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Molecular Endocrinology 18 (2): 269-278
Copyright © 2004 by The Endocrine Society

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Making Sense of Cross-Talk between Steroid Hormone Receptors and Intracellular Signaling Pathways: Who Will Have the Last Word?

Carol A. Lange

University of Minnesota Cancer Center, Departments of Medicine (Division of Hematology, Oncology, and Transplant) and Pharmacology, Minneapolis Minnesota 55455

Address all correspondence and requests for reprints to: C. A. Lange, University of Minnesota Cancer Center, Departments of Medicine (Division of Hematology, Oncology, and Transplant) and Pharmacology, Minneapolis Minnesota 55455. E-mail: Lange047{at}tc.umn.edu.


   ABSTRACT
TOP
 ABSTRACT
CLASSICAL ACTIONS OF...
 PR ISOFORMS ARE PHOSPHOPROTEINS
 MULTIPLE FUNCTIONS OF PR...
 NOVEL EXTRANUCLEAR ACTIONS OF...
 CONCLUDING REMARKS
 REFERENCES
 
In classical models of nuclear steroid hormone receptor function, ligand binds receptor, heat shock proteins dissociate, and receptor dimers enter or are withheld in the nucleus and interact with coregulatory molecules to mediate changes in gene expression. The footnotes, "receptors become phosphorylated" and "dynamic nucleo-cytoplasmic shuttling occurs" describe well-accepted, but less well-understood aspects of receptor action. Recently, the idea that several protein kinases are activated in response to steroid hormone binding to cognate cytoplasmic or membrane-associated receptors has become fashionable. However, the precise role of steroid hormone receptor phosphorylation and our understanding of which cytoplasmic kinases are activated and their functional significance remain elusive. This review provides an overview of the primary ways in which steroid hormone receptor and growth factor cross-talk occurs, using the human progesterone receptor (PR) as a model. The functional consequences of PR phosphorylation by protein kinases classically activated in response to peptide growth factors and novel extranuclear or nongenomic functions of PR as potential independent initiators of signal transduction pathways are discussed. Intracellular protein kinases are emerging as key mediators of steroid hormone receptor action. Cross-talk between steroid receptor- and growth factor-initiated signaling events may explain how gene subsets are coordinately regulated by mitogenic stimuli in hormonally responsive normal tissues, and is suspected to play a role in their cancer biology.


   CLASSICAL ACTIONS OF PROGESTERONE RECEPTORS (PRs)
TOP
 ABSTRACT
 CLASSICAL ACTIONS OF...
PR ISOFORMS ARE PHOSPHOPROTEINS
 MULTIPLE FUNCTIONS OF PR...
 NOVEL EXTRANUCLEAR ACTIONS OF...
 CONCLUDING REMARKS
 REFERENCES
 
THE ACTION OF THE OVARIAN steroid hormone, progesterone, is mediated through binding to its cognate receptors; the full length B- and N-terminally-truncated A isoforms of the PR are members of the type I family of nuclear hormone receptors and are classically defined as ligand-activated transcription factors (1). In the absence of hormone, PRs are complexed with several chaperone molecules including heat shock protein (hsp) 90, hsp70, hsp40, Hop, and p23; these interactions are requisite for proper protein folding and assembly of stable steroid receptor-hsp90 heterocomplexes that are competent to bind ligand (2, 3). hsps also function to connect steroid receptors to protein trafficking systems. Upon exposure to progesterone, the ligand-activated receptor undergoes a conformational change, dissociates from hsps, dimerizes, and can directly interact with specific progesterone response elements (PREs) in the promoter regions of target genes, including c-myc (4), fatty acid synthetase (5), and the mouse mammary tumor virus promoter (6, 7). However, treatment with progesterone also leads to an up-regulation of key regulatory molecules such as cyclin D1 (8), signal transducer and activator of transcription 5, c-fos, and p21 (9) that do not contain a classical PRE in their promoter regions. Without canonical PREs, progesterone regulation of these genes may occur through nonclassical mechanisms, as has been reported in the case of PR tethering to the transcription factor Sp1 to promote p21 transcription in the presence of progestin (10). When bound to DNA, either directly or otherwise, PRs interact with components of the basal transcription machinery, assisted by nuclear receptor coregulatory molecules, including steroid receptor coactivators (SRCs), E3 ubiquitin-protein ligases, thyroid receptor-associated proteins (known as DRIPs; vitamin D receptor-interacting proteins), p300/CBP, and transcription intermediary factors (11, 12). Coactivator molecules interact with liganded nuclear receptors via conserved LXXLL amphipathic helix or nuclear-receptor box motifs, which make initial contacts with several helices (notably helix 12) in the activation function (AF)-2 (activation function) region of the ligand binding-domain of the nuclear receptor (12). The regulation of steroid hormone receptors by coactivators has been expertly reviewed (12, 13).


   PR ISOFORMS ARE PHOSPHOPROTEINS
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 ABSTRACT
 CLASSICAL ACTIONS OF...
 PR ISOFORMS ARE PHOSPHOPROTEINS
MULTIPLE FUNCTIONS OF PR...
 NOVEL EXTRANUCLEAR ACTIONS OF...
 CONCLUDING REMARKS
 REFERENCES
 
Phosphorylation-dephosphorylation events provide an added level of complexity to PR action. PRs exist as either A (97 kDa) or B (120 kDa) isoforms created from the same gene by the use of alternate promoters (Fig. 1Go) (14). Each isoform contains a C-terminal hormone-binding domain, a DNA-binding domain, a hinge region (H), and at least two transcriptional activation function (AF) domains, located within the hormone-binding domain (AF-2) and N termini (AF-1); PR-B contains an additional AF (AF-3) within the unique 164-amino acid B-upstream segment. Like other steroid hormone receptor family members, PR isoforms are heavily phosphorylated by multiple protein kinases; phosphorylation occurs primarily on serine residues throughout each molecule but is concentrated within the amino termini (15, 16). PRs contain a total of 14 known phosphorylation sites (17, 18, 19, 20). Serines at positions 81, 162, 190, and 400 are defined as "basal" sites (19), constitutively phosphorylated in the absence of hormone (Fig. 1Go). Serines 102, 294, and 345 are hormone-induced sites that are maximally phosphorylated 1–2 h after progestin treatment (18). Specific kinases responsible for phosphorylation of selected sites have been identified, whereas others remain unknown. For example, the serines at positions 81 and 294 have been demonstrated to be phosphorylated by casein kinase II (17) and MAPK (21, 22), respectively; progestins can also stimulate Ser294 phosphorylation independently of MAPKs by activation of an unknown kinase(s) (23). Eight of the total 14 sites (serines 25, 162, 190, 213, and 400; Thr 430, 554, and 676) have been demonstrated to be phosphorylated by cyclin A/cyclin-dependent protein kinase 2 (CDK2) complexes in vitro (19, 20). Five of these sites (serines 162, 190, 213, 400, 676) have been confirmed as authentic in vivo phosphorylation sites (17, 19, 20).



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  		Fig. 1. Phosphorylation Sites in Human PR

Thirteen serine residues and one threonine residue in human PR have been shown to represent basal (constitutive) and hormone-induced phosphorylation sites (20 ) and may contribute to PR regulation by MAPK (21 22 23 ), casein kinase II (17 ), and CDK2 (19 20 ). Individual PR phosphorylation sites may be regulated by multiple protein kinases (23 ) and/or in a sequential manner (97 ), illustrating the complexity of PR regulation by phosphorylation.

 
Although the role of PR phosphorylation is not fully understood, it may influence aspects of transcriptional regulation such as interaction with coregulators, as has been found for estrogen receptor (ER){alpha} (24) and recently for PR (25). Regulation of ligand-dependent (22) and -independent (26) PR transcriptional activities, and receptor turnover (21) have all been shown to involve phosphorylation. Recently, MAPK-dependent PR Ser294 phosphorylation has been shown to be required for rapid nuclear translocation of unliganded PR, suggesting that MAPK signaling may regulate PR action by altering nucleo-cytoplasmic shuttling (23). The functional significance of PR nuclear sequestration in response to MAPK activation is unknown. However, it is tempting to speculate that it may serve to concentrate inactive receptors, as PRs are degraded in the cytoplasm (23). Alternatively, unliganded phosphorylated PR may regulate genes via nonclassical mechanisms. For example, Bamberger et al. (27) demonstrated ligand-independent transcriptional activity of PR using an activating protein-1-driven promoter in human endometrial adenocarcinoma cells; addition of progestins blunted this effect.

Phosphorylation is generally accepted as a positive regulator of steroid receptor function and may serve to integrate signals initiated by growth factors with responses to steroid hormones in endocrine tissues. As has been reported for ER{alpha} (28, 29), phosphorylated PRs are ultrasensitive to subphysiological levels (0.1 nM range) of progestin relative to their underphosphorylated counterparts (30). This may explain, in part, how epidermal growth factor (EGF) potentiates the proliferative effects of progesterone and estrogen and causes ductal side branching and lobuloalveolar development of the mature mammary gland (31). In addition, EGF and progestins synergistically up-regulate mRNA or protein levels for a number of growth-regulatory genes (9), including cyclin D1 and cyclin E (32); the regulation of cyclins by progestins is MAPK dependent. Cyclins, in turn, regulate progression of cells through the cell cycle by interaction with CDKs. Progestins activate CDK2 (8), and PRs are predominantly phosphorylated by CDK2 at proline-directed (S/TP) sites (19, 20), perhaps allowing for the coordinate regulation of PR action during cell cycle progression. In support of this idea, Narayanan and co-workers (25) have recently found that PR activity is highest in S phase and lower in the G0/G1 phases of the cell cycle, but impaired during G2/M concomitant with lowered PR phosphorylation. Overexpression of either cyclin A or CDK2 enhanced both PR and androgen receptor transcriptional activity; whereas cyclin A interacts with the N terminus of PR, CDK2 seems to alter PR function indirectly by increasing the recruitment of SRC-1 to liganded PR (25). These exciting results await further investigation aimed at understanding the details of PR regulation by cyclin-CDK complexes.


   MULTIPLE FUNCTIONS OF PR Ser294 PHOSPHORYLATION
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 ABSTRACT
 CLASSICAL ACTIONS OF...
 PR ISOFORMS ARE PHOSPHOPROTEINS
 MULTIPLE FUNCTIONS OF PR...
NOVEL EXTRANUCLEAR ACTIONS OF...
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PR Ser294 is a ligand-inducible phosphorylation site, becoming rapidly phosphorylated upon exposure to hormone (18). Ser294 is also a proline-directed or MAPK consensus site (PXXSP), and extensive studies have uncovered a role for MAPK phosphorylation in the regulation of numerous functions of PR (21, 22, 23). In contrast to progestin-induced Ser294 phosphorylation, which occurs within 30–60 min independently of MAPK activation, growth factor-induced Ser294 phosphorylation occurs within 3–5 min and is MAPK dependent (23). Ser294 phosphorylation is required to sustain transcriptional synergy in the presence of progestins and growth factors (22), and to signal for ligand-induced receptor down-regulation by the ubiquitin-proteasome pathway (21). After ligand binding, PR undergo rapid down-regulation (33). This process is dependent on phosphorylation of Ser294, which targets liganded PR for ubiquitination and degradation by the 26S-proteosome pathway (21). Mutant PR, in which Ser294 has been replaced by alanine (S294A), binds ligand and like wild-type (wt) PR, undergoes a characteristic upshift in gel mobility (due to phosphorylation at other sites), enters the nucleus, and binds to PRE elements (21, 22, 23, 30). However, it fails to be ubiquitinated and is thus highly stable in the presence of progestins relative to wt PR (21). Phosphorylation of PR Ser294 by MAPK also induces greatly increased transcriptional activity of liganded PR at PRE-containing promoters (22). Interestingly, liganded mutant S294A PR is a weak transcriptional activator when stably expressed in breast cancer cells, and does not undergo synergistic regulation in response to agents that activate MAPK (22). Thus, phosphorylation of PR Ser294 couples increased transcriptional activity to rapid down-regulation of PR protein by the ubiquitin-proteosome pathway.

Indeed, the classic view of proteasome-mediated protein down-regulation predicts that ubiquitination serves to tag regulatory proteins for destruction by the multisubunit proteasome complex to rapidly attenuate the signal. However, several recent studies suggest other exciting functional roles for ubiquitination and/or proteasome subunits as regulators of transcription. Salghetti et al. (34, 35) first noted overlap between the activation domains and destruction elements or "degrons" of several unstable transcription factors, including E2F-1, fos, jun, and p53. Furthermore, a close correlation exists between the ability of an acidic activation domain to both activate transcription and signal proteolysis. Phosphorylation is a prerequisite for degron function of the yeast cyclins, Cln2 (36) and Cln3 (37). These elements activated transcription when fused to a DNA-binding domain (35). Thus, short-lived transcription factors may be rapidly destroyed because of their ability to efficiently activate transcription, perhaps through a common cellular machinery triggered by phosphorylation events. In support of this idea, the ubiquitin-protein ligases, hRPF1 (38) and E6-AP (39), have been shown to function as coactivators for liganded steroid hormone receptors, including PR. The Met30 ubiquitin-ligase is required for the transcriptional activity of the VP16 transcriptional activation domain; fusion of ubiquitin to the VP16 activator bypassed this requirement, indicating that ubiquitination is essential for transcriptional activation (40). Most recently, the related p300/CBP transcriptional coactivators originally identified as enzymes that acetylate histones and other proteins were shown to possess ubiquitin-ligase activity (41). It will be important to define the role of phosphorylation/dephosphorylation events and their prevalence in recruitment of coactivators to liganded steroid receptor transcription factors.

Phosphorylation of Ser294 may also mediate some aspects of ligand-independent PR action. Growth factors, such as EGF, are strong activators of p42/p44 MAPKs, and interestingly, within 5 min of EGF treatment but in the absence of progestins, PR Ser294 becomes phosphorylated, and rapid nuclear accumulation of PR occurs, as measured by both fluorescence microscopy of intact cells and cellular fractionation experiments (23). Mutation of the consensus MAPK site, Ser294 to Ala (S294A), abolished EGF-mediated translocation; however, the ability of progestin (R5020) to induce nuclear localization of S294A PR was unaffected. Thus, the nuclear localization of the PR is regulated by multiple mechanisms; progestin-induced translocation is independent of Ser294 phosphorylation and MAPKs. In contrast, EGF-induced nuclear accumulation requires p42/p44 MAPK activation and phosphorylation of Ser294, and occurs independently of progestins, suggesting a mechanism for ligand-independent transcriptional activation of PR. Ligand-independent PR effects in neuroendocrine cells have been shown to mediate sexual behavior in rodents in response to dopamine (42, 43); dopaminergic ligand-independent action of chick PR maps to a required serine residue (44). Additionally, Labriola et al. (26) have recently reported that the EGF family member, heregulin, can stimulate nuclear localization, DNA binding, and transcriptional activity of PR in T47D breast cancer cells in the absence of hormone. This was accompanied by activation of MAPK and PR Ser294 phosphorylation. Qiu et al. (23) reported that similar changes map to PR Ser294 phosphorylation, but growth factors alone did not stimulate PR transcriptional activity or alter PR down-regulation in T47D cell variants (22). These events required ligand binding, but were greatly augmented by MAPK activation (22, 23). The reasons for these differences are unknown, but may involve differential expression of EGF receptor family members expressed on the cell surface between T47D cell line clones, leading to differences in the activation of downstream intracellular kinases. In any case, these exciting data (23, 26) suggest that regulation of the PR by alternate signaling pathways, including the elevated MAPK activity, often exhibited by breast tumors, is likely to contribute to disregulated gene expression and changes in cell growth.

CDK2 REGULATION OF LIGAND-DEPENDENT AND -INDEPENDENT PR TRANSCRIPTIONAL ACTIVITIES: ROLE OF Ser400
In contrast to Ser294, Ser400 of human PR is both basally phosphorylated and regulated by ligand in vivo, and Ser400 phosphorylation has been demonstrated to be mediated by CDK2 in vitro (19). Like mitogens, progestins regulate CDK2 activity (8, 45). Similarly, recent evidence suggests that, in addition to progestins, mitogenic stimuli also induce rapid and robust phosphorylation of PR Ser400 (46). The functional consequence of PR Ser400 phosphorylation by CDK2 is unclear. However, this event suggests a mechanism of cell cycle-dependent regulation of PR. In contrast to Ser294, phospho-Ser400 PRs undergo delayed ligand-induced down-regulation relative to PRs that are not phosphorylated on Ser400, suggesting that phosphorylation of Ser400 may stabilize PR protein (46). In addition, CDK2 overexpression increased PR transcriptional activity in the presence or absence of ligand. Mutation of Ser400 to alanine selectively blocked the increase in PR transcriptional activity observed in the absence of ligand but had little effect on that induced by progestin binding (46). This result suggests that CDK2 may positively regulate unliganded PR (i.e. basal transcriptional activity) by acting at Ser400 and exert positive effects in the presence of ligand, perhaps in cooperation with other CDK2 sites on PR (47) or its coactivators (25). Interestingly, Ser400 is adjacent to a nine-amino acid destruction (D)-box motif, indicating that alteration of PR turnover may be a primary means by which PR transcriptional activities are regulated in response to phosphorylation (22). Whereas the role of CDK2 and PR Ser400 in the control of PR stability/turnover and transcriptional activity awaits further examination, it is clear that modulation of protein turnover is emerging as a key means by which nuclear receptors are regulated (48). In addition to PR (22), an inverse relationship has been demonstrated between receptor stability and transcriptional activity for ER{alpha} (49, 50, 51) and thyroid receptor (52), whereas glucocorticoid receptor (GR) does not appear to fall into this category (53, 54). Similar to human PR, the phosphorylation status of mouse GR has a profound effect on the stability of the receptor protein: GR containing seven or eight mutated serine-phospho sites have a markedly extended half-life and do not show the ligand-dependent destabilization seen with wt receptor (53). However, in contrast to PR, the transcriptional activity of human GR was enhanced by proteasome inhibition, and mutation of Lys426 within an N-terminal proline, glutamic acid, serine, and threonine (PEST) element both abrogated ligand-dependent down-regulation of GR protein and simultaneously enhanced GR-induced transcriptional activity (54).


   NOVEL EXTRANUCLEAR ACTIONS OF PR
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 ABSTRACT
 CLASSICAL ACTIONS OF...
 PR ISOFORMS ARE PHOSPHOPROTEINS
 MULTIPLE FUNCTIONS OF PR...
 NOVEL EXTRANUCLEAR ACTIONS OF...
CONCLUDING REMARKS
 REFERENCES
 
In contrast to the classically defined function of PRs as ligand-activated nuclear transcription factors, studies of amphibian oocyte maturation make it clear that PRs can also mediate the activation of cytoplasmic signaling pathways. Progesterone induces germinal vesicle breakdown and activation of phospholipases C and the cell cycle kinase Cdc2 in enucleated amphibian eggs (55, 56, 57, 58, 59). These effects appear to be mediated through a cytosolic PR that has transcriptional activity, although transcription is not required for germinal vesicle breakdown (60, 61). Instead, evidence suggests that progesterone acts through the amphibian PR to activate kinase pathways that, in turn, mediate progesterone’s oocyte maturation activity. Briefly, progesterone activates the Eg2 protein kinase that phosphorylates cytoplasmique polyadenylation element binding protein (CPEB), a protein bound to the 3'-untranslated region of several mRNAs including Mos (amphibian MAPK/ERK kinase kinase) mRNA; CPEB phosphorylation stimulates Mos mRNA translational activation, and newly translated Mos activates MAPK/ERK kinase (MEK), leading to MAPK activation. This results in the activation of the cyclin B-Cdc-2 complex, which catalyzes entry into M phase of meiosis I (reviewed in Ref. 59). Expression of exogenous constitutively active MEK causes oocyte maturation in the absence of progesterone, and antibodies that bind MEK block progesterone-mediated maturation (62, 63). Additionally, progesterone activates MAPK and c-jun N-terminal kinase in amphibian oocytes, and exogenous PR accelerates MAPK activation in enucleated oocytes (60, 64). Amphibian PR has also been shown to physically associate with cytosolic kinases, including p42 MAPK and phosphatidylinositol-3 kinase (65). These data suggest that in nonmammalian systems, PR is able to regulate cell cycle progression by kinase signaling independently of its transcriptional activity. Interestingly, a novel membrane progesterone receptor (mPR) gene was recently cloned from spotted sea trout ovaries, and structural analyses indicates that it encodes a seven transmembrane-spanning G protein-coupled receptor (66). Breast cancer cells transfected with the fish mPR demonstrated progestin-induced MAPK activation and inhibition of cAMP production that was pertussis toxin sensitive. The mPR in fish oocytes was up-regulated in response to progestin treatment, suggesting a role for nuclear PR in this process (66). The relative contributions of nuclear PR vs. mPR to oocyte maturation are unknown.

PR can also function as a signaling molecule independently of its transcriptional activity in mammalian systems (Fig. 2Go). Indeed, the extranuclear actions of steroid hormone receptors have become an intense area of investigation, particularly in breast cancer cell models (67, 68, 69). Migliaccio et al. (70) were the first to report p60-Src kinase and MAPK activation by estradiol in MCF-7 cells and interaction between PR, ER, and p60-Src kinase in T47D cells (71). Progestin treatment of breast cancer cells causes a rapid and transient activation of MAPK signaling that is PR dependent but independent of PR transcriptional activity (71, 72). Whereas the genomic effects of progestin treatment are latent (i.e. several minutes to hours after transcription and translation), the extranuclear or nongenomic effects occur rapidly (few minutes). For example, maximal activation of p60-Src kinase is observed within 2–5 min, and downstream activation of p42/p44 MAPKs occurs within 5–10 min of progestin treatment (71, 72). Human PR contains a proline-rich (PXXP) motif that mediates direct binding to the Src homology 3 (SH3) domains of signaling molecules in the p60-Src kinase family in a ligand-dependent manner (72). Purified liganded PR-A and PR-B activated the Src-related protein kinase, HcK in vitro. Mutation of the PXXP sequence in PR abolished the ability of progestins to bind c-Src and activate both c-Src (or HcK) and p42/p44 MAPKs, indicating that MAPKs are likely activated by a c-Src-dependent mechanism involving Ras activation via phosphorylation of the c-Src substrates p190 and Shc, followed by Grb-2 and Sos binding. Mutation of the PR DNA-binding domain abolished PR transcriptional activity but had no effect on c-Src or MAPK kinase activation. A polyproline-rich sequence in the androgen receptor interacts with c-Src by a similar mechanism (73).



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  		Fig. 2. Functional Significance of PR Phosphorylation

Phosphorylation (P) of specific sites in PR couples multiple PR functions, including nuclear localization (shuttling) in response to MAPK activation, transcriptional synergy in the presence of progestins and growth factors predicted to activate MAPK and/or CDK2, and rapid ligand-dependent PR down-regulation by the ubiquitin-proteasome pathway (degradation). Phosphorylated PR may recruit regulatory molecules that function in one or more of these interconnected processes, perhaps linked by a common cellular machinery. PR and growth factors activate MAPKs independently, and this may result in positive regulation of PR action via feed-back regulation (i.e. direct phosphorylation of PR), occurring in both the absence and presence of steroid hormone ligands and on PRE-containing or other PR-regulated gene promoters. Activation of MAPKs by PR provides for regulation of gene targets the promoters of which do not contain PREs and are otherwise independent of PR-transcriptional activities but utilize PR-activated MAPKs, such as regulation of the cyclin D1 promoter by Ets factors.

 
Ballare et al. (74) recently reported that MAPK activation by progestins is blocked by antiprogestins and antiestrogens in COS-7 cells transfected with both PR and ER. They propose that c-Src/MAPK activation by PR is mediated indirectly by the interaction of the Src-homology (SH2) domain of c-Src with phosphotyrosine 537 of ER{alpha} (74). In their model, activation of c-Src and the MAPK pathway by progestins depends upon the presence of unliganded ER{alpha}, which interacts with PR-B via two domains that flank PR’s proline-rich sequence. In contrast to the studies conducted by Boonyaratanakornkit et al. (72), Ballare and co-workers (74) found that deletion of either of these two ER interacting domains in PR-B, but not mutation of its PXXP domain, blocked c-Src/MAPK activation by progestins in the presence of ER{alpha}. However, Boonyaratanakornkit et al. (72) found that PR expression increased c-Src activity in COS-7 cells in the absence of progestins and independently of added ER; coexpression of both PR and ER{alpha} reduced basal levels of c-Src activity, enabling a clear stimulation of c-Src by progestin. In addition, progestins activated c-Src in PR-null MCF12A cells transduced with wt PR but not PXXP-mutant PR adenoviruses. Although the reasons for these discrepancies must be resolved, it is possible that overexpression of steroid receptors in COS-7 cells leads to concentration-dependent effects resulting in the formation of subtly different signaling complexes that depend on the presence of other signaling and adaptor molecules. In support of this idea, Wong et al. (75) have recently identified an additional ER-interacting adaptor protein, termed "modulator of nongenomic activity of ER," that contains both LXXLL (nuclear receptor binding) and PXXP (SH3-domain binding) motifs and is essential for the ER-Src interaction; modulator of nongenomic activity of ER is not required for progestin/PR-dependent activation of c-Src (Edwards, D. P., personal communication). Taken together, these data indicate that multiple and complex interactions contribute to direct protein kinase activation by steroid hormone receptors and suggest that at least some of the nongenomic signaling functions of amphibian PR have been conserved in mammals. A role for a separate gene product encoding the putative mammalian homolog of mPR (76) remains to be determined.

The physiological role(s) of steroid hormone receptor-mediated activation of cytoplasmic signaling molecules is not yet clear in mammalian systems but could theoretically serve to potentiate several functions of PR (Fig. 2Go). One prospective role of this cytoplasmic activation is to provide a mechanism for rapid, direct phosphorylation of PR coincident with ligand binding, thus allowing a form of PR-induced amplification of the signal or positive feedback. This feedback has a dramatic influence on PR function (Fig. 2Go). In addition to MAPK-dependent regulation of PR subcellular localization in the absence of ligand (23), several progestin-dependent functions of PR are also MAPK-dependent (21, 22). It is interesting to speculate that the extranuclear actions of progestins may contribute to disregulated breast cancer cell growth (8, 77) and/or increased breast cancer risk (78), perhaps by linking progestin action to the regulation of MAPK-regulated genes (discussed below). In support of this idea, microinjection of breast cancer cell lines with cDNA of catalytically inactive c-Src or anti-Ras antibodies blocked estradiol and progestin-dependent cell cycle progression (79). In addition, the extranuclear actions of liganded ER{alpha} are thought to induce a state of adaptive hypersensitivity during endocrine therapy in which growth factor signaling pathways are co-opted by up-regulated ER{alpha} (80). In this model of ER-dependent MAPK activation, liganded cell membrane-associated ER{alpha} molecules interact with the adapter protein Shc and induce its phosphorylation, leading to recruitment of Grb-2 and Sos, followed by activation of Ras and the Raf-1/MEK/MAPK module. ER{alpha} activation of MAPK may explain why many tumors respond well to aromatase inhibitors, but not selective ER modulators, which inhibit ER transcriptional activity, but can act as partial agonists and may not block ER-dependent MAPK activation (80). The role of cytoplasmic signaling cascades and estrogen action has been expertly reviewed (81, 82, 83).

PR REGULATION OF MAPK-DEPENDENT GENE PROMOTERS: STUDIES WITH CYCLIN D1
In addition to the direct regulation of gene transcription by PR (i.e. the classical pathway), rapid activation of cytoplasmic signaling cascades by liganded PR may provide for a separate mechanism by which transcriptional regulation in the nucleus is achieved (Fig. 2Go). For example, growth factor stimulation of the p42/p44 MAPK signaling module results in phosphorylation and activation of numerous transcription factors, including c-Ets-2, a member of the large family of ETS transcription factors. ETS proteins promote G1 phase progression by the regulation of a number of genes involved in control of cell proliferation, including the cell cycle regulator cyclin D1 (84). Cyclin D1 activates and defines the substrate specificity of CDKs 4 and 6, whose activity is required for progression from G1 to S phase (85, 86). CDKs are inactive in the absence of cyclins; thus the availability or abundance of cyclin D1 is a major determinant for cell cycle progression through G1 (87). P42 and p44 MAPKs can also function posttranslationally to regulate the assembly and activation of cyclin D1/CDK complexes (88).

Cyclin D1 is ubiquitously expressed in most cell types; however, higher protein levels are found in normal breast epithelial cells, and cyclin D1 expression is elevated in approximately 45% of breast tumor samples (89). Interestingly, cyclin D1 null mice exhibit deficiencies in mammary gland development, including specific defects in alveolar growth (90, 91), a phenotype similar to that exhibited in adult female mice lacking PR (92, 93). Recently, the oncogenic effects of cyclin D1 overexpression were found to be mediated by a novel interaction with the transcription factor CCAAT enhancer binding protein (94). CCAAT enhancer binding protein-ß knockout mice also exhibit similarly compromised mammary alveolar development (95, 96), indicating the importance of coordinate expression of these gene products.

The cyclin D1 promoter is complex, containing multiple target elements for transcription factors inputs, but lacks a canonical PRE. Cyclin D1 protein levels, however, do increase in response to progestin treatment alone (8) and are synergistically regulated by progestins and growth factors in a MAPK-dependent manner (32, 77), a phenomenon that may partially explain the mitogenic actions of progestins. Transcriptional regulation of the cyclin D1 promoter by progestins is also MAPK dependent (Faivre, E., and C. Lange, unpublished results). Thus, the activation of cytoplasmic signaling pathways by liganded PR provides both enhanced PR action at specific PR-regulated genes (9, 22) and couples this to the regulation of additional growth-regulatory genes, the promoters of which function independently of PR but utilize PR-activated MAPK pathways (Fig. 2Go).


   CONCLUDING REMARKS
TOP
 ABSTRACT
 CLASSICAL ACTIONS OF...
 PR ISOFORMS ARE PHOSPHOPROTEINS
 MULTIPLE FUNCTIONS OF PR...
 NOVEL EXTRANUCLEAR ACTIONS OF...
 CONCLUDING REMARKS
REFERENCES
 
Rather than acting in an obligatory or switch-like manner, phosphorylation is generally thought to exert subtle effects on nuclear steroid hormone receptors. However, one caveat of this conclusion is that it is based largely on observations made with liganded receptors in the absence of controlled activation of alternate signaling pathways. Transcriptional readouts have focused primarily on steroid receptor function at cognate response elements as part of artificial promoter-reporter gene contexts in which maximal reactions are driven. Indeed, studies with human PR reviewed herein suggest that the effects of phosphorylation are somewhat more profound in the context of multiple signaling inputs. Perhaps a more accurate conclusion is that the phosphorylation status of a particular steroid hormone receptor serves as a direct sensor to cellular kinase activities to coordinate responses to growth factors and steroid hormones. In the absence of alternate stimuli, independent activation of MAPKs by extranuclear liganded steroid hormone receptors may result in positive regulation of receptor action via feed-back regulation by direct phosphorylation. Theoretically, this may occur in both the presence and absence of steroid hormone ligands and on diverse gene promoters. In addition, activation of cytoplasmic kinase cascades including MAPK modules by liganded receptors provides for regulation of gene targets the promoters of which are entirely independent of steroid receptor transcriptional activities, but rely on the activity of classical MAPK-targeted transcription factors such as the Ets family members, Elk-1, c-myc, fos, and jun. This important linkage provides for well-integrated control of a large number of genes or gene subsets coordinately regulated in response to convergence of growth factor and steroid hormone receptor signaling. Finally, the newly discovered ability of steroid receptors to activate kinase pathways classically defined as key regulators of cell growth is likely to play an important role in the development of resistance to endocrine therapies (80).


   ACKNOWLEDGMENTS
 
I thank Emily Faivre, Lisa Piers-Mullany, Abby Olsen, Andy Skildum, and Doug Yee for helpful comments during the writing of this manuscript.


   FOOTNOTES
 
This work was supported by NIH Grant DK53825.

Abbreviations: AF, Activation function; CBP, cAMP responsive element binding protein (CREB)-binding protein; CDK2, cyclin-dependent protein kinase 2; EGF, epidermal growth factor; ER, estrogen receptor; GR, glucocorticoid receptor; H, hinge; Hsp, heat shock protein; MEK, MAPK/ERK kinase; mPR, membrane progesterone receptor; PR, progesterone receptor; PRE, progesterone response element; SH2, Src-homology 2 domain (interaction with phospho-tyrosine residues); SH3, Src-homology 3 domain (interaction with proline-rich regions); SRC, steroid receptor coactivator; wt, wild-type.

Received for publication August 29, 2003. Accepted for publication October 6, 2003.


   REFERENCES
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 ABSTRACT
 CLASSICAL ACTIONS OF...
 PR ISOFORMS ARE PHOSPHOPROTEINS
 MULTIPLE FUNCTIONS OF PR...
 NOVEL EXTRANUCLEAR ACTIONS OF...
 CONCLUDING REMARKS
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NURSA Molecule Pages Link:

Nuclear Receptors:   PR
Coregulators:   SRC-1  |  PELP1
Ligands:   Progesterone



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