Unfolding the Action of Progesterone Receptors*

Progesterone regulates a plethora of biologically distinct processes in a broad range of tissues through the action of the progesterone receptor (PR). With the identification of PR target genes and membrane-associated PR, its actions span normal homeostatic functions and a wide variety of seemingly unlinked biological processes. In recent years, significant progress has been made in the molecular aspects of PR function. Intense interest in defining the action of progesterone has revealed important mechanisms involving multiple layers of regulation in transcription. Here, we will review recent studies regarding genetic, biochemical, and molecular aspects of PR function.

a tissue-specific manner mediating a subset of the reproductive functions. Selective knockout of PR-B results in reduced mammary ductal morphogenesis but does not affect ovarian, uterine, or thymic responses to progesterone. Thus, PR-A is both necessary and sufficient to elicit the progesterone-dependent reproductive responses required for female fertility, whereas PR-B is required for normal proliferative responses to progesterone in the mammary gland (6).
Other PR variants have been described in several reports. A third PR isoform, PR-C, has been identified in some tissues including decidual cells (Ref. 8 and reviewed in Ref. 9). PR-C is an N-terminally truncated form of PR ( Fig. 1) with a molecular mass of about 60 kDa and is restricted primarily to the cytosolic fraction (8). PR variants encoded with transcripts lacking one or more exons also have been isolated from normal and malignant breast cells (10). These "anomalous" variants may modify progesterone action by serving as decoys for PR-A and PR-B, rendering the transcriptional capabilities of these isoforms less efficient (9).
Rapid, non-genomic actions of progesterone and other steroids have been characterized that may be mediated by interactions with membrane-associated receptors. Membrane-mediated effects of progesterone have been studied most intensively in human spermatozoa and in the Xenopus oocyte (reviewed in Ref. 11). Membrane-associated, progesterone-specific receptors have been isolated and cloned from a range of tissues in a number of species (11). The prototypical example is the resumption of meiosis in amphibian oocytes, triggered by progesterone at the plasma membrane level (12); this action is mediated by a G protein-coupled receptor distinct from the classic intracellular PR (13). The putative membrane progestin receptors ␣, ␤, and ␥ in human have distinct distributions in reproductive, neural, kidney, and intestinal tissues, with the ␣ and ␤ subtypes displaying high affinity saturable binding for progesterone (13). Despite accumulating evidence for cell-surface membrane actions of progesterone and other steroids, further studies are needed to understand this aspect of progesterone-mediated signaling as well as the characteristics of these membrane receptors to establish the basis for their cellular function.

Target Genes of PR Activation
Another recent advance in PR biology has been the identification of numerous PR target genes from different laboratories. Kester et al. (14) have identified several PR-regulated genes from a T47-D cell line using a differential display strategy. Putative target genes induced by progesterone in the presence of cycloheximide, which should represent genes directly induced by PR, are involved in regulation of transcription and cell differentiation. These genes include TSC-22, a putative transcription factor, CD-9/MRP-1 (motility-related protein 1), Na ϩ /K ϩ -ATPase ␣1, desmoplakin, CD-59/ protectin, and FKBP51, an immunophilin.
A systematic large scale investigation of gene regulation by PRs using a unique human breast cancer cell model that expresses either the PR-A or PR-B isoforms exclusively demonstrates that PR-A and PR-B regulate different subsets of genes involved in particular functional pathways (2). It is interesting that in breast cancer cells, although some genes are regulated by progesterone through both PR isoforms, most genes are uniquely regulated through one or the other isoform, predominantly through PR-B. A subset of these genes is involved in breast cancer and mammary gland development, including STAT5A, MSX-2, and C/EBP␤. Surprisingly, a significant number of genes are involved in membraneinitiated events, including proteins involved in cell adhesion, membrane-bound receptors, calcium-binding proteins, and signaling molecules. These genes represent almost half of all progesteroneregulated genes identified in this breast cancer cell model, clearly pointing to the cell membrane as an important downstream target of progesterone action (2). Another gene cluster involved in metabolism includes genes in cholesterol/steroid, fatty acid, nucleotide, or amino acid metabolism. Also, up-regulation of the BCL-X L gene in PR-A expressing T47-D cells suggests a role for progesterone in blocking apoptosis, which may explain the resistance to apoptosis in transgenic mice that overexpress PR-A in the mammary gland (15). Other newly found PR target genes have functions in transcription, cell growth, and protein processing, indicating a broad range of genes regulated by PRs.
Using endometrial carcinoma cell lines stably transfected with PR-A or PR-B, Smid-Koopman et al. (16) have observed a different panel of genes regulated by PRs. Among the progesterone-regulated genes in PR-A-positive Ishikawa cells, retinoic acid receptor ␥, integrin ␤4/␣7, mitogen-activated protein kinase P97, and p16-INK4 are up-regulated, whereas cytokeratin 8 and cyclin D1 are down-regulated. In contrast, Ishikawa cells expressing PR-B exhibited only down-regulation of three genes: IGFBP-3, fibronectin, and replication protein A. Although these genes may not be direct targets of PRs, these observations suggest cell-and tissue-specific distinctions in target gene regulation between the two PR isoforms.
In an effort to explore the downstream events of progesterone action in vivo, Takamoto et al. (17) used uteri of wild type and PR null mutant mice as a starting material and screened for differentially expressed genes using cDNA expression arrays. One of the genes identified was Indian hedgehog (Ihh), which was rapidly induced (within 3 h) after a single administration of progesterone to ovariectomized mice. Furthermore, the expression profile of several genes known to be in the hedgehog signaling pathway, including patched-1, followed the expression pattern of Ihh during the peri-implantation period in other tissues. Continuing studies using microarrays in the same group have revealed more than 100 genes regulated by progesterone within 4 h of treatment. 2 Analysis of the impact of the induction of these genes will be important in understanding the physiological role of progesterone in regulating a broad range of biologically distinct processes.

Transcriptional Mechanisms and the Action of PR Cofactors
The molecular mechanisms by which progesterone regulates the transcription of target genes through PRs have been actively in-vestigated over several decades. The traditional ligand-dependent mechanism of receptor activation after binding of hormone to the ligand binding domain involves multiple steps, including a conformational change and dissociation from a multiprotein sequestering complex consisting of protein chaperones including heat shock proteins and immunophilins. Receptors then dimerize and bind to progesterone-responsive elements (reviewed in Refs. 18 and 19). The DNA-bound receptors can increase rates of target gene transcription through additional interactions with steroid receptor coactivators (reviewed in Ref. 20) and the general transcription machinery, which facilitates the assembly of the preinitiation complex at the promoter.
Initial contact between the activated receptor and coactivators is mediated in large part by an amphipathic helix conserved on the surface of most coactivators, the LXXLL motif, or NR box (21). These factors include the steroid receptor coactivator (SRC)/p160 family (22), CBP/P300 (23), CIA (24), ASC-2/TRBP/AIB3/RAP250/ PRIP/NCR (25,26), and PBP/DRIP205/TRAP220 (27)(28)(29). The SRC family is composed of three distinct but structurally and functionally related members: SRC-1, SRC-2 (TIF-2/GRIP1), and SRC-3 (p/CIP/RAC3/ACTR/TRAM-1/AIB1) (20). Although all of these coactivators are implicated in PR-mediated gene activation, not all are functionally equivalent in vivo nor are they expressed in the same manner in all cells. For example, SRC-3 has a more restricted pattern of expression than SRC-1 or SRC-2 (30). A recent study in our laboratory demonstrates that PR preferentially recruits SRC-1, SRC-3, and CBP, but not SRC-2 or pCAF, leading to specific histone modification of the mouse mammary tumor virus promoter and suggesting differential recruitment of coactivators by nuclear receptors as a mechanism to mediate specific hormone signaling (31). The SRC-1 null mice exhibit partial hormone resistance in progesterone target tissues, such as mammary gland and uterus, further substantiating the importance of SRC-1 for the biological actions of progesterone (32). ASC-2 recently has been identified as a coactivator involved in PR-mediated transcription. 3 ASC-2 belongs to a steady-state complex of ϳ2 MDa (ASCOM) in HeLa cell nuclei (33). ASCOM has been reported to contain retinoblastoma-binding protein RBQ-3, ␣/␤-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. Interestingly, ALR-1/2 and HALR contain a highly conserved SET domain, which was recently implicated in lysine-specific methylation of histone H3. ASC-2 also interacts with SRC-1 and CBP/P300 to stimulate ligand-dependent transactivation by nuclear receptors (33). Whether ASC-2 plays an important physiological role in the progesterone pathway in vivo will require further investigation in ASC-2 gene-targeted animal models (34).
A different group of coactivators is specialized for interaction with the DBD of receptors. The DBD of PR is required for binding to specific progesterone-responsive element DNA sequences, but much less is known about the function of nuclear cofactors that bind to the DBD. This includes small nuclear RING finger protein (SNURF), GT198, a tissue-specific coactivator, and high mobility group (HMG) proteins. SNURF is a ubiquitously expressed nuclear protein capable of activating steroid receptor-dependent transcription (35). It forms a functional link between steroid-and Sp1regulated transcription (36), demonstrating a mechanism by which DBD-interacting coactivators synergize transactivation by allowing cooperation between nuclear receptors and other transcription factors. HMG-1 and -2 are members of a family of non-histone chromatin proteins produced by two separate genes (37). HMG proteins do not recognize specific DNA sequences but rather bind to DNA in the minor groove, recognizing bends in DNA. PR appears to utilize HMG-1 or -2 proteins for high affinity interaction with DNA in vitro and for full transcription activity in vivo. However, HMG-1 and -2 do not influence the activities of non-steroid nuclear receptors. The DBD of class II nuclear receptors, but not steroid receptors, contains a C-terminal extension that mediates high affinity interactions with DNA. The specificity of HMG-1/2 coactivation seems to be achieved by functionally substituting for the C-terminal extension and facilitating DNA binding by steroid nuclear receptors (37).
Compared with AF-2 and DBD interacting coregulators, cofactor interactions with the AF-1 are less well characterized. AF-1s are 2 F. DeMayo, personal communication. 3  the least conserved regions among PRs from different species and are likely associated with the differential ability of PR-A and PR-B to recruit specific coregulator proteins (38). A recent study has identified several N-terminal domain interacting factors, including Jun dimerization protein-2 (JDP-2), nuclear receptor coactivator-62 (NCOA-62, also known as Ski interacting protein), and APE2, a base excision repair protein (39). JDP-2, initially defined as a repressor of Jun and other bZIP transcription factors, functions as an AF-1 coactivator of PR. It has been shown that endogenous JDP-2 and PR are recruited in a hormone-dependent manner to a progesterone-responsive promoter in the context of chromatin in vivo. AF-1 cofactors may potentiate PR function by recruiting or stabilizing other coactivators in an alternative pathway independent of the AF-2 and SRC coactivators (39).
The recent description of PR coactivators, which influence RNA splicing, has revealed another mechanism by which PR regulates gene expression. Transcription and mRNA processing are coupled events in vivo, but the mechanisms that coordinate these processes were largely unknown until recently. Studies in our laboratory have demonstrated that the promoter-dependent effect of progesterone on alternative splicing relies on the PR-mediated recruitment of coregulators that are involved in both stimulating transcription and regulating alternative mRNA splicing (40). One of these coregulators is coactivator activator (CoAA), which contains two highly conserved RNA recognition motifs (RRM) commonly found in ribonucleoproteins. In addition, CoAA potently coactivates transcription mediated by multiple hormone-response elements and acts synergistically with TRBP and CBP (41). Other coactivators of nuclear receptors have been shown previously to regulate RNA splicing (42). Furthermore, PR may influence splicing by promoting the expression of a splicing factor. For example, Kester et al. (14) identified a novel progesterone target gene with homology to members of the SR (serine/arginine-rich) protein family of splicing factors. Therefore, activated steroid hormone receptors coordinately control gene transcription activity and RNA processing, and PR appears to play a prominent role in this regard.
The precise mechanisms by which these different classes of cofactors coordinately regulate transcription are not fully understood. However, a number of studies have indicated several aspects of coactivators that appear to involve multiple cooperative mechanisms. First, the structural properties of coactivators allow for multiple interactions among receptor and coactivator complexes (20). Also, the coupling of interaction domains with various enzymatic activities within coactivators determines the recruitment of distinct acetyltransferases (CBP/P300, pCAF), methyltransferases (CARM1, PRMT1) (43,44), kinases (Rsk-2, Msk-1) (45), ubiquitin ligases (E6-AP, p300) (46,47), ATP-dependent chromatin remodeling complexes (SWI/SNF) (48), and RNA splicing factors, facilitating the formation of a preinitiation complex and contributing to downstream events in transcription as well. In addition, preferential recruitment of specific cofactors to particular promoters and in a specific cellular environment creates various distinct patterns of gene expression. Competition between cofactors binding to recep-tors may antagonistically influence the interaction with other complexes (49). Although beyond the scope of this review, corepressors also play a role in the regulation of coactivator function. For example, coactivator/corepressor ratios have been reported to modulate PR-mediated transcription (50). Accumulating evidence highlights the functional significance of specific covalent modifications of cofactors in the process of transcriptional activation. Phosphorylation of SRC-1 and SRC-3 at specific sites potentiates PR-mediated transcription, probably because of enhanced interaction with other histone acetyltransferases such as CBP (51). 3 Acetylation of SRC-3 by p300/CBP at lysine residues adjacent to NR boxes disrupts its association with the receptor (52), providing another example of modulating cofactor interaction by covalent modification. Sumoylation of SRC-1 has been shown to increase the PR/SRC-1 interaction and to prolong SRC-1 retention in the nucleus (53). It is reasonable to imagine that similar to histone proteins, modification of cofactors may preclude or enhance interactions of other coactivators. Taken together, these potential mechanisms provide effective means of enhancing the functional plasticity of coregulators that will eventually result in reorganization of protein-protein or protein-DNA contacts and receptor-mediated transcription.

Ligand-independent Activation and PR-mediated
Cross-talk In addition to regulation by ligand, activation of cellular signaling pathways can be sufficient to activate PR in the absence of hormone. Intracellular kinases modulate the function of PR. Elevation of intracellular cyclic AMP, a common second messenger for a number of hormones and a direct activator of protein kinase A, can induce ligand-independent activation of chick PR (54). Phosphorylation seems to be an important step in ligand-independent gene activation, because PR-dependent transcription in the absence of ligand also is stimulated by okadaic acid, an inhibitor of protein phosphatases PP1 and PP2A (55). Ligand-independent activation of PR has been demonstrated by treatment with dopamine or dopamine agonists, which result in the translocation of the receptor from the cytoplasm to the nucleus (56). A number of in vivo studies show this to be a prominent pathway for PR regulation and induction of reproductive behavior in rats and mice (57,58). Studies on growth factor-mediated activation of chick PR also suggest that ligand-independent activation of PR is mediated via membrane-associated signaling cascades (59).
Progesterone signaling often converges or is intertwined with other hormones, growth factor, or cytokine signaling. For example, estrogen induces the expression of PR, and therefore many of the in vivo effects attributed to progesterone are dependent on estrogens. In contrast, progesterone attenuates the proliferative effect of estrogen in the uterus (reviewed in Ref. 60). Hormone-induced prevention of breast cancer in animals involves combinatorial actions of estrogen and progesterone (reviewed in Ref. 61). A recent report demonstrated that PR is involved in progesterone activation of the c-Src/Erk pathway in mammalian cells, either alone (62) or in combination with endoplasmic reticulum (63). It is well docu-FIG. 2. Summary of PR-mediated actions. Progesterone (Prog)-mediated, ligand independent (membrane signaling), or non-genomic actions of PR are represented. The genomic function of PR recruits different coactivator complexes to facilitate transcription initiation as well as splicing. Non-genomic modulation of cellular activities may ultimately influence transcription. It is not clear whether PR is directly involved in the regulation of elongation or termination of transcription. mented that progesterone/PR up-regulate epidermal growth factor receptors and other tyrosine kinase receptor family members. Progesterone also amplifies at least two cytokine signaling pathways: one involving mitogen-activated protein kinase (MAPK) and the other involving signal transducers and activators of transcription (STATs) (64). Modulation of coactivators provides another example of cross-talk because coregulators are themselves targets of multiple signal transduction pathways. In response to different signals, posttranslational modifications of cofactors at specific amino acid residues serve as crucial molecular switches to modulate the processes of progesterone and other steroid hormone actions.

Concluding Remarks
PR biology has been extensively dissected in the past three decades. The identification of distinct roles for PR isoforms and characterization of their physiological function have benefited from molecular and genetic studies of their actions in promoting gene expression. The identification of numerous endogenous PR target genes should make it possible to understand how progesterone signaling ultimately leads to biological actions in reproductive and other tissues. PR-mediated transcriptional regulation encompasses many layers of complexity and intricate mechanisms, involving distinguishable receptor variants, a broad range of coregulators, a combinatorial network of multiple modifications among the coregulators, and ligand-dependent, ligand-independent, genomic, and non-genomic actions. In addition to transcriptional initiation, PRcontrolled gene expression involves regulation of mRNA splicing and may involve transcriptional elongation and termination processes as well (Fig. 2). To unfold the diverse array of physiological roles involved in PR action, it is critically important to understand how distinct cellular signaling pathways converge on the processes of PR-mediated transactivation. Future studies will focus on how coregulators modulate their targeted molecules through recruitment of additional interacting proteins or enzyme substrates within distinct cofactor complexes. Complete understanding of PR functions in transcriptional regulation remains an important but formidable task.