Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells

Norihiro Sugino,1, Ayako Karube-Harada, Aki Sakata, Shuji Takiguchi and Hiroshi Kato

Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Minamikogushi 1–1–1, Ube 755-8505, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The present study was undertaken to investigate the cAMP-dependent regulation of copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese SOD (Mn-SOD) by ovarian steroids in human endometrial stromal cells (ESC). METHODS and RESULTS: To examine the effect of cAMP on SOD expression, ESC were incubated with dibutyryl-cAMP (db-cAMP, 0.5 mmol/l), forskolin (25 µmol/l), or estradiol (E2, 10-8 mol/l) + medroxyprogesterone acetate (MPA, 10-6 mol/l), for 18 days. E2 + MPA significantly increased Cu,Zn-SOD activity and mRNA concentrations, whereas db-cAMP and forskolin had no effect. On the other hand, Mn-SOD activity and mRNA concentration were significantly increased by all of these treatments. Insulin-like growth factor-binding protein-1, a marker of decidualization, was clearly induced by db-cAMP, forskolin or E2 + MPA, accompanied by morphological changes characteristic of decidualization. To study whether the increase in Mn-SOD by db-cAMP or E2 + MPA was mediated by cAMP-dependent protein kinase A (PKA), ESC were incubated with protein kinase inhibitor (PKI) (10 µg/ml), an inhibitor of PKA, in the presence of db-cAMP or E2 + MPA. The increase in Mn-SOD activity following db-cAMP or E2 + MPA was completely inhibited by PKI. CONCLUSIONS: In the process of decidualization, E2 + MPA increases Mn-SOD expression via a cAMP-dependent pathway. Cu,Zn-SOD is also up-regulated by E2 + MPA, but via a different pathway from that involving cAMP.

Key words: cAMP/endometrial stromal cell/progesterone/superoxide dismutase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
It is well known that oxygen radicals, including superoxide anions, cause cell damage, whereas superoxide dismutase (SOD) protects cells by scavenging superoxide. Recently, much attention has been focused on reports showing that oxygen radicals and SOD regulate cellular function by controlling production or activation of biologically active substances (Feng et al., 1995Go; Li and Karin, 1999Go; Sugino et al., 1999Go; Buhimschi et al., 2000Go; Marshall et al., 2000Go; Sugino et al., 2001Go). In the human endometrium, oxygen radicals are generated, and their production changes during the menstrual cycle and in pregnancy (Sugino et al., 1996Go). Oxygen radicals stimulate human endometrial stromal cells (ESC) to produce prostaglandin F2{alpha} (PGF2{alpha}), which is involved in endometrial breakdown (Baird et al., 1996Go; Sugino et al., 2000bGo, 2001Go). On the other hand, two types of SOD, copper-zinc SOD (Cu,Zn-SOD), located in the cytosol and manganese SOD (Mn-SOD), located in the mitochondria, are present in the human endometrium (Narimoto et al., 1990Go; Sugino et al., 1996Go). We have suggested that Cu,Zn-SOD controls PGF2{alpha} production by preventing accumulation of oxygen radicals in the cytosol (Sugino et al., 2000bGo, 2001Go) and Mn-SOD works for cell survival to scavenge oxygen radicals generated in the mitochondria (Karube-Harada et al., 2001Go).

Estrogen and progesterone increase both Cu,Zn-SOD and Mn-SOD expression in human ESC, accompanied by decidualization in vitro (Sugino et al., 2000aGo). However, we recently found that Cu,Zn-SOD and Mn-SOD expression were differently regulated (Sugino et al., 2002Go): in human ESC treated with estrogen and progesterone, Cu,Zn-SOD gradually declined after withdrawal of these ovarian steroids, whereas Mn-SOD did not. Accumulating data have also shown that Cu,Zn-SOD and Mn-SOD are differently regulated in the ovary and uterus (Sugino et al., 1998aGo, 2000bGo, 2000cGo, 2002Go; Takiguchi et al., 2000Go; Karube-Harada et al., 2001Go). However, little information is available regarding the intracellular signalling pathway that regulates Cu,Zn-SOD and Mn-SOD in human ESC. It has been reported that estrogen and progesterone induce decidualization of ESC via cyclic adenosine 3',5'-monophosphate (cAMP) (Tang et al., 1993Go; Brar et al., 1997Go; Telgmann et al., 1997Go; Kasahara et al., 2001Go). The promoters of human Cu,Zn-SOD and Mn-SOD genes have binding sites for cAMP response element-binding protein (CREB) (Kim et al., 1994Go; Wan et al., 1994Go). However, there are also some binding sites for glucocorticoid receptor/progesterone receptor (GR/PR) in the promoter regions of both SOD genes (Kim et al., 1994Go; Wan et al., 1994Go). Therefore, there is a possibility that estrogen and progesterone regulate Cu,Zn-SOD and Mn-SOD through the different pathways, e.g. directly or via a cAMP-dependent pathway. The present study was therefore undertaken to investigate whether the effects of estrogen and progesterone on both types of SOD are mediated by cAMP during decidualization of human ESC.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This project was reviewed and approved by the committee of investigations involving human subjects of Yamaguchi University School of Medicine. Informed consent from the patient was obtained before collection of any tissue samples for this study.

Materials
Phenol red-free Dulbecco's modified Eagle's medium (DMEM) and glutamine were purchased from ICN Biomedical Inc. (Aurora, OH, USA). Streptomycin, penicillin, 1xtrypsin-EDTA, deoxynucleotide triphosphate and Moloney murine leukaemia virus reverse transcriptase were from Life Technologies Inc. (Grand Island, NY, USA). Collagenase (type I), estradiol (E2), medroxyprogesterone acetate (MPA), protein kinase inhibitor (PKI, a synthetic 20-amino acid peptide, rabbit sequence), dibutyryl cyclic AMP (db-cAMP) and forskolin were obtained from Sigma Chemical Co. (St Louis, MO, USA). Fetal calf serum (FCS) was from PAA Laboratories GmbH (Linz, Austria). Tissue flasks and nylon mesh were from Becton Dickinson Co. (Franklin lakes, NJ, USA). Random hexamer and Taq DNA polymerase were from Perkin-Elmer Co. (Foster City, CA, USA). [{alpha}-32P]-deoxycitidine triphosphate (dCTP) was from Amersham (Arlington Heights, IL, USA). Isogen was from Wako Pure Chemical Industries Ltd. (Osaka, Japan).

ESC isolation
Human endometrium was obtained at hysterectomy from normally cycling pre-menopausal women, aged 42–49 years, who underwent surgery for fibroids. Endometrial samples were histologically diagnosed as late proliferative phase according to published criteria (Noyes et al., 1950Go). Tissue samples were washed with phenol red-free DMEM containing 4 mmol/l glutamine, 50 µg/ml streptomycin and 50 IU/ml penicillin, and minced into small pieces of <1 mm3. ESC were isolated as reported previously (Sugino et al., 2000aGo). In brief, after the enzymatic digestion of minced tissues with 0.2% collagenase in a shaking water bath for 2 h at 37°C, stromal cells were separated by filtration through a 70 µm nylon mesh. The filtrates were washed three times, and the number of viable cells was counted by trypan blue dye exclusion. The homogeneity of the stromal cell preparation was verified by the immunocytochemistry using the specific antibody against stromal cells, vimentin (data not shown). Cells were seeded at 105 cells/cm2 in 75 cm2 tissue culture flasks and incubated in phenol red-free DMEM containing glutamine, antibiotics and 10% dextran-coated charcoal-stripped FCS at 37°C, 95% air and 5% CO2. At confluence, cells were treated with 1xtrypsin-EDTA and subcultured into 25 cm2 tissue culture flasks. At ~80% confluence after the first passage, the cell culture medium was changed to the treatment medium.

Cell culture
To examine the effects of cAMP on SOD activity and mRNA concentration in ESC, cells were incubated with treatment medium (phenol red-free DMEM supplemented with glutamine, antibiotics, 2% stripped FCS) containing db-cAMP (0.5 mmol/l) and forskolin (25 µmol/l), that increases intracellular cAMP via adenylate cyclase, or a combination of E2 (10-8 mol/l) and MPA (10-6 mol/l) for 18 days at 37°C, 95% air and 5% CO2. The concentrations of ovarian steroids and the period of incubation were based on our previous report (Sugino et al., 2000aGo) and those of other researchers (Irwin et al., 1989Go; Brar et al., 1997Go; Cicinelli et al., 2000Go; Kasahara et al., 2001Go). Secondly, we studied whether the change in SOD expression caused by cAMP analog or E2 + MPA is mediated by cAMP-dependent protein kinase, protein kinase A (PKA) (Telgmann et al., 1997Go). ESC were incubated with treatment medium containing PKA inhibitor (PKI, 10 µg/ml) in the presence of db-cAMP (0.5 mmol/l) or E2 (10-8 mol/l) + MPA (10-6 mol/l) for 18 days at 37°C, 95% air and 5% CO2. Decidualization was confirmed by morphology and mRNA expression of insulin-like growth factor-binding protein-1 (IGFBP-1), which is a specific marker of decidualization (Giudice et al., 1992Go; Kim et al., 1998Go; Sugino et al., 2000aGo). The medium was changed every other day. Three different experiments were performed in triplicate.

SOD assay
After incubation, cells were washed with PBS, resuspended in Tris–HCl buffer (0.01 mol/l, pH 7.4) and sonicated. Cu,Zn-SOD activity and Mn-SOD activity were determined as reported previously (Sugino et al., 1993Go). The amount of protein required for 50% inhibition in the absorbance at 550 nm was defined as one unit (nitrite unit = NU) of SOD activity. All data were expressed in NU of SOD activity per mg protein. Protein concentrations were determined by a published method (Lowry et al., 1951Go).

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from the cultured cell with Isogen by the method provided by the manufacturer. For mRNA analysis, RT-PCR was performed with the oligonucleotide primers for Cu,Zn-SOD(5'-CGAGCAGAAGGAAAGTAATG-3' and 5'-TAGCAGGATAACAGATGAGT-3') and for Mn-SOD (5'-AGTTCAATGGTGGTGGTCATA-3' and 5'-CAATCCCCAGCAGTGGAATAA-3') as reported previously (Sugino et al., 2000aGo). Direct sequence analyses of the PCR products were performed for sequence verification (Sugino et al., 2000aGo). The oligonucleotide primers (5'-TGCTGCAGAGGCAGGGAGCCC-3' and 5'-AGGGATCCTCTTCCCATTCCA-3') were used for IGFBP-1 as a marker of decidualization (Kim et al., 1998Go; Sugino et al., 2000aGo). Two oligonucleotide primers (5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-GGACAGAGTCTTGATGATCTC-3') were also used to amplify ribosomal protein L19 as an internal control as reported previously (Sugino et al., 1998bGo). Briefly, 3 µg of total RNA was reverse-transcribed at 42°C in a reaction mixture (single-strength PCR buffer, 2.5 µmol/l deoxynucleotide triphosphates, 5 µmol/l random hexamer primer, 1.5 µmol/l MgCl2, and 200 IU Moloney murine leukaemia virus reverse-transcriptase). The RT product was divided into two equal aliquots (one tube was for L19 primers), and PCR was performed. For PCR amplification, a mixture containing the oligonucleotide primers (50 pmol), [{alpha}-32P]-dCTP (2 mCi at 3000 Ci/mmol), and Taq DNA polymerase (2.5 IU) was added to each reaction. Amplification was carried out for 25 cycles consisting of 95°C (1 min), 52°C (1 min) and 72°C (1 min) for Cu,Zn-SOD, 25 cycles consisting of 95°C (1 min), 54°C (1 min) and 72°C (1 min) for Mn-SOD, and 24 cycles consisting of 94°C (1 min), 60°C (2 min) and 72°C (3 min) for IGFBP-1 followed by 10 min of final extension at 72°C in a programmed temperature control system PC-800 (ASTEC, Fukuoka, Japan). The predicted sizes of the PCR-amplified products were 455 bp for Cu,Zn-SOD, 282 bp for Mn-SOD, 379 bp for IGFBP-1 and 194 bp for L19. Reaction products were electrophoresed on an 8% polyacrylamide non-denaturing gel. After autoradiography, band intensities were analysed using a bioimaging analyser BAS2000 (Fuji Photo Film Co, Tokyo, Japan). For quantification, the density of the signals of Mn-SOD, Cu,Zn-SOD, and IGFBP-1 was normalized to that of the internal control L19.

Statistical analysis
Data were examined by analysis of variance and Duncan's new multiple range test. Differences were considered significant at P < 0.05.


    Results
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 Results
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 References
 
After ESC were treated with db-cAMP, forskolin, or E2 + MPA for 18 days, decidualization was determined by IGFBP-1 mRNA expression and morphology. As shown in Figure 1Go, db-cAMP, forskolin or E2 + MPA clearly induced IGFBP-1 mRNA expression, accompanied by morphological changes characteristic of decidualization as described previously (Irwin et al., 1989Go).



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Figure 1. In-vitro decidualization of human endometrial stromal cells (ESC) induced by estradiol and medroxyprogesterone acetate(E2 + MPA), dibutyryl cyclic AMP (db-cAMP), and forskolin. (A) Insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA expression in human ESC treated with E2 (10-8 mol/l) + MPA (10-6 mol/l), db-cAMP (0.5 mmol/l), and forskolin (25 µmol/l) for 18 days. mRNA expression was analysed by reverse transcription-polymerase chain reaction (RT-PCR). Ribosomal protein L19 is used as an internal control. (B) Morphology of human ESC treated with E2 + MPA, db-cAMP, and forskolin for 18 days (original magnification x90).

 
The effects of db-cAMP, forskolin or E2 + MPA on Cu,Zn-SOD and Mn-SOD activities in human ESC are shown in Figure 2Go. E2 + MPA significantly increased Cu,Zn-SOD activity whereas db-cAMP and forskolin had no significant effect (Figure 2Go). On the other hand, Mn-SOD activity was significantly increased by db-cAMP, forskolin and E2 + MPA, and there was a significant difference between forskolin and E2 + MPA groups (Figure 2Go). The effects of db-cAMP, forskolin or E2 + MPA on mRNA concentrations of Cu,Zn-SOD and Mn-SOD are shown in Figure 3Go. E2 + MPA significantly increased Cu,Zn-SOD mRNA concentrations, whereas db-cAMP and forskolin had no significant effect (Figure 3AGo). On the other hand, Mn-SOD mRNA concentrations were significantly increased by db-cAMP, forskolin and E2 + MPA (Figure 3BGo).



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Figure 2. Effects of dibutyryl cyclic AMP (db-cAMP), forskolin, and estradiol and medroxyprogesterone acetate (E2 + MPA) onCu,Zn-SOD and Mn-SOD activities in human endometrial stromal cells (ESC). ESC were incubated as described in the legend to Figure 1Go. Values are mean ± SEM of three different experiments. aP < 0.01 versus control (C) and bP < 0.05 versus forskolin.

 


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Figure 3. Effects of dibutyryl cyclic AMP (db-cAMP), forskolin, and estradiol and medroxyprogesterone acetate (E2 + MPA) on mRNA concentrations of Cu,Zn-SOD (A) and Mn-SOD (B) in human endometrial stromal cells (ESC). ESC were incubated as described in the legend to Figure 1Go. mRNA expression was analysed by RT-PCR. The intensity of the signals of Cu,Zn-SOD or Mn-SOD was normalized to that of the internal control L19. The quantification data (the ratio of Cu,Zn-SOD or Mn-SOD to L19) represent the mean ± SEM of three different experiments. bP < 0.05 versus control (C).

 
To study whether the increase in Mn-SOD expression induced by db-cAMP or E2 + MPA was mediated by cAMP, ESC were incubated with PKI, which inhibits cAMP-dependent protein kinase, in the presence of db-cAMP or E2 + MPA. PKI had no significant effect on the increase in Cu,Zn-SOD activity caused by E2 + MPA (Figure 4AGo). On the other hand, stimulatory effects of db-cAMP or E2 + MPA on Mn-SOD activity were completely suppressed by PKI (Figure 4BGo). PKI alone had no effect on Cu,Zn-SOD and Mn-SOD activities (Figure 4A and BGo).



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Figure 4. Effects of protein kinase inhibitor (PKI) on the increases in Cu,Zn-SOD and Mn-SOD activities caused by estradiol and medroxyprogesterone acetate (E2 + MPA) or dibutyryl cyclic AMP (db-cAMP) in human endometrial stromal cells (ESC). ESC were incubated with PKI (10 µg/ml) in the presence of db-cAMP (0.5 mmol/l) or E2 (10-8 mol/l) + MPA (10-6 mol/l) for 18 days. Values are mean ± SEM of three different experiments. (A) Cu,Zn-SOD activity; aP < 0.01 versus control, (B) Mn-SOD activity, aP < 0.01 versus control, db-cAMP + PKI, and E2 + MPA + PKI.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Both Cu,Zn-SOD and Mn-SOD expression in human ESC were increased by estrogen and progesterone in vitro (Sugino et al., 2000aGo). The present study demonstrates that estrogen and progesterone increase Mn-SOD expression via a cAMP-dependent pathway, whereas Cu,Zn-SOD is, at least in part, regulated in a different way from the cAMP-dependent pathway. Cu,Zn-SOD may be regulated directly by progesterone in human ESC, which may be supported by the findings that the promoter of human Cu,Zn-SOD gene has binding sites for GR/PR (Kim et al., 1994Go) and that progesterone was important in the stimulatory effect of estrogen and progesterone on Cu,Zn-SOD mRNA expression in human ESC (Sugino et al., 2002Go). In fact, we found that Cu,Zn-SOD, but not Mn-SOD, rapidly declined in ESC after withdrawal of estrogen and progesterone (Sugino et al., 2002Go). However, since the promoter of Mn-SOD gene also has binding sites for CREB and GR/PR, we cannot discount the presence of additional mechanisms modulating the expression of Cu,Zn-SOD and Mn-SOD genes.

These different regulations of Cu,Zn-SOD and Mn-SOD may reflect the different roles of these two types of SOD in ESC undergoing decidualization. We have reported that Cu,Zn-SOD is closely involved in the regulation of cellular function, e.g. progesterone production in luteal cells (Sugino et al., 1999Go) and PGF2{alpha} production in ESC (Sugino et al., 2000bGo, 2001Go), whereas Mn-SOD works for cell survival by scavenging oxygen radicals generated in the mitochondria (Sugino et al., 1998aGo; Karube-Harada et al., 2001Go). The fact that cAMP, which is a powerful factor in the induction of decidualization, increased only Mn-SOD suggests that Mn-SOD protects ESC for successful decidualization by scavenging oxygen radicals generated in the mitochondria. Also, the present result may raise another possibility that substances that elevate intracellular cAMP concentrations have the potential to induce Mn-SOD. For example, human chorionic gonadotrophin, which is often used for luteal support, may protect ESC from oxygen radicals by directly inducing Mn-SOD expression. On the other hand, Cu,Zn-SOD, which is not associated with decidualization, may be involved in the cellular function of ESC such that Cu,Zn-SOD in the cytosol prevents accumulation of oxygen radicals responsible for PGF2{alpha} production.

The present result showed that forskolin increased Mn-SOD activities more than E2 + MPA did. This is consistent with the report that substances that elevate intracellular cAMP concentrations can induce decidualization much more effectively than progesterone (Tang et al., 1993Go), which is also shown in Figure 1Go of the present study. Mn-SOD activities in the db-cAMP group tended to be higher than those in the E2 + MPA group, but there was no significant difference between them. This may be due to the difference in intracellular cAMP concentrations. Also, there seems to be a discrepancy in this study between enzyme activities and mRNA concentrations of Mn-SOD. This may be due to the different sensitivities of RT-PCR and SOD activity assay.

In conclusion, the present study has shown that in the process of decidualization, estrogen and progesterone increase Mn-SOD expression via a cAMP-dependent pathway but Cu,Zn-SOD via a different pathway. These different regulations of Cu,Zn-SOD and Mn-SOD may reflect the different roles of these two types of SOD in ESC undergoing decidualization.


    Acknowledgements
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported in part by a grant from Grant-in-Aid for Scientific Research (11671623 and 13671721) from the Ministry of Education, Science, and Culture, Japan.


    Notes
 
1 To whom correspondence should be addressed. E-mail: obgyn{at}po.cc.yamaguchi-u.ac.jp Back


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 Discussion
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Submitted on January 14, 2002; accepted on March 18, 2002.