17β-Estradiol Inhibits Oxidative Stress-Induced Apoptosis in Keratinocytes by Promoting Bcl-2 Expression
2003; Elsevier BV; Volume: 121; Issue: 6 Linguagem: Inglês
10.1111/j.1523-1747.2003.12617.x
ISSN1523-1747
AutoresNaoko Kanda, Shin‐ichi Watanabe,
Tópico(s)Cell death mechanisms and regulation
ResumoWe examined in vitro effects of 17β-estradiol on H2O2-induced apoptosis in human keratinocytes. 17β-estradiol prevented the H2O2-induced apoptosis. H2O2 decreased, whereas 17β-estradiol increased Bcl-2 protein and mRNA levels in keratinocytes, and H2O2 plus 17β-estradiol led to basal levels. Overexpression of Bcl-2 protected keratinocytes against H2O2-induced apoptosis, indicating the anti-apoptotic effect of Bcl-2. H2O2 suppressed, whereas 17β-estradiol enhanced bcl-2 promoter activity, and H2O2 plus 17β-estradiol led to basal activity. Cyclic adenosine monophosphate (cAMP) response element on bcl-2 promoter was responsible for the effects of 17β-estradiol and H2O2. Bcl-2 expression was enhanced by membrane-impermeable bovine serum albumin-conjugated 17β-estradiol, indicating the effects via membrane 17β-estradiol-binding sites. H2O2 decreased, whereas 17β-estradiol increased the amount of phosphorylated cAMP response element-binding protein and cAMP response element-dependent transcriptional activity, and H2O2 plus 17β-estradiol led to basal levels. H-89, an inhibitor of cAMP-dependent protein kinase A, suppressed basal and 17β-estradiol-induced cAMP response element-binding protein phosphorylation, cAMP response element-dependent transcriptional activity, Bcl-2 expression, and apoptosis resistance. The cAMP analog, dibutyryl cAMP, enhanced cAMP response element-binding protein phosphorylation, cAMP response element-dependent transcriptional activity, Bcl-2 expression, and apoptosis resistance. 17β-estradiol increased intracellular cAMP level and protein kinase A activity, whereas these were not altered by H2O2. Keratinocytes expressed mRNA for estrogen receptor β and guanine nucleotide-binding protein-coupled receptor, GPR30. GPR30 anti-sense oligonucleotide did, but anti-sense estrogen receptor β did not suppress 17β-estradiol-induced cAMP signal, cAMP response element-binding protein phosphorylation, Bcl-2 expression, and apoptosis resistance. These results suggest that 17β-estradiol may enhance Bcl-2 expression and prevent H2O2-induced apoptosis by phosphorylating cAMP response element-binding protein via cAMP/protein kinase A pathway in keratinocytes. These effects of 17β-estradiol may be mediated via membrane GPR30. We examined in vitro effects of 17β-estradiol on H2O2-induced apoptosis in human keratinocytes. 17β-estradiol prevented the H2O2-induced apoptosis. H2O2 decreased, whereas 17β-estradiol increased Bcl-2 protein and mRNA levels in keratinocytes, and H2O2 plus 17β-estradiol led to basal levels. Overexpression of Bcl-2 protected keratinocytes against H2O2-induced apoptosis, indicating the anti-apoptotic effect of Bcl-2. H2O2 suppressed, whereas 17β-estradiol enhanced bcl-2 promoter activity, and H2O2 plus 17β-estradiol led to basal activity. Cyclic adenosine monophosphate (cAMP) response element on bcl-2 promoter was responsible for the effects of 17β-estradiol and H2O2. Bcl-2 expression was enhanced by membrane-impermeable bovine serum albumin-conjugated 17β-estradiol, indicating the effects via membrane 17β-estradiol-binding sites. H2O2 decreased, whereas 17β-estradiol increased the amount of phosphorylated cAMP response element-binding protein and cAMP response element-dependent transcriptional activity, and H2O2 plus 17β-estradiol led to basal levels. H-89, an inhibitor of cAMP-dependent protein kinase A, suppressed basal and 17β-estradiol-induced cAMP response element-binding protein phosphorylation, cAMP response element-dependent transcriptional activity, Bcl-2 expression, and apoptosis resistance. The cAMP analog, dibutyryl cAMP, enhanced cAMP response element-binding protein phosphorylation, cAMP response element-dependent transcriptional activity, Bcl-2 expression, and apoptosis resistance. 17β-estradiol increased intracellular cAMP level and protein kinase A activity, whereas these were not altered by H2O2. Keratinocytes expressed mRNA for estrogen receptor β and guanine nucleotide-binding protein-coupled receptor, GPR30. GPR30 anti-sense oligonucleotide did, but anti-sense estrogen receptor β did not suppress 17β-estradiol-induced cAMP signal, cAMP response element-binding protein phosphorylation, Bcl-2 expression, and apoptosis resistance. These results suggest that 17β-estradiol may enhance Bcl-2 expression and prevent H2O2-induced apoptosis by phosphorylating cAMP response element-binding protein via cAMP/protein kinase A pathway in keratinocytes. These effects of 17β-estradiol may be mediated via membrane GPR30. adenylate cyclase cAMP response element CRE-binding protein 17β-estradiol 17β-estradiol 6-(O-carboxymethyl)oxime:bovine serum albumin estrogen receptor extracellular signal-regulated kinase glyceraldehyde-3-phosphate dehydrogenase guanine nucleotide-binding protein Keratinocyte basal median mitogen-activated protein kinase MAPK-activated protein MAPK or ERK kinase phosphatidylinositol 3-OH kinase protein kinase A It has been reported that estrogen manifests protective effects on the skin, and may aid in maintaining skin homeostasis (Shah and Maibach, 2001Shah M.G. Maibach H.I. Estrogen and skin. An overview.Am J Clin Dermatol. 2001; 2: 143-150Crossref PubMed Scopus (150) Google Scholar). Estrogen is known to sustain skin thickness and water-holding capacity (Shah and Maibach, 2001Shah M.G. Maibach H.I. Estrogen and skin. An overview.Am J Clin Dermatol. 2001; 2: 143-150Crossref PubMed Scopus (150) Google Scholar), and also accelerates cutaneous wound healing (Ashcroft et al., 1997Ashcroft G. Dodsworth J. van Boxtel E. Tarnuzzer R.W. Horan M.A. Schultz G.S. Ferguson W.J. Estrogen accelerates cutaneous wound healing associated with an increase in TGF-β1 levels.Nature Med. 1997; 3: 1209-1215Crossref PubMed Scopus (439) Google Scholar). These effects of estrogen are mostly exerted by its direct action on dermal fibroblasts; estrogen induces fibroblasts to produce transforming growth factor β1, which promotes the maturation of fibroblasts and synthesis of the extracellular matrix (Ashcroft et al., 1997Ashcroft G. Dodsworth J. van Boxtel E. Tarnuzzer R.W. Horan M.A. Schultz G.S. Ferguson W.J. Estrogen accelerates cutaneous wound healing associated with an increase in TGF-β1 levels.Nature Med. 1997; 3: 1209-1215Crossref PubMed Scopus (439) Google Scholar). On the other hand, little is known about the direct effects of estrogen on epidermal keratinocytes. We recently found that 17β-estradiol (E2) acted on human keratinocytes and inhibited their production of chemokines, IP-10, MCP-1, and RANTES, which recruit activated T helper-1 cells or macrophages (Kanda and Watanabe, 2003aKanda N. Watanabe S. 17β-estradiol inhibits the production of interferon-induced protein of 10 kDa by human keratinocytes.J Invest Dermatol. 2003; 120: 411-419Crossref PubMed Scopus (25) Google Scholar,Kanda and Watanabe, 2003bKanda N. Watanabe S. 17β-estradiol inhibits the production of RANTES in human keratinocytes.J Invest Dermatol. 2003; 120: 420-427Crossref PubMed Scopus (51) Google Scholar,Kanda and Watanabe, 2003cKanda N. Watanabe S. 17β-estradiol inhibits MCP-1 production in human keratinocytes.J Invest Dermatol. 2003; 120: 1058-1066Abstract Full Text Full Text PDF PubMed Google Scholar). These results indicate that E2 may suppress the development of inflammatory skin diseases, such as psoriasis or lichen planus.Urano et al., 1995Urano R. Sakabe K. Seiki K. Ohkido M. Female sex hormone stimulates cultured human keratinocyte proliferation and its RNA- and protein-synthetic activities.J Dermatol Sci. 1995; 9: 176-184Abstract Full Text PDF PubMed Scopus (36) Google Scholar also reported that E2 stimulated proliferation of human keratinocytes; however, it has not been examined if E2 may promote the survival of keratinocytes. Keratinocytes apt to be exposed to a variety of environmental or endogenous stresses, such as oxidative stress, inflammatory cytokines such as tumor necrosis factor-α, ultraviolet (UV) light, or ionizing irradiation (Qin et al., 2001Qin J.-Z. Bacon P. Chaturvedi V. Nickoloff B.J. Role of NF-κB activity in apoptotic response of keratinocytes mediated by interferon-γ, tumor necrosis factor-α, and tumor-necrosis factor-related apoptosis-inducing ligand.J Invest Dermatol. 2001; 117: 898-907Crossref PubMed Google Scholar;Chang et al., 2002Chang H. Sander C.S. Muller C.S.L. Elsner P. Thiele J.J. Detection of poly(ADP-ribose) by immunocytochemistry: A sensitive new method for the early identification of UVB- and H2O2-induced apoptosis in keratinocytes.Biol Chem. 2002; 383: 703-708Crossref PubMed Scopus (25) Google Scholar). To prevent the stress-induced apoptosis may thus maintain the survival of keratinocytes and contribute to skin homeostasis. Previous studies reported that E2 inhibited staurosporine-induced apoptosis in rat cortical neurons (Honda et al., 2001Honda K. Shimohama S. Sawada H. Kihara T. Nakamizo T. Shibasaki H. Akaike A. Nongenomic antiapoptotic signal transduction by estrogen in cultured cortical neurons.J Neurosci Res. 2001; 64: 466-475Crossref PubMed Scopus (119) Google Scholar) or tumor necrosis factor-α-induced apoptosis in human breast cancer MCF-7 cells (Burow et al., 1999Burow M.E. Tang Y. Collins-Burow B.M. Krajewski S. Reed J.C. McLachlan J.A. Beckman B.S. Effects of environmental estrogens on tumor necrosis factor α-mediated apoptosis in MCF-7 cells.Carcinogenesis. 1999; 20: 2057-2061Crossref PubMed Scopus (64) Google Scholar). On the other hand, E2 enhanced tamoxifen-induced apoptosis in hypothalamic neuronal cells (Hashimoto et al., 1997Hashimoto M. Inoue S. Muramatsu M. Masliah E. Estrogens stimulate tamoxifen-induced neuronal cell apoptosis in vitro: A possible nongenomic action.Biochem Biophys Res Commun. 1997; 240: 464-470Crossref PubMed Scopus (28) Google Scholar). Though E2 inhibited vitamin E succinate-induced apoptosis, it promoted spontaneous apoptosis in MCF-7 cells (Tesarik et al., 1999Tesarik J. Garrigosa L. Mendoza C. Estradiol modulates breast cancer cell apoptosis: A novel nongenomic steroid action relevant to carcinogenesis.Steroids. 1999; 64: 22-27Crossref PubMed Scopus (18) Google Scholar). Thus E2 may either suppress or promote apoptosis dependently on cell types or experimental conditions. It is reported that hydrogen peroxide, tumor necrosis factor-α, or cell-permeable ceramides suppressed the expression of anti-apoptotic protein Bcl-2 and thus induced apoptosis in leukemia HL-60 or U937 cells or hippocampal neurons (Chen et al., 1995Chen M. Quintans J. Fuks Z. Thompson C. Kufe D.W. Weichselbaum R.R. Suppression of Bcl-2 messenger RNA production may mediate apoptosis after ionizing radiation, tumor necrosis factor α, and ceramide.Cancer Res. 1995; 55: 991-994PubMed Google Scholar;Pugazhenthi et al., 2003Pugazhenthi S. Nesterova A. Jambal P. et al.Oxidative stress-mediated down-regulation of bcl-2 promoter in hippocampal neurons.J Neurochem. 2003; 84: 982-996Crossref PubMed Scopus (95) Google Scholar), whereas overexpression of Bcl-2 counteracted apoptosis in B-lineage cells (Vaux et al., 1988Vaux D.L. Cory S. Adams J.M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells.Nature. 1988; 335: 440-442Crossref PubMed Scopus (2634) Google Scholar;Alam et al., 1997Alam M.K. Davison S. Siddiqui N. Norton J.D. Murphy J. Ectopic expression of Bcl-2, but not Bcl-xL rescues Ramos B cells from Fas-mediated apoptosis.Eur J Immunol. 1997; 27: 3485-3491Crossref PubMed Scopus (25) Google Scholar). Bcl-2 prevents the activation of caspases, inhibits membrane lipid peroxidation, and blocks the actions of pro-apoptotic bcl-2 family members such as Bax or Bad (Merry and Korsmeyer, 1997Merry D.E. Korsmeyer S.J. Bcl-2 gene family in the nervous system.Annu Rev Neurosci. 1997; 20: 245-267Crossref PubMed Scopus (536) Google Scholar). Thus E2 may either suppress or promote apoptosis by regulating the expression of these pro-apoptotic or anti-apoptotic bcl-2 family proteins. It is known that the effects of E2 are mediated by genomic or nongenomic mechanisms (Beato, 1989Beato M. Gene regulation by steroid hormones.Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2807) Google Scholar;Kelly and Levin, 2001Kelly M.J. Levin E.R. Rapid actions of plasma membrane estrogen receptors.Trends Endocrinol Metab. 2001; 12: 152-156Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). E2 may thus regulate apoptosis by either or both mechanisms. The genomic mechanism is that E2-bound nuclear estrogen receptor (ER) α or β stimulates or inhibits gene expression by binding to estrogen response element of the target genes or by interacting with other transcription factors (Beato, 1989Beato M. Gene regulation by steroid hormones.Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2807) Google Scholar;Kanda and Watanabe, 2003cKanda N. Watanabe S. 17β-estradiol inhibits MCP-1 production in human keratinocytes.J Invest Dermatol. 2003; 120: 1058-1066Abstract Full Text Full Text PDF PubMed Google Scholar). On the other hand, the nongenomic mechanism is that E2 interacts with cell surface E2-binding sites and rapidly induces a variety of intracellular signals (Kelly and Levin, 2001Kelly M.J. Levin E.R. Rapid actions of plasma membrane estrogen receptors.Trends Endocrinol Metab. 2001; 12: 152-156Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar), such as activation of adenylate cyclase (AC) (Aronica et al., 1994Aronica S.M. Kraus W.L. Katzenellenbogen B.S. Estrogen action via the cAMP signaling pathway: Stimulation of adenylate cyclase and cAMP-regulated gene transcription.Proc Natl Acad Sci USA. 1994; 91: 8517-8521Crossref PubMed Scopus (591) Google Scholar), phospholipase C (Razandi et al., 1999Razandi M. Pedram A. Greene G.L. Levin E.R. Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: Studies of ERα and ERβ expressed in Chinese hamster ovary cells.Mol Endocrinol. 1999; 13: 307-319Crossref PubMed Scopus (982) Google Scholar), or extracellular signal-regulated kinase (ERK) (Migliaccio et al., 1996Migliaccio A. Di Domenico M. Castoria G. de Falco A. Bontempo P. Nola E. Auricchio F. Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.EMBO J. 1996; 15: 1292-1300Crossref PubMed Scopus (847) Google Scholar). Membrane E2-binding sites are not a unique molecule; some are post-translationally modified forms of nuclear ERα or β, whereas others are structurally different from classical nuclear ER (Papas et al., 1995Papas T.C. Gametchu B. Watson C.S. Membrane estrogen receptors identified by multiple antibody labeling and impeded ligand binding.FASEB J. 1995; 9: 404-410PubMed Google Scholar;Razandi et al., 1999Razandi M. Pedram A. Greene G.L. Levin E.R. Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: Studies of ERα and ERβ expressed in Chinese hamster ovary cells.Mol Endocrinol. 1999; 13: 307-319Crossref PubMed Scopus (982) Google Scholar;Benten et al., 2001Benten W.P.M. Stephan C. Lieberherr M. Wunderlich F. Estradiol signaling via sequesterable surface receptors.Endocrinology. 2001; 142: 1669-1677Crossref PubMed Scopus (134) Google Scholar). It is reported that E2 interacts with several guanine nucleotide-binding protein (G-protein)-coupled receptors such as steroid hormone-binding globulin receptor (Fissore et al., 1994Fissore F. Fortunati N. Comba A. Fazzari A. Gaidano G. Berta L. Frairia R. The receptor-mediated action of sex steroid binding protein (SPB, SHBG): Accumulation of cAMP in MCF-7 cells under SBP and estradiol treatment.Steroids. 1994; 59: 661-667Crossref PubMed Scopus (66) Google Scholar;Nakla et al., 1994Nakla A.M. Khan M.S. Romas N.A. Rosner W. Estradiol causes the rapid accumulation of cAMP in human prostate.Proc Natl Acad Sci USA. 1994; 91: 5402-5405Crossref PubMed Scopus (127) Google Scholar), or an orphan receptor GPR30 (Filardo et al., 2002Filardo E.J. Quinn J.A. Frackelton Jr., A.R. Bland K.I. Estrogen action via the G protein-coupled receptor, GPR30: Stimulation of adenylyl cyclase and cAMP-mediated attenuation of the epidermal growth factor receptor-to-MAPK signaling axis.Mol Endocrinol. 2002; 16: 70-84Crossref PubMed Scopus (557) Google Scholar). In this study, we examined the in vitro effects of E2 on H2O2-induced apoptosis in human keratinocytes. We used H2O2 as an apoptotic model as reactive oxygen species, including H2O2 are produced and act as apoptotic regulators in keratinocytes exposed to UV, tumor necrosis factor-α, bacteria, or viruses (Kohen and Gati, 2000Kohen R. Gati I. Skin low molecular weight antioxidants and their role in ageing and in oxidative stress.Toxicology. 2000; 148: 149-157Crossref PubMed Scopus (135) Google Scholar). We found that E2 prevented H2O2-induced apoptosis in keratinocytes. We further studied the mechanism for the effects, focusing on the regulation of Bcl-2 expression by E2. E2-bovine serum albumin (E2-BSA) and 17α-estradiol were purchased from Sigma (St Louis, Missouri). SQ22536, LY294002, SB202190, PD98059, H-89, and W-7 were obtained from Calbiochem (La Jolla, California). Monoclonal antibodies against cyclic adenosine monophosphate (cAMP) response element (CRE) binding protein (CREB) and Ser133-phosphorylated CREB were purchased from New England Biolabs (Beverly, Massachusetts). Anti-Bcl-2 monoclonal antibody was from Transduction Laboratories (Lexington, Kentucky). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody was from American Research Products (Belmont, Massachusetts). Human neonatal foreskin keratinocytes were cultured in serum-free keratinocyte growth medium (Clonetics, Walkersville, Maryland) consisting of basal medium MCDB153 supplemented with 0.5 μg hydrocortisone per mL, 5 ng epidermal growth factor per mL, 5 μg insulin per mL, and 0.5% bovine pituitary extract. The cells in third passage were used. Apoptosis was measured by quantification of cytoplasmic histone-associated DNA fragments using the cell death detection kit ELISA Plus (Roche, Indianapolis, Indiana) according to the manufacturer's instructions. Keratinocytes (5×103 per well) were seeded in triplicate into 96-well plates in 200 μL of keratinocyte growth medium, adhered overnight, then the medium was changed to phenol red-free, basal KBM depleted of growth supplements, and incubated for 24 h. The medium was removed and the cells were incubated with 30 μM H2O2 in the presence or absence of indicated hormones in phenol red-free KBM for 5 h. The cell lysates were centrifuged, and the amount of apoptotic nucleosomes in the supernatants was measured. The extent of apoptosis was presented as enrichment factor defined as absorbance at 405 nm minus absorbance at 492 nm divided by the same absorbance difference in controls with medium alone. Preliminary dose–response (10–200 μM) and kinetic (1–10 h) experiments led to the determination of the optimal conditions, i.e., 30 μM H2O2 for 5 h. Mitochondrial membrane potential was measured by rhodamine 123 fluorescence as described (Sitailo et al., 2002Sitailo L.A. Tibudan S.S. Denning M.F. Activation of caspase-9 is required for UV-induced apoptosis of human keratinocytes.J Biol Chem. 2002; 277: 19346-19352Crossref PubMed Scopus (132) Google Scholar). H2O2-treated keratinocytes were trypsinized and incubated for 20 min in KBM containing 5 μM rhodamine 123 (Sigma) at room temperature. The cells were washed and analyzed by flow cytometry for reduced rhodamine 123 fluorescence, indicating loss of mitochondrial membrane potential. We performed semiquantitative reverse transcription–PCR to determine relative changes in mRNA levels by densitometric analysis. Keratinocytes were incubated as above, and total cellular RNA was isolated using TRIzol reagent (Invitrogen, Rockville, Maryland). We reverse-transcribed 0.5 μg of total RNA to produce cDNA as described (Tjandrawinata et al., 1997Tjandrawinata R.R. Dahiya R. Hughes-Fulford M. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells.Br J Cancer. 1997; 75: 1111-1118Crossref PubMed Scopus (209) Google Scholar), and 1/30 of synthesized cDNA was thermocycled for PCR amplification with 1 μM each primer and 1.5 U of Taq polymerase (Invitrogen). The sequences of PCR primers for ERα, ERβ, GPR30, bcl-2, bax, bcl-xL, bad, bak, and GAPDH were previously described (Carmeci et al., 1997Carmeci C. Thompson D.A. Ring H.Z. Francke U. Weigel R.J. Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer.Genomics. 1997; 45: 607-617Crossref PubMed Scopus (402) Google Scholar;Dong et al., 1999Dong L. Wang W. Wang F. et al.Mechanisms of transcriptional activation of bcl-2 gene expression by 17β-estradiol in breast cancer cells.J Biol Chem. 1999; 274: 32099-32107Crossref PubMed Scopus (233) Google Scholar;Dubal et al., 1999Dubal D.B. Shughrue P.J. Wilson M.E. Merchenthler I. Wise P.M. Estradiol modulates bcl-2 cerebral ischemia: A potential role for estrogen receptors.J Neurosci. 1999; 19: 6385-6393PubMed Google Scholar;Vegeto et al., 2001Vegeto E. Boninconto C. Pollio G. et al.Estrogen prevents the lipopolysaccharide-induced inflammatory response in microglia.J Neurosci. 2001; 21: 1809-1818PubMed Google Scholar;Chernavsky et al., 2002Chernavsky A.C. Rubio A.E. Vanzulli S. Rubinstein N. de Rosa S. Fainboim L. Evidence of the involvement of Bak, a member of the bcl-2 family of proteins, in active celiac disease.Autoimmunity. 2002; 35: 29-37Crossref PubMed Scopus (13) Google Scholar;Kanda and Watanabe, 2003cKanda N. Watanabe S. 17β-estradiol inhibits MCP-1 production in human keratinocytes.J Invest Dermatol. 2003; 120: 1058-1066Abstract Full Text Full Text PDF PubMed Google Scholar). PCR was performed by one denaturing cycle of 95°C for 3 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, and a final extension at 72°C for 3 min. In preliminary experiments using control cDNA from human spleen, 30 cycles lay in the linear range of PCR amplification for each gene examined. The PCR products were analyzed by electrophoresis on 2.5% agarose gels and stained with ethidium bromide, viewed by UV light. Densitometric analysis of the bands was performed by NIH Image Software. The mRNA levels of bcl-2, bax, bcl-xL, bad, and bak were normalized for that of GAPDH. The mRNA level for bcl-2 was further measured by real-time quantitative reverse transcription–PCR using a TaqMan PCR Reagent Kit (Perkin-Elmer Applied Biosystems, Foster City, California) according to the manufacturer's protocol. The sequences of forward and reverse primers and TaqMan fluorogenic probe for bcl-2 and GAPDH were previously described (Ikeguchi et al., 2002Ikeguchi M. Hirooka Y. Kaibara N. Quantitative analysis of apoptosis-related gene expression in hepatocellular carcinoma.Cancer. 2002; 95: 1938-1945Crossref PubMed Scopus (66) Google Scholar). The mRNA level of bcl-2 was normalized for that of GAPDH. Keratinocytes incubated under the indicated conditions were lyzed and centrifuged as described (Kanda and Watanabe, 2002Kanda N. Watanabe S. Ketoconazole suppresses prostaglandin E2-induced cyclooxygenase-2 expression in human epidermoid carcinoma A-431 cells.J Invest Dermatol. 2002; 119: 174-181Crossref PubMed Scopus (9) Google Scholar). Protein concentration in the supernatant was determined by Bio-Rad DC reagent (Bio-Rad Laboratories, Hercules, California). For western analysis, 20 μg of proteins were resolved on a 10% sodium dodecyl sulfate–polyacrylamide gel. The proteins were transferred to a PVDF membrane. The membrane was blocked, and exposed to primary antibodies, followed by peroxidase-conjugated secondary antibodies (Bio-Rad). The blots were developed with an enhanced chemiluminescence kit (Amersham, Arlington Heights, Illinois). Densitometric analysis of the bands was performed by NIH Image Software. The firefly luciferase reporter plasmids driven by human bcl-2 P1 promoter (–1640/–1287 bp relative to the transcriptional start site) were constructed by PCR and insertion into pGL3 basic vector (Promega, Madison, Wisconsin) as described (Wilson et al., 1996Wilson B.E. Mochon E. Boxer L.M. Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis.Mol Cell Biol. 1996; 16: 5546-5556Crossref PubMed Scopus (368) Google Scholar), and was denoted as pbcl-2 luc. Site-specific mutation of the promoter was created by multiple rounds of PCR using primers with altered bases as described (Dong et al., 1999Dong L. Wang W. Wang F. et al.Mechanisms of transcriptional activation of bcl-2 gene expression by 17β-estradiol in breast cancer cells.J Biol Chem. 1999; 274: 32099-32107Crossref PubMed Scopus (233) Google Scholar). p4xCRE-TATA-luc was constructed by inserting four copies of CRE-like element (5′-ACGTGTGACGTTACGCAC-3′) from bcl-2 promoter in front of TATA box upstream of firefly luciferase reporter as described (Kanda and Watanabe, 2003cKanda N. Watanabe S. 17β-estradiol inhibits MCP-1 production in human keratinocytes.J Invest Dermatol. 2003; 120: 1058-1066Abstract Full Text Full Text PDF PubMed Google Scholar). Transient transfections were performed with Effectene (Qiagen, Tokyo, Japan) as described (Zellmer et al., 2001Zellmer S. Gaunitz F. Salvetter J. Surovoy A. Ressig D. Gebhardt R. Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes.Histochem Cell Biol. 2001; 115: 41-47Crossref PubMed Scopus (25) Google Scholar). The efficiency of transfection into keratinocytes by this method was mean±SEM 28.5±3.1% (n=9) as determined by flow cytometry using β-galactosidase vector. Keratinocytes were plated in 10 cm dishes and grown to about 60% confluence. Twenty-four hours before the transfection, the medium was changed to phenol red-free KBM. Keratinocytes were incubated for 6 h with 5 μg of pbcl-2 luc or p4xCRE-TATA-luc and 1 μg of SV40 promoter-linked β-galactosidase reporter vector, pCH110 (Amersham), premixed with enhancer, transfection buffer, and Effectene. The transfected cells were washed and incubated in fresh phenol redfree KBM for 18 h, then incubated with 30 μM H2O2 in the presence or absence of indicated hormones. After 5 h, luciferase activities of the cell extracts were quantified by luciferase assay system (Promega). The same cell extracts were assayed for β-galactosidase activity using chemiluminescent Galacto-Light kit (Tropix, Bedford, Massachusetts). All readings were taken using a Lumat 9501 luminometer (Berthold, Wildbach, Germany). The results obtained in each transfection were normalized for β-galactosidase activity and expressed as relative luciferase activity. In our preliminary experiments, H2O2 did not alter the control β-galactosidase activity (data not shown). Human bcl-2 expression vector, pSV2-neo-bcl-2 was constructed by inserting human bcl-2 cDNA (Tsujimoto and Croce, 1986Tsujimoto Y. Croce C.M. Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma.Proc Natl Acad Sci USA. 1986; 83: 5214-5218Crossref PubMed Scopus (1053) Google Scholar) into pSV2-neo vector as described (Fernandez et al., 1994Fernandez A. Marin M.C. McDonnell T. Ananthaswamy H.N. Differential sensitivity of normal and Ha-ras-transformed C3H mouse embryo fibroblasts to tumor necrosis factor: Induction of bcl-2, c-myc, and manganese superoxide dismutase in resistant cells.Oncogene. 1994; 9: 2009-2017PubMed Google Scholar,Fernandez et al., 1995Fernandez A. Fosdick L.J. Marin M.C. Diaz C. McDonnell T.J. Ananthaswamy H.N. McConkey D.J. Differential regulation of endogenous endonuclease activation in isolated murine fibroblast nuclei by ras and bcl-2.Oncogene. 1995; 10: 769-774PubMed Google Scholar). Ten micrograms of the expression vector or empty vector were transfected into 107 keratinocytes using Effectene as described above. After 2 d of growth, individual bcl-2-overexpressing or control cell clones were isolated following G418 selection (400 μg per mL for 2 wk) as described (Fernandez et al., 1994Fernandez A. Marin M.C. McDonnell T. Ananthaswamy H.N. Differential sensitivity of normal and Ha-ras-transformed C3H mouse embryo fibroblasts to tumor necrosis factor: Induction of bcl-2, c-myc, and manganese superoxide dismutase in resistant cells.Oncogene. 1994; 9: 2009-2017PubMed Google Scholar,Fernandez et al., 1995Fernandez A. Fosdick L.J. Marin M.C. Diaz C. McDonnell T.J. Ananthaswamy H.N. McConkey D.J. Differential regulation of endogenous endonuclease activation in isolated murine fibroblast nuclei by ras and bcl-2.Oncogene. 1995; 10: 769-774PubMed Google Scholar). Anti-sense oligonucleotides were synthesized as described (O'Dowd et al., 1998O'Dowd B.F. Nguyen T. Marchese A. et al.Discovery of three novel G-protein-coupled receptor genes.Genomics. 1998; 37: 310-313Crossref Scopus (239) Google Scholar;Lau et al., 2000Lau K.-M. LaSpina M. Long J. Ho S.-M. Expression of estrogen receptor (ER)-α and ER-β in normal and malignant prostatic epithelial cells: Regulation by methylation and involvement in growth regulation.Cancer Res. 2000; 60: 3175-3182PubMed Google Scholar). The oligonucleotides were ERβ, 5′-CATCACAGCAGGGCTATA-3′; GPR30, 5′-TTGGGAAGTCACATCCAT-3′; random control, 5′-GATCTCAGCACGGCAAAT-3′. For anti-sense experiments, keratinocytes were washed twice in phosphate-buffered saline, then transfected, finally, with 0.2 μM of the indicated oligonucleotides premixed with Superfect Reagent (Qiagen) in phenol red-free KBM for 4 h. The medium was aspirated and fresh medium containing E2 was added. The efficiency of transfection was 89.3±9.5% (n=9) as examined microscopically using fluorescein isothiocyanate-labeled oligonucleotides. Keratinocytes were
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