Mullerian Inhibiting Substance, a member of the TGF-beta superfamily of growth and differentiation factors, is produced by Sertoli cells during embryonal development and is required for normal reproductive development in male embryos. The signal activity of MIS is the regression of the Mullerian duct, the precursor of the uterus, fallopian tubes and upper vagina. MIS is also produced both in the adult testis by Sertoli cells and in the ovary by granulosa cells, where its exact role remains to be fully explored. Based on in vitro and in vivo evidence, it is our hypothesis that signal transduction by MIS, via its heteromeric serine/threonine kinase receptor, is required to maintain reproductive competence of the gonad and to prevent hyperplastic growth. The goal of this proposal is to (I) understand the developmental, cell- specific and sexually dimorphic molecular mechanisms regulating the expression of the MIS type II receptor (MISrII) in Leydig cells, Sertoli cells, and granulosa cells during different stages of the development and also in the mesenchymal cells surrounding the Mullerian duct during embryonal development and (II) to uncover target genes whose expression is regulated by MIS signal transduction. Since we have cloned the MISrII gene with its TATA-less promoter and have identified cell lines expressing endogenous MISrII, we now have the tools to study expression of MISrII and its downstream target genes. To analyze the MISrII promoter, we will express chimeric promoter/ reporter constructs in MIS type II receptor expressing cells, then perform DNase I footprinting and gel shift analysis, with nuclear extracts prepared from those cells, to determine the cis-acting DNA elements necessary and sufficient for transcription and to examine the role of TFII-I in assembly of the pre-initiation complex. Sequences identified will be used for oligoaffinity purification of trans-acting factors which bind to those sequences. We will also immortalize the Mullerian duct mesenchymal cells which undergo apoptosis and regression in response to MIS to provide cell lines in which to identify downstream, transcriptionally regulated target genes of MIS participating in this important process. We will approach this by studying candidate genes that we might expect to be regulated. We will also perform subtractive hybridization with both R2C cells which respond to MIS, express the receptor, and from which we recently constructed a cDNA library. Information uncovered by these studies will contribute to our understanding of how MIS initiates apoptosis and causes G1 arrest and that these molecular mechanisms can be harnessed for the control of tumors known to respond to MIS, such as human ovarian cancer.
