Progesterone Receptor Deficient in Chromatin Binding Has an Altered Cellular State

Abstract

Our previous work has shown that the progesterone receptor (PR) can exist in two distinct functional states in mammary adenocarcinoma cells. The differences in function included the ability to activate a promoter in organized chromatin, sensitivity to ligand, and ligandindependent activation. To determine whether these functional differences were because of altered cellular processing, we carried out biochemical analyses of the functionally distinct PRs. Although the majority of PR is localized to the nucleus, biochemical partitioning resulted in a loosely bound (cytosolic) fraction, and a tightly bound (nuclear) fraction. In the absence of progestins, the functionally distinct PRs differed significantly in partitioning between the two fractions. To characterize these fractions further, we analyzed interactions of unliganded PR with chaperones by coimmunoprecipitation. We determined that PR in the cytosolic fraction associated with hsp90 and p23. In contrast, PR in the nuclear fraction consisted of complexes containing hsp90, p23, and FKBP51 as well as PR that was dimerized and highly phosphorylated. Hormone treatment significantly reduced the formation of all PR-chaperone complexes. The hsp90 inhibitor, geldanamycin, similarly blocked transcriptional activity of both functionally distinct receptors. However, the two forms of the PR differed in their ability to associate with the mouse mammary tumor virus promoter in organized chromatin. These findings provide new information about the composition and distribution of mature progesterone receptor complexes in mammary adenocarcinoma cells, and suggest that differences in receptor subcellular distribution have a significant impact on their function. These findings also reveal that transiently expressed steroid receptors may not always be processed like their endogenous counterparts.Progesterone receptor (PR) 1 functions in the development of lobular alveolar structures in the normal mammary gland (1, 2). Data from in vitro studies using cultured breast cancer cells suggest that PR exerts its effects on mammary epithelium by regulating cell cycle progression. PR positive breast cancer cells exhibit a biphasic response to progestins, initial cell division followed by long term G 1 phase growth arrest (3, 4). In mammary adenocarcinoma, PR-mediated growth regulation is frequently lost. However, loss of function through receptor deletion or receptor mutation occurs only in a subset of tumors (5). Therefore, in the majority of cases, other mechanisms must be involved in loss of PR function. A better understanding of the mechanisms by which PR function can be modulated would be beneficial to developing therapy for tumors in which PR function has been lost or altered.In vivo, steroid receptors must interact with transcriptionally inactive promoters having complex chromatin structure. Accordingly, their transcriptional activity is dependent upon a variety of proteins that modify chromatin structure, such as ATP-dependent chromatin remodelers, histone acetyltransferases, and...

Effect of Initial Subcellular Localization of Progesterone Receptor on Import Kinetics and Transcriptional Activity

“…First, wt PRA is localized more in the nucleus than wt PRB, suggesting that the import rate of wt PRA is faster than export rate even in the absence of hormone. A second reason could be stronger binding of wt PRB with hsp90 than wt PRA in the cytoplasm . Another possible reason could be the differences in binding affinity of wt PRA and wt PRB with cofactors (coactivators and corepressors) …”

Progesterone receptors, their isoforms and progesterone regulated transcription

“…3) The origin of PR is important. Endogenous PR activate transcription driven by the MMTV-LTR in the context of chromatin, while transiently expressed PR fail to do so (Botos et al , 2004). 4) Many studies use the pGL3/luc vector for promoter construction.…”

Impact of chromatin structure on PR signaling: Transition from local to global analysis

“…In the absence of ligand it is restricted from chromatin interaction through interaction with specific chaperone proteins (Botos et al, 2004). Binding of ligand results in phosphorylation of the receptor and strong nuclear localization, where the receptor engages the chromatin embedded genome to bind specific DNA response elements.…”