The long term objective of this proposal is to delineate the molecular mechanisms by which estradiol (E2) and estrogen receptors (ER) regulate gene expression in normal mammary development and differentiation.
The specific aims of this proposal are: (1) to isolate and purify from E2-insensitive lactating mammary glands the cellular component responsible for impeding the activation and/or interaction of mammary ER with DNA nd chromatin; (2) to characterize the mammary progesterone receptor (PgR) in detail with emphasis on receptor heterogeneity and subunit composition; (3) to examine whether the modulation in mammary PgR levels (measured by steroid binding) due to E2 and mammary development and differentiation is associated with differences in the immunoreactive PgR and (4) determine whether the modulation in mammary PgR levels observed due to E2 and as a function of mammary development and differentiation are associated with differences in the accumulation of mRNA levels for PgR. the """"""""putative"""""""" inhibitor of ER interaction with nuclear components will be purified using biochemical techniques and the activity of the factor will be monitored by its ability to inhibit binding of activated ER to DNA and chromatin and also by its ability to impair activation of ER. Mammary PgR will be characterized both by biochemical and immunological techniques using a specific monoclonal antibody raised against avian PgR with cross-reactivity in murine tissues. the PgR levels in tissues obtained from control and E2 treated animals and from different developmental states will be analyzed both by steroid binding, and immunoprecipitation. The mRNA for PgR from these various tissues will be isolated and purified by a powerful polysome immunoadsorption technique using the antibody to PgR. Subsequently, these mRNAs will be translated in vitro in a rabbit reticulocyte system; and the translation products will be immunoprecipitated with the antibody to PgR. The relative level of mRNA in various tissues will then be estimated from the relative rates of PgR synthesis. These studies will provide valuable information concerning the molecular mechanisms underlying E2 action in normal mammary glands, its alteration during mammary development and differentiation and its relationship to ER. And ultimately, these studies using normal mammary gland in an experimental system may also provide critical insights into mechanisms regulating endocrine control of human mammary neoplasia.
| Shyamala, G; Schneider, W; Guiot, M C (1992) Estrogen dependent regulation of estrogen receptor gene expression in normal mammary gland and its relationship to estrogenic sensitivity. Receptor 2:121-8 |
| Schneider, W; Ramachandran, C; Satyaswaroop, P G et al. (1991) Murine progesterone receptor exists predominantly as the 83-kilodalton 'A' form. J Steroid Biochem Mol Biol 38:285-91 |
| Schott, D R; Shyamala, G; Schneider, W et al. (1991) Molecular cloning, sequence analyses, and expression of complementary DNA encoding murine progesterone receptor. Biochemistry 30:7014-20 |
| Shyamala, G; Schneider, W; Schott, D (1990) Developmental regulation of murine mammary progesterone receptor gene expression. Endocrinology 126:2882-9 |
