Project Description:
The primary focus of my research program is the study of gene regulatory mechanisms and transcription factors responsible for promoting phenotypic changes in cardiovascular cell types during development and in disease. We are particularly interested in understanding how smooth muscle cells and fibroblasts trans-differentiate in response to environmental signals promoting wound repair and vascular remodeling.

Arteriosclerosis (arterial wall thickening) is a progressive tissue remodeling process that is caused by chronic inflammatory disease and acute vascular injury. Restriction of blood flow due to luminal expansion of the vessel wall can lead to lethal outcomes such as heart attack or stroke. In human beings and in animal models, a variety of regulatory factors have been linked to aberrant stimulation of stromal fibroblast and/or smooth muscle cell proliferation, migration, and resultant neointimal hyperplasia. Importantly, down-regulation of contractile protein expression accompanies this switch to a fibroproliferative phenotype and may in fact be a requisite event in disease progression. To better understand the molecular biology underlying phenotypic modulation of smooth muscle cells and fibroblasts in the context of cardiovascular disease or injury, we aspire to delineate the mechanism(s) responsible for the remarkable plasticity of smooth muscle a-actin (SMaA) gene expression in these cell types. This line of research has led to the identification of several novel single-stranded DNA (ssDNA)/mRNA-binding proteins that possess the capacity to alter SMaA expression at the level of transcription and translation. We are using techniques of protein biochemistry and biophysics coupled with cell and molecular biology to test hypothetical models of SMaA gene repression by certain members of the Pur and Y-box families of ssDNA/RNA proteins (see Figure below). Experiments are designed to acquire both qualitative and quantitative information about the physical and functional properties of Pura, Purb, and MSY1 in the test tube and in specific cell types. We are also pursuing in vivo studies to better assess the physiological roles of these proteins in mouse models of vascular disease and injury. We anticipate that our findings will lead to an improved understanding of the molecular events underlying cardiovascular dysfunction and may reveal novel targets of diagnostic and/or therapeutic utility in the early detection and/or treatment of coronary artery disease in man.

Conceptual model of cryptic enhancer regulation by Pura, Purb, and MSY1. An asymmetric polypurine/polypyrimidine-rich element (Pur/Pyr) containing a core muscle-CAT (MCAT) enhancer motif is proposed to exist in two distinct conformations. In the ssDNA conformation, disruption of core MCAT enhancer base pairing is stabilized by interaction of Pura, Purb, and MSY1 with opposite strands of the Pur/Pyr element spanning -195 to -165. This arrangement necessarily precludes double-stranded DNA-binding by transcription enhancer factor 1 (TEF-1), a well-known MCAT interacting activator protein.

Enhancer repression by ssDNA-binding proteins
(Adapted from Carlini et al. J. Biol. Chem. 277:8682-8692, 2002)

Knapp, A. M., Ramsey, J. E., Wang, S. X., Godburn, K. E., Strauch, A. R., and Kelm, R. J., Jr. (2006) Nucleoprotein interactions governing cell type-specific repression of the mouse smooth muscle a-actin promoter by single-stranded DNA-binding proteins Pura and Purb. J. Biol. Chem. 281:7907-7918

Wang, S. X., Elder, P. K., Zheng, Y., Strauch, A. R., and Kelm, R. J., Jr. (2005) Cell cycle-mediated regulation of smooth muscle a-actin gene transcription in fibroblasts and vascular smooth muscle cells involves multiple adenovirus E1A-interacting cofactors. J. Biol. Chem. 280:6204-6214

Ramsey, J.E., Daugherty, M.A., and Kelm, R.J., Jr.. (2007) Hydronamic studies on the quarternary structure of recombinant mouse PurB. J. Biol. Chem. 282:1552-1560.