Recent insights into the mechanism of steroid hormone action have advanced our understanding of PRA action significantly and suggested how the two forms of PR, hPRA and hPRB, manifest their unique regulatory activities in target cells. Specifically, it has been demonstrated that nuclear hormone receptors, upon binding their cognate ligands, undergo distinct conformational changes. This event permits the dissociation of the receptors from corepressor complexes, possessing histone deacetylase activity and facilitates their interaction with coactivator complexes, which display histone acetylase activity. As a consequence, the DNA-bound receptor is able to positively regulate target gene transcription (19
). In support of this model, it has been shown that the ability of nuclear receptors to repress target gene transcription correlates with their ability to bind to the corepressors NCoR and SMRT (21
). Conversely, transcriptional activation by nuclear hormone receptors was observed to correlate with the recruitment of coactivators to the promoter region of target genes (19
). To determine whether the opposing transcriptional activities of hPRA and hPRB were due to differential cofactor association, we examined the abilities of hPRA and hPRB to interact with different coactivators and corepressors and assessed the effects of these associations on the receptors' transcriptional activities. Using both in vivo and in vitro methodologies, we found that antagonist-bound hPRA has a higher affinity for the NR box of SMRT (C'SMRT) than antagonist-bound hPRB (Fig. and ). This interaction appears to be physiologically relevant since overexpression of C'SMRT (a dominant negative SMRT) effectively reverses hPRA-mediated transrepression of ERα transcriptional activity. In addition, overexpression of SMRT enhanced the ability of hPRA to inhibit hER-mediated transcriptional activity. Significantly, we also observed that unlike hPRB, hPRA did not associate efficiently with the coactivators SRC-1 and GRIP1. Thus, the robust interaction of hPRA with SMRT together with its inability to efficiently engage coactivators appears to explain why hPRA is a repressor of progesterone-responsive promoters.
Initially, it was proposed that the differences in the transcriptional activities of hPRA and hPRB were due to a third activation function, AF-3, present within the extreme amino terminus of hPRB, a region that is absent in hPRA (47
). Thus, it was considered that functional synergy between the activation functions located in the amino terminus (AF-3 and AF-1) and the carboxyl terminus (AF-2) was required for maximal hPRB transcriptional activity. However, unlike AF-1 and AF-2, AF-3 does not demonstrate autonomous activity when fused to a heterologous DBD (40
), suggesting that instead of functioning as a classical AF, AF-3 might be required for proper AF-1 and AF-2 transcriptional activity. For instance AF-3 may contribute to hPRB transcriptional activity directly, by enhancing the activity of AF-1 or AF-2, or indirectly, by suppressing an inhibitory function contained within sequences common to both hPRA and hPRB (18
). Evidence in support of the latter hypothesis came from our studies, as well as those of others, which identified an ID within the amino terminus of hPRA which, when deleted, resulted in a receptor mutant functionally indistinguishable from hPRB (18
). Specifically, it was demonstrated that the first 140 aa of hPRA are necessary for its ability to function as a transcriptional inhibitor as well as a transrepressor of heterologous steroid receptor transcriptional activity (18
). Thus, one role of AF-3 is to override the function of the ID present within the amino terminus of the receptor, allowing hPRB to activate transcription (18
In addition to hPR, several other transcription factors have been shown to contain both activation and repression functions (2
). Of particular relevance to our studies of hPRA, it has been shown in vitro that the ability of RORα to repress transcription correlates with the ability of the inhibitory domain within RORα to recruit the corepressors NCoR and SMRT (21
). In addition, RORα was shown to preferentially associate with NCoR and not SMRT in vivo. When we tested the ability of hPRA and hPRB to interact with NCoR and SMRT in the presence of antagonist, we found that while both receptors associate with NCoR, hPRA has a higher affinity for SMRT than hPRB (Fig. C). Furthermore, a deletion mutant lacking the inhibitory domain, ΔhPRA, does not interact efficiently with SMRT. This implies that like the case for RORα, a specific domain within hPRA is required for corepressor interactions.
The ability of agonist-activated nuclear receptors to activate transcription correlates with their ability to displace corepressors and engage coactivators (reviewed in reference 55
). Not surprisingly, therefore, we were able to show in this study that agonist-bound hPRB, but not hPRA, can form a productive interaction with coactivators, thus allowing hPRB to activate transcription from progesterone-responsive promoters. This suggested that hPRA may be unable to completely dissociate from corepressors and thus may not be able to recruit coactivators. However, the fact that R5020-bound hPRA was unable to activate transcription in cells expressing a dominant negative variant of SMRT, or in the presence of the histone deacetylase inhibitor TSA, suggests that dissociation from corepressors is not sufficient for hPRA to activate transcription (Fig. ). This observation, together with the mammalian two-hybrid data, implies that agonist-bound hPRA, unlike hPRB, does not efficiently recruit coactivators. It appears, therefore, that the unique sequences present at the amino terminus of hPRB are required for proper transcriptional activation.
In most cell and promoter contexts, the transcriptional activity of steroid hormone receptors appears to require the functional synergy between the amino and carboxyl termini of each individual receptor (6
). This synergy occurs as a consequence of an agonist-dependent association between the amino and carboxyl AFs of ERα (31
), the androgen receptor (5
), and hPRA and hPRB, respectively (54
). Interestingly, in the case of hPRA and hPRB, the amino terminus of hPRB containing AF-3 was shown to interact more efficiently with the carboxyl terminus of the receptor than the amino terminus of hPRA lacking AF-3 (54
). This agonist-dependent interaction was enhanced by the addition of SRC-1 and CBP, while dominant negative variants of SRC-1 and CBP completely abolished this interaction, suggesting that these coactivators may be required for transcriptional synergy between the amino-terminal and carboxyl-terminal AFs of the receptor (54
). Interestingly, a role for coactivators as bridging factors between the amino and carboxyl AFs of receptors is supported by the observation that SRC-1 can interact with both the amino and carboxyl termini of PR (44
Previously, we have shown that the agonist-dependent interaction of the PR carboxyl terminus with the amino terminus of hPRB is more robust than that with the amino terminus of hPRA, an activity which mirrors their activity as transcriptional activators (18
). Thus, the ability of hPRB to function as an activator of transcription could be due to the fact that hPRB, but not hPRA, undergoes a conformational change which is conducive to coactivator binding. The ability of hPRA and hPRB to adopt different conformations within the cell is also supported by our peptide analysis (Fig. and ). The peptide competition data presented in Fig. also suggest that the two receptors are bound to different cellular factors which may, in turn, explain their distinct functions within the cell. For example, hPRB, unlike hPRA, is likely to be associated with AF-2-type coactivators. It is not surprising, then, that the LX-H10 peptide, which contains an LXXLL motif common to these coactivators, was an efficient inhibitor of hPRB activity but had no effect on hPRA activity when overexpressed in cells along with the receptors (Fig. ). This hypothesis is further supported by additional findings which show that agonist-bound hPRB, but not agonist-bound hPRA, directly interacts with the NR boxes of the coactivators GRIP1 and SRC-1 in vitro (Fig. B). Although our studies focused on the ability of hPRA to interact specifically with the previously defined NR boxes of SRC-1 and GRIP1, it has been reported previously that both hPRA and hPRB interact with full-length SRC-1A in the presence of agonist (44
). However, in these latter studies it was not determined whether hPRA and hPRB can bind directly to the sequences of SRC-1 used in this study. In addition, the SRC-1(NR) protein used in our studies did not contain the fourth LXXLL motif found in SRC-1A. Together, these observations suggest that the two PR isoforms do not interact in the same manner with SRC-1.
Our working model to explain the opposing transcriptional activities of hPRA and hPRB is depicted in Fig. A. We propose that hPRB is a transcriptional activator of progesterone-responsive promoters, since upon binding hormone, hPRB undergoes a conformational change which allows it to dissociate from corepressor proteins and recruit coactivators. This productive interaction with the coactivators allows the receptor to activate transcription from the promoters of target genes. Conversely, under the same conditions, hPRA is transcriptionally inactive because, unlike hPRB, it does not effectively recruit coactivators to the promoters of target genes. Thus, the inability of hPRA to activate target gene transcription does not appear to be related to its ability to associate with corepressors such as SMRT. However, our data reveal a central role for SMRT in hPRA-mediated repression of ERα transcriptional activity. Thus, we propose that the hPRA-SMRT complex blocks estrogen action by interfering with the assembly or function of the ERα-coactivator complex.
FIG. 12 Two distinct models are required to describe the molecular mechanism of action of hPRA. (A) Transcriptional activation. Based on the in vivo and the in vitro binding studies, we propose that hPRA interacts more efficiently with corepressors and less efficiently (more ...)
Whereas the results of these studies explain why hPRB acts as a strong transcriptional activator of progesterone-responsive promoters and why hPRA is transcriptionally inactive in these contexts, it remains to be determined how the hPRA-SMRT complex can transrepress ERα transcriptional activity. We believe that agonist-bound hPRB can interfere with ERα transcriptional activity by squelching a required coactivator protein (i.e., p160 family of coactivators). It does not appear, however, that hPRAs transrepression function involves a direct competition between hPRA and ERα for coactivators. It may well be that hPRA inhibits the activity of a cofactor required for ERα action by binding directly at a site distinct from the ERα-interacting site or indirectly by binding to other proteins within the ERα-coactivator complex. Distinguishing between these possibilities is the subject of our current investigations.