The abnormal motility of inflamed colon reflects changes in both smooth muscle and its regulatory components, namely the enteric nervous system, ICC networks, and various humoral mediators. The focus of this proposal is on the molecular mechanisms that mediate hypocontractility of circular smooth muscle, a well-established feature of colonic inflammation. We and others have identified three major signaling targets whose expression is altered by inflammation resulting in hypocontractility of circular smooth muscle: RGS4, which regulates G?q activity, and CPI-17 and Rho kinase, which regulate MLC phosphatase activity. The mechanisms eliciting these changes in expression are not known. We hypothesized that caveolin (Cav-1) acts as a pro-contractile and anti-proliferative regulatory molecule that promotes normal smooth muscle function, and that a decrease in Cav-1 expression during inflammation mediates the changes in expression of signaling targets and contractile proteins that determine colonic circular smooth muscle hypocontractility. Preliminary studies show that a decrease in Cav-1 expression initiates a regulatory cascade involving co-transcription factor myocardin and target- specific miRs that mediate the changes in expression of signaling targets. The same Cav-1-dependent regulatory cascade triggers a concomitant decrease in contractile protein expression. Preliminary studies also show that treatment of mouse and human colonic circular smooth muscle with IL-1? or TNF-? in vitro and acute induction of TNBS colitis in vivo decreased Cav-1 expression; a similar decrease in Cav-1 was present in colonic circular smooth muscle from Crohn's disease. The decrease in Cav-1 was mediated by NF-?B/ERK1/2 and was accompanied by changes in expression of myocardin, miRs, signaling targets, and contractile proteins that closely mimicked those in colonic smooth muscle of Cav-1-/- mice and human colonic muscle after Cav-1 silencing with siRNA. These studies provide strong evidence for the novel concept that a cytokine-mediated, NF-?B/ERK1/2-dependent decrease in Cav-1 expression acts via myocardin and miRs to regulate changes in expression of signaling targets and contractile proteins that determine hypocontractility of circular smooth muscle. The hypotheses are:
Aim 1. Cav-1-dependent myocardin determines expression of RGS4, Rho kinase, and CPI-17 via miR-1, miR-148a and miR-145, respectively.
Aim 2. Cav-1-dependent myocardin determines expression of contractile proteins directly and via miR-145 and miR-221.
Aim 3. Activation of NF-?B/ ERK1/2 pathways by cytokines in vitro and in vivo (TNBS colitis; Crohn's disease) decreases Cav-1 expression leading to changes in expression of myocardin and miRs that regulate signaling targets and contractile proteins. These studies will expand our understanding of the mechanisms underlying abnormal motility during colonic inflammation in humans and offer avenues for the development of novel therapies.
Gastrointestinal motility disorders are common in diseases related to gut inflammation. Caveolin-1, the principal scaffolding protein in caveolar membranes, plays an important role in lipid homeostasis and signal transduction, and is commonly deleted in various cancers. The objective of the proposal to understand the mechanisms by which caveolin-1 regulates signaling targets and contractile proteins that promote normal smooth muscle function, and mediates the alterations in expression of signaling targets that underlie circular smooth muscle hypocontractility during inflammation.
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