The overall goal of this grant application is to understand how polyamines influence the migration of GI mucosal cells during the process of early mucosal restitution. Experiments supported during the past 10 years have shown that: 1) polyamines are essential to the healing of gastric and duodenal mucosal, 2) polyamines are required for the mucosal restitution that depends on cell migration, 3) polyamines are essential to migration in a normal line (IEC-6) of intestinal epithelial cells in a model that mimics mucosal restitution, and 4) polyamines alters some components of the cytoskeleton that are essential for cell migration. The first set of specific aims is designed to define the manner in which polyamines interact with the cytoskeleton. These studies will examine the effect of polyamines on the equilibrium between and distribution of, F- and G-actin, determine whether polyamines alter the amounts of distribution of G-actin binding proteins, determine whether polyamines are involved in the attachment of cells to their substrates, and then whether they alter the amounts and distributions of integrins and attachment proteins. Most of these studies will be carried out using immunocytochemical techniques. Preliminary evidence from the PI's laboratory indicates that the small GTP binding protein Rho regulates cell migration in IEC-6 cells. The second set of specific aims tests this hypothesis by inhibiting Rho function and examining the effects on cell migration and the cytoskeleton. Rho function will be inhibited with toxins A and B from C. difficile, C3 toxin from C. botulinum, and recombinant dominant negative Rho(A) T19N protein. These experiments will establish the role of Rho in the regulation of GI epithelial cell migration. In the third set of specific aims, studies will determine whether and how polyamines affect Rho function. Western blot analysis of preliminary results indicates that polyamine depletion decreases the amount of Rho protein. These studies will be pursued along with those involving Rho mRNA and Rho GTPase activity. The experiments outlined will explain the original observations of the PI on mucosal healing in whole animals in terms of cell function and describe, in turn, how it is regulated at the molecular level. These results will provide the first description of the involvement of polyamines in the migration of any cell type, and be the first to indicate how Rho regulates GI mucosal cell migration and how it in turn is dependent on polyamines. These studies have clinical application in peptic ulcer disease, cancer, colitis and all conditions where cell migration is important.
| Ray, Ramesh M; Johnson, Leonard R (2014) Regulation of intestinal mucosal growth by amino acids. Amino Acids 46:565-73 |
| Ray, Ramesh M; Li, Chunying; Bhattacharya, Sujoy et al. (2012) Spermine, a molecular switch regulating EGFR, integrin ýý3, Src, and FAK scaffolding. Cell Signal 24:931-42 |
| Ray, Ramesh M; Viar, Mary Jane; Johnson, Leonard R (2012) Amino acids regulate expression of antizyme-1 to modulate ornithine decarboxylase activity. J Biol Chem 287:3674-90 |
| Ray, Ramesh M; Bavaria, Mitulkumar N; Bhattacharya, Sujoy et al. (2011) Activation of Dbl restores migration in polyamine-depleted intestinal epithelial cells via Rho-GTPases. Am J Physiol Gastrointest Liver Physiol 300:G988-97 |
| Bavaria, Mitulkumar N; Ray, Ramesh M; Johnson, Leonard R (2011) The phosphorylation state of MRLC is polyamine dependent in intestinal epithelial cells. Am J Physiol Cell Physiol 300:C164-75 |
| Elias, Bertha C; Bhattacharya, Sujoy; Ray, Ramesh M et al. (2010) Polyamine-dependent activation of Rac1 is stimulated by focal adhesion-mediated Tiam1 activation. Cell Adh Migr 4:419-30 |
| Ray, Ramesh M; Bhattacharya, Sujoy; Johnson, Leonard R (2007) EGFR plays a pivotal role in the regulation of polyamine-dependent apoptosis in intestinal epithelial cells. Cell Signal 19:2519-27 |
| Ray, Ramesh M; Guo, Huazhang; Patel, Minesh et al. (2007) Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 292:G983-95 |
| Ray, Ramesh M; Vaidya, Rajiv J; Johnson, Leonard R (2007) MEK/ERK regulates adherens junctions and migration through Rac1. Cell Motil Cytoskeleton 64:143-56 |
| Bhattacharya, Sujoy; Ray, Ramesh M; Johnson, Leonard R (2006) Integrin beta3-mediated Src activation regulates apoptosis in IEC-6 cells via Akt and STAT3. Biochem J 397:437-47 |
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