DIRECT AND INDIRECT MECHANISM FOR THE INHIBITION OF SECRETORY ACTIVATION BY PROGE

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Progesterone plays a central role in the development and differentiation of the mammary gland during pregnancy. It stimulates extensive epithelial cell proliferation leading to ductal side branching and alveolar morphogenesis, and late in pregnancy, has the additional function of inhibiting secretory activation, defined as prolactin (PRL) and glucocortiocoid (GC) stimulation of milk protein synthesis and tight junction closure. The mechanism for this important inhibitory action of progesterone is not well defined and is the overall objective of this proposal. Our preliminary studies taken together with the fact that progesterone receptor (PR) is expressed in only a fraction of epithelial cells, indicates that both direct and indirect paracrine mechanisms mediate progesterone-dependent inhibition of secretory activation. Our Preliminary results show that PR can directly repress beta-casein gene transcription induced by PRL/GC by interfering with StatS transactivation at the level of the beta-casein promoter and they suggest that TGFbeta acts as a paracrine factor mediating progesterone-dependent inhibition of beta-casein synthesis and tight junction closure. In this proposal we seek to test and define these direct and indirect mechanisms with the following aims. AIM#1 will define the molecular mechanisms by which PR directly inhibits PRL/GC induction of beta-casein transcription in primary mouse mammary epithelial cells. We will determine the extent to which progesterone inhibits transcriptional vs mRNA translational control of beta-casein expression stimulated by PRL/GC, and explore the hypothesis that PR blocks the transcriptional activity of StatS by disrupting the normal balance of transcriptional coactivators and corepressors at the promoter. AIM#2 will test the hypothesis that TGFbeta is a paracrine mediator of progesterone-dependent inhibition of casein synthesis and tight junction closure. Mammary gland explants and cell cultures will be used for in vitro studies and transgenic mice expressing a dominant negative TGFbeta receptor under the control of MMTV will be used for in vivo analysis. AIM#3 will confirm that direct and indirect mechanisms established from Aims #1 and #2 occur in vivo by use of PR-LacZ and beta-casein-EGFP reporter gene mice and crosses of these transgenic animals. We will determine the spatial and temporal expression patterns of beta-casein and PR promoters, along with endogenous TGFbeta, in response to short term endocrine ablation/hormone replacement protocols.