Silencing of the JNK pathway maintains progesterone receptor activity in decidualizing human endometrial stromal cells exposed to oxidative stress signals

Abstract

Survival of the conceptus is dependent on continuous progesterone signaling in the maternal decidua but how this is achieved under conditions of oxidative stress that characterize early pregnancy is unknown. Using primary cultures, we show that modest levels of reactive oxygen species (ROS) increase sumoylation in human endometrial stromal cells (HESCs), leading to enhanced modification and transcriptional inhibition of the progesterone receptor (PR). The ability of ROS to induce a sustained hypersumoylation response, or interfere with PR activity, was lost upon differentiation of HESCs into decidual cells. Hypersumoylation in response to modest levels of ROS requires activation of the JNK pathway. Although ROS-dependent JNK signaling is disabled on decidualization, the cells continue to mount a transcriptional response, albeit distinct from that observed in undifferentiated HESCs. We further show that attenuated JNK signaling in decidual cells is a direct consequence of altered expression of key pathway modulators, including induction of MAP kinase phosphatase 1 (MKP1). Overexpression of MKP1 dampens JNK signaling, prevents hypersumoylation, and maintains PR activity in undifferentiated HESCs exposed to ROS. Thus, JNK silencing uncouples ROS signaling from the SUMO conjugation pathway and maintains progesterone responses and cellular homeostasis in decidual cells under oxidative stress conditions imposed by pregnancy.

Figure 1.

ROS effects on sumoylation and PR-A activity in undifferentiated and decidualizing HESCs. A, B) Primary HESC cultures, either maintained undifferentiated or decidualized with 8-br-cAMP and MPA for 3 d, were exposed to 0.1, 0.25, 0.5, 1, and 10 mM of H₂O₂ for 1 h (A), or to 250 μM H₂O₂ over a time course of 24 h (B). Whole-cell protein extracts were analyzed by Western blotting with anti-SUMO-1 and anti-β-actin antibodies. C) HESCs were treated with 8-br-cAMP and MPA as indicated for 3 d, while being transfected with PR-A (500 ng) and EGFP-SUMO-1 (50 ng) expression vectors and exposed to 0 or 250 μM H₂O₂ for the last 24 h. Whole-cell extracts were subjected to Western blot analysis with anti-PR, GFP, and β-actin antibodies. D, E) Primary HESCs were transfected with expression vectors for PR-A wild-type (D) or sumoylation-deficient K388R mutant (400 ng) (E) and PRE₂-luciferase reporter (100 ng), and treated with 8-br-cAMP and MPA in the indicated combinations, for 3 d. In the last 24 h, cells were exposed to 0, 100, or 250 μM H₂O₂, and harvested for luciferase assay. Data are expressed as fold induction with regard to control-treated sample and represent means ± sd of triplicate experiments.

Figure 2.

Modest levels of ROS do not trigger oxidative modifications of SUMO enzymes. A) HESCs were transfected with full-length immature SUMO-1 (SUMO-1-GG-HSTV) (500 ng), and at 24 h post-transfection were treated with 250 μM H₂O₂ for the indicated times. Some cultures were exposed for 30 min to a concentration of 0.1 M H₂O₂, known to inactivate SUMO proteases. Whole-cell protein extracts were resolved on a 7.5% SDS-polyacrylamide gel and immunoprobed with anti-SUMO-1 antibody to visualize SUMO-1 conjugates. For detection of free SUMO-1, samples were separated on a 12% gel. In vitro expression using vectors encoding the immature (SUMO-1-GG-HSTV) and matured form (SUMO-1-GG) of SUMO-1 were used as positive controls. Open arrowhead indicates the position of C-terminally processed SUMO-1; solid arrowhead indicates the immature form. B) HESCs were decidualized with 8-br-cAMP and MPA for 3 d and subjected to time-course treatment with 250 μM H₂O₂ over a 24-h period. As a positive control for Ubc9-SAE2 cross-linking, some cultures were treated with 1 mM H₂O₂ for 15 min. Total protein harvested in Laemmli buffer not containing β-mercaptoethanol was analyzed by Western blotting with anti-Ubc9, SAE2, and β-actin antibodies. Open arrowheads indicate positions of the uncoupled proteins; solid arrowheads show Ubc9-SAE2 cross-linked species; asterisks indicate nonspecific bands detected by the anti-Ubc9 antibody.

Figure 3.

JNK signaling mediates ROS-dependent hypersumoylation. A) Undifferentiated and decidualizing primary HESCs were exposed to 250 μM H₂O₂ for the indicated times, and DNA binding of NF-κB contained in the nuclear extracts was analyzed by EMSA. Nuclear lysate from myometrial cells stimulated with 1 ng/ml IL-1β for 30 min was used as a positive control. Myometrial lysates were also incubated with 100-fold excess of unlabeled NF-κB (xs spec) and Oct1 (xs nonspec) oligonucleotides prior to the addition of the [³²P]-labeled probe, as DNA binding competitors to verify binding specificity. For supershift analysis, myometrial nuclear extracts were incubated with an antibody against p65 prior to addition of the hot probe. Solid arrowheads indicate position of specific complexes; solid bullet indicates position of nonspecific complexes; open arrowhead indicates the position of uncomplexed DNA probe. B) Primary HESC cultures were kept undifferentiated or decidualized with 8-br-cAMP and MPA for 3 d and then exposed to 250 μM H₂O₂ for 0, 15, 30, or 90 min. Whole-cell extracts were subjected to Western blot analysis with antibodies against the total and phosphorylated forms of Akt, JNK, p38, and ERK1/2. C) Undifferentiated HESC cultures were either kept untreated or treated with 36 μM of JNKVIII inhibitor for 2 h before exposure to 250 μM H₂O₂ for 10, 30, or 60 min. Whole-cell extracts were subjected to Western blot analysis with antibodies against SUMO-1, total and phosphorylayed forms of c-jun, and β-actin as loading control. D) HeLa cells were transfected with PR-A (500 ng) and EGFP-SUMO-1 (50 ng), with or without ΔMEKK1:ER*-myc (500 ng). Twenty-four hours later, cells were treated with MPA and 4-hydroxytamoxifen (4-OHT) for the times indicated. Western blotting was performed on whole-cell protein extracts by using antibodies against PR, GFP, myc tag, β-actin, and total or phosphorylated forms of JNK, p38, and ERK1/2.

Figure 4.

ROS triggers distinct gene expression regulation in undifferentiated and decidualizing HESCs. Primary HESCs were maintained undifferentiated or decidualized with 8-br-cAMP and MPA for 3 d and exposed or not to 250 μM H₂O₂ for 8 h. RNA extracts were utilized for gene expression profiling using genome-wide arrays (A) or RTQ-PCR (B). A) Venn diagram shows numbers of significantly down-regulated and up-regulated genes in undifferentiated and decidualizing cells. B) Data are depicted as fold induction relative to the transcript levels of undifferentiated and decidualizing untreated samples and represent means ± sd of triplicate determinations.

Figure 5.

MKP1 expression is induced upon decidualization. Primary HESC cultures were maintained undifferentiated or decidualized with 8-br-cAMP and MPA for 1, 2, 4, and 8 d. Cells were harvested for RNA or protein, and MKP1 expression was profiled by RTQ-PCR (A) or Western blot analysis (B), respectively. A) Data are presented as mean ± sd fold induction relative to transcript levels in untreated cells at d 1 of triplicate experiments. C) RNA was extracted from proliferative (P; n=9) and secretory (S; n=10) endometrial biopsies, and RTQ–PCR was used to determine levels of MKP1 transcripts. Statistical significance was determined using Student’s t test. D) MKP1 expression in secretory endometrium. MKP1 immunoreactivity was apparent not only in luminal and glandular epithelial cells (LE and GE, respectively) but also in the stromal compartment, with staining intensity varying in different cell populations from weak to intense (arrowheads; original view: ×20). Inset: negative control (no primary antibody).

Figure 6.

MKP1 overexpression in undifferentiated HESCs protects against ROS effects. A) Undifferentiated HESC cultures were either mock or MKP1-myc transfected (500 ng) and exposed to 250 μM H₂O₂ over a time course of 24 h. Whole-cell extracts were subjected to Western blot analysis with antibodies against SUMO-1, total- and phospho-JNK, myc tag, and β-actin. B) Undifferentiated HESC cultures were either mock or MKP1-myc transfected (500 ng) and exposed to 250 μM H₂O₂ for 8 h. RNA was extracted, and RTQ-PCR was used to determine the abundance of GADD45A, RRAD, and FOXO1 transcripts, relative to L19. Data are presented as fold induction relative to the transcript levels in untreated mock transfected cells and depict means ± sd triplicate determinations. Results are representative of 3 independent experiments. C) Primary HESCs were transfected with PR-A (400 ng) and PRE2-luciferase reporter (100 ng) with or without MKP1 expression vector (500 ng), and treated for 24 h with MPA and 0, 100, 250, and 500 μM H₂O₂, as indicated. Cells were harvested, and luciferase activity was assayed. Data are expressed as fold induction with regard to −MPA treatment and represent means ± sd of triplicate determinations.