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Epidermal Growth Factor Increases Cortisol Production and Type II 3ß-Hydroxysteroid Dehydrogenase/⁵-⁴-Isomerase Expression in Human Adrenocortical Carcinoma Cells: Evidence for a Stat5-Dependent Mechanism

Departments of Obstetrics and Gynecology (F.A.F., N.A.D., M.H.M.) and Cell Biology (F.A.F., S.K.N., N.A.D., M.H.M.), and Division of Endocrinology (W.J.K., W.N.), Vanderbilt University School of Medicine, and Veterans Affairs (W.J.K., W.N.), Nashville, Tennessee 37232; and Department of Internal Medicine, University of Virginia (C.M.S.), Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Dr. Michael H. Melner, Department of Obstetrics and Gynecology, B-1100 Medical Center North, Vanderbilt University, Nashville, Tennessee 37232. E-mail: mike.melner{at}vanderbilt.edu.

	Abstract

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We tested the ability of epidermal growth factor (EGF) to regulate a key enzyme in the adrenal synthesis of glucocorticoids: human type II 3ß-hydroxysteroid dehydrogenase/⁵-⁴-isomerase (3ßHSD). EGF treatment (25 ng/ml) of human adrenocortical carcinoma cells (H295R) resulted in a 5-fold increase in cortisol production and a corresponding 2-fold increase in 3ßHSD mRNA. Experiments were performed to determine whether EGF is acting through a previously identified signal transducer and activator of transcription 5 (Stat5)-responsive element located from -110 to -118 in the human type II 3ßHSD promoter. A Stat5 expression construct was cotransfected with a 3ßHSD-chloramphenol acetyltransferase (CAT) reporter construct comprised of nucleotides -301+45 of the human type II 3ßHSD promoter linked to the CAT reporter gene sequence. The addition of EGF at doses as low as 10 ng/ml resulted in an 11- to 15-fold increase in CAT activity. The introduction of 3-bp point mutations into critical nucleotides in the Stat5 response element obviated the EGF response. Either Stat5a or Stat5b isoforms induced CAT reporter expression upon treatment with EGF. These results demonstrate the ability of EGF to regulate the expression of a critical enzyme (3ßHSD) in the production of cortisol and suggest a molecular mechanism by which this regulation occurs.

	Introduction

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A CRITICAL STEP in the adrenal production of glucocorticoids and mineralocorticoids is the conversion of pregnenolone into progesterone by the enzyme, 3ß-hydroxysteroid dehydrogenase/⁵-⁴-isomerase (3ßHSD). 3ßHSD exists as two isoforms encoded by separate genes in humans. Type I 3ßHSD (1) is primarily expressed in the placenta and at a lower level in other tissues, such as prostate, breast, and skin, whereas type II 3ßHSD (2, 3) is expressed in adrenal, ovary, and testis. Type II 3ßHSD is known to be regulated by ACTH (4) in adult human adrenal cells, and the mechanism by which this occurs is thought to require the orphan nuclear receptor, steroidogenic factor-1 (SF-1; Ref. 5). However, ACTH-independent regulation of 3ßHSD expression has been demonstrated in the adrenal (6, 7), and alternative regulatory pathways may exist.

The primate fetal adrenal is composed of an inner fetal zone and an outer neocortical (definitive) zone. The definitive zone differentiates into the zona glomerulosa, zona fasciculata, and zona reticularis, whereas the fetal zone regresses after birth. A third zone, termed the transitional zone, arises from the outer edge of the fetal zone late in gestation (8). As the fetal zone does not express 3ßHSD, steroids are metabolized via the ⁵-steroid pathway, leading to the production of dehydroepiandrosterone and dehydroepiandrosterone sulfate, which are the primary steroid products of this zone; these precursor steroids serve as a reservoir for placental estrogen biosynthesis (9). The definitive and transitional zones express 3ßHSD and are thought to be the sites of fetal aldosterone and cortisol biosynthesis, respectively (10, 11). Neocortical expression of 3ßHSD appears at 22 wk, and by 28 wk gestation, 3ßHSD is widely distributed throughout the neocortex (12).

The mechanisms responsible for the differential expression of 3ßHSD in the fetal adrenal are unknown, but evidence exists for the control of 3ßHSD expression in the primate fetal adrenal to occur in the absence of ACTH. Induction of neocortical 3ßHSD expression in the fetal baboon adrenal occurs in the absence of ACTH at midgestation (13), and the anencephalic human fetus, which lacks ACTH production, exhibits normal definitive zone development (14). Thus, fetal adrenal growth and function are not completely dependent on ACTH until late in gestation, and definitive zone (a site of 3ßHSD expression) development occurs independently of ACTH.

Evidence suggests that epidermal growth factor (EGF) can affect adrenal development and steroid output. EGF is mitogenic in human fetal adrenals (6, 7) and has been shown to increase 3ßHSD activity in rat granulosa (15) and porcine Leydig (16) cells. Infusion of EGF into ewes for 24 h resulted in a 700% increase in cortisol levels (17). Macaque fetuses treated with EGF increased 3ßHSD protein in the adrenal with an induction in the transitional zone and an increase in adrenal weight due to hypertrophy of the definitive zone. Forskolin or 12-O-tetradecanoyl phorbol 13-acetate (TPA) induce 3ßHSD activity in human fetal adrenocortical cells; either angiotensin II (A-II) or EGF can enhance the forskolin effect (18). These studies all point to a role for EGF in adrenal development and function.

Previous work has identified a functional signal transducer and activator of transcription 5 (Stat5) response element in the 5'-flanking region of the human type II 3ßHSD gene (19). As EGF has been shown to be a potent activator of Stat5 in the mouse liver (20), it is possible that EGF could induce 3ßHSD mRNA through a Stat5-mediated mechanism. EGF activation of Stat5 is thought to occur by ligand-dependent activation of the tyrosine kinase, Src, resulting in recruitment of latent Stat5 molecules via Src homology 2 domains from the cytoplasm to the receptor complex (21). Stat5 becomes phosphorylated on tyrosine 694 (22), and the Stat5 molecules then dimerize via association with Src homology 2 domains, translocate to the nucleus, and bind to Stat5 response elements in the regulatory regions of target genes, thereby activating transcription. We demonstrate here that EGF increases cortisol production and 3ßHSD expression in H295R cells, and that this effect can be transduced via Stat5.

	Materials and Methods

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Cell culture
HeLa (human cervical carcinoma) cells were maintained in DMEM/Ham’s F-12 (Life Technologies, Inc., Grand Island, NY) with 10% fetal calf serum (Hyclone Laboratories, Inc., Logan, UT) and 50 µg/ml gentamicin (Sigma-Aldrich Corp., St. Louis, MO). H295R (human adrenocortical carcinoma) cells were cultured as described above, but in 2% fetal bovine serum (FBS) and 1% ITS-X (Life Technologies, Inc.). EGF treatment was performed with 25 ng/ml purified murine EGF (gift from Stanley Cohen, Vanderbilt University, Nashville, TN). Treatment was for 24 h for chloramphenol acetyltransferase (CAT) assays and for 48 h for Northern and cortisol measurements.

Cortisol RIA
Cortisol was measured by a specific liquid phase RIA using antisera from IgG Corp. (Nashville, TN). Reference standard cortisol was from Sigma-Aldrich Corp., and radiolabeled [¹²⁵I]cortisol was from Diagnostic Products (Los Angeles, CA). Cross-reaction in the assay was 0.02% for progesterone, 0.4% for 17-hydroxyprogesterone, and 5.9% for 11-deoxycortisol.

Total RNA isolation and Northern analysis
Isolation of H295R total RNA was performed using an RNeasy Mini Kit (QIAGEN) following the protocol supplied with the kit. Before RNA extraction, samples in 100-mm tissue culture dishes were washed with 1x PBS and trypsinized, and pellets were quick-frozen in dry ice. After extraction and column purification, samples were eluted in ribonuclease-free water, quantified by absorbance at A₂₆₀, and stored under excess ethanol at -20 C before analysis. Northern analysis was conducted using Me₂SO-glyoxal RNA sample preparation and agarose gel electrophoresis as previously described (23). Samples (20 µg) or RNA size standards (Life Technologies, Inc.) were denatured in 1.08 M deionized glyoxal, 54% (vol/vol) Me₂SO, and 10 mM sodium phosphate (pH 7.0) in a reaction volume of 30 µl for 1 h at 50 C and resolved using agarose [1.5% (wt/vol) in 10 mM sodium phosphate, pH 7.0] gel electrophoresis with 10 mM sodium phosphate (pH 7.0). RNA was transferred to a nylon membrane (Duralon-UV, Stratagene, La Jolla, CA) by capillary action with 20x SSC (1 x SSC = 0.15 M and, 15 mM Na₃ citrate) and fixed by UV cross-linking and baking for 1 h at 80 C. The blot was deglyoxalated in 0.1 M sodium phosphate (pH 9.0) for 1 h at 65 C, followed by prehybridization in 100 ml hybridization buffer [50% (vol/vol) deionized formamide, 5x SSC, 0.025 M sodium phosphate buffer (pH 7.0), 5% (wt/vol) sodium dodecyl sulfate, 0.1% (wt/vol) BSA, 0.1% (wt/vol) Ficoll 400, 0.1% (wt/vol) polyvinylpyrrolidone, and 0.05% (wt/vol) salmon sperm DNA] for 1 h at 42 C before the addition of 50 µCi [-³²P]deoxy-ATP-labeled human type II 3ßHSD cDNA probe. cDNA probe was generated using random primers and exo^- Klenow fragment (Prime-It II kit, Stratagene). Probes were purified from unincorporated nucleotides using Nuc-Trap columns (Stratagene). After hybridization (19 h), the blot was washed once with 2x SSC/1% (wt/vol) sodium dodecyl sulfate at 45 C for 15 min, twice with 2x SSC/0.1% (wt/vol) sodium dodecyl sulfate at 45 C for 15 min, and once with 0.1x SSC/0.1% (wt/vol) sodium dodecyl sulfate for 15 min at 55 C. Autoradiography was performed at -70 C with intensifying screens using Kodak BioMax MR film (Eastman Kodak Co., Rochester, NY). After autoradiography, blots were stripped of probe by washing for 5 min at 90 C in 10 mM sodium phosphate (pH 7.0), and rehybridized (19 h) with a [-³²P]deoxy-ATP-labeled constitutive probe (625-bp fragment of CHO-B) encoding ribosomal protein S2 (24). After hybridization, the blot was washed as described above. Autoradiographs were scanned using an Agfa Arcus II (Agfa Corp., Ridgefield Park, NJ) flatbed scanner and saved as TIFF (Tag Image File Format) files. RNA expression quantification was performed using Image software (Scion Corp., Frederick, MD).

Transient Transfection
HeLa cells were transiently transfected using a modification of the calcium phosphate coprecipitation method (25). Plasmid constructs employed were oStat5 (26), HA-hStat5A, and HA-Stat5B. Human type II 3ßHSD promoter fragments were inserted into pCAT-Basic (Promega Corp., Madison, WI) reporter plasmids as described previously (5). The construct containing the Stat5 response element (Stat5RE) point mutations (5'-TTCTGAGAA-3' to 5'-TTTTGATTA-3') was generated as previously described (underlined nucleotides are mutated; Ref. 19). Adherent HeLa cells were cultured to 55–65% confluence in 100-mm tissue culture dishes (Corning, Inc., Corning, NY) in 10 ml of the appropriate medium. Calcium phosphate-DNA coprecipitates were formed by dropwise addition of equal volumes (0.5 ml) of solution A (0.24 M CaCl₂ containing 15 µg of the appropriate plasmid constructs) to solution B (2x HEPES-buffered saline: 50 mM HEPES; 1.4 mM Na₂HPO₄; and 0.28 M NaCl, pH 7.1). Calcium phosphate:DNA precipitates were incubated at 23 C for at least 20 min and added to single 100-mm dishes of cells containing 9 ml fresh medium. HeLa cells were then incubated with precipitate for 4 h at 37 C (5% CO₂ and 95% air), shocked for 1 min with 15% (vol/vol) glycerol in Dulbecco’s PBS (0.137 M NaCl, 0.137 M NaCl, 0.5 mM MgCl₂, 6.45 mM Na₂HPO₄, and 1.5 mM K₂HPO₄), washed three times with Dulbecco’s PBS, and incubated at 37 C for 24 h. During the final 24 h of incubation, cells were cultured in the presence or absence of appropriate treatments. Cells were then harvested using trypsin/EDTA (Life Technologies, Inc.), pelleted, resuspended in 0.25 M Tris-HCl (pH 7.4), and stored at -70 C until assayed for CAT activity. Transfections were performed in triplicate with mock negative controls. Internal transfection efficiency was monitored by cotransfection of 0.5 µg cytomegalovirus promoter-ß-galactosidase and measurement of ß-galactosidase activity. Individual samples were corrected for transfection efficiency based upon their relative ß-galactosidase activity.

CAT assays
Frozen cell pellets were thawed on ice and lysed by sonication. Extracts were heated to 60 C for 5 min to denature any endogenous acetylase/deacetylase enzymes. Soluble extracts were then separated from cell debris by centrifugation, divided into aliquots for CAT assays, and stored at -70 C before use. Fluorescent CAT assays were performed as previously described (27) with some modifications using the FLASH CAT assay kit (Stratagene). Acetyl coenzyme A (CoA) was synthesized by reaction of CoA (Pharmacia Biotech, Piscataway, NJ) with acetic anhydride (Sigma-Alrich Corp.) as described previously (28) and stored at -70 C until use. Cell extracts (10–20 µl) were incubated in 0.25 M Tris-HCl (pH 7.4) in a total reaction volume of 50 µl with acetyl-CoA (8.2 µM) and fluorescent borondipyrromethene difluoride chloramphenicol (BODIPY CAM) substrate (1:12.5 dilution) at 37 C for 4–8 h. Reactions were terminated by the addition of cold ethyl acetate (850 µl), followed by vigorous vortexing. An aliquot (800 µl) of extracted substrate and acetylated products was removed (organic phase), dried under vacuum, and resuspended in ethyl acetate (20 µl) before separation on thin layer chromatography plates (LK6, Whatman, Clifton, NJ) with chloroform/methanol (9:1) for 30 min. Substrate and products were visualized under long-wave UV light (366 nm) and photographed (type 55 positive/negative film, Polaroid, Cambridge, MA). Substrate and combined product bands were scraped from the plates, extracted, and diluted 1:10 in methanol before quantification by fluorescence spectrophotometry at excitation and emission wavelengths of 490 and 512 nm, respectively, using a fluorometer. The percent conversion of BODIPY CAM substrate to 1-, 3-, and 1,3-acetylated BODIPY CAM products was computed.

Western blot analysis
HeLa nuclear and whole cell extracts were prepared as previously described (19). The indicated volumes of nuclear and whole cell extracts were separated by 12% SDS-PAGE (29), immunoblotted with antibodies to human (h) Stat5A or hStat5B, and detected by enhanced chemiluminescence as previously described (30, 31). The Stat5b antibody (polyclonal) was obtained from Santa Cruz Biotechnology, Inc. (catalog no. SC-835), and the Stat5a (monoclonal) was obtained from Transduction Laboratories, Inc. (catalog no. S21520, Lexington, KY).

Statistical analysis
Statistical significance was determined by single-factor ANOVA, followed by Bonferroni correction for multiple comparisons. Sample differences were not considered statistically significant unless P < 0.05 (divided by the number of treatment groups) according to the Bonferroni correction for multiple comparisons.

	Results

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EGF enhances cortisol production and 3ßHSD mRNA expression in a human adrenocortical carcinoma cell line (H295R)
Treatment of H295R cells with 200 nM 12-O-tetradecanoyl phorbol 13-acetate (PMA) resulted in a 9.6-fold increase in cortisol production relative to basal, whereas 25 ng/ml EGF treatment resulted in a 5.5-fold increase (Fig. 1). Increases in cortisol production correlate with increases in 3ßHSD mRNA expression. Northern analysis showed an increase in 3ßHSD expression after PMA and EGF treatment (Fig. 2A). Densitometric analysis of band intensities showed that EGF treatment resulted in a 2.6-fold increase in mRNA, whereas PMA treatment resulted in a 7-fold increase (Fig. 2B). These data demonstrate the ability of EGF and phorbol esters to increase 3ßHSD mRNA and cortisol output by H295R cells.

View larger version (10K): [in this window] [in a new window]   Figure 1. EGF and phorbol esters increase cortisol output by human adrenocortical carcinoma cells. H295R cells were grown to 60–70% confluence, washed repeatedly, and cultured for 48 h in DMEM/Ham’s F-12 medium supplemented with 0.1% FBS, 1% ITSX, antibiotics, and either 200 nM PMA or 25 ng/ml EGF. Medium was collected, and cortisol concentrations were determined by RIA as described in Materials and Methods.

View larger version (36K): [in this window] [in a new window]   Figure 2. EGF and phorbol esters increase 3ßHSD mRNA in human adrenocortical carcinoma cells by Northern analysis. Triplicate dishes of H295R cells were grown to 60–70% confluence, washed repeatedly, and cultured for 48 h in DMEM/Ham’s F-12 medium supplemented with 0.1% FBS, 1% ITSX, antibiotics, and either 200 nM PMA or 25 ng/ml EGF. A, Total RNA was isolated, and 20 µg were run on a 1.5% agarose gel. RNA was transferred to a nylon membrane, hybridized with 32P-labeled human type II 3ßHSD cDNA probe, washed, and autoradiographed. Blots were stripped and reprobed for the constitutive message, ribosomal protein S2 (CHO-B) as described in Materials and Methods. B, Ratio of 3ßHSD/ribosomal protein S2 band intensities from A. An asterisk represents statistical significance (P < 0.002) relative to the unstimulated control.

View larger version (16K): [in this window] [in a new window]   Figure 3. Expression of Stat5 is required for the induction of type II 3ßHSD reporter activity by EGF. HeLa cells were cotransfected with a -301+45 fragment of the type II 3ßHSD promoter fused to a CAT reporter gene (-301 CAT; 5 µg), ovine Stat5, or empty vector (5 µg), ß-galactosidase (0.5 µg), and control DNA for a total of 15.5 µg using the calcium phosphate precipitation method, followed by treatment for 24 h with EGF (25 ng/ml) as described in Materials and Methods. , EGF treatment. The number above the bar is fold activation compared with the identically transfected group minus EGF. Data represent the mean ± SE of triplicate cultures after correction for transfection efficiency from a representative experiment of two performed.

View larger version (16K): [in this window] [in a new window]   Figure 4. Increasing concentrations of EGF result in increased type II 3ßHSD reporter activity. HeLa cells were cotransfected with a -301+45 fragment of the type II 3ßHSD promoter fused to a CAT reporter gene (-301 CAT; 5 µg), ovine Stat5 (5 µg), ß-galactosidase (0.5 µg), and control DNA for a total of 15.5 µg using the calcium phosphate precipitation method, followed by treatment for 24 h with increasing doses of EGF (0100 ng/ml) as described in Materials and Methods. Data represent the mean ± SE of triplicate cultures after correction for transfection efficiency from a representative experiment of two performed.

View larger version (16K): [in this window] [in a new window]   Figure 5. hStat5A and hStat5B isoforms in activation of 3ßHSD reporter activity. HeLa cells were cotransfected with a -301+45 fragment of the type II 3ßHSD promoter fused to a CAT reporter gene (-301 CAT; 5 µg), hStat5A or hStat5B (5 µg), ß-galactosidase (0.5 µg), and control DNA for a total of 15.5 µg using the calcium phosphate precipitation method, followed by treatment for 24 h with EGF (25 ng/ml) as described in Materials and Methods. , EGF treatment. The number above the bar is fold activation compared with the identically transfected group minus EGF. Data represent the mean ± SE of triplicate cultures after correction for transfection efficiency from a representative experiment of two performed.

View larger version (36K): [in this window] [in a new window]   Figure 6. Endogenous expression levels of Stat5A and Stat5B isoforms in HeLa cells. HeLa nuclear extracts (NE) and whole cell extracts (WCE) were prepared as described in Materials and Methods. Five, 10, 15, and 20 µl of nuclear extracts (lanes 1–4) and 5 and 10 µl (lanes 6 and 7) were separated by 12% SDS-PAGE, immunoblotted with antibodies to hStat5A or hStat5B, and detected by ECL.

View larger version (14K): [in this window] [in a new window]   Figure 7. A Stat5 regulatory element confers EGF responsiveness to the human type II 3ßHSD promoter region. HeLa cells were cotransfected with -301 (wild-type) or -301 (mutant) CAT reporter constructs (5 µg), ovine Stat5 (5 µg), ß-galactosidase (0.5 µg), and control DNA for a total of 15.5 µg using the calcium phosphate precipitation method, followed by treatment for 24 h with EGF (25 ng/ml) as described in Materials and Methods. , EGF treatment. Data represent the mean ± SE of triplicate cultures after correction for transfection efficiency from a representative experiment of two performed.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A number of growth factors have been implicated in the control of adrenal development. These include EGF, fibroblast growth factor, IGF-I, IGF-II, inhibin, activin, and TGFß (8). EGF has been shown to enhance 3ßHSD activity in human fetal adrenal cells (18), and EGF treatment of human fetal zone and definitive zone cells results in proliferation with a higher mitogenic response seen in the definitive zone cells (6). As 3ßHSD is expressed in definitive, but not fetal, zone cells, it is possible that EGF is mediating this effect. Other studies have shown that injection of EGF into the fetus of rhesus monkeys results in hypertrophy of the definitive zone with an increase in 3ßHSD protein in the definitive and transitional zones (34). The mitogenic effect of EGF in the adrenal appears to be species dependent, as EGF treatment of bovine adrenocortical cells does not result in a mitogenic response (35). It should be noted that TGF, but not EGF, is expressed in adult and fetal human adrenals (36), and activation of the EGF receptor by TGF (or other EGF receptor ligands) might mediate the induction of 3ßHSD in the adrenal gland in vivo.

As EGF has been shown to be a potent activator of Stat5 (20), Stat5 activation could be the mechanism by which EGF increases 3ßHSD transcription in the adrenal. Previous studies on type II 3ßHSD regulation have examined a functional Stat5RE located from -110 to -118 (5'-TTCTGAGAA-3') in the type II 3ßHSD promoter region (19). In these studies it was shown that PRL-activated Stat5 binds to the Stat5RE and functionally activates 3ßHSD transcription. Evidence that EGF is activating 3ßHSD through the Stat5RE presented here suggests a molecular mechanism for EGF regulation of adrenal 3ßHSD, yet it cannot be ruled out that parallel signal transduction cascades initiated by EGF in vivo could also be affecting 3ßHSD transcription.

A-II also has the potential for regulation of 3ßHSD through a Stat5-mediated pathway. A-II acts through the AT₁ and AT₂ receptors and activates cAMP/protein kinase A, phosphoinositol turnover/Ca²⁺ flux/protein kinase C (PKC), and Janus kinase/Stat signaling pathways (37). In the zona glomerulosa, A-II modulates aldosterone production by activating AT₁ receptors which involves increases in PKC activity (38). Phorbol ester increases PKC activity, and induction of the human type II 3ßHSD gene maps to a response element (5'-TCAAGGTAA-3') that binds the orphan nuclear receptor, SF-1, and is located from -64 to -56 in the 5'-flanking sequence of the transcription initiation (5). Increases in 3ßHSD mRNA by A-II and TPA in H295R cells have been demonstrated (39). Interestingly, AT₁ receptors that transduce A-II signals in zona glomerulosa cells have been shown to activate Stat5 in cardiac myocytes via the Janus kinase/Stat pathway (40). This opens the possibility that A-II could regulate 3ßHSD directly through a Stat5 mechanism independently or in parallel with PKC mechanisms.

Besides EGF and PRL induction of 3ßHSD transcription via Stat5, it has been shown that the cytokine IL-4 requires the Stat5RE for functional activity in conjunction with a Stat6 response element located 5' of the Stat5RE from -160 to -151 (Cote, S., S. Gingras, F. A. Feltus, M. Freeman, M. H. Melner, and J. Simard, manuscript in preparation). Cytokine activation of 3ßHSD has important implications for the regulation of steroidogenesis in the ovary due to changes in the immune cell complement during the follicular cycle. Multiple extracellular factors therefore are capable of activating Stat proteins and converging at the Stat5 element in the human type II 3ßHSD gene. This element is independent of the region of trophic hormone action, which occurs through the SF-1 response element.

Regulation of the steroidogenic pathway by Stat proteins is not limited to 3ßHSD. Stat3 has been shown to induce aromatase activity in human adipose tissue (41). This suggests that control of the steroidogenic pathway can occur through Stat protein binding in the regulatory regions of steroid enzyme genes at multiple levels in the pathway. In the case of adrenal steroidogenesis, an important study would be to examine the localization patterns of the Stat5 isoforms in a primate fetal adrenal to track expression changes during 3ßHSD induction in the definitive and transitional zones as well as expression differences in other steroidogenic enzymes. In addition, as knockout mice exist for both Stat5A (42) and Stat5B (43), defects in adrenal development and steroid production could be examined with the caveat that compensatory changes may occur.

The increase in cortisol synthesis observed could reflect changes in the expression of multiple steroidogenic enzyme genes, including 3ßHSD. However, it is also possible EGF could alter the expression of other genes that could either directly or indirectly influence steroid synthesis. For example, the expression of steroidogenesis acute regulatory protein could be altered directly by EGF. Alternatively, EGF could alter the availability of cholesterol substrate via elevation of low density lipoprotein receptor expression or increases in the de novo synthesis of cholesterol. We show that EGF treatment increases 3ßHSD expression, but the exact steps responsible for increased cortisol production will require a systematic search.

In conclusion, these data demonstrate the ability of EGF to induce 3ßHSD mRNA and cortisol biosynthesis in human adrenocortical carcinoma (H295R) cells. This model system was used to test the capacity of EGF to directly induce the expression of a steroidogenic enzyme in the human adrenal gland. We have also presented a molecular mechanism by which EGF can transduce its signal to the type II 3ßHSD gene through the activation of Stat5. Induction of 3ßHSD reporter activity required the expression of Stat5, and this increase in transcriptional activity is not isoform specific. Stat5 mediation of EGF signaling requires an intact Stat5-responsive element located from -110 to -118 bp in the regulatory region of the type II 3ßHSD gene, as point mutations in this element abrogated EGF induction. These data confirm the ability of EGF to regulate a gene that is critical to the production of adrenal steroids.

	Footnotes

 
This work was supported by NIH Training Grant 5T3-HD-07043 (to F.A.F.) and in part by NIH Grants U54-HD-37321 and EPA G7041337 (to M.H.M.). W.J.K. is the recipient of a Career Development Award (Clinical Investigator) from the U.S. Department of Veterans Affairs.

Abbreviations: A-II, Angiotensin II; BODIPY CAM, borondipyrromethene difluoride chloramphenicol; CAT, chloramphenol acetyltransferase; CoA, coenzyme A; EGF, epidermal growth factor; FBS, fetal bovine serum; h, human; 3ßHSD, 3ß-hydroxysteroid dehydrogenase/⁵-⁴-isomerase; PKC, protein kinase C; PMA, 12-O-tetradecanoyl phorbol 13-acetate; SF-1, steroidogenic factor-1; Stat5, signal transducer and activator of transcription 5; Stat5RE, Stat5 response element.

Received December 13, 2000.

Accepted for publication January 9, 2003.

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