NADPH oxidase-derived reactive oxygen species mediate decidualization of human endometrial stromal cells in response to cyclic AMP signaling

Abstract

Differentiation of human endometrial stromal cells into specialized decidual cells is critical for embryo implantation and survival of the conceptus. Initiation of this differentiation process is strictly dependent on elevated cAMP levels, but the signal intermediates that control the expression of decidual marker genes, such as prolactin (PRL) and IGFBP1, remain poorly characterized. Here we show that cAMP-dependent decidualization can be attenuated or enhanced upon treatment of primary cultures with a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenylen iodonium) or activator (apocynin), respectively. Time-course analysis demonstrated that cAMP enhances endogenous reactive oxygen species production, apparent after 12 h of stimulation, which coincides with a dramatic increase in decidual PRL and IGFBP1 expression. Knockdown of the Rho GTPase RAC1, which disables activation of the NADPH oxidase homologs NADPH oxidase (NOX)-1, NOX-2, and NOX-3, had no effect on PRL or IGFBP1 expression. In contrast, silencing of NOX-4, or its cofactor p22(PHOX), inhibited the expression of both decidual markers. Finally, we show that the NOX-4/p22(PHOX) complex regulates the DNA-binding activity of CCAAT/enhancer binding protein-β, a key regulator of human endometrial stromal cell differentiation. Thus, NOX-4 activation and reactive oxygen species signaling play an integral role in initiating the endometrial decidual response in preparation of pregnancy.

Fig. 1.

NADPH-dependent ROS production upon initiation of the decidual process. A, Undifferentiated primary HESCs were loaded with DCFH-DA for 30 min and then pulsed with apocynin (Apo) or DPI. DCF fluorescence was measured every 2 min over a 30-min period. The data represent the mean DCF oxidation level (±sem) at each interval. B, HESCs were maintained untreated or treated with 8-br-cAMP in the presence or absence of Apo or DPI for 24 h. Quantitative RT-PCR (RTQ-PCR) analysis was carried out for PRL and IGFBP1 mRNA levels normalized to L19 mRNA. Data are depicted as fold induction relative to transcript levels of untreated samples and represent means (±sd) of triplicate determinations. C, HESCs were either maintained untreated or treated with 8-br-cAMP or MPA in the presence or absence of Apo or with Apo alone for 24 h. Total RNA was harvested and RTQ-PCR analysis was carried out for IGFBP1 and PRL, normalized to L19 transcript levels. Data are depicted as fold induction relative to transcript levels of untreated samples and represent means (±sd) of triplicate determinations. D and E, Primary HESCs were maintained untreated or treated with 8-br-cAMP in the presence or absence of Apo, allopurinol, L-NAME, or AEBSF. RTQ-PCR analysis was carried out for PRL and IGFBP1 mRNA levels normalized to L19 mRNA. Data are depicted as fold induction relative to transcript levels of undifferentiated samples and represent means (±sd) of triplicate determinations. F, Primary cultures were transfected with a catalase expression vector or empty vector. The cells were treated 2 d later with 8-br-cAMP for 24 h. Total mRNA was harvested and PRL and IGFBP1 transcript levels were measured using RTQ-PCR analysis. Data are depicted as fold induction relative to transcript levels of undifferentiated samples and represent means (±sd) of triplicate determinations. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 2.

Kinetics of cAMP-dependent ROS generation in differentiating HESCs. A and B, Cultured HESCs were treated or untreated with 8-br-cAMP in the presence or absence of apocynin (Apo) for 3, 6, 12, and 24 h. PRL (A) and IGFBP1 (B) transcript levels were normalized to L19 mRNA. Data are depicted as fold induction relative to transcript levels of untreated samples and represent means (±sd) of triplicate determinations. C, HESCs were treated or untreated with 8-br-cAMP for 3, 6, 12, and 24 h. Cultures were loaded with DCFH-DA (30 μm) 25 min before terminating the treatments. DCF fluorescence measurement was taken every 2 min over a 30-min period using a fluorometer. Data are depicted as the difference in ROS production between 8-br-cAMP treated and untreated cultures and represent means (±sem) of sextuplicate determinations. D, Primary cultures were untreated or treated with 8-br-cAMP for 24 h and then pulsed with Apo or DPI for 30 min. Subsequently the cells were loaded with DCF-DA for 25 min and DCF fluorescence was measured using flow cytometry. *, P < 0.01; **, P < 0.001.

Fig. 3.

RAC1 is not required for the induction of the decidual markers. A, Cultured HESCs were untreated or treated with 8-br-cAMP for 24 h. Whole-cell lysates were extracted and blotted for RAC1 expression. β-Actin served as a loading control. B, The left panel shows quantitative RT-PCR (RTQ-PCR) analysis of RAC1 transcript levels, normalized to L19 mRNA, in cells transfected with NT or RAC1 siRNAs and treated 2 d later with 8-br-cAMP for 24 h. Data are depicted as fold change relative to transcript levels of undifferentiated samples and represent means (±sd) of triplicate determinations. The right panel shows Western blot analysis of RAC1 expression in protein lysates from parallel cultures. β-Actin served as a loading control. C, RTQ-PCR analysis for PRL and IGFBP1 levels, normalized to L19 mRNA, upon RAC1 silencing. B, The results are fold change relative to transcript levels of undifferentiated samples transfected with NT siRNA and represent means (±sd) of triplicate determinations. *, P < 0.01; **, P < 0.001.

Fig. 4.

p22^PHOX and NOX4 knockdown perturbs cAMP-dependent HESC decidualization. A, Cultured HESCs were untreated or treated with 8-br-cAMP for 6, 8, 10, 16, and 24 h. Whole-cell lysates were extracted and blotted for p22^PHOX and NOX4 expression. β-Actin served as a loading control. B, Validation of p22^PHOX knockdown at mRNA (left) and protein level (right) in HESCs transfected with NT or p22^PHOX siRNAs. C, Quantitative (RTQ-PCR) analysis for both PRL and IGFBP1 levels, normalized to L19 mRNA upon p22^PHOX silencing. D, Validation of NOX4 knockdown at mRNA (left) and protein level (right) in HESCs transfected with NT or NOX4 siRNAs. E, RTQ-PCR analysis for both PRL and IGFBP1 levels, normalized to L19 mRNA upon NOX4 silencing. Data in C and E are depicted as fold change relative to transcript levels of samples transfected with NT siRNA and represent means (±sd) of triplicate determinations. F, Primary cultures were transfected with NT siRNA or siRNA targeting p22^PHOX and NOX4 and treated 2 d later with 8-br-cAMP for 24 h. The cells were then loaded with DCF-DA for 30 min and DCF fluorescence was measured over a 30-min period. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 5.

C/EBPβ is a downstream mediator of NADPH oxidase produced ROS signaling in HESCs. A, Cultured HESCs were untreated or treated for 24 h with 8-br-cAMP in the presence or absence of apocynin (Apo) and/or DPI. Cytosolic/nuclear extracts were prepared and blotted for SRC, STAT5, FOXO1, and C/EBPβ. The expression of lamin B and α-tubulin was assessed to confirm the integrity of the cell fractionation. β-Actin was used as a loading control. B, HESCs were treated for 24 h with 8-br-cAMP in the presence or absence of Apo, and DNA binding of CEBPβ contained in the nuclear extracts was analyzed by EMSA. Nuclear lysates were incubated with excess of [³²P]-labeled C/EBP, OCT1 (xs nonspecific) and unlabeled C/EBP (xs specific) oligonucleotides. Both OCT1 and unlabeled C/EBP oligonucleotides served as DNA binding competitors to verify binding specificity. For supershift analysis, nuclear extracts were incubated with an antibody against C/EBPβ before addition of the hot probe. Solid arrowhead indicate position of specific complexes; open arrowhead marks the position of uncomplexed DNA probe. C, Undifferentiated HESCs were transfected with NT siRNA or siRNA targeting p22^PHOX and NOX4 and treated 2 d later with 8-br-cAMP. DNA binding of C/EBPβ contained in the nuclear extracts was analyzed by EMSA. To verify DNA binding, binding specificity and for supershift analysis, nuclear lysates were incubated with excess of [³²P]-labeled C/EBP, OCT1, and unlabeled C/EBP oligonucleotides. For supershift analysis, nuclear extracts were incubated with an antibody against C/EBPβ before addition of the hot probe. Solid arrowhead indicate position of specific complexes; open arrowhead marks the position of uncomplexed DNA probe. D, Primary cultures were transfected with C/EBP-pGL4, pSG₅-C/EBPβ (encoding for full length C/EBPβ), and pCH110 (encoding for β-galactosidase). The cells were left untreated or treated with 8-br-cAMP, Apo, or the combination of 8-br-cAMP and Apo for 24 h. Subsequently firefly luciferase activity was measured. *, P < 0.01; **, P < 0.001.