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Biol. Pharm. Bull. 35(9) 1588–1593 (2012)
Note
Vol. 35, No. 9
Interferon Gamma Induces Steroid Sulfatase Expression in Human Keratinocytes Kenji Hattori,a Nozomi Yamaguchi,a Kazuo Umezawa,b,† and Hiroomi Tamura*,a a
Graduate School of Pharmaceutical Sciences, Keio University; 1–5–30 Shibakoen, Minato-ku, Tokyo 105–8512, Japan: and b Graduate School of Science and Technology, Keio University; 3–14–1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223–8522, Japan. Received January 6, 2012; accepted June 5, 2012 Steroid sulfatase (STS) plays an important role in steroid metabolism in which estrogens and dehydroepiandrosterone (DHEA) are produced from their sulfates. However, little is known about the transcriptional regulation of the STS gene in keratinocytes. Since keratinocytes are thought to be a primary target of interferon gamma (IFNγ) in inflammatory and immune responses, we assessed the effects of this cytokine upon STS gene expression in the human keratinocyte cell line SVHK and in normal human keratinocytes (NHEK). Stimulation of SVHK cells with 50 ng/mL of IFNγ for 24 h induced an approximately three-fold increase in STS activity and in its mRNA levels compared to non-treated cells. IFNγ treatment also induced an approximately 1.5-fold increase in STS mRNA levels in NHEK cells. This induction was completely inhibited by treatment with phosphatidylinositol (PI) 3-kinase inhibitors such as LY294002 or wortmannin, and by the nuclear factor-kappa B (NF-κB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). These data suggest that activation of the PI 3-kinase signal transduction pathway mediates induction of STS gene expression by IFNγ through activation of NF-κB. The anti-inflammatory agent dexamethasone inhibited IFNγ induction of STS gene expression, suggesting involvement of a glucocorticoid receptor in the regulation of STS gene expression in keratinocytes. Regulation of STS gene expression in skin as a novel target of drugs for therapy of psoriasis in the skin is discussed. Key words
steroid sulfatase; keratinocyte; interferon gamma; nuclear factor-kappa B; dexamethasone
Steroid sulfatase (STS) is a microsomal enzyme that catalyzes the hydrolysis of sulfated 3-hydroxysteroids to the corresponding free active 3-hydroxysteroids.1) This enzyme is widely distributed throughout the body, and its actions are implicated in both physiological processes and pathological conditions. In many peripheral organs such as skin STS play a key role in the production of dehydroepiandrosterone (DHEA) from DHEA sulfate, which is abundant in the circulation. DHEA is then used as a major precursor for the synthesis of potent androgens in the skin.2) Early studies in vitro and in vivo have shown that the human STS activity appears in specific skin types, such as in foreskin and infantile abdominal skin, after birth.3) It also has been shown that STS plays an important functional role in the skin as this enzyme is deficient in X-linked ichthyosis (X-LI).4,5) It has been further demonstrated that both the expression of STS mRNA and its enzymatic activity are increased in malignant breast and endometrial tissues compared with nonmalignant tissues and that this increased STS affects active estrogen levels within cells by hydrolysis of inactive estrogen sulfates.6) We recently reported that estradiol sulfate, whose formation is catalyzed by estrogen sulfotransferase, is a major metabolite of estradiol in human epidermal keratinocytes.7) Since estrogens have a significant effect on skin differentiation and development,8,9) the activity of STS, as well as of estrogen sulfotransferase in the skin must be properly regulated to maintain cutaneous homeostasis. However, little is known about the regulation of STS gene expression or activity in skin. It has been reported previously that interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) act synergistically to The authors declare no conflict of interest. † Present address: Department of Molecular Target Medicine Screening, Aichi Medical University; Nagakute 480–1195, Japan. * To whom correspondence should be addressed.
increase STS activity in breast cancer cells,10,11) and that this effect on STS activity is regulated by posttranslational modification of a cysteine residue in the catalytically active site of this enzyme to formyl glycine. In contrast, the inflammatory cytokine IL-1β decreases STS activity and the expression of STS mRNA in human endometrial stromal cells.12) In psoriatic plaques in skin, there is a predominance of T helper 1 cytokines, most notably interferon gamma (IFNγ).13) IFNγ is a cytokine that is produced by activated T cells and natural killer cells, and induces a variety of effects including anti-viral responses, cell growth and differentiation.14) In the skin, IFNγ is known to play a pivotal role in the development of inflammatory and immune responses.15–17) However, no information is available concerning the effect of IFNγ on STS expression. IFNγ has been reported to stimulate involucrin gene expression via STAT1 signaling in cells of the SVHK cell line that was established from SV40-transformed human keratinocytes.18,19) To determine the effect of IFNγ on STS expression in the human epidermis, we investigated the effects of IFNγ on STS gene expression in both SVHKs and normal human epidermal keratinocytes (NHEKs).
MATERIALS AND METHODS Materials [3H]Dehydroepiandrosterone sulfate was purchased from PerkinElmer Life Sciences (Shelton, CT, U.S.A.). Dehydroepiandrosterone sulfate was purchased from Sigma (St. Louis, MO, U.S.A.). The other chemicals used were purchased from Wako Pure Chemical, Ind., Ltd. (Osaka, Japan). LY294002 was purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.). Wortmannin and rapamycin were purchased from Nacalai Tesque (Kyoto, Japan). The Akt inhibitor (sc-221226) and the src tyrosine kinase inhibitor PP2 were purchased from Santa Cruz (Santa Cruz, CA, U.S.A.).
e-mail:
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© 2012 The Pharmaceutical Society of Japan
September 2012
Dehydroxymethylepoxyquinomicin (DHMEQ) was synthesized as previously described.20) Cell Culture The SVHK human keratinocyte cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (low glucose) supplemented with 10% fetal calf serum (FCS) and the cells were grown to subconfluence at 37°C in a humidified atmosphere of 95% air and 5% CO2. The culture medium was replaced with DMEM containing 1% FCS one day prior to stimulation with IFNγ (Pepro Tech EC Ltd., London, U.K.) and inhibitors. The cells were stimulated with IFNγ and/or individual inhibitors, which were added simultaneously. Second-passage neonatal foreskin NHEK were purchased from Kurabo Industries (Osaka, Japan) and cultured in serumfree keratinocyte growth medium, HuMedia-KG2 (Kurabo Industries) containing human epidermal growth factor (0.1 ng/ mL), insulin (10 µg/mL), gentamicin (50 µg/mL), amphotericin B (50 ng/mL), and bovine brain pituitary extract (0.4%, v/v), at 37°C in a humidified atmosphere of 95% air and 5% CO2. The Ca2+ concentration in HuMedia-KG2 was 0.15 m M. Cells were passaged at 60–70% confluence to avoid differentiation, and the experiments were conducted using subconfluent cells at passage 3 in the proliferative phase at 60–80% confluence. RNA Extraction and Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total RNA was isolated from keratinocytes by acid guanidinium thiocyanate phenol-chloroform extraction using ISOGEN (Nippon Gene, Tokyo, Japan). The first cDNA strand was synthesized from 1 µg of total RNA using 1 unit of ReverTra Ace (Toyobo, Tokyo, Japan) with random primers, in accordance with the manufacturer’s protocol. After incubation, the reaction mixtures were diluted 10-fold with TE buffer (10 m M Tris–HCl, pH 8.5; 1 m M ethyelnediaminetetraacetic acid (EDTA)). Aliquots (2.5 µL) of this diluted solution were then added to a PCR reaction mixture (7.5 µL) consisting of 5 µL of SYBR green master mix (Roche Diagnostics, Basel, Switzerland) and 0.25 µM of both sense and antisense primers. The levels of RNA encoding the genes of interest were standardized using transcripts encoding 18S ribosomal RNA in each sample. The primers used to amplify the target cDNAs were: 18S ribosomal RNA, 5′-TGG TTG CAA AGC TGA AAC TTA AAG-3′ (forward) and 5′-AGT CAA ATT AAG CCG CAG GC-3′ (reverse); STS, 5′-TCA AGG CCG AAC ATC ATC CT-3′ (forward) and 5′-GGT ACC GGC CAG TCA TGA AG-3′ (reverse). The amplification conditions were: 30 s at 94°C and 1 min at 60°C for 40 cycles and the reactions were followed in real time using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, U.S.A.). The relative levels of each target gene were calculated using the 2CT method (ABI PRISM 7700 Sequence Detection System User Bulletin 5). The relative efficiencies of target and reference gene amplification were then assessed in accordance with the manufacturer’s instructions (ABI PRISM 7700 Sequence Detection System User Bulletin 2). Assay of STS Activity SVHK cells were harvested and lysed in phosphate buffered saline (PBS) containing 1.5 m M dithiothreitol by sonication. The microsomal fraction was prepared as follows: a supernatant of the lysate was obtained by centrifugation at 8000×g for 10 min at 4°C and the resulting supernatant was then urther centrifuged for 1 h at 105000×g for 60 min at 4°C. The obtained pellet was then suspended
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in PBS containing 1.5 m M dithiothreitol to yield the microsomal fraction. The protein concentration of this fraction was determined using the Bradford method with bovine serum albumin standards. The STS assay was carried out according to the method described by Epstein and Leventhal 21) with slight modifications. Briefly, 30 µg of microsomal protein and 2.5 pmol of [3H]dehydroepiandrosterone (DHEA) sulfate (Perkin Elmer, Boston, MA, U.S.A.) were mixed in a 0.25 mL volume of 50 m M Tris–HCl buffer, pH 7.4. The reaction was started by adding the enzyme preparation and was stopped after 1 h incubation at 37°C by the addition of 1 mL of toluene. The contents were mixed by vortexing and the phases were separated within 1 min. A 0.6 mL aliquot of the upper toluene phase was then removed and added to 3 mL of scintillation fluid (Clear-sol I, Nacalai Tesque) to determine the quantity of [3H]DHEA extracted. Statistical Analysis Statistical comparisons of two groups were undertaken using two-tailed and unpaired ttests. In the multigroup experiments, the data were compared using Tukey’s test. Differences were considered significant at p
