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To determine the effect of progesterone on nitric oxide synthase (NOS) expression in human endometrial epithelial cells.
University based research institute.
The effect of progesterone on the expression of NOS protein isoforms was examined in an in vitro preparation.
The expression of NOS and phosphorylated endothelial NOS (peNOS) protein in human endometrial derived epithelial cells (HES cells) and mRNA in human primary endometrial cell culture.
Progesterone induced a concentration and time-dependant stimulation of endothelial NOS (eNOS), inducible NOS (iNOS) and peNOS protein in HES cells. Progesterone also stimulated eNOS and iNOS mRNA in human primary endometrial cells. The effect of progesterone on eNOS/iNOS was completely blocked by RU486 but was partially blocked in case of phosphorylated eNOS. RU486 alone had an inhibitory effect on expression of eNOS but not iNOS protein at concentrations of progesterone greater than 10−5 M. Progesterone stimulated phosphorylation of eNOS within 30 minutes and this effect was completely blocked by an inhibitor of PI3/Akt pathway, wortmanin and by the extracellular signal-regulated kinase (ERK) 1, 2 pathway blocker UO126.
Progesterone has both genomic and non-genomic effects to stimulate the expression of NOS in HES cells. The non-genomic action of progesterone on NOS phosphorylation is mediated by the PI3/Akt and ERK1, 2 pathways.
Nitric oxide (NO) is a free radical that is derived from L-arginine by the action of nitric oxide synthase (NOS). Nitric oxide has diverse physiological functions and is involved in cellular injury, regulation of vascular resistance, and as a signal transduction molecule (1). The synthesis of NO is catalyzed by 3 isoforms of NOS, neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). The activity of the NOS is also regulated by pos-translational modification, including kinase-mediated protein phosphorylation (2; 3). Phosphorylation of NOS can occur at a number of different amino acids each of which has differing physiological consequences, in most instances resulting in activation of the enzyme (2). Significant amount of research has been devoted to understanding the regulation of NOS expression by ovarian steroids in both vascular and reproductive tissues (4). Our prior research indicated that the predominant isoform of NOS in the endometrium is eNOS and this is predominantly localized in the epithelial cell layer (5). We also showed that estrogen through both genomic and non-genomic mechanisms up-regulates eNOS/iNOS and phosphorylated eNOS in primary endometrial epithelial cells, and HES cells, a cell line derived from uterine surface epithelial layer (6). These observations strongly suggested that sex hormones regulate expression of NOS in the endometrium just as has been demonstrated in other sites such as the vascular endothelium (4). The purpose of this study was to determine the regulation of NOS by progesterone, especially in light of the fact that several studies have suggested that in vascular tissue progesterone opposes the stimulatory effect of estrogen on eNOS (7; 8). Understanding the regulation of uterine NOS is important as marked changes in the expression of eNOS have recently been demonstrated in the endometrium of patients with endometriosis (9; 10) and adenomyosis (10).
The details of HES and primary endometrial cell culture have previously been published (6). HES cells were chosen because they express both the estrogen and progesterone receptor and functionally respond in the same manner as epithelial cells of endometrium (11; 12). Briefly, HES cells provided by Dr. Douglas A. Kniss (Ohio State University, Columbus, OH.) were grown in Medium 199 (M199, GIBCO ) supplemented with 10% fetal bovine serum, 10 mM L-glutamine and 0.5 mM L-Arg in a 5% CO2 humidified atmosphere. After 24 hours of serum starvation, medium was replaced with experimental medium, M199 with 2% charcoal treated FBS supplemented with 10mM L-glutamine and 0.5mM L-Arg. To determine the effects of estrogen on NOS expression, cells were treated with vehicle or progesterone (P4), (10−12 to 10−6M) and harvested at 30 minutes in case of short term experiments and up to 48 hours for long term cultures. In experiments using an antagonist the drug was added 2 hours prior to addition of progesterone. Control wells contained the vehicle diluent.
Endometrial cell preparation and culture was performed according to Gebel et al. (13). Tissue was obtained from hysterectomy specimens (proliferative phase) from reproductive aged women undergoing surgery for benign gynecologic indications under an approved IRB protocol. The endometrium was scraped and placed into a tube containing Hank’s Balanced Salt Solution (HBSS, phenol red free) with 1% Penicillin/Streptomycin and 2 ug/ml Fungizone. Tissue specimens were cut into 2-mm pieces and washed twice in HBSS. After removing the supernatant, the specimens were incubated in 10ml of HBSS containing collagenase (0.02%) and dexyribonuclease (DNase I; 0.01%) in a 37°C water bath for 40 minutes. After digestion, cells were collected by centrifuging for 5 min at 1000 rpm and resuspended in RPMI 1640 medium supplemented with 10% FBS, 1 % Penicillin/Streptomycin and 2 ug/ml Fungizone. 2× 105 of cells (stromal and epithelial) were placed in 6-well plates and medium was changed after 24 hours of incubation before addition of estradiol.
In brief, samples after collection were treated with RNA later preservative (Ambion/Applied Biosystems, Foster City, CA), and total RNA was isolated using the RNAqueous-4PCR kit (Ambion). RNA was DNase-treated and quantitated by absorbance using a nanodrop spectrophotometer (Nanodrop Instruments, Wilmington, DE). 1 ug of total RNA was reverse-transcribed into single-stranded cDNA using the Omniscript Reverse Transcription kit (QIAGEN, Valencia, CA) at 37°C for 60 min in a total volume of 20 uls. The P CR reaction mix consisted of 1 ul of 10-fold diluted cDNA, qPCR MasterMix Plus for SYBR green I reagent (EUROGENTEC, San Diego, CA) and optimized forward and reverse gene specific primers (300 nM each). Real-Time PCR reactions in triplicate were run on 96 well plates using an ABI PRISM 7000 HT Sequence Detection System (Applied Biosystems). Reactions were started by activation of DNA polymerase at 95°C for 10 min followed by 40 PCR cycles of den aturing at 95°C for 15 sec and annealing/extension at 60°C for 1min. Normaliz ation control was the 18S ribosomal RNA. Data was analyzed to select a threshold level of fluorescence that was in the linear phase of the PCR product accumulation (the threshold cycle (CT) for that reaction. The CT value for 18S was subtracted from the CT value of the gene to obtain a delta CT (ΔCT) value. The relative fold change for each gene was calculated using the ΔΔCT method (14).
Cells were sonicated in protein lysis buffer and protein concentrations determined by the BCA assay (Pierce, Rockford, IL). For each sample 70 ug of protein was separated on a 7.5% polyacrylamide gel. The separated proteins were transferred electrophoretically to Nitrocellulose membranes (Bio-Rad, Hercules, and CA). Membranes were blocked for 2 h in a 5% milk buffer before incubation with a mouse monoclonal antibody against human eNOS (Transduction Laboratories, Lexington, KY), iNOS (Santa Cruz Laboratories, Santa Cruz, CA and Transductions Laboratories) and phosphoeNOS (peNOS), which recognizes eNOS phosphorylated on Ser 1177(Cell Signaling, Danvers, MA) at 4 °C overnight at a dilution of 1:1000. The blots were subjected to enhanced chemiluminescence (ECL Western Blotting Detection System, Amersham Corp, Arlington Heights, IL); with enzyme conjugate anti-mouse IgG horseradish peroxidase as a secondary antibody. Blots were then exposed to autoradiography film. The resulting bands of 140 kDa and 135 kDa corresponding to eNOS and iNOS respectively were compared by scanning densitometry. To ensure equal loading of protein, blots were stripped and re-probed for Glyceryl-aldehyde 3-phophate dehydrogenase (GAPDH). All data were normalized to GAPDH.
The statistical software SigmaStat was used for data analysis. The results were analyzed by ANOVA in case of parametric data and Wilcoxon ranked-sum test in case of non-parametric data. Post hoc testing was by the Student-Newman-Keuls test. Significance was established at P<0.05.
The effect of progesterone on expression of eNOS, iNOS, and peNOS protein is shown in Fig. 1. Progesterone stimulated the expression of eNOS in a concentration and time-dependant manner. At concentrations of 10−8M and greater progesterone stimulated the expression of eNOS (Fig 1A), iNOS (Fig.1B) and peNOS protein (Fig. 1C) at 24 hours of culture. At 48 hours of culture no significant effects of progesterone were detected on NOS isoform expression (data not shown). When NO was measured in the media by chemiluminescence (Sievers NO Analyzer; Boulder, CO), significantly higher level of NO could be detected in media from cells treated with progesterone (10−6 M) (control: 4.4±0.3 µmole vs. progesterone 6.9±0.65 µmole; P< .001) after 24 hours of culture. The effect of progesterone on eNOS was completely blocked by the progesterone nuclear receptor blocker, RU486 as shown in Fig. 2A. However, the effect of progesterone on peNOS expression was reduced but not completely blocked (P<.05 comparing Ru486 with P+RU486 groups) suggesting the involvement of other mechanisms by which progesterone stimulates phosphorylation of eNOS. RU486 had effects of its own on eNOS expression, and inhibited eNOS protein expression at concentrations of 10−5 M (Fig. 2C). However, RU486 had no effect on iNOS protein expression at concentrations as high as 10−5 M (data not shown).
The effect of progesterone on eNOS was not limited to HES cells and as shown progesterone up-regulated eNOS (Fig. 3A), and iNOS (Fig. 3B) mRNA in human primary endometrial cells and this effect could be blocked by RU486.
In order to determine if the effect of progesterone on phosphorylation of eNOS was non-genomic, HES cells were exposed to progesterone for short term (30 minutes). As shown in Fig. 4 within 30 minutes progesterone induced the expression of peNOS. Inhibitors of PI3/Akt kinase (wortmanin) and the ERK1, 2 pathways (UO126) completely blocked the effect of progesterone on phosphorylation of eNOS.
We have demonstrated the progesterone induces the expression of eNOS and iNOS through a nuclear progesterone receptor-mediated mechanism in both an human endometrial epithelial derived cell line (HES cells) and primary human endometrial cells (containing both epithelial cells and stromal cells) in vitro. Progesterone was also demonstrated to have rapid effects on HES cells to phosphorylate eNOS on Ser 1177 position through kinase-mediated pathways involving both the PI3/Akt and Erk 1, 2 pathways.
The regulation of NOS by ovarian steroids has been investigated primarily in endothelial cells and reproductive tissues. In previous work we reported that the expression of endometrial eNOS was higher in the luteal phase of cycling women (5) and rhesus monkeys (15), and in postmenopausal women on continuous hormone replacement therapy (5), implicating the importance of progesterone in the regulation of uterine NOS expression. More recently we reported that progesterone augmented the effects of estrogen to up-regulate eNOS/peNOS expression in endometrial epithelial cells in vitro (6). The current study supports our previous findings and indicates that in human primary endometrial cells or in an endometrial-derived epithelial cell line (HES cells) progesterone has a stimulatory effect on eNOS/iNOS expression and like estrogen (6) stimulates the phosphrylation of eNOS in a non-genomic fashion. Work by others also has shown a positive stimulatory effect of different hormone replacement therapy regimens on circulating NO in humans (16). Zervou et al demonstrated that progesterone up-regulated myometrial smooth muscle eNOS which could be blocked by RU486 in a primary human myocyte cell culture system (17). Several studies have addressed the role of progesterone on uterine NOS expression in the rat, and both positive and negative influences of progesterone have been reported in various in vivo and in vitro models. Buhimschi et al (18) reported administration of progesterone in rats has a negative effect on uterine NO levels and cGMP, whereas Al-Hijji et al (19) and Ogando et al (20) both reported stimulatory effects of progesterone administration on uterine NOS. Similar to uterine tissue, progesterone has been reported to have either positive (21) or negative (22; 23) effects on NOS expression in endothelial cells. These contradictory effects of progesterone on NOS expression could be attributed to the use of different types of cell preparations and animal models.
The results of our study demonstrate for the first time non-genomic action of progesterone to induce phosphorylation of eNOS in HES cells. Several studies predominantly in the cardiovascular system have also demonstrated non-genomic effect of progesterone on NOS expression. In the heart progesterone was shown to regulate cardiac repolarization (24) and in the aorta it simulated NOS activity and inhibited platelet aggregation (25; 26). Progesterone was shown to have rapid effects on the contraction of rat aorta in vitro, and cultured endothelial cells from these vessels released NO after short exposure to progesterone (27).
Phosphorylation of NOS can occur at different amino acids. Phosphorylation at certain amino acids such as Ser 1177 induced eNOS activation whereas phosphorylation on Thr 495 resulted in NO synthesis inhibition (28). Our findings demonstrate progesterone stimulates phosphrylation of eNOS on Ser 1177 which is known to result in activation of eNOS. This phophorylation involved both the PI3K/Akt pathway and the ERK1, 2 pathways. Most studies in endothelial cells have shown the importance of the Akt pathway in eNOS phosphorylation (29; 30); however in response to epoxyeicosatrienoic acids phosphphorylation on Ser 1177 was shown to involve both the PI3/Akt and MAPK pathways similar to our findings with progesterone-induced phophorylation in HES cells.
The progesterone receptor antagonist RU486 was found to have an inhibitory effect on eNOS in HES cells at high concentrations. Similar results were reported by Sun et al showing that administration of RU486 during the implantation phase in normal cycling women induced an inhibition of eNOS in endometrial epithelial but not in endometrial endothelial cells (31). This inhibitory effect of RU486 on eNOS may be due to the mixed agonist and antagonist properties of RU486 (32). At high concentrations RU486 can have agonistic effects on the glucocorticoid receptors (33), and as previously demonstrated by others glucocorticoids can inhibt the expression of eNOS (34) and iNOS (35) in endothelial cells and iNOS in the rat uterus (36). The lack of effect of RU 486 on iNOS expression is similar to our previously reported finding in HES cells, where ICI 182,780, an estrogen receptor antagonist in high concentrations stimulated the expression of iNOS protein while inhibiting eNOS expression (6).
In summary, this study demonstrates that progesterone directly acts on epithelial cells of the endometrium to stimulate the expression of eNOS/iNOS through a nuclear progesterone receptor-mediated mechanism. Progesterone also has rapid non-genomic effects on endometrial epithelial cells to stimulate phosphorylation of eNOS through both the PI3/Akt and MAPK pathways.
The authors wish to thank Ms Hye Jin Park for manuscript preparation.
Financial Support: Supported by NIH grant RO3 HD41409-01 to O. Khorram
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