The mesangial cell plays an important role as an effector of glomerulosclerosis in progressive kidney disease. Although TGF-beta has been implicated in this process, the mechanisms mediating fibrogenesis are not well understood. During the previous funding period, we determined that the Smad signal transduction pathway is a critical mediator of TGF-beta1-stimulated human mesangial cell collagen I expression. A number of additional signaling mechanisms contribute to optimal collagen-producing responses. Of particular note, we defined a role for the ERK MAP kinase pathway. We found that TGF-beta1 activation of ERK requires Smad3 and, conversely, that the ERK pathway plays a role in phosphorylation of the linker region of Smad3. Although this phosphorylation site is outside of the C-terminal Smad3 domain that is classically associated with R-Smad activation, it clearly contributes to collagen I expression. These results suggest the hypothesis that in mesangial cells, Smad signaling and the ERK MAP kinase cascade interact at multiple levels to amplify collagen I gene activation by TGF-beta1. To test this hypothesis, we propose three specific aims.
In aim 1, we will determine the mechanism by which Smad3 participates in ERK activation by identifying the pathway(s) through which TGF-beta1 stimulates ERK activity and determining which are inactivated by blocking Smad3 activity.
In aim 2, we will determine the significance and mechanism of Smad3 linker reqion phosphorylation by testing the effect of mutating the linker region on Smad translocation, transactivation activity and COL1A1 or COL1A2 promoter activation, and by identifying proteins that bind to Smad in an ERK-dependent manner.
In aim 3, we will determine how Smad3-ERK pathway interactions regulate collagen I gene expression by examining how and where Smad and ERK interact in the cell, and how inhibiting Smad3-ERK pathway interactions affects the activation of collagen I gene transcription by TGF-beta1. These experiments will provide new information regarding basic mechanisms of Smad action and collagen expression, and could identify potential targets for therapeutic intervention in glomerulosclerosis.
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