This application for a Mentored Scientist Development Award is submitted by a junior faculty member who trained in Molecular Biology with a focus on gene promoter regulation but has since become interested in understanding the cell biology of renal disease. We propose to study signal transduction mechanisms by which TGF-beta stimulates mesangial cell collagen production. Previously, we have shown that the Smad pathway is activated by TGF-beta1 in human mesangial cells and plays a role in TGF-beta1-induced type I collagen expression; and that Smad proteins and Sp1 cooperate to mediate TGF-beta1-increased alpha2(I) collagen (COL1A2) promoter activity. We also showed that the initiation of type I collagen mRNA expression in response to TGF-beta1 is independent of new protein expression, whereas the sustained response (24 h) requires de novo protein synthesis. Here, we propose to investigate TGF-beta1 activation of pathways that are less typically related to TGF-beta signal transduction. Our preliminary results suggest that a PI3K inhibitor and a PKCdelta inhibitor decrease basal and TGF-beta1-induced type I collagen production, block TGF-beta1-stimulated COL1A2 promoter activity and decrease Smad transcriptional activity. We propose to test the hypothesis that both PI3K and PKC pathways are activated by TGF-beta1 in mesangial cells, interact with the Smad pathway and play a role in the initiation and the sustained response to TGF-beta1 leading to collagen accumulation. First, we will assess activation of the PI3K pathway and certain of its downstream targets by TGF-beta1. We will examine the role of PI3K, and study the effect of PI3K-Smad pathway interactions, in collagen expression. Second, we will determine the role of PKC activation and PKC-Smad interactions in collagen production, and the interaction between the PKC and PI3K pathways. Third, we will characterize the signaling mechanisms supporting the sustained collagen response and identify proteins produced after TGF-beta1 treatment that mediate sustained collagen expression. Carrying out this project, the PI will improve her skills in microscopy and the molecular dissection of signaling pathways. Didactic activities will broaden her knowledge in nephrology and fibrogenesis. Support from a K01 award would permit expansion of her ability to supervise others, and facilitate graduated steps toward becoming an independent investigator in renal cell biology.
|Hayashida, Tomoko; Wu, Ming-Hua; Pierce, Amy et al. (2007) MAP-kinase activity necessary for TGFbeta1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397. J Cell Sci 120:4230-40|
|Poncelet, Anne-Christine; Schnaper, H William; Tan, Ruoyun et al. (2007) Cell phenotype-specific down-regulation of Smad3 involves decreased gene activation as well as protein degradation. J Biol Chem 282:15534-40|
|Runyan, Constance E; Poncelet, Anne-Christine; Schnaper, H William (2006) TGF-beta receptor-binding proteins: complex interactions. Cell Signal 18:2077-88|
|Runyan, Constance E; Schnaper, H William; Poncelet, Anne-Christine (2005) The role of internalization in transforming growth factor beta1-induced Smad2 association with Smad anchor for receptor activation (SARA) and Smad2-dependent signaling in human mesangial cells. J Biol Chem 280:8300-8|