Chronic accumulation of myofibroblasts in healing wounds is associated with hypercontractility, excessive deposition of interstitial collagens, and destructive tissue remodeling. Vascular smooth muscle alpha-actin (SMalphaA) is a contractile protein transiently expressed by differentiated myofibroblasts for generating tensile force required to close open wounds. In chronic fibrotic disease, myofibroblast differentiation is dysfunctional and we discovered that molecular signaling required for activation of both the SMaA and type I collagen genes in these cells also provides negative feedback that could potentially limit the recruitment of hyperactive myofibroblasts during wound healing and destructive remodeling. Studies outlined in this proposal are expected to reveal novel forms of functional interplay of the DNA- and mRNA-binding proteins YB-1, Pur alpha, and Pur beta with the SMalphaA and type I collagen promoters and clarify how these proteins are affected by pro-fibrotic agents such as TGFbeta1 and thrombin that, if unchecked, may cause myofibroblast progression to hypertrophic scarring. Experiments are designed to initiate, amplify, or attenuate myofibroblast differentiation to better understand strategies for controlling SMalphaA and type I collagen gene output at the transcriptional and translation levels as well as reveal novel interventional strategies that might be useful for minimizing aberrant wound healing outcomes.
Aim 1 will examine TGFbeta1-regulated interaction of YB-1 and Pur protein repressors with SMalphaA and collagen promoter DNA and the transcriptional activators Sp1, SRF, and Smads 2,3, delineate regions of repressor polypeptide chains required for this functional interplay, and attempt to disrupt complex formation and disable pathobiologic myofibroblast differentiation using peptide decoys and small molecule pharmacologic inhibitors.
Aim 2 will determine if thrombin potentiates myofibroblast differentiation at the level of translational control thus functioning as a TGFbeta1 supplement or instead antagonizes this growth factor by blocking transcription and myofibroblast recruitment by inducing the anti-fibrotic transcriptional regulatory protein, Egr-1.
Aim 3 studies will explore alternative, Smad-independent mechanisms of myofibroblast differentiation and fibrosis. Loss-of-function approaches based on pharmacologic inhibition of TGFbeta1/Smad kinase- or phosphatidylinositol-3-kinase (PI3K)/Akt kinase signaling will be used to evaluate their possible suppressive effect on myofibroblast activation in vitro and cardiac fibrosis in mice after ischemia/reperfusion injury. The ability of TGFbeta1 and thrombin to exploit the unique DNA-, RNA-, and protein-binding properties of YB-1 and Pur proteins adds a new dynamic perspective to control of gene expression during myofibroblast differentiation that may reveal optimum strategies for therapeutic management of chronic fibrotic diseases.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Lung Injury, Repair, and Remodeling Study Section (LIRR)
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Reynolds, Herbert Y
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Ohio State University
Schools of Medicine
United States
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Hariharan, Seethalakshmi; Kelm Jr, Robert J; Strauch, Arthur Roger (2014) The Pur?/Pur? single-strand DNA-binding proteins attenuate smooth-muscle actin gene transactivation in myofibroblasts. J Cell Physiol 229:1256-71
Willis, William L; Hariharan, Seethalakshmi; David, Jason J et al. (2013) Transglutaminase-2 mediates calcium-regulated crosslinking of the Y-box 1 (YB-1) translation-regulatory protein in TGF?1-activated myofibroblasts. J Cell Biochem 114:2753-69
David, Jason J; Subramanian, Sukanya V; Zhang, Aiwen et al. (2012) Y-box binding protein-1 implicated in translational control of fetal myocardial gene expression after cardiac transplant. Exp Biol Med (Maywood) 237:593-607
Cho, Ji-Hoon; Gelinas, Richard; Wang, Kai et al. (2011) Systems biology of interstitial lung diseases: integration of mRNA and microRNA expression changes. BMC Med Genomics 4:8
Liu, Xiaoying; Kelm Jr, Robert J; Strauch, Arthur R (2009) Transforming growth factor beta1-mediated activation of the smooth muscle alpha-actin gene in human pulmonary myofibroblasts is inhibited by tumor necrosis factor-alpha via mitogen-activated protein kinase kinase 1-dependent induction of the Egr-1 transcr Mol Biol Cell 20:2174-85
Bringardner, Benjamin D; Baran, Christopher P; Eubank, Timothy D et al. (2008) The role of inflammation in the pathogenesis of idiopathic pulmonary fibrosis. Antioxid Redox Signal 10:287-301
Zhang, Aiwen; David, Jason J; Subramanian, Sukanya V et al. (2008) Serum response factor neutralizes Pur alpha- and Pur beta-mediated repression of the fetal vascular smooth muscle alpha-actin gene in stressed adult cardiomyocytes. Am J Physiol Cell Physiol 294:C702-14