Leiomyomas are benign uterine smooth muscle tumors, which depend in part, on ovarian steroids for growth. Clinical and basic science research shows that gonadotropin releasing hormone analogues (GnRHa), acting through suppression of the pituitary-gonadal axis cause leiomyoma to regress by affecting uterine arteriole size, blood flow, cellular content and matrix accumulation. The cellular and molecular mechanisms of how GnRHa might regulate these changes at the tumor level are not known. The PI and coworkers have established that leiomyomas and their smooth muscle cells (LSMC) over-express transforming growth factor-beta (TGFBETA), a multifunctional cytokine that controls an array of cellular functions and its overexpression causes tissue fibrosis. In addition, GnRHa therapy causes downregulation of TGFBETA and its receptor expression at the tumor level in vivo, and in LSMC in vitro through a receptor mediated processes. The core hypothesis is that TGFBETA is a key regulator of leiomyoma growth and GnRH/GnRH receptor expression, implying the presence of a regulatory system that provide an additional therapeutic site of direct action for GnRHa on leiomyoma cells. Activation of this system causes down-regulation of TGFBETA/TGFBETA receptor expression and interruption of their downstream signal transduction contributing to leiomyoma regression. To test this core hypothesis investigators will determine if constitutive overexpression of TGFBETA in LSMC causes a differential gene expression pattern that differs from that found in MSMC. Inhibition/reduction of TGFBETA expression with the use of antisense oligomers will induce LSMC to adopt MSMC-like gene expression pattern determined by gene microarray technique (Aim #1). We will determine if the effect of TGFBETA is transmitted through TGFBETA receptor signaling pathways that involve Smads and MEK/ERK (Aim #2), and if GnRHa can directly inhibit the effect of TGFBETA overexpression in LSMC by altering the activation of Smads or MEK/ERK (Aim #3). We will focus on TGFBETA activation of Smad-3 and Smad-4, and MEK/ERK, signal transduction pathways that activate plasminogen activator inhibitor (PAI-1) and fibronectin expression. By inhibition/reduction of TGFBETA type II receptor expression/action with the use of antisense and neutralizing antibodies, Smad 4 antisense and MEK/ERK specific inhibitors, and GnRHa action with the use of antagonist, the biological significance of TGFBETA and GnRHa receptor signaling, and their interaction will be determined. Activation of Smad 7, which presents Smad3/4 association and transmission from the cytoplasm to the nucleus, and expression of PAI-1, fibronectin, c-fos and TGFBETA1 will be measured as indicator of TGFBETA and GnRHa actions. To achieve these Aims, the investigators will use LSMC and MSMC cell cultures, gene microarray, antisense oligomers, neutralizing antibodies, specific kinase inhibitors, immunoprecipitation, immunoblotting, quantitative RT-PCR, ELISA, and immunocytochemistry. The investigators anticipate that the proposed research will lead to the identification of novel interactions in signal transduction pathways and improved understanding of regulatory molecular mechanisms involved in growth and regression of leiomyoma. This is the second revision of an application. It was proposed to investigate the effects of TGF and GnRH analogs on differential gene expression and signaling pathways involving Smads and MEK/ERK in human leiomyoma smooth muscle cells (LSMC). Previous criticisms focused primarily on the lack of a clear hypothesis as to the roles of GnRH and antiprogestins in the growth and treatment of leiomyomas. To address these concerns the PI revised the application substantially and removed the portion regarding antiprogestins. More preliminary data regarding GnRH and TGF action were added. The PI also included results of the previously proposed PhosphoSpots assay to demonstrate the regulation of phosphorylation of substrates for Raf-1, MEK and ERK by TGF1 and a GnRH agonist in LSMC. The PI now proposes three new or revised aims, which are substantially different from the previous ones. The current proposal still continues to suffer from the lack of a clear hypothesis and an integrated direct approach to the determination of the roles of TGF and GnRH action in the growth and treatment of leiomyomas.
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