., Takakura, A. Denker, B.M., Venugopal, B., Zhou. J. Polycystin-1L2 is a Novel G-protein Binding Protein. Genomics, 2004; 84:126-138.

Yao, J.,  Tu, C.L., Kos, C.H. Henderson, J.M., Allen, P.G., Denker, B.M., Pollak, M.R.  a-actinin-4 mediated FSGS: an autosomal dominant kidney disease caused by a misfolded and rapidly degraded cytoskeletal protein. PLOS, 2004; 2:787-794.

Zhu, D., Kosik, K.S., Meigs, T.E., Yanamadala, V., Denker, B.M.  Ga12 Directly Interacts with PP2A;  Evidence for Ga12-Dependent Stimulated PP2A Phosphatase Activity and Dephosphorylation of  Microtubule Associated Protein, Tau. J. Biol. Chem. 2004; 279: 54983-54986.

Hunt, J.L., Pollak, M.R., and Denker, B.M.  Cultured Podocytes Establish a Size Selective Barrier Regulated by Specific Signaling Pathways and Demonstrate Synchronized Barrier Assembly in a Calcium Switch Model of Junction Formation. J. Am. Soc. Neph. 2005; 16:1593-1602.

Guan, J., Luo, Y, and Denker, B.M.  Purkinje Cell Protein-2 (Pcp2) Stimulates Neurite Differentiation and Growth in PC12 Cells Through Activation of P38 Map Kinase. 2005; Bochem. J. 392:389-397.

Zhu, D., Tate, R.I., Ruediger, R., Meigs, T.E. and Denker, B.M. Domains Necessary for Ga12 Binding and Stimulation of PP2A:  is Ga12 a Novel Regulatory Subunit of PP2A? 2007; Mol. Pharmacol. 71:1-9.

Yamandadala, V., Kong, T., Negoro, H. and Denker, B.M. Ga12 Stimulates  Apoptosis in Epithelial Cells Through JNK1 Mediated BCL-2 Degradation and  Upregulation of IkB-a.  2007, J. Biol. Chem. 282:24352-24362

Weins, A., Schlondorff, J.S., Nakamura, F., Denker, B.M., Hartwig, J.H., Stossel, T.P.and Pollak, M.R. A disease-associated mutant a-actinin-4 reveals a novel mechanism for regulating its F-actin binding affinity.  2007; Proc. Nat. Acad. Sci, 104;16080-16085.

Sabath, E., Negoro, H., Beaudry, S., Paniagua, M., Angelow, S., Shah, J., Grammatikakis, N., Yu, A.S.L., and Denker, B.M.  Ga12 Regulates Protein Interactions Within the MDCK Cell Tight Junction and Inhibits Tight Junction Assembly.  2008; Journal of Cell Science, in press.

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