Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Minamikogushi 111, Ube 755-8505, Japan
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Abstract |
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Key words: cAMP/endometrial stromal cell/progesterone/superoxide dismutase
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Introduction |
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Estrogen and progesterone increase both Cu,Zn-SOD and Mn-SOD expression in human ESC, accompanied by decidualization in vitro (Sugino et al., 2000a). However, we recently found that Cu,Zn-SOD and Mn-SOD expression were differently regulated (Sugino et al., 2002
): 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., 1998a
, 2000b
, 2000c
, 2002
; Takiguchi et al., 2000
; Karube-Harada et al., 2001
). 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., 1993
; Brar et al., 1997
; Telgmann et al., 1997
; Kasahara et al., 2001
). The promoters of human Cu,Zn-SOD and Mn-SOD genes have binding sites for cAMP response element-binding protein (CREB) (Kim et al., 1994
; Wan et al., 1994
). 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., 1994
; Wan et al., 1994
). 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.
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Materials and methods |
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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). [-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 4249 years, who underwent surgery for fibroids. Endometrial samples were histologically diagnosed as late proliferative phase according to published criteria (Noyes et al., 1950). 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., 2000a
). 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., 2000a) and those of other researchers (Irwin et al., 1989
; Brar et al., 1997
; Cicinelli et al., 2000
; Kasahara et al., 2001
). 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., 1997
). 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., 1992
; Kim et al., 1998
; Sugino et al., 2000a
). 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 TrisHCl 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., 1993). 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., 1951
).
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., 2000a). Direct sequence analyses of the PCR products were performed for sequence verification (Sugino et al., 2000a
). The oligonucleotide primers (5'-TGCTGCAGAGGCAGGGAGCCC-3' and 5'-AGGGATCCTCTTCCCATTCCA-3') were used for IGFBP-1 as a marker of decidualization (Kim et al., 1998
; Sugino et al., 2000a
). 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., 1998b
). 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), [
-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.
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Results |
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Discussion |
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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., 1999) and PGF2
production in ESC (Sugino et al., 2000b
, 2001
), whereas Mn-SOD works for cell survival by scavenging oxygen radicals generated in the mitochondria (Sugino et al., 1998a
; Karube-Harada et al., 2001
). 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
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., 1993), which is also shown in Figure 1
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.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 14, 2002; accepted on March 18, 2002.