Leiomyomas are proliferative/fibrotic tumors characterized by the presence of an unusually large number of mast cells (MC). What regulates MC recruitment into leiomyoma and their biological significance is unknown although MC and their secretory products are directly linked to angiogenesis and tissue fibrosis. In previous funding periods of this project we discovered that interleukine 13 (IL-13) and transforming growth factor beta (TGF-B), which are 2 major secretory products of MC with profibrotic activities, are overexpressed in leiomyomas and their smooth muscle cells (LSMC). We also showed that GnRHa therapy, which regresses leiomyoma growth, downregulates expression of IL-13 and TGF-B at tumor level in vivo, and in LSMC in vitro. We determined that IL-13 acts as a key regulator of TGF-B expression which is highly regulated by ovarian steroids in LSMC. The interactions between TGF-B and GnRH receptors signaling pathways also result in differential regulation of extracellular matrix (ECM) and protease expression, whose products are involved in tissue remodeling and angiogenesis. The results lead us to hypothesize that MC/LSMC-derived IL-13, directly and/or indirectly through a mechanism involving TGF-B directs transcription of specific genes whose products promote leiomyoma growth. GnRHa, SERM and SPRM, and Tranilast, a synthetic mast cell stabilizer, alter the expression profile of these genes by inhibiting IL-13 and TGF-B resulting in leiomyoma regression. To further understand to what extent and by what mechanisms MCs and MC-derived IL-13 and TGF-B influence fibrotic responses in leiomyoma we propose the following Aims.
Aim #1 to test the hypothesis that MC-derived profibrotic mediators alter the gene expression profile of myometrial SMC (MSMC) to adopt LSMC-like characteristics. We will co-culture MSMC with MC and/or expose MSMC to MCsecretory products and compare them to LSMC using protein microarrays.
Aim #2 will characterize IL-13 independent and dependent mechanisms involving TGF- b in regulating profibrotic gene expression in LSMC, through signaling pathways of JAK/Stat6, Smad (independent) and MAPKs (common) and transcriptional activation of c-fos/c-jun. By silencing TGF-B and IL-13 receptors expression, preventing their autocrine/paracrine actions, we will determine the contribution of IL-13-independent actions on regulating the expression of these genes.
Aim #3 will characterize the effectiveness of GnRHa, SERM, SPRM and Tranilast in down-regulating MC-derived IL-13 and TGF-B, and target genes downstream from these mediators, resulting in leiomyoma regression. To achieve these Aims, we will use primary cultures of LSMC and MSMC, gene and protein microarrays, Realtime PCR, immunoblotting, ELISA, lentiviral vector SiRNAs and specific kinase inhibitors. Achieving these aims will provide direct evidence for the biological significance of MC, and novel molecular mechanisms that converge the actions of IL-13 and TGF- b leading to leiomyoma growth. We will also identify molecular regulatory pathways utilized by GnRHa, SERMs and SPRMs to regress leiomyoma growth and through the introduction of SiRNA technology and Tranilast, will work toward the development of a novel approach to prevent growth and to medically treat leiomyomas.
Showing the most recent 10 out of 33 publications
