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Although involved in processes leading to the emergence and development of hormone-dependent breast cancers, the estrogen receptor alpha (ERalpha) also prevents transformed cells from progressing toward a more aggressive phenotype. The transcriptional activity of ERalpha is mediated through two transactivation functions, called activation function 1 and 2, whose respective involvement varies in a cell-specific manner. Here, we identify the Rho/megakaryoblastic leukemia 1 (MKL1) signaling pathway as a main actor in controlling the cell-specific activity of both transactivation functions of ERalpha. Notably, we show that, when the coregulator MKL1 is sequestered in an inactive form by unpolymerized actin, the transcriptional activity of ERalpha mainly relies on the activation function 1. The activation of MKL1, which results from its dissociation from unpolymerized actin, promoted by the ability of Rho to support polymeric actin accumulation, silences the activation function 1 of ERalpha and allows the receptor to mainly act through its activation function 2. Importantly, this switch in the respective contribution exerted by both transactivation functions is correlated with an impaired ability of ERalpha to efficiently transactivate estrogen-regulated reporter genes. MKL1 is further shown to be present on estrogen-responsive genes in vivo. Interestingly, the Rho/MKL1 signaling pathway is activated during the epithelial-mesenchymal transition. A reduced transactivation efficiency of ERalpha, resulting from the activation of this pathway, may therefore suppress the protective role exerted by ERalpha toward tumor progression and invasiveness.

The estrogen receptor alpha (ER alpha) is key in regulating normal breast development and function and is closely involved in the onset and progress of cancers. ER alpha transcriptional activity is mediated through two activation functions, AF1 and AF2, whose activity is tightly regulated in a cell-specific manner through yet unknown processes. Here, we demonstrate that cell-cell junctions generate cell permissiveness to AF1 through an up-regulation of the activity of an AF1 sub-region termed box 1. Moreover, the loss of E-cadherin expression is shown to silence the AF1 activity of ER alpha, allowing the receptor to mainly act through its AF2. This switch from an AF1 to an AF2 cell permissiveness also consequently results in the attenuation of ER alpha activity. Therefore, a loss of cell-cell junctions, a key process that occurs during the epithelial-mesenchymal transition, should have a broad impact on ER alpha transcriptional functions.

The expression of two human estrogen receptor-alpha (hERalpha) isoforms has been characterized within estrogen receptor-alpha-positive breast cancer cell lines such as MCF7: the full-length hERalpha66 and the N terminally deleted hERalpha46, which is devoid of activation function (AF)-1. Although hERalpha66 is known to mediate the mitogenic effects that estrogens have on MCF7 cells, the exact function of hERalpha46 in these cells remains undefined. Here we show that, during MCF7 cell growth, hERalpha46 is mainly expressed in the nucleus at relatively low levels, whereas hERalpha66 accumulates in the nucleus. When cells reach confluence, the situation reverses, with hERalpha46 accumulating within the nucleus. Although hERalpha46 expression remains rather stable during an estrogen-induced cell cycle, its overexpression in proliferating MCF7 cells provokes a cell-cycle arrest in G(0)/G(1) phases. To gain further details on the influence of hERalpha46 on cell growth, we used PC12 estrogen receptor-alpha-negative cell line, in which stable transfection of hERalpha66 but not hERalpha46 allows estrogens to behave as mitogens. We next demonstrate that, in MCF7 cells, overexpression of hERalpha46 inhibits the hERalpha66-mediated estrogenic induction of all AF-1-sensitive reporters: c-fos and cyclin D1 as well as estrogen-responsive element-driven reporters. Our data indicate that this inhibition occurs likely through functional competitions between both isoforms. In summary, hERalpha46 antagonizes the proliferative action of hERalpha66 in MCF7 cells in part by inhibiting hERalpha66 AF-1 activity.

In this study, we hypothesized that many of the reported effects of phthalate esters and other peroxisome proliferators (PPs) in the testis are mediated by members of the PP- activated receptor (PPAR) family of transcription factors through alterations in proteins involved in steroidogenesis. Exposure of Leydig cells to PPs prevented cholesterol transport into the mitochondria after hormonal stimulation and inhibited steroid synthesis, without altering total cell protein synthesis or mitochondrial and DNA integrity. PPs also reduced the levels of the cholesterol-binding protein peripheral-type benzodiazepine receptor (PBR) because of a direct transcriptional inhibition of PBR gene expression in MA-10 Leydig cells. MA-10 cells contain mRNAs for PPARalpha and PPARbeta/delta, but not for PPARgamma. In vivo treatment of mice with PPs resulted in the reduction of both testis PBR mRNA and circulating testosterone levels, in agreement with the proposed role of PBR in steroidogenesis. By contrast, liver PBR mRNA levels were increased, in agreement with the proposed role of PBR in cell growth/tumor formation in nonsteroidogenic tissues. However, PPs did not inhibit testosterone production and testis PBR expression in PPARalpha-null mice. These results suggest that the antiandrogenic effect of PPs is mediated by a PPARalpha-dependent inhibition of Leydig cell PBR gene expression.