Mü llerian inhibiting substance (MIS), a transforming growth factor-␤ family member, causes regression of the Mü llerian duct in male embryos. MIS overexpression in transgenic mice ablates the ovary, and MIS inhibits the growth of ovarian cancer cell lines in vitro, suggesting a key role for this hormone in postnatal development of the ovary. This report describes a mechanism for MISmediated growth inhibition in both a human epithelial ovarian cancer cell line and a cell line derived from normal ovarian surface epithelium, which is the origin of human epithelial ovarian cancers. MIS-treated cells accumulated in the G 1 phase of the cell cycle and subsequently underwent apoptosis. MIS up-regulated the cyclin-dependent kinase inhibitor p16 through an MIS type II receptor-mediated mechanism and inhibited growth in the absence of detectable or inactive Rb protein. Prolonged treatment with MIS down-regulated the Rb-related protein p130 and increased the Rb familyregulated transcription factor E2F1, overexpression of which inhibited growth. These findings demonstrate that p16 is required for MIS-mediated growth inhibition in ovarian epithelial cells and tumor cells and suggest that up-regulation of E2F1 also plays a role in this process.MIS, 1 a member of the TGF-␤ family of hormones, induces regression of the epithelial-mesenchymal unit of the Mü llerian duct in the embryonic urogenital ridge in males. In the absence of MIS, differentiation of the Mü llerian duct into the uterus, fallopian tubes, and upper vagina in female embryos occurs autonomously (1). The 140-kDa MIS homodimer is enzymatically cleaved into two distinct fragments. The carboxylterminal fragment composed of a dimer with subunits of M r 12,500 retains bioactivity, whereas a noncleavable mutant is devoid of biological function (2). MIS is produced at high levels by Sertoli cells of the testis even after the regression of the Mü llerian duct and decreases at adolescence. In females, it is synthesized by granulosa cells of the ovary. Measurement of circulating serum MIS levels in females indicates that MIS in females is produced postnatally, increases at the onset of puberty, and is undetectable at menopause (3, 4). It is hypothesized that binding of MIS ligand to the MIS type II receptor, a serine threonine kinase (5-7), leads to heterodimerization with a type I receptor, initiating a signaling cascade.The MIS type II receptor gene contains 11 exons and encodes a 63-kDa protein, which is expressed at very high levels in the uterus, testis, and ovary (5-7). Male mice that lack both alleles of the MIS type II receptor have a persistent Mü llerian duct, which differentiates into a uterus and oviducts, a phenotype reminiscent of MIS ligand null mice (8). Imbeaud et al. (9) have identified MIS type II receptor mutations in male patients with Persistent Mü llerian Duct syndrome, which reaffirms the developmental significance of its expression in humans. Transgenic female mice that overexpress MIS ligand, demonstrate complete ablation of the ovary, along wit...

Mü llerian inhibiting substance (MIS) is a key element required to complete mammalian male sex differentiation. The expression pattern of MIS is tightly regulated in fetal, neonatal, and prepubertal testes and adult ovaries and is well conserved among mammalian species. Although several factors have been shown to be essential to MIS expression, its regulatory mechanisms are not fully understood. We have examined MIS promoter activity in 2-day postnatal primary cultures of rat Sertoli cells that continue to express endogenous MIS mRNA. Using this system, we found that the region between human MIS؊269 and ؊192 is necessary for full MIS promoter activity. We identified by DNase I footprint and electrophoretic mobility-shift analyses a distal steroidogenic factor-1 (SF-1)-binding site that is essential for full promoter activity. Mutational analysis of this new distal SF-1 site and the previously identified proximal SF-1 site showed that both are necessary for transcriptional activation. Moreover, the proximal promoter also contains multiple GATA-4-binding sites that are essential for functional promoter activity. Thus multiple SF-1-and GATA-4-binding sites in the MIS promoter are required for normal tissue-specific and developmental expression of MIS.Mü llerian inhibiting substance promoter M üllerian inhibiting substance (MIS), also called antiMüllerian hormone, a glycoprotein homodimer belonging to the transforming growth factor ␤ superfamily, is a critical component of sex differentiation responsible for regression in the male embryo of the Müllerian ducts, which in a normal female embryo become the uterus, fallopian tubes, and upper vagina (1). In the rat, MIS is expressed in fetal Sertoli cells from the time of testis differentiation (13 days postcoitum) (2). Both MIS mRNA and protein remain high after birth and fall precipitously after day 5 to a low level, where they remain throughout adult life. Conversely, MIS mRNA is undetectable in the fetal ovaries but becomes increasingly expressed after birth in the granulosa cells of developing follicles (3-5).The complex expression pattern of MIS suggests that it is tightly regulated. The MIS genes from mouse (6), rat (7), bovine (8), porcine (9), chicken (10), and human (8, 11) have been cloned, and all mammalian proximal promoters show regions of evolutionary conservation (Fig. 1). The mouse MIS transcriptional start site is located only 328 bp downstream of the SAP62 gene, suggesting that the region conferring critical regulation of MIS expression is located within close proximity of the transcriptional start site (12).Several factors important for sex determination have been proposed to regulate MIS expression, including SRY (13), SOX9 (14, 15), SF-1 (16, 17), WT-1 (18, 19), Dax-1 (19), and 21), and all except Dax-1 and possibly WT-1 can bind to the Ϫ180 bp region upstream of the transcriptional start site of MIS. Moreover, evidence is convincing that transcriptional upregulation of MIS requires coordinate interactions between SF-1 and SOX9 (15), GATA-4 (21), an...

Signal reception of Müllerian inhibiting substance (MIS) in the mesenchyme around the embryonic Müllerian duct in the male is essential for regression of the duct. Deficiency of MIS or of the MIS type II receptor, MISRII, results in abnormal reproductive development in the male due to the maintenance of the duct. MIS is a member of the transforming growth factor-beta (TGFbeta) superfamily of secreted protein hormones that signal through receptor complexes of type I and type II serine/threonine kinase receptors. To investigate candidate MIS type I receptors, we examined reporter construct activation by MIS. The bone morphogenetic protein (BMP)-responsive Tlx2 and Xvent2 promoter-driven reporter constructs were stimulated by MIS but the TGFbeta/activin-induced p3TP-lux or CAGA-luc reporter constructs were not. The induction of Tlx2-luc was dependent upon the kinase activity of MISRII and was blocked by a dominant negative truncated ALK2 (tALK2) receptor but not by truncated forms of the other BMP type I receptors ALK1, ALK3, or ALK6. MIS induced activation of a Gal4DBD-Smad1 but not a Gal4DBD-Smad2 fusion protein. This activation could also be blocked by tALK2. The BMP-induced inhibitory Smad, Smad6, was up-regulated by MIS endogenously in Leydig cell-derived lines and is expressed in male but not female Müllerian duct mesenchyme. ALK6 has been shown to function as an MIS type I receptor. Investigation of the pattern of ALK2, MISRII, and ALK6 in the developing urogenital system demonstrated overlapping expression of ALK2 and MISRII in the mesenchyme surrounding the duct while ALK6 was observed only in the epithelium. Examination of ALK6 -/- male animals revealed no defect in duct regression. The reporter construct analysis, pattern of expression of the receptors, and analysis of ALK6-deficient animals suggest that ALK2 is the MIS type I receptor involved in Müllerian duct regression.

Observations of patients deficient in the ste-