We show that PR is a component of several distinct pathways that function both directly and indirectly to positively upregulate E2F1 expression in breast cancer cells (Fig. ). First, PR directly regulates E2F1 transcription by binding to proximal and distal enhancer sites located near E2F1. Second, progestin induces the hyperphosphorylation of Rb, which results in increased recruitment of E2F1 to its own promoter, thereby activating a positive feedback loop that further amplifies its transcription. Finally, PR induces expression of KLF15 and potentially other Sp/KLF family members, which bind to GC-rich regulatory regions within the E2F1 promoter and further activate transcription. Together, these pathways represent a complex multimodal regulatory system in which the combined actions of each component are required for maximal progestin-mediated upregulation of E2F1 transcription.
FIG. 7. Model of multimodal regulation of E2F1 by progestins. Ligand-bound PR can bind to proximal and distal enhancer sites located near E2F1 and directly regulate E2F1 transcription. PR can also act indirectly through hyperphosphorylation of Rb and induction (more ...)
In most breast cancer cell lines, estrogens are important for regulation of PR expression; however, the estrogen receptor (ER) has previously been shown to induce expression of E2F1, and we wanted to concentrate solely on PR-specific regulation of E2F1 expression. Therefore, we chose T47D cells as a model system for our studies because in this cell line, PR expression is uncoupled from ER signaling (14
). Given that progestins can stimulate proliferation of T47D cells in vitro
and when propagated as xenografts in vivo
, it was not unexpected to see that PR also modulates expression of E2F1, a transcription factor that controls cell cycle progression. However, we noted that E2F1 expression was also induced in response to progestins in BT483 breast cancer cells (Fig. ) and in ER-negative/PR-negative human mammary epithelial cells (HMECs) infected with a PR-B adenovirus (Fig. ), model systems where progestins do not stimulate proliferation. Importantly, the downstream biological effects of E2F1 are not limited to regulation of cell proliferation; indeed, E2F1 has been implicated in other critical processes such as DNA damage response, checkpoint control, and apoptosis (4
). Defining the role(s) of these additional processes in PR biology is an area of continued exploration in our group. Additionally, the microarray analysis showed that treatment of T47D cells with R5020 stimulated the expression of E2F2 and E2F7; further studies are necessary to explore the roles of other E2F family members in PR signaling.
The initial purpose of our microarray study was to determine the overall involvement of the MAPK signaling pathway in PR regulation of target gene transcription. We were surprised to find that the expression levels of almost 80% of the 1,794 PR target genes identified in this analysis were affected by pretreatment with the MEK 1/2 inhibitor U0126 (Fig. ). Of course, since inhibition of MAPK reduces progestin-mediated upregulation of E2F1 expression (Fig. ), any PR target genes that are coregulated by this protein would be correspondingly affected. One explanation for the inhibitory effect of U0126 on progestin-mediated induction of E2F1 expression is the observation that MAPK inhibition partially suppressed PR-mediated hyperphosphorylation of Rb (Fig. ), which is necessary for release of E2F and activation of the positive feedback loop (Fig. ).
While the mechanism(s) by which progestins induce hyperphosphorylation of Rb has not been fully elucidated, it has been established that treatment of T47D cells with progestin leads to induction of cyclins D1 and E and increased activity of the cyclin D1/cdk4 complex (31
), which has been implicated in phosphorylation of several sites on Rb (37
). Previous studies have reported that progestin induction of cyclin D1 is dependent on rapid PR activation of the Src/MAPK pathway (2
); therefore, we initially hypothesized that direct interactions between PR and Src family kinases might activate MAPK and contribute to progestin regulation of E2F1. However, we determined that R5020 effectively induces expression of E2F1 mRNA in cells expressing either wild-type PR-B or the mutant PR-BmPro (Fig. ), which cannot directly interact with c-Src or mediate rapid, nongenomic activation of Src/MAPK signaling.
However, other studies have proposed an alternative mechanism for rapid activation of MAPK signaling by progestins, whereby PR interacts with unliganded ER, which in turn activates the Src/MAPK signaling pathway (1
). Furthermore, a recent study reported that progestin induction of cyclin D1 requires both the DNA-binding domains of PR, which allow PR to bind directly to distal regions of the cyclin D1 promoter, and the two ER-interacting domains (ERID) of PR, which allow PR to interact with ER to achieve rapid activation of Src/MAPK (27
). Additional studies are necessary to determine whether PR activation of MAPK through this alternative, ER-dependent pathway and subsequent induction of cyclin D1 is the mechanism leading to progestin-mediated hyperphosphorylation of Rb, and subsequent induction of the positive feedback loop that amplifies E2F1 expression. Interestingly, we noted that the magnitude of PR-mediated induction of E2F1 expression in ER-negative cell lines, such as T47D:C42 cells (Fig. ) or HMECs (Fig. ), was not as great as that achieved by progestins in ER-positive cell lines, such as T47D:A18 cells (Fig. ) or BT483 cells (Fig. ). The significance of this observation is currently under investigation.
Bioinformatic analyses revealed a 277-gene subset of progestin-regulated transcripts that was enriched for E2F-binding sites (Fig. ); this subset includes classic E2F1 target genes such as those for CDC6, cyclin E, and CDK2. However, it is currently unclear whether the effects of progestins on these genes and others are mediated solely by secondary E2F1 actions or whether PR also directly regulates their transcriptional activity. Analyses with Patser showed that 99 progestin-regulated genes contain both putative PREs and E2F1-binding sites within their promoters (see Fig. S8 at http://mcdonnelllab.duhs.duke.edu
), and this may indicate a trend of coregulation of target genes by direct actions of PR and E2F1. Interestingly, since the expression of as many as 277 R5020-regulated genes may be modulated by E2F1, a target of PR-B but not PR-A (Fig. ), it is possible that regulation of E2F1 by the PR-B isoform could be an important factor that contributes to the vastly different profiles of PR-A and PR-B as transcriptional regulators.
Similarly, several pieces of data suggest a trend of coregulation of target genes by PR and members of the Sp/KLF superfamily. For instance, pretreatment with mithramycin A affected R5020-mediated induction of many downstream PR target genes that we examined; moreover, we observed that knockdown of KLF15 inhibited R5020 induction of several PR target genes (data not shown). Bioinformatic analyses using Patser revealed that out of the 1,794 PR target genes detected in our microarray study, the promoters of 1,372 genes contain putative GC-rich binding sites for Sp/KLF family members (see Fig. S8 at http://mcdonnelllab.duhs.duke.edu
). Studies are currently ongoing to determine whether cooperation between PR and KLF15 and/or other SP/KLF family members in the regulation of gene transcription constitutes a more global model of PR function.
While the extent to which PR engages in multimodal regulation of target genes remains to be determined, the data we have generated in this study indicate that the ability of PR to induce the expression of E2F and Sp/KLF family members and their resulting impact on gene expression provides a mechanism to explain secondary, cycloheximide-sensitive responses to progestins. In general, the indirect secondary responses that are stimulated by progestins have been less studied than primary transcriptional responses; however, this area of PR signaling deserves more attention, since the regulation of target gene expression by PR-stimulated transcription factors can dramatically influence the overall transcriptional program set into motion by progestins. In the context of PR regulation of E2F1 transcription, secondary factors such as E2F1 and KLF15 act to reinforce progestin-mediated induction of E2F1 expression, but E2F and Sp/KLF family members may act to suppress PR actions on other target genes.
Finally, induction of KLF15 expression by PR has ramifications that extend beyond its role in progestin-mediated regulation of E2F1. KLF15 is a recently discovered transcription factor, and the transcriptional mechanisms that regulate KLF15 promoter activity are poorly understood; however, several recent studies support a role for NRs in regulation of KLF15 expression. In ovariectomized mice, treatment with estradiol and progesterone upregulates KLF15 expression in the uterine epithelium (24
). In addition, dexamethasone treatment induces KLF15 expression in chondrocytes (12
), and both corticosterone and the glucocorticoid receptor-specific agonist cortivazol upregulate KLF15 expression in cardiomyocytes (39
). Furthermore, little is known about the biological function(s) of KLF15 in the breast. In our qPCR analysis of breast cancer cells, we observed that basal transcription of KLF15 was low; in contrast, KLF15 is highly expressed in the liver, kidney, heart, and skeletal muscle (35
). Studies involving KLF15 in other tissues have revealed an emerging role for KLF15 in regulation of metabolic processes such as glucose homeostasis (9
) and lipid accumulation (5
). It is clear that further studies are warranted to determine how progestin-mediated activation of KLF15 signaling may affect metabolic signaling processes in the breast.
In conclusion, although E2F1 transcription is affected by the direct interaction of PR with the regulatory regions near E2F1, we also established that maximal induction of E2F1 expression by progestins requires the actions of additional transcription factors, such as E2F1 and KLF15, on the E2F1 promoter. The same may be true for a much larger subset of PR target genes. In fact, we suspect that PR often acts in concert with these and other secondary factors to coregulate target gene expression, depending on the cell- or tissue-specific context. These results suggest a paradigm for multimodal PR gene regulation that entails cooperation between direct and indirect pathways of PR signaling to achieve the desired downstream transcriptional cascade.