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Proc Natl Acad Sci U S A. Oct 31, 2006; 103(44): 16272–16277.
Published online Oct 19, 2006. doi:  10.1073/pnas.0603002103
PMCID: PMC1637572
Cell Biology

Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone

Abstract

cAMP is required for differentiation of human endometrial stromal cells (HESCs) into decidual cells in response to progesterone, although the underlying mechanism is not well understood. We now demonstrate that cAMP signaling attenuates ligand-dependent sumoylation of the progesterone receptor (PR) in HESCs. In fact, decidualization is associated with global hyposumoylation and redistribution of small ubiquitin-like modifier (SUMO)-1 conjugates into distinct nuclear foci. This altered pattern of global sumoylation was not attributable to impaired maturation of SUMO-1 precursor or altered expression of E1 (SAE1/SEA2) or E2 (Ubc9) enzymes but coincided with profound changes in the expression of E3 ligases and SUMO-specific proteases. Down-regulation of several members of the protein inhibitors of activated STAT (PIAS) family upon decidualization pointed toward a role of these E3 ligases in PR sumoylation. We demonstrate that PIAS1 interacts with the PR and serves as its E3 SUMO ligase upon activation of the receptor. Furthermore, we show that silencing of PIAS1 not only enhances PR-dependent transcription but also induces expression of prolactin, a decidual marker gene, in progestin-treated HESCs without the need of simultaneous activation of the cAMP pathway. Our findings demonstrate how dynamic changes in the SUMO pathway mediated by cAMP signaling determine the endometrial response to progesterone.

Keywords: endometrium, protein inhibitor of activated STAT 1, decidualization, progesterone receptor

The success of pregnancy depends on the growth and protection of the semiallogenic fetus within the maternal uterine environment. The maternal response to pregnancy is characterized by profound morphological and biochemical differentiation of the endometrial stromal cells into decidual cells (1, 2). In humans, the decidual process is initiated during the midsecretory phase of the menstrual cycle in response to elevated progesterone levels (1, 2). Progesterone exerts its actions on endometrial cells predominantly through activation of the progesterone receptor (PR), a member of the superfamily of ligand-activated transcription factors. Two isoforms exist, PR-A and PR-B, which arise from different promoter usage in a single gene. PR-A, which lacks the N-terminal 164 aa of PR-B, is the predominant isoform in differentiating human endometrial stromal cells (HESCs) (3). Furthermore, gene ablation studies in mice have shown that PR-A, in contrast to PR-B, is indispensable for decidualization (4).

Although endometrial differentiation requires activation of PR-A, expression of decidua-specific genes in vivo is only apparent ≈10 days after the postovulatory rise in circulating progesterone levels (2, 5, 6). Similarly, progesterone, alone or in combination with oestradiol, induces decidualization of HESCs in culture but again this process requires at least 8–10 days of stimulation. Progesterone-dependent HESC differentiation coincides with a gradual increase in intracellular cAMP levels and is abrogated in the presence of a PKA inhibitor (5). Conversely, treatment of primary cultures with factors capable of activating the PKA pathway sensitizes HESCs to progesterone signaling, leading to rapid and sustained expression of the decidual phenotype (2, 7, 8). These observations demonstrate that decidualization requires convergence of cAMP and progesterone signaling pathways.

Like many other transcription factors and cofactors, PR-A is subject to small ubiquitin-like modifier (SUMO)-1 modification upon activation (9). SUMO proteins (SUMO-1, -2, -3, and -4) are posttranslational modifiers whose dynamic and reversible attachment to other proteins proceeds through a sequence of enzymatically directed steps (10). First, proteolytic cleavage of four C-terminal amino acids from the precursor gives rise to mature SUMO-1. The E1 activating enzyme, a heterodimer consisting of SAE1/SAE2, then binds the modifier and transfers it in an ATP-dependent manner to the E2 conjugase, Ubc9. Ubc9 has the ability to convalently conjugate SUMO at lysine residues of target proteins. Conjugation, however, is aided by the activity of E3 ligases, which include the protein inhibitor of activated STAT (PIAS) family of proteins, RanBP2 and Pc2 (1113). Besides stimulating the rate of SUMO-1 modification, E3 ligases also confer substrate specificity to the reaction. Members of the SUMO-specific protease (SENP) family of SUMO-specific isopeptidases can reverse the reaction by cleaving SUMO-1 from its substrates, while being the same enzymes responsible for the initial maturation step of the SUMO cycle (10).

Sumoylation profoundly modulates the function of many diverse target proteins by altering protein stability, protein–protein interactions, and cellular localization. Sumoylation of transcription factors, including PR, generally bestows repressive properties, although there are some exceptions (9, 14, 15). We now demonstrate that cAMP sensitizes HESCs to progesterone, at least in part, by attenuating PR-A sumoylation. We further show that cAMP and progesterone signaling induces profound changes in the expression of E3 ligases and SENPs in decidualizing cells. As a consequence, hyposumoylation upon endometrial differentiation is not restricted to PR but involves many target proteins, indicating that dynamic changes in the SUMO cycle play an integral part in decidual reprogramming of HESCs.

Results

cAMP Attenuates Hormone-Dependent Sumoylation of PR-A.

We sought to determine whether activation of the cAMP pathway in HESCs impacts the sumoylation status of PR-A. To test this, primary cultures were cotransfected with expression vectors that encode for EGFP-tagged SUMO-1, WT PR-A, or a PR-A mutant in which the lysine required for covalent binding of SUMO-1 was replaced by an arginine (PR-AK388R) (9). Subsequently, cells remained untreated or were treated for 72 h with a cAMP analogue [8-bromo-cAMP (8-br-cAMP)], a progestin [medroxyprogesterone acetate (MPA)], either alone or in combination. Immunoblotting of cell lysates with an anti-PR antibody demonstrated the presence of a slower migrating form of PR-A in MPA-treated cultures transfected with WT PR-A (Fig. 1A). This band was not detected in cells transfected with PR-AK388R mutant, indicating that the higher molecular weight species consists of EGFP-SUMO-1-modified receptor. PR-A was not sumoylated in untreated cells or cells treated with 8-br-cAMP alone. However, 8-br-cAMP markedly inhibited MPA-dependent PR-A sumoylation (Fig. 1A Top). Notably, exogenous expression of both receptor and modifier was required for detecting sumomoylation because we did not observe modification of the endogenous PR-A or PR-B (Fig. 1A and data not shown).

Fig. 1.
Attenuated sumoylation in decidual HESCs enhances PR activity. (A) WT PR-A or K388R mutant (400 ng) were cotransfected with EGFP-SUMO-1 (200 ng) in primary HESCs. Cultures were treated with cAMP and MPA as indicated and harvested 72 h later. Whole-cell ...

Activation of the cAMP pathway is known to enhance the transcriptional activity of PR in many cell systems, including HESCs (2, 6). To determine whether sumoylation of the receptor is implicated in this process, primary cultures were transfected with a luciferase reporter gene driven by two consensus palindromic PR response elements (PREs) and WT PR-A or PR-AK388R. Subsequently, cultures were treated with 8-br-cAMP and/or MPA for 72 h. As shown in Fig. 1B, the K388R mutation in PR-A greatly enhanced its activity upon hormone binding. Activation of the cAMP pathway further enhanced the transcriptional activity of the liganded SUMO-deficient receptor (2.1-fold) but consistently less so when compared with the WT PR-A (3.6-fold). Together, the data suggest attenuated sumoylation as one of the mechanisms whereby cAMP enhances progesterone-dependent transcription in HESCs.

Expression of SUMO Pathway Enzymes Is Regulated upon HESC Decidualization.

Interestingly, the changes in PR sumoylation observed in cells treated with 8-br-cAMP or MPA for 72 h took place against a wider background of altered SUMO-1 conjugation. As shown in Fig. 1A Middle, most SUMO-1 in undifferentiated cells was conjugated to a variety of proteins with little or no unconjugated modifier. Upon decidualization of cells with 8-br-cAMP and MPA there was a loss of high molecular weigh conjugates and simultaneous accumulation of free SUMO-1. This effect was also observed in cells treated with only 8-br-cAMP but not in cultures treated with only MPA. Time-course analysis of endogenous SUMO conjugates revealed that global desumoylation upon HESC differentiation is a gradual process, apparent upon 48 h of treatment with 8-br-cAMP and MPA (Fig. 2A Upper). Dot blot analysis did not reveal a change in total SUMO-1 levels (Fig. 2A Lower), suggesting that decidualization is associated with altered utilization rather than reduced expression of this modifier. In support of this idea, we found little or no change in the abundance of SUMO-1 transcripts in HESCs treated with 8-br-cAMP and MPA over an 8-day time course (data not shown).

Fig. 2.
Regulated expression of the SUMO pathway enzymes during HESC decidualization. (A) Primary HESC cultures were kept untreated or stimulated with 8-br-cAMP and MPA for 2, 4, or 8 days. Whole-cell lysates were subjected to Western blot (Upper) and dot blot ...

Conjugation of SUMO-1 first requires its maturation from a precursor form by proteolytic cleavage of four C-terminal amino acids (HSTV), a role undertaken by SENPs (16). We reasoned that reduced ability to mature SUMO-1 upon HESC differentiation could account for the observed hyposumoylation of target proteins and accumulation of free SUMO-1. However, overexpression of SUMO-1 precursor in undifferentiated primary HESCs or cells decidualized with 8-br-cAMP and MPA for 72 h resulted in increased levels of free but mature SUMO-1 (Fig. 2B). Notably, the levels of conjugated SUMO-1 remained unaltered in both undifferentiated and decidualized cells (Fig. 2B). The results demonstrate that neither the expression nor the maturation of SUMO-1 are rate limiting upon HESC differentiation.

These findings prompted us to examine the expression of enzymes involved in SUMO activation, conjugation, ligation, and deconjugation in differentiating cells treated with 8-br-cAMP and MPA over a time course lasting 8 days. As shown in Fig. 2C, we found no evidence of down-regulation of E1 (SAE1/SAE2) or E2 (Ubc9) enzymes upon decidualization. In contrast, SUMO E3 ligases were highly regulated in decidualizing cells. Expression of PIAS1, PIAS3L, PIASxβ, and RanBP2 declined upon 2–4 days of treatment with 8-br-cAMP and MPA, whereas the levels of PIAS3, PIASy, and Pc2 increased. The levels of the SUMO proteases also changed upon HESC differentiation, characterized by a reduction in SENP1 and a reciprocal increase in the expression of SENP2 and SENP6. Treatment of HESCs for 8 days with either 8-br-cAMP, MPA, or a combination demonstrated that, with the exception of PIAS3, the changes in E3 ligases and SENP expression are the consequence of cAMP or cAMP plus MPA signaling (Fig. 2C). Taken together, the data demonstrate that the profound changes in SUMO conjugation/deconjugation upon HESC differentiation concur with altered expression of E3 ligases and proteases.

cAMP Signaling Alters the Subcellular Distribution of SUMO-1.

Given that SENPs are expressed in different cellular compartments and that E3 ligases target distinct substrates (16), we postulated that the altered expression of these enzymes upon decidualization of HESCs may lead to redistribution of endogenous SUMO-1 conjugates. To test this idea, cultures were treated with 8-br-cAMP, MPA, or a combination for 4 days and stained with an anti-SUMO-1 antibody. In untreated cells, SUMO-1 immunostaining was diffusely nuclear and perinuclear (Fig. 3). The nuclear staining was further characterized by the presence of several more intense bodies. This pattern was drastically altered in cells treated with 8-br-cAMP and MPA where SUMO-1 staining was confined to distinct nuclear punctate structures. Treatment with 8-br-cAMP alone also elicited relocalization of SUMO-1 conjugates into nuclear speckles although these were bigger and fewer than in cells cotreated with MPA. In contrast, MPA alone had little effect on the subcellular distribution of SUMO-1 conjugates. These findings are in agreement with the observation that cAMP signaling elicits global hyposumoylation in differentiating HESCs (Fig. 1A) by regulating expression of several SUMO cycle enzymes (Fig. 2C). Nevertheless, cotreatment with MPA is required for regulation of a subset of the enzymes, which could account for the more discrete changes in the subnuclear localization of SUMO-1 conjugates.

Fig. 3.
Subcellular localization of SUMO-1 is altered upon HESC decidualization. Primary HESCs were treated with 8-br-cAMP, MPA, or both for 4 days. Methanol/acetone-fixed cells were immunostained with an antibody to SUMO-1, and the distribution of endogenous ...

PIAS1 Is a SUMO E3 Ligase for PR-A.

In contrast to many other steroid receptors, the identity of the E3 ligase responsible for PR sumoylation is unknown. We therefore transfected COS-1 cells with expression vectors encoding for EGFP-SUMO-1 and WT PR-A or PR-AK388R mutant. In addition, cells were cotransfected with increasing amounts of different E3 ligases. Cultures were treated with MPA for 24 h, and protein lysates were subjected to Western blot analysis. We found no evidence that PIAS3, PIAS3L, PIASxα, PIASxβ, PIASy, or Pc2 are involved in sumoylation of PR-A. In fact, PR-A sumoylation was attenuated in the presence of increasing amounts of these E3 ligases (Fig. 6, which is published as supporting information on the PNAS web site), suggesting sequestering of EGFP-SUMO-1 to other target proteins. In contrast, increasing amount of PIAS1 enhanced conjugation of EGFP-SUMO-1 to PR-A in MPA-treated cells (Fig. 4A).

Fig. 4.
PIAS1 interacts with and enhances PR-A sumoylation. (A) COS1 cells were transfected with PR-A WT or K388R mutant (500 ng), EGFP-SUMO-1 (50 ng), Ubc9 (500 ng), and Flag-PIAS1 (100, 500, and 1,000 ng) and treated with MPA for 36 h. Western blotting was ...

As a putative SUMO ligase for PR, PIAS1 is required to interact directly with its substrate. Lysates prepared from COS-1 cells transfected with PR-A and enhanced yellow fluorescent protein (EYFP)-tagged PIAS1 and treated with or without MPA for 36 h were subjected to immunoprecipitation with a PR antibody. Subsequent immunoblotting with an antibody against the EYFP tag demonstrated coprecipitation of PIAS1 with PR-A, irrespective of treatment with MPA (Fig. 4B). Furthermore, confocal microscopy demonstrated colocalization of endogenous PR-A and PIAS1 in untreated HESCs although this overlap was only partial, suggesting that a significant pool of receptor is not bound to this E3 ligase (Fig. 4C). Collectively, the data demonstrate that PIAS1 can function as a SUMO E3 ligase for PR-A. As PR sumoylation is ligand-dependent (9), the result also suggests that the conformation of the activated receptor is required for access and modification of the N-terminal sumoylation motif.

PIAS1 Modulates PR-A Transcriptional Activity.

We next explored the functional consequences of PIAS1 and PR-A interaction. First, HESCs were transfected with a PRE-driven reporter, WT PR-A or PR-AK388R and WT PIAS1 or a PIAS1 mutant (C351S, W372A) devoid of E3 ligase activity (17, 18). Subsequently, cultures were treated for 24 h with MPA. As shown in Fig. 5A, WT PIAS1 but not the E3 ligase-deficient mutant inhibited the activity of PR-A by ≈70%. PIAS1-mediated transrepression was less pronounced in cells transfected with the PR-AK388R mutant (20%), although not entirely eliminated. This finding suggests that PIAS1 inhibits PR-dependent transcription not only by sumoylating the receptor but possibly also by targeting cofactors. Conversely, silencing of endogenous PIAS1 expression in HESCs by siRNA triggered a 2-fold increase in the activity of the WT receptor but had little or no effect on the activity of the sumoylation-deficient PR-AK388R mutant (Fig. 5 B and C).

Fig. 5.
PIAS1 modulates PR-A activity. (A) Primary HESC cultures were transfected with 500 ng of WT PR-A or K388R mutant, 100 ng of EGFP-SUMO-1, and 400 ng of PRE2-luciferase in the presence or absence of 500 ng of WT PIAS1 or the E3 ligase-deficient mutant PIAS1 ...

These observations indicated that cAMP-mediated down-regulation of PIAS1 could play a role in sensitizing HESCs to progesterone (6). To test this idea, we monitored the expression of decidual prolactin (PRL) mRNA, a cardinal marker of HESC differentiation (6, 19), in primary cultures that were either mock-transfected or transfected with PIAS1 siRNA. As expected, treatment with MPA alone for 72 h was insufficient to induce PRL mRNA expression in control cells (Fig. 5D). In contrast, silencing of PIAS1 expression produced a 9-fold increase in the PRL mRNA levels in response to MPA treatment. Notably, the induction of PRL transcripts in response to 8-br-cAMP treatment was significantly higher and not further enhanced by PIAS1 siRNA. Together, the data suggest that PIAS1 negatively regulates PR-A activity in accordance with its role as a SUMO ligase for PR-A.

Discussion

Microarray studies have shown that decidualization involves sequential reprogramming of functionally related families of genes involved in extracellular matrix organization, cell adhesion, cytoskeletal organization, signal transduction, metabolism, differentiation, and apoptosis (2022). This reprogramming of endometrial cells is thought to be essential for controlled trophoblast invasion and the formation of a functional placenta (1). Although decidualization depends on elevated progesterone levels, there is overwhelming evidence to suggest that initiation of this process requires elevated intracellular cAMP levels (1, 2, 6–8, 23, 24).

Convergence of cAMP and progesterone signaling in HESCs is complex and appears to occur at multiple levels. First, cAMP activation of the PKA pathway has been shown to disrupt the interaction of PR with the corepressors NCoR and SMRT (25), thereby facilitating recruitment of the coactivator SRC-1 (26). Second, cAMP induces the expression or activation of several transcription factors, including FOXO1, STAT5, and C/EBPβ, capable of interacting directly with PR (8, 24, 27–29). This finding has led to the hypothesis that PR-A may serve as a platform in endometrial cells for the formation of multimeric transcriptional complexes that regulate the expression of decidua-specific genes (2). Our observation that down-regulation of PIAS1 in response to cAMP signaling enhances PR-A-dependent transcription suggest yet another level of cross-talk. Interestingly, PIASy has been shown to interact with PR (30), although we found that PIAS1 is the only family member capable of enhancing sumoylation of the receptor.

Hyposumoylation of the liganded PR-A in response to activation of the cAMP pathway took place within the context of a general decline in SUMO-1 conjugation and confinement of modified proteins to distinct nuclear foci. This dramatic change in global sumoylation occurred without affecting the levels of SUMO-1 or its maturation from the precursor form. However, altered expression of E3 ligases and simultaneous regulation of SUMO proteases may account for the redistribution and differential targeting of SUMO-1 substrates upon decidualization. An important feature of the SENP family of isopeptidases is that several members are expressed in distinct cellular compartments. For examples, SENP1 is found in punctate nuclear foci, SENP2 associates with the nuclear pore, and SENP6 resides in the cytoplasm (3133). Hence, up-regulation of SENP2 and SENP6 could account for reduced levels of SUMO-1 conjugates in the cytoplasm and at the nuclear envelope where these enzymes localize. The punctate appearance of nuclear SUMO-1 in decidual cells is reminiscent of PcG bodies, of which the Pc2 ligase is a member (13), perhaps reflecting the up-regulation of this E3 ligase and therefore increased recruitment and sumoylation of its substrates to these locations. If confirmed, this finding would implicate Pc2 as an important repressor in differentiated HESCs.

An interesting observation is the up-regulation of PIAS3 in decidualizing cells, while its splice variant PIAS3L is down-regulated (34). We also observed accumulation of PIASxβ, and to a lesser extent PIASxα, in undifferentiated but not decidualizing cells (Fig. 2C), which may reflect growth arrest in confluent undifferentiated cultures. Notably, the levels of some PIAS proteins have been shown to fluctuate through the cell cycle independently of mRNA levels (35). Hence, it appears likely that transcriptional and posttranslational events are involved in regulating the expression of SUMO enzymes in decidualizing cells.

We report on the regulation of the SUMO pathway during a process of cellular differentiation. However, global perturbations of the SUMO cycle have been linked to pathological events and cellular stress responses. Certain viral infections have been shown to disrupt promyelocytic leukemia bodies, which represent nuclear foci of sumoylation (36), or to inhibit the SUMO pathway by inactivating and degrading E1/E2 enzymes (37). Both scenarios are thought to result in a transcriptionally permissive environment that facilitates viral replication. Many types of cellular stress, including temperature shock, toxic metals, and osmotic and oxidative stress, cause rapid changes in SUMO conjugation (3840). Recently, a molecular mechanism has been described that explains hyposumoylation at low doses of reactive oxygen species and explains hypersumoylation at high doses. Here, E1/E2 enzymes are inhibited by cross-linking of their catalytic cysteines in the first instance, whereas oxidation and inactivation of SUMO proteases occur in the second instance (41). However, both viral infections and cellular stresses, through acting on E1/E2 enzymes or all SUMO proteases, will indiscriminately affect sumoylation of substrates. In contrast, fine-tuning of the SUMO pathway by regulating the expression of individual ligases and proteases suggests a selective mechanism of targeting specific cellular processes involved in differentiation.

Several transcription factors linked to decidualization, such as C/EBPβ and p53, and many nuclear receptor cofactors, including SRC-1, p300/CBP, GRIP1, CtBP, and NCoR, are also subject to SUMO modification (13, 4247). It is therefore conceivable that PR activity upon decidualization is further modulated indirectly by altered sumoylation of its interacting partners. The global nature of the changes in sumoylation pattern is likely to have functional consequences for many other processes. Sumoylation also targets proteins involved in RNA processing and transport, subcellular trafficking, cytoskeletal regulation, metabolism, and DNA repair and nuclear organization (38, 39). Our findings therefore suggest that dynamic changes in the SUMO cycle may serve as an overarching regulatory mechanism in decidual reprogramming of HESCs.

In summary, pregnancy requires coordinated interaction between the implanting embryo and the decidualizing maternal endometrium. Timing of the decidual response in the secretory phase of the cycle is therefore crucial and depends on the convergence of the cAMP and progesterone pathways. We have now shown that cAMP signaling profoundly alters the SUMO cycle in HESCs in a time-dependent manner, leading to hyposumoylation of the activated PR-A and transcriptional activation of decidua-specific genes.

Materials and Methods

Primary Cell Culture.

The study was approved by Local Research and Ethics Committee at Hammersmith Hospitals NHS Trust, and patient consent was obtained before tissue collection. Endometrial samples were obtained from cycling women without uterine pathology, and HESCs were isolated, established in culture, maintained, and decidualized with 0.5 mM 8-br-cAMP (Sigma, St. Louis, MO) and 1 μM MPA (Sigma) as described (23). All primary culture experiments were carried out before the third cell passage.

Expression Vectors.

The expression plasmids for PR-A, PR-AK388R, and EGFP-SUMO-1 have been described (9). PRE2/-32dPRL/luciferase was obtained from B. Gellersen (University of Hamburg, Hamburg, Germany). pSG5-SUMO-1 WT, pSG5-SUMO-1-GG, and pSG5 were gifts from H. Hurst (Barts' and the London School of Medicine, London, U.K.), and V. De Laurenzi (Leicester University, Leicester, U.K.) kindly provided the pCMV5-Flag-PIAS1 and pEYFP-PIAS1 constructs. Vectors for Flag-PIAS3/L, Flag-PIASxα/β, PIASy and Pc2 were donated by H. Boeuf (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France), J. Palvimo (University of Helsinki, Helsinki, Finland), K. Shuai (University of California, Los Angeles CA), and D. Wotton (University of Virginia, Charlottesville, VA), respectively. The E3-ligase deficient PIAS1 mutant was generated by introducing two point mutations (C351S, W372A) with a QuikChange Site-Directed Mutagenesis Kit (Strategene, La Jolla, CA).

Transient Transfections and Luciferase Reporter Assays.

Primary HESCs were transfected with DNA vectors or siRNA by the calcium phosphate coprecipitation method using the Profection mammalian transfection kit (Promega, Madison, WI). PIAS1 siGenome smart-pool and a nontargeting control siRNA were purchased from Dharmacon (Lafayette, CO) and used at 100 nM. Transfections of COS-1 cells were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Luciferase reporter assays were performed on cells cultured in 24-well plates and harvested in reporter lysis buffer (Promega). Luciferase activity was measured with a luciferase assay reagent (Promega). Transfections were performed in triplicate and repeated at least three times. Representative experiments are shown (mean ± SD). The amounts of DNA transfected are indicated in the figure legends.

Western Blotting, Dot Blot Analysis, and Immunoprecipitation.

Whole-cell protein extracts were obtained by scraping cells cultured in six-well plates in reducing SDS/PAGE loading buffer heated to 85°C, followed by sonication. Proteins were resolved by SDS/PAGE and transferred to PVDF membrane (Amersham, Piscataway, NJ). Signal visualization was performed with a the ECL+ kit (Amersham). The following primary antibodies were used: anti-PR (NCL-L-PGR-312; NovoCastra, Newcastle, U.K.; of note, the antibody recognizes both denatured PR-A and PR-B in SDS/PAGE analysis but only the native conformation of PR-A under nondenaturing conditions used for immunoprecipitation and confocal microscopy), anti-SUMO-1 (33–2400; Zymed, Carlsbad, CA), anti-SAE1 (ab5645; Abcam, Cambridge, MA), anti-SAE2 (U10820; BD Biosciences, Franklin Lakes, NJ), anti-Ubc9 (sc-5231; Santa Cruz Biotechnology, Santa Cruz, CA), anti-PIAS1 (8152; Santa Cruz Biotechnology), anti-PIAS3 (sc-14017; Santa Cruz Biotechnology), anti-PIAS3L (a gift from H. Boeuf), anti-PIASx (AP1248a; Abgent, San Diego, CA), anti-PIASy (IMG-290; Ingenex, Sorrento Valley, CA), anti-Pc2 (AP2514b; Abgent), anti-RanBP2 (a gift from F. Melchior, University of Goettingen, Goettingen, Germany), anti-SENP1 (IMG-521, Ingenex), anti-SENP2 (ab3660; Abcam), anti-SENP6 (AP1239a; Abgent), anti-β-actin (ab6276; Abcam), anti-Flag M5 (F4042; Sigma), and anti-GFP (8371; BD Biosciences). For dot blot analysis, serial dilutions of total cell lysates (10, 2, and 0.4 μg) were spotted on a nitrocellulose membrane and probed with anti-SUMO-1. For immunoprecipitation, cells were lysed in high-salt extraction buffer [20 mM Tris·HCl, pH 7.5/300 mM NaCl/1% Nonidet P-40, with Complete protease inhibitors (Roche, Indianapolis, IN) and 10 mM N-ethylmaleimide]. After removal of cell debris by cetrifugation, lysates containing 1 mg of protein were precleared with Protein G Plus-Agarose (Santa Cruz Biotechnology) and incubated with anti-PR antibody (NovoCastra). Proteins were recovered by precipitation with Protein G Plus-Agarose, washed three times with PBS, and analyzed by Western blot.

Confocal Immunofluorescence Microscopy.

Primary HESCs were cultured on four-well multispot glass slides (CA Hendley-Essex, Loughton, U.K.), fixed in 1:1 methanol/acetone for 10 min, and permeabilized with 0.5% Triton X-100 in PBS for 30 min. Endogenous proteins were stained with mouse anti-SUMO-1 (Zymed), mouse anti-PR (NovoCastra), and rabbit anti-PIAS1 (sc-14016; Santa Cruz Biotechnology) followed by anti-mouse FITC (F0479; Dako, Glostrup, Denmark) anti-mouse Alexa Fluor 594 (Invitrogen), or anti-rabbit FITC (F0205; Dako). Cells were mounted in Vectashield with DAPI (Vector Labs, Burlingame, CA) and visualized under a Zeiss (Thornwood, NY) Meta 512 confocal microscope.

Supplementary Material

Supporting Figure:

Acknowledgments

We thank H. Boeuf, B. Gellersen, H. Hurst, V. De Laurenzi, F. Melchior, J. Palvimo, K. Shuai, and D. Wotton for reagents and G. Zoumpoulidou and S. Fernandez de Mattos for technical assistance.

Abbreviations

8-br-cAMP
8-bromo-cAMP
EYFP
enhanced yellow fluorescent protein
HESC
human endometrial stromal cell
MPA
medroxyprogesterone acetate
PIAS
protein inhibitor of activated STAT
PR
progesterone receptor
PRE
PR response element
PRL
prolactin
SENP
sentrin-specific protease
SUMO
small ubiquitin-like modifier.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS direct submission. W.P.T. is a guest editor invited by the Editorial Board.

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