Differential regulation of 3ß-hydroxysteroid dehydrogenase type II and 17a-hydroxylase/lyase P450 in human adrenocortical carcinoma cells by epidermal growth factor and basic fibroblast growth factor
J Doi, H Takemori, M Ohta1, Y Nonaka2 and M Okamoto
Department of Molecular Physiological Chemistry, Osaka University Medical School, Suita, Osaka, Japan
1Laboratory of Nutrition, Koshien College, Nishinomiya, Hyogo, Japan
2College of Nutrition, Koshien University, Takarazuka, Hyogo, Japan
(Requests for offprints should be addressed to M Okamoto, Department of Molecular Physiological Chemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan; Email: mokamoto@mr-mbio.med.osaka-u.ac.jp)
Abstract
Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are pluripotent growth factors that stimulate both the proliferation and steroidogenesis of adrenocortical cells. Here we demonstrate that EGF and bFGF specifi- cally induce mRNA of 3ß-hydroxysteroid dehydrogenase type II (3ßHSD II) and suppress that of 17a-hydroxylase/ lyase P450 (CYP17) in human adrenocortical H295R cells. The induction of 3ßHSD II mRNA did not occur until 6 h after the growth factor treatment and was completely abolished in the presence of a protein synthesis inhibitor, cycloheximide (CHX), suggesting that the induction required de novo protein synthesis. The CYP17 mRNA suppression began at almost the same time as the induction of the 3ßHSD II mRNA. Interestingly, the CYP17 mRNA level was increased by the CHX treat-
ment. Both the 36HSD II and CYP17 mRNAs were repressed by treatment with a calmodulin kinase II (CaMK II) inhibitor, KN-93, and were enhanced by a mitogen- activated protein kinase (MAPK) inhibitor, PD98059. The PD98059-mediated induction of the 30HSD II mRNA was completely blocked by the CHX treatment. Interestingly, treatment with EGF in the presence of both PD98059 and CHX produced a greater increase in the CYP17 mRNA than did treatment in the presence of PD98059 alone. These results suggest that CHX-sensitive factor(s) and CaMK II- and MAPK-signaling pathways may have important roles in both induction of 3ßHSD II and suppression of CYP17 by EGF or bFGF in H295R cells.
Journal of Endocrinology (2001) 168, 87-94
Introduction
Numerous investigators have reported that epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) influence steroidogenesis in adrenocortical cells (Gospodaeowicz & Handley 1975, 1986, McAllister & Hornsby 1987a, Fisher & Lakshmanan 1990, Mesiano et al. 1993, Mesiano & Jaffe 1997). Singh and coworkers reported that EGF stimulated cortisol secretion from cultured bovine (Singh & Waters 1983) and sheep (Singh et al. 1985) adrenal cortical cells. The stimulation was abolished when inhibitors of cholesterol biosynthesis, com- pactin and AY9944, were added. Further studies showed that the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and the rate of cholesterol synthesis were increased in the EGF-treated cells. In contrast, the rates of conversion of radioactive pregne- nolone to steroid intermediates were little influenced by the growth factor. On the basis of these results, they
concluded that EGF activated the biosynthesis of the steroid hormone precursor, rather than the steroidogenic enzymes themselves.
bFGF has been suggested as a physiological regulator of adrenocortical functions both in vivo (Mesiano et al. 1991, Basile & Holzwarth 1994) and in vitro (Crickard et al. 1981, Hornsby et al. 1983, Hotta & Baird 1986, Ho & Vinson 1995). It was purified from extracts of bovine adrenals, and a large amount was detected in the zona fasciculata and the medulla (Basile & Holzwarth 1993). It was also detected immunochemically and by northern blot analysis in bovine (Gospodarowicz et al. 1986), rat (Basile & Holzwarth 1993) and human (Mesiano et al. 1991) adrenal cortex. Basile & Holzwarth (1993) observed that, in a unilaterally adrenalectomized rat, the remaining adrenal gland underwent compensatory hypertrophy, and they suggested that bFGF might be involved in this phenomenon. Recently, Thomas et al. (1997) showed that transplantation of bovine adrenocortical cells with
bFGF-overproducing mouse fibroblasts into immuno- deficient mice resulted in both cell proliferation and activation of steroidogenesis of the transplants. Receptors for bFGF were also shown to exist in the rat zona glomerulosa (Basile & Holzwarth 1994). These previous reports strongly suggest that EGF and bFGF act as para- crine effectors in the adrenal cortex and may modulate steroidogenesis in adrenocortical cells.
H295R cells were originally isolated from human adrenal carcinoma cells (Gazdar et al. 1990, Rainey et al. 1994). The cells were reported to possess five steroidogenic P450s - namely, side-chain cleavage P450 (CYP11A), 17a-hydroxylase/lyase P450 (CYP17), 21- hydroxylase P450 (CYP21), 11ß-hydroxylase P450 (CYP11B1) and aldosterone synthase P450 (CYP11B2) - and 3ß-hydroxysteroid dehydrogenase type II (3ßHSD II) (Bird et al. 1996). Because of this, these cells have often been used for studying the molecular mechanism under- lying human adrenocortical steroidogenesis. In the present study, we investigated the effects of EGF and bFGF on gene expression of steroidogenic enzymes in H295R cells.
Materials and Methods
Cell culture and treatment with EGF and bFGF
H295R cells were generous gifts from J Ian Mason at University of Edinburgh, UK. The cells were maintained in Dulbecco’s Modified Eagle’s/Ham’s F-12 medium (DMEM/F-12; Gibco BRL, Grand Island, NY, USA) containing 1% ITS plus (insulin/transferrin/selenium/ linoleic acid; Becton Dickinson Labware, Lincoln Park, NJ, USA), 2% Ultroser G (BioSepra Inc., France) and antibiotics, at 37 ℃ under an atmosphere of 5% CO2-95% air, as described before (Bird et al. 1996). The cells (5 × 105) were cultured in 100 mm dishes for 48 h and then incubated for further 24 h in serum-free medium (DMEM/F-12 containing antibiotics and 0-01% BSA). To initiate the experiments, the medium was exchanged for one containing EGF (Austral Biochemicals, San Ramon, CA, USA) or bFGF (Austral Biochemicals) at the final concentration of 10 ng/ml. After the 24 h incubation, the cells were washed with PBS, and the total RNA was extracted. Protein kinase inhibitors (bisindolylmaleimide I, H-8, H-89, PD98059 and KN-93) and a protein synthesis inhibitor (cycloheximide; CHX) were purchased from Calbiochem Ltd (La Jolla, CA, USA) and Sigma, respect- ively, dissolved in dimethylsulfoxide, and stored at - 20 ℃ until required for use. Cells that had been maintained in the serum-free medium for 24 h, were first incubated with or without bisindolylmaleimide I, H-8, H-89, PD98059 or KN-93 for 1 h, and then stimulated with EGF or bFGF dissolved in the freshly prepared inhibitor-containing medium.
RNA extraction
Isolation of the total RNA was performed according to the method of Chomczynski & Sacchi (1987) with some modifications (Takemori et al. 1997). Briefly, the cells were washed with 1 ml PBS, and then dissolved in 1.6 ml guanidine isothiocyanate solution (Trizol reagent; Gibco BRL). The cell lysates were transferred into micro- centrifuge tubes and mixed with 0-4 ml chloroform. After the centrifugation, an aqueous phase was taken, to which ethanol was added to precipitate total RNAs. The RNAs were washed with 70% ethanol, dried and dissolved in 200 ul distilled water. DNA contaminant in the RNA preparation was digested by 1 U DNase I (Gibco BRL) using the buffer supplied by the manufacturer. The RNAs were again extracted by phenol-chloroform (1:1) and reprecipitated by ethanol.
Northern blot analysis
To prepare a cDNA probe for 3ßHSD II, a DNA fragment of the exon 3 was amplified by PCR using primers shown in Table 1 and ligated into the Smal site of pUC18. cDNA fragments of CYP11A and CYP21 were also prepared, using RT-PCR with sets of primers listed in Table 1, and introduced into pT7R (Novagen, Madison, WI, USA). A NdeI-HindIII cDNA fragment of CYP17 was excised from pCW (Katagiri et al. 1995) that had been kindly provided by M Katagiri at Osaka Kyoiku University, Japan. We utilized a rat glyceraldehyde-3- phosphate dehydrogenase (G3PDH) cDNA fragment (Halder et al. 1998) as the control probe. Total RNAs (5 µg) were separated by electrophoresis in 1% agarose gel in the presence of 6% formaldehyde and then transferred onto nylon membrane (Hybond-N+; Amersham, Arlington Heights, IL, USA) by capillary action in 20 × SSPE (0.2 M phosphate buffer, pH 7.7, containing 3.6 M NaCl and 0-02 M EDTA). The RNA on the membrane was hybridized with [32P]-labeled DNA frag- ments in 5 × SSPE containing 50% formamide, 0-5% SDS, 0.1% polyvinylpyrrolidone, 0-1% Ficoll and 0.1% BSA at 42 ℃ for 16 h. After the hybridization, the membrane was washed sequentially in 2 x SSPE/0.1% SDS, 1 × SSPE/ 0.1% SDS, and 0-2 × SSPE/0.1% SDS at 60 ℃, and then exposed to X-ray film at - 80 ℃ with an intensifying screen. The radioactivity in hybridized signals was quantified with a phosphoimager (BAS2000; Fuji-film, Tokyo, Japan).
Determination of mRNAs for CYP11B1 and CYP11B2 by RT-PCR followed by Southern blot analysis
Total RNAs (2 µg) were reverse-transcribed by 200 U reverse transcriptase (Super Script II; Gibco BRL) in 20 ul reaction mixture containing 150 ng random hexamer. An aliquot (5 ul) of the reaction mixture was used for PCR
| Forward primers (5'->3') | Reverse primers (5'->3') | |
|---|---|---|
| Enzyme | ||
| 3ßHSD type II | GCA CAT GGA TCT GTG CAT GTG GTT GCA Gt | GAC CTG GGC TTG TGC CCC TGT TGC Ct |
| 3ßHSD type I-specific | TGG TCC GCC TGT TGG TGG AA# | 1 CTA CCT CTA TGC TAC TGG TGT AG# |
| 3ßHSD type II-specific | TCA TCC GCC TCT TGG TGA AG# | 1 (common to 3ßHSD types I and II) |
| CYP11A | TTG CCT TTG AGT CCA TCA CT+ | GAG CAG GAC TTG GGA CAG ACt |
| CYP21 | CTC AGC TGC CTT CAT CAG TTCt | CAC CCC TTG GAG CAT GTA GTt |
| Common to CYP11B and CYP11B2 | CAA ATG TGG CGT GTT CTT GT# | AGT TGC TGG CTT CTA TGG# |
| CYP11B1-specific | CCC AAC GCT | GTG CAS |
| CYP11B2-specific | CCC AAG GCC | GTG CAS |
+Primers to generate cDNA probes for northern blot analysis.
Primers for RT-PCR analysis.
§Oligonucleotide probes for Southern blot analysis after RT-PCR.
amplification with 20 pmol primer sets (common primer) shown in Table 1 in 30 cycles of 94 ℃ for 30 s, 58 ℃ for 30 s and 72 ℃ for 30 s. The amplified cDNA fragments were separated in 1-5% agarose gel and transferred onto a nylon membrane. To distinguish the cDNA of CYP11B1 from that of CYP11B2, the membrane was subjected to Southern blot analyses using [32P]-labeled oligonucleotide probes specific to the respective P450s (Table 1). The membrane was hybridized in 5 x SSPE containing 0-5% SDS, 0-1% polyvinylpyrrolidone, 0-1% Ficoll and 0.1% BSA at 40 ℃ for 16 h, and washed three times in 0.2 × SSPE containing 0-1% SDS at room temperature for 20 min.
Results
Effect of EGF and bFGF on expression of steroidogenic mRNAs in H295R cells
Northern blot analyses were performed on the growth- factor-treated H295R cells to examine the level of mRNAs of 3ßHSD, CYP17, CYP11A and CYP21. To distinguish between CYP11B1 and CYP11B2 mRNAs, both were first amplified by RT-PCR using primers common to the two mRNAs and then the respective cDNAs were detected by Southern blot analysis using specific oligonucleotide probes. As shown in Fig. 1A and C, the level of mRNA of 3ßHSD markedly increased in the EGF- or bFGF-treated cells, whereas that of CYP17 was decreased substantially by the EGF and bFGF treat- ment. The maximal stimulation of 3ßHSD expression and the strongest suppression of CYP17 were found with a concentration of 10 ng/ml of either growth factor (data not shown). In contrast, the mRNA levels of the other steroidogenic P450s - CYP11A, CYP21, CYP11B1 and CYP11B2 - showed little change with the growth factor treatment (Fig. 1A, B and C). The RT-PCR analyses using the primers specific to either the type I or the type
II isoform of 3ßHSD revealed that only the type II cDNA was clearly amplified from the total RNAs extracted from H295R cells (Fig. 1D).
Time courses of 3ßHSD and CYP17 mRNA expression
Time courses of the EGF- and bFGF-mediated induction of 3ßHSD mRNA and suppression of CYP17 mRNA were examined (Fig. 2). A distinct increase in 3ßHSD mRNA was seen after the 6 h incubation with EGF, its level becoming maximal after 12 h and gradually dimin- ishing thereafter. When bFGF instead of EGF was used as the stimulator, the mRNA level continued to increase after 12 h. In contrast, EGF or bFGF decreased the level of CYP17 mRNA, which reached the lowest point after 24 h. There was no significant change in the cell number under these growth factor treatments.
Effect of various protein kinase inhibitors on EGF- and bFGF-mediated gene expression
Numerous investigators have reported that protein kinases are involved in the signal transduction system of 3ßHSD and CYP17 gene expression (McAllister & Hornsby 1987b, Brentano et al. 1990, Chris et al. 1990, Bird et al. 1996). Therefore, several protein inhibitors were tested to explore the signaling pathway occurring in the EGF- and bFGF-treated H295R cells. As shown in Fig. 3, protein kinase A inhibitors, H-8 (15 µM) and H-89 (20 µM), or a protein kinase C (PKC) inhibitor, bisindolylmaleimide I (10 µM), seemed to have no effect on the levels of 3ßHSD and CYP17 mRNAs in both EGF- and bFGF-treated cells. A mitogen-activated protein kinase (MAPK) inhibi- tor, PD98059 (20 µM), in contrast, further increased the growth factor-dependently increased 3ßHSD mRNA level. PD98059 appeared to restore the growth factor- mediated suppression of CYP17 mRNA, but had little effect on the basal levels of expression of both genes (data not shown).
Next, the inhibitor of Ca2+/calmodulin-dependent kinase II (CaMK II), KN-93, was tested. KN-93 (5 µM) markedly decreased the level of 3ßHSD mRNA in the EGF- and bFGF-treated cells. It exerted little effect on the basal mRNA expression in the non-treated cells (data not
A
cont EGF
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mRNA level (fold change)
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3BHSD CYP17 CYP11A CYP21 CYP11B1 CYP11B2
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shown). Another CaMK II inhibitor, KN-62 (10 uM), or a calmodulin antagonist, W-7 (30 µM), also inhibited the 3ßHSD expression data (not shown). It should be noted that KN-93 further decreased the growth-factor- dependent repressed mRNA level of CYP17.
Effect of CHX on EGF- and bFGF-mediated gene expression
Because the induction of 3ßHSD and suppression of CYP17 appeared to be late events of growth factor treatment, we examined whether de novo protein biosyn- thesis was required for this phenomenon. An inhibitor of protein synthesis, CHX (10 µg/ml), was added to the culture medium together with EGF and bFGF. As shown in Fig. 4A, CHX completely suppressed the EGF- and bFGF-mediated induction of 3ßHSD mRNA, suggesting that de novo protein synthesis is required for the induction of 3ßHSD mRNA. The CHX treatment also com- pletely abolished the PD98059-mediated enhancement of 3ßHSD mRNA in EGF-stimulated H295R cells (Fig. 4B). To our surprise, CHX seemed to prevent the EGF- and bFGF-mediated repression of CYP17 mRNA. The treatment with EGF in the presence of both PD98059 and CHX produced a more increased level of CYP17 mRNA than did treatment in the presence of PD98059 alone (Fig. 4B). Similar results were obtained by treatment with the two inhibitors in the bFGF-stimulated cells (data not shown).
Discussion
Previous studies of bovine (Singh & Waters 1983) and sheep (Singh et al. 1985) adrenal cortical cells demon- strated that the addition of EGF to culture media resulted in stimulated cortisol secretion from and cholesterol syn- thesis in the cells, without significant effect on the production rate of steroid intermediates. Abolition of the EGF-mediated stimulation of cortisol secretion by inhibi- tors of cholesterol synthesis led to the idea that HMG-CoA
A
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reductase, the rate-limiting enzyme of cholesterol syn- thesis, may be the major target of EGF action in the cells. The presence of EGF receptor has been reported in human normal, tumor (Sasano et al. 1994), and fetal (Smikle et al. 1996) adrenocortical cells. Coulter et al. (1996b) reported that the administration of EGF increased the volume of the definitive zone of fetal adrenals of rhesus monkeys in late gestation. This zone-specific increase seemed to be the result of cellular hypertrophy, not of cell proliferation. It was also found that EGF treatment induced 3ßHSD II protein in the definitive and transitional zones without significant effect on CYP17. Similar induction of 3ßHSD II occurred in fetal rhesus monkeys in which endogenous adrenocorticotrophic hormone (ACTH) secretion was stimulated by administration of an inhibitor of CYP11B1, metyrapone (Coulter et al. 1996a). Coulter and colleagues were unable to determine whether the effect on adreno- cortical function that was produced by the EGF adminis- tered to the animals had occurred as the result of the activation of the hypothalamic-pituitary-adrenal axis, as described by Luger et al. (1988) and Polk et al. (1987), or as the result of the direct action of EGF on adrenocortical cells. Our present study with H295R cells, however, demonstrated that treatment of the cells with EGF or bFGF stimulated transcription of the 3ßHSD II gene in the absence of ACTH or cAMP, the second messenger of ACTH.
Interestingly, the EGF or bFGF treatments resulted in a decrease in CYP17 mRNA levels in H295R cells. Welsh & Hsueh (1982) also reported that gonadotropin- stimulated production of testosterone, androstenedione and 17a-hydoxyprogesterone in cultured testicular cells was inhibited by EGF. Because EGF also inhibited the conversion of exogenous 17a-hydoxyprogesterone to androstenedione, they proposed that the EGF effect occurred via inhibition of 17a-hydoxylase and 17,20- lyase. These results suggested that EGF and bFGF could directly modulate the gene expression of steroidogenic enzymes in steroidogenic cells.
Hornsby et al. (1983) reported that bFGF acted as a mitogen in human fetal adrenal cortex, and that the mitogenic effect of bFGF was greater in the definitive zone than in the fetal zone. The mRNA of bFGF in cultured human adrenocortical cells was increased by about threefold by the addition of ACTH. These results suggest that bFGF may be one of the autocrine factors of adrenocortical cells, in which secretion is regulated by ACTH. Li et al. (1998) reported that bFGF treatment increased the level of CYP17 mRNA in cultured bovine fasciculata cells. Our results, in contrast, showed that both bFGF and EGF decreased the level of CYP17 mRNA in H295R cells. This discrepancy may have resulted from the species difference or from the difference in the zonal origin of the cells.
Membrane-associated receptor tyrosine kinases such as EGF/TGFa receptor and bFGF receptor are believed to
stimulate several protein-kinase-mediated signaling path- ways, such as those involving protein kinase C (PKC) and MAPK (Kim & Muller 1999, Rowan et al. 2000). Several investigators (Leers et al. 1997, Bird et al. 1998) have demonstrated that a PKC agonist, phorbol-12-myristate- 13-acetate (PMA), increased 3ßHSD II mRNA expres- sion. MAPK was immunochemically detected in the rat zona glomerulosa, and MAPK seemed to be involved in gene expression of 3ßHSD II, and thus activate the production of aldosterone (McNeil et al. 1998). These findings indicate that PKC- and MAPK-mediated signal- ing pathways may be involved in regulating 3ßHSD II gene expression, suggesting the possibility that the effect of EGF and bFGF on 3ßHSD II gene expression described here could be mediated via these signaling systems. The PKC inhibitor, bisindolylmaleimide I, however, did not block the EGF- or bFGF-mediated induction of 3ßHSD II mRNA in a concentration of 10 uM - a concentration sufficient to block the PMA-mediated induction of 3ßHSD II mRNA (data not shown). Moreover, the level of 3ßHSD II mRNA was further increased, not inhibited, by the addition of the MAPK inhibitor PD98059 in the presence of EGF or bFGF. These results suggest that a signaling system mediated by a member(s) other than PKC or MAPK may be important for the EGF- and bFGF- stimulated induction of 3ßHSD II mRNA. A possible candidate signal would be CaMK II, because KN-93, an inhibitor of CaMK II, markedly inhibited the EGF- and bFGF-stimulated induction of 3ßHSD II mRNA. How- ever, this inhibitor also inhibited the expression of CYP17 mRNA. This indicates that the CaMK II-mediated path- way may be essential for the gene expression of 3ßHSD II, but this signaling system could not account for the repressive effects of EGF and bFGF on the expression of the CYP17 gene.
It took several hours for EGF or bFGF to increase the 3ßHSD II mRNA level in H295R cells (Fig. 2). As in the PKC-mediated induction of 3ßHSD II mRNA (Leers et al. 1997), CHX blocked the effect of EGF and bFGF on 3ßHSD II mRNA expression (Fig. 4), indicating that de novo protein synthesis is required for the EGF- and bFGF-mediated 3ßHSD II gene expression. In contrast, EGF or bFGF decreased the level of CYP17 mRNA in H295R cells (Fig. 1). Interestingly also, the EGF- or bFGF-mediated suppression of CYP17 mRNA was abolished by CHX treatment (Fig. 4). These results may suggest the presence of a CHX-sensitive protein factor(s) that is induced in H295R cells by the growth factor treatment, and which stimulates the expression of the 3ßHSD II gene but inhibits that of the CYP17 gene.
Treatment of the cells with EGF in the presence of both CHX and PD98059, however, revealed the complicated involvement of the MAPK signaling pathway in the expression of these genes. The incubation with both CHX and PD98059 resulted in further increase in the level of CYP17 mRNA in EGF-stimulated H295R cells, whereas
the presence of PD98059 seemed not to influence the blocking effect of CHX on 3ßHSD II gene expression (Fig. 4B). These results indicate that more than one factor - one sensitive to the CHX-treatment and the other related to the MAPK signaling pathway - is involved in the regulation of 3ßHSD II and CYP17 gene expression. In the EGF-mediated activation of the 3BHSD II gene, the CHX-sensitive factor may act more strongly than that involved in the MAPK pathway, because treatment with both CHX and PD98059 completely abolished expression of the gene. In contrast, in the EGF-mediated suppression of the CYP17 gene, the CHX-sensitive factor and the factor involved in the MAPK signaling pathway seemed to repress the gene independently. To date, we have failed in our attempts to search the database of the promoter regions of the 3HSD II and CYP17 genes for sequences typical of cis elements of transcription factors that may act downstream of either the MAPK or CaMK II-mediated signaling pathway.
Further investigation is in progress in our laboratory to elucidate the precise mechanism underlying the enhance- ment of 3ßHSD II mRNA and suppression of CYP17 mRNA by EGF and bFGF.
Acknowledgements
We thank Dr J Ian Mason at Edinburgh University for the generous gift of H295R cells and Dr Masanao Katagiri at Osaka Kyoiku University for pCW plasmid. We also thank Dr Hiromasa Tojo at Osaka University Medical School, Drs Michael R Waterman and Norio Kagawa at Vanderbilt University School of Medicine and Dr William E Rainey at University of Texas Southwestern Medical Center for their helpful discussion. A part of this work was supported by Health Sciences Research Grants from the Ministry of Health and Welfare of Japan.
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Received 3 May 2000 Revised manuscript received 3 August 2000 Accepted 31 August 2000