Differential Control of 17a-Hydroxylase and 3B- Hydroxysteroid Dehydrogenase Expression in Human Adrenocortical H295R Cells*
IAN M. BIRD, MARK M. PASQUARETTE, WILLIAM E. RAINEY, AND J. IAN MASON
The Cecil H. and Ida Green Center for Reproductive Biology Sciences (I.M.B., M.M.P., W.E.R., J.I.M.) and Departments of Obstetrics and Gynecology (I.M.B., M.M.P., W.E.R., J.I.M.) and Biochemistry (J.I.M.), University of Texas Southwestern Medical Center, Dallas, Texas 75235; Department of Obstetrics / Gynecology (I.M.B.), University of Wisconsin, 7E Meriter Hospital Park, Madison, Wisconsin 53715; Department of Clinical Biochemistry (J.I.M.), University of Edinburgh, Royal Infirmary, Edinburgh EH3, 9YW, Scotland, United Kingdom
ABSTRACT
Previous studies of human adrenocortical cells have given incon- sistent findings concerning the effects of angiotensin II (AII) alone or in combination with activators of the protein kinase A-signaling path- way on expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17a-hydroxylase cytochrome P450 (P450c17), and 3ß-hy- droxysteroid dehydrogenase (33-HSD), as well as the corresponding effects on adrenocortical cell steroid secretory products. We have used the human adrenocortical carcinoma H295R cell to evaluate further this question and determine the role of protein kinase C in each of these responses to AII. Treatment with AII alone (10 nmol/L, 48 h) resulted in a significant increase in cortisol production (1.8-fold), as well as a much greater effect on aldosterone production. This in- creased formation of 17a-hydroxysteroids was accompanied by in- creased expression of P450c17 as determined at the level of messenger RNA (mRNA) and enzyme activity. Similar increases in expression of P450scc were observed at the level of mRNA. Increases in 36-HSD expression were also seen at the level of mRNA and, to a lesser extent, at the level of enzyme activity. Because of the comparatively low basal 17a-hydroxylase and high basal 36-HSD activity of H295R cells, how- ever, the overall effect of AII treatment was actually a rise in the 17a-hydroxylase/38-HSD activity ratio, resulting in increased forma- tion of 17a-hydroxysteroids such as cortisol. Whereas treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) reproduced the effect of AII on 33-HSD expression, TPA failed to reproduce the effects of AII on P450c17 and P450scc and even resulted in a marked decrease in expression of P450c17. Thus, the stimulatory effect of AII alone on
P450c17 expression was not mediated via protein kinase C but, like the action of K+, was probably mediated via the Ca2+-signaling path- way. Treatment with forskolin (10 umol/L, 48 h) resulted in a dra- matic increase in both cortisol and dehydroepiandrosterone produc- tion together with increases in expression of P450c17, P450scc, and 38-HSD as measured at the level of mRNA and activity. Consistent with the increase in 17a-hydroxysteroid formation, the effect on 17a- hydroxylase expression was greater than that on 33-HSD at the level of enzyme activity, so a larger 17a-hydroxylase/3-HSD activity ratio was achieved. Cotreatment with forskolin and AII, however, resulted in a dose-dependent reduction in cortisol and DHEA production con- comitant with a marked attenuation of P450scc and P450c17 expres- sion. Although forskolin-induced expression of 33-HSD was not fur- ther increased at the level of mRNA by cotreatment with AII, additivity was observed at the level of changes in enzyme activity. Thus, AII cotreatment resulted in a marked reduction of the forskolin- induced increase in 17a-hydroxylase/38-HSD activity ratio, and so, 17a-hydroxysteroid synthesis was attenuated. These effects of AII cotreatment on expression of P450c17 and P450scc were reproduced by cotreatment with TPA (10 nmol/L), suggesting the involvement of protein kinase C in these attenuative responses. Furthermore, the effect of AII cotreatment on changes in forskolin-induced 17a-hy- droxylase and 36-HSD activities were blocked by the AII Type 1 (AT1) receptor antagonist DuP753 (Losartan), confirming the involvement of an AT1 receptor-linked phospholipase C in activating protein ki- nase C. (J Clin Endocrinol Metab 81: 2171-2178, 1996)
T HE INITIAL conversion of cholesterol to pregnenolone by the enzyme cholesterol side-chain cleavage cyto- chrome P450 (P450scc) is the rate-limiting step of the steroi- dogenic process. It is the relative levels of 17a-hydroxylase and 3ß-hydroxysteroid dehydrogenase (36-HSD), however, both of which can act directly on the newly formed preg- nenolone, that have a profound effect in determining the relative amounts and nature of the subsequent adrenocor-
Received October 27, 1995. Revision received December 20, 1995. Accepted December 22, 1995.
Address all correspondence and requests for reprints to: Ian M. Bird, Department of Obstetrics/Gynecology, University of Wisconsin, 7E Meriter Hospital Park, 202 South Park Street, Madison, Wisconsin 53715.
* This study was supported in part by awards (to W.E.R.) from Merck, Sharp and Dohme, the American Heart Association (Texas Affiliate 93R-082), and the NIH (DK-43140) and by NIH Training Grant T32- HD-07190 (to I.M.B.).
tical steroid products. Upon 17a-hydroxylation, preg- nenolone is irreversibly committed to cortisol and/or C19 steroid production and away from aldosterone synthesis. In contrast, 33-HSD action combined with low 17a-hydroxy- lase activity favors aldosterone formation and opposes cor- tisol or C19 steroid production. Thus, a high 17a-hydroxy- lase/3B-HSD activity ratio supports C19 steroid and cortisol biosynthesis but not aldosterone synthesis, whereas a low 17a-hydroxylase/3ß-HSD ratio favors mineralocorticoid production and opposes C19 steroid and cortisol synthesis. Consistent with this hypothesis, the mammalian adrenal zona glomerulosa does not express 17«-hydroxylase cyto- chrome P450 (P450c17), but expression is high in the zona fasciculata and reticularis; on the other hand, 30-HSD is expressed at a high level in the zona glomerulosa and fas- ciculata but to a lesser extent in the zona reticularis (1, 2).
The classical model of adrenocortical function in vivo is
that the zona glomerulosa is primarily controlled by angio- tensin II (AII) to secrete mineralocorticoids, whereas the zona fasciculata and reticularis are controlled by ACTH to secrete cortisol and C19 steroids, respectively. With the application of molecular techniques to the study of bovine and ovine adrenocortical cells, it is now established that both P450c17 and 3ß-HSD gene expression are directly stimulated by ACTH acting through the intermediary of a cAMP-depen- dent pathway (3). Expression of 30-HSD in the adrenal cor- tex, however, has been shown to be stimulated not only by ACTH but also, to a lesser extent, by AII (4). Type 1 AII (AT1) receptors are expressed in both the zona glomerulosa and zona fasciculata of the bovine, ovine, and human adrenal cortex and are functionally coupled to phospholipase C (5- 8). Recent reports have suggested that, in bovine adrenocor- tical cells, AII alone can also stimulate P450c17 expression (9), albeit to a lesser extent than ACTH. Furthermore, in com- bination with ACTH, AII can attenuate the production of cortisol through long-term alteration of the expression of P450c17 and 30-HSD, resulting in a reduced 17a-hydroxy- lase/36-HSD activity ratio in both ovine and bovine adre- nocortical cells (4, 6, 10, 11). In both species, these effects of AII seem to be mediated via protein kinase C. An under- standing of the effects of AII on expression of P450c17 in human adrenocortical cells is more limited, partially caused by the paucity of available human adrenocortical cells for study. Results from studies of fetal human adrenocortical cells in extended culture (12, 13) or adult human adrenocor- tical cells in primary culture (14) suggest AII may also be able to regulate both P450c17 and 3ß-HSD expression, but results are inconsistent and may reflect the differences of the various models used. Both studies confirmed the positive effect of AII on 36-HSD expression and additivity of this response with that of ACTH or forskolin. The data from fetal adrenal cells either did not examine the singular effect of AII alone on P450c17 expression (12, 13) or reported a negative effect (15). The ability of AII to attenuate forskolin-induced P450c17 expression was, however, confirmed (12, 13). In contrast, a more recent study on adult human adrenocortical cells (14) reported a positive effect of AII on P450c17 expression but failed to demonstrate an attenuation of the ACTH-induced P450c17 expression. Because of the inconsistencies regarding the control of human adrenocortical function by AII, together with the limited availability of human adrenocortical cells, we have used the H295R adrenocortical tumor cell model (16) to examine the differential effect of AII, alone or in combi- nation with forskolin, on steroid production and changes in expression of P450c17 and 3B-HSD in each case. We report here the results of these studies and the dependence of these effects of AII alone or in combination with forskolin on pro- tein kinase C activation.
Materials and Methods
Cell culture
H295R cells were initially obtained as NCI-H295 cells from the Amer- ican Type Culture Collection (Rockville, MD) and then selected as de- scribed previously (16, 17). Because of growth and culture differences between the original ATCC cells and the selected subpopulation, these cells are designated as H295R cells. Cells were maintained in a 1:1 mixture of DMEM and Ham’s F12 medium (DMEM/F12 containing
pyridoxine HCI, L-glutamine, and 15 mmol/L Hepes; Gibco BRL, Gaith- ersburg, MD; catalog no. 11331-014) supplemented with insulin (6.25 µg/mL), transferrin (6.25 µg/mL), selenium (6.25 ng/ml), linoleic acid (5.35 µg/mL)(1% ITS plus, Collaborative Research, Bedford, MA), 2% low-protein serum replacement-1 (LPSR-1, Sigma, St. Louis, MO), and antibiotics. Cells were maintained and grown on 75 cm2 flasks at 37 C under an atmosphere of 5% CO2/95% air. Cells were subcultured to 100-mm dishes or 24-well plates as required and, after 48 h, medium removed and replaced with serum-free medium (DMEM/F12 contain- ing antibiotics and 0.01% BSA). Cells were cultured for a further 24 h before treatment in the same medium.
Analysis of steroids
The cortisol contents of media were determined against cortisol stan- dards prepared in defined (serum-free) medium using a coated tube [125I]cortisol-linked immunoassay (ICN Biomedicals, Costa Mesa, CA). DHEA and aldosterone were determined using assay kits from Diag- nostic Products Corp. (Los Angeles, CA). Results of steroid assays were expressed as nmol steroid per mg cell protein.
Analysis of 17a-hydroxylase activity
Cells were rinsed in DMEM-F12 and incubated for 4 h at 37 C with 0.5 mL medium consisting of DMEM-F12/0.01% BSA supplemented with pregnenolone (2.5 µmol/L), 200,000 dpm/mL [7-3H]pregnenolone (New England Nuclear-DuPont, Boston, MA), and a potent 5a-reduc- tase/3ß-hydroxysteroid dehydrogenase inhibitor, 17B-N, N-diethylcar- bamoyl-4-diethyl-4-aza-5@-androstane-3-one (4MA); 1 µmol/L; Merck, Sharp & Dohme, West Point, PA). At the end of the incubation, the medium was recovered and extracted into dichloromethane (2 × 4 mL). Samples were then concentrated, applied to TLC plates, and developed twice in chloroform/ethyl acetate (9:1). 17a-Hydroxylase activity was computed from the fractional conversion of pregnenolone to 17a-hy- droxypregnenolone and DHEA as identified against authentic stan- dards. Results were expressed as nanomoles per milligram cell protein per hour.
Analysis of 3B-hydroxysteroid dehydrogenase activity
Cells were rinsed in DMEM-F12 and incubated for 4 h at 37 C with 1 mL serum-free medium consisting of DMEM-F12/0.01% BSA supple- mented with 5a-androstane-33,173-diol (2.5 umol/L) and 150000 dpm/mL [3a-3H]5a-androstane-30,17ß-diol (18). At the end of incuba- tion, 0.9 mL medium was removed and combined with an equal volume of water. Radiolabeled steroids were then extracted by mixing with chloroform (3 mL), and phase separation achieved by brief centrifuga- tion. An aliquot (1.5 mL) of the upper phase was recovered and mixed with an equal volume of charcoal/dextran (5%/0.5% wt/vol). After centrifugation to pellet the charcoal, 2 mL aqueous phase were removed, and radioactivity determined in a liquid scintillation counter. The 38- HSD enzymic activity caused release of the tritium into products that were recovered in the aqueous phase of the final extract, and activity was then calculated after appropriate correction for volume and expressed as nanomoles per milligram cell protein per hour.
Protein determination
Cells were solubilized in Tris-HCI (50 mmol/L, pH 7.4) containing NaCl (150 mmol/L), SDS (1%), EGTA (5 mmol/L), MgCl2 (0.5 mmol/L), MnCl2 (0.5 mmol/L), and phenylmethylsulfonylfluoride (0.2 mmol/L) and stored frozen at -70 C. Protein content of samples was then de- termined by bicinchoninic acid protein assay using the BCA assay kit (Pierce, Rockford, IL).
Northern analysis
Cells on 100-mm culture dishes were lysed at 4 C into 1 mL RNAzol B solution (Cinna Biotecx, Houston, TX) and transferred to a microfuge tube. Phase separation was achieved by mixing with 0.15 mL CHCI3, incubation at 4 C for 5 min, and centrifugation (12,000 × g; 20 min; 4 C). The upper phase (0.7 mL) was transferred to a second microfuge tube, and RNA was then precipitated by the addition of 0.8 mL isopropanol
and standing for 1 h at -20 C. RNA was recovered by centrifugation (30 min; 12,000 × g; 4 C) and the recovered pellet was washed once in 75% ethanol (1.0 mL), before drying under air and dissolving in 1 mm EDTA, pH 7.0 (0.1 mL). After determination of recovery and purity by mea- suring absorbance at 260 and 280 nm, samples were precipitated by the addition of 1 mL absolute ethanol and 0.01 mL sodium acetate (3 m; pH 5.2) and stored at -70 C before analysis.
Samples were separated by electrophoresis on gels containing 1.1% agarose in the presence of formaldehyde. The presence and integrity of the major RNA species were examined under UV light to ensure con- sistency between lanes. RNA was transferred to a Magna NT membrane (Molecular Separations Inc., Westboro, MA) by pressure blotting (75 psi, 1 h; PossiBlot Pressure Blotter, Stratagene, La Jolla, CA) and cross-linked under UV light. Prehybridization was carried out at 42 C overnight in a final buffer composition of 50% formamide, 5 × SSC, 1 X PE, and 50 ug/mL transfer RNA. [20 x SSC contains 3.0 mol/L NaCl and 0.3 mol/L trisodium citrate, pH 7.0; 5 x PE contains 250 mm Tris-HCI (pH 7.5), 0.5% sodium pyrophosphate, 5% SDS, 1% polyvinylpyrrolidone, 1% Ficoll, 25 mm EDTA, and 1% BSA]. Hybridizations were performed in the same buffer at 42 C for 16-24 h using antisense probes, which were labeled with [32P] by asymmetrical PCR in the presence of [32P]dCTP (3000 Ci/mmol, Amersham, Arlington Heights, IL). The blots were then washed in 2 × SSC containing 0.1% SDS at room temperature for 15 min, and in 0.1 × SSC containing 0.1% SDS at room temperature for 2 × 30 min before drying, direct radioimaging, and exposure to film (Hyper- film, Amersham). Blots were subsequently stripped by repeated wash- ing in 0.1 × SSC/0.5% SDS at 65 C and checked for lack of radioactivity before reprobing. Finally, all blots were probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) using an antisense probe generated by asymmetrical PCR against bases 39-900 of the human cDNA and bound probe quantified as above. Binding of GAPDH probe per lane was then used to normalize data for P450c17, P450scc, and 3BHSD mRNA against minor variations in lane loading.
Probe preparation
Antisense probes were prepared by PCR in a 50-uL volume under standard conditions but with the following modifications: forward to reverse primers were added at a 1:100 ratio (0.3 and 30 pmol), the free dCTP concentration reduced 40-fold, and the addition of 50 uCi [32P]dCTP (3000 Ci/mmol, Amersham). Template was added at 10 ng/ kilobase. Labeling was performed through 40 cycles. Incorporation of label was routinely 60-75% by this procedure. Templates and oligonu- cleotides were as follows: human P450c17 probe template was pcD- 17aH (19) and the forward and reverse oligonucleotides were 5’-GCAC- CAAGACTACAGTG-3’ and 5’-ACTGACGGTGAGATGAG-3’. Human 3B-HSD probe template was the human Type II cDNA (in PCR1000), and the forward and reverse oligonucleotides were 5’-CTCTCCAGCATCT- TCTG-3’ and 5’-TCACTACTTCCAGCAGG-3’. Human cytochrome P450scc probe template was a complete cDNA in Bluescript, kindly provided by Dr. M. Waterman (Vanderbilt University, Nashville, TN). Forward and reverse oligonucleotides were 5’-TCTCCTGGTGA- CAATGG-3’ and 5’-CTTGCACCAGTGTCTTG-3’ respectively.
Statistical analysis
Statistical analysis of the data was accomplished using ANOVA, followed by Student-Newman-Keuls multiple comparison analysis.
Results
The effects of AII alone or in combination with dibutyryl cAMP (dbcAMP) and forskolin on steroid secretory re- sponses of H295R cells are shown in Fig. 1. An incubation time of 48 h was chosen because previous studies have shown that cortisol and DHEA production in response to forskolin, dbcAMP, (17) are linear up to this time but not beyond, whereas aldosterone production in response to AII continues to increase linearly for 72 h (20). Consistent with our previous findings (17, 20), treatment of H295R cells with AII had a marked effect on the production of aldosterone but
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no measurable effect on DHEA production (Fig. 1). There was also a small (1.8-fold) but significant (P < 0.05) increase in cortisol production. In contrast, both forskolin and db- cAMP promoted marked increases in cortisol and DHEA production with a lesser but significant (P < 0.05) increase in aldosterone production. When AII treatment was carried out in combination with forskolin or dbcAMP, the net result was potentiation of aldosterone production over that with fors- kolin or dbcAMP treatment alone but an attenuation of the effect on cortisol or DHEA production relative to the effects of forskolin or dbcAMP alone. These effects of AII treatment in conjunction with forskolin or dbcAMP were dose depen- dent from 0.1-10 nmol/L AII and were reversed by the addition of the selective AT1 receptor antagonist, DuP 753
(Losartan, Dupont; 10 µmol/L) but not by the selective AT2 receptor antagonist, PD123319 (10 umol/L). This suggests the effects of AII were mediated via the AT1 receptor, which we have previously shown is both expressed (8, 21) and coupled to phosphoinositidase C (20) in H295R cells.
To clarify the role of changes in activities of 17a-hydrox- ylase and 3ß-HSD on the steroid products formed, activity assays also were performed on the same cells at the end of the 48-h treatment period. Changes in 17@-hydroxylase ac- tivity (Fig. 2) largely paralleled changes in the formation of C19 and C21 steroids, i.e. a small but significant (P < 0.05) increase in 17«-hydroxylase activity was observed in re- sponse to AII whereas much larger increases were observed in response to forskolin or dbcAMP. Combined treatment with AII and forskolin or dbcAMP resulted in an attenuation (maximum 46%) of the response to forskolin or dbcAMP alone and again was dose dependent over the range 0.1-10 nmol/L AII. Furthermore, the attenuative effect of AII was blocked by addition of the AT1 receptor antagonist DuP 753 (10 umol/L) but not by addition of the AT2 receptor antag- onist PD123319 (10 µmol/L). Studies of changes in activity of 3ß-HSD in the same cells after 48 h treatment is shown in Fig. 3. Whereas basal 17a-hydroxylase activity was found to be very low or undetectable in H295R cells, basal 3ß-HSD activity was readily measurable. Treatment with AII alone resulted in a small but significant (P < 0.05) increase in 3B-HSD activity that was greater than that seen in response to forskolin or dbcAMP treatment. Combined treatment with AII and forskolin or dbcAMP also resulted in a further in- crease in activity that was significantly greater than that in response to forskolin or dbcAMP treatment alone and seemed to be additive. This action of AII to further increase 38-I ISD activity in response to forskolin treatment was also blocked by the selective AT1 receptor antagonist DuP 753 but not by the selective AT2 antagonist PD123319. Thus, the AT1 receptor mediates the effects of AII in altering both steroid production and expression of key steroidogenic enzymes.
To confirm that changes in enzyme activity could be ac- counted for through altered expression at the level of mRNA and to establish which of these effects of AII could be re- produced by 12-O-tetradecanoylphorbol 13-acetate (TPA), Northern analysis was performed on total RNA recovered from H295R cells after treatment for 20 h (Fig. 4). The data represent means from combined experiments after normal- ization for GAPDH mRNA levels. A stimulation period of 20 h was chosen based on previous time-course studies of the effects of forskolin, dbcAMP or AII treatment alone on the expression of P450scc, P450c17, and 30-HSD mRNA. Where observed, message levels progressively increased over 24 h but not always beyond that time (22). Treatment with AII promoted a small but significant (1.5-fold, n = 13, P < 0.05) increase in level of message for P450c17, whereas a far greater increase was seen in response to forskolin or dbcAMP (6.2- and 6.9-fold, respectively) (Fig. 4). Treatment with AII and forskolin combined resulted in an attenuation (43%) of the response to forskolin alone, a finding in close agreement with the effect seen on cortisol production and 17a-hydroxylase activity. When comparing the actions of AII with those of TPA, the action of AII alone on 17a-hydroxylase was not reproduced by TPA alone at the level of mRNA (Fig. 4),
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unlike our findings in the fetal bovine adrenocortical cells. In fact, a drop in message level was observed in response to TPA treatment (0.5 of basal, n = 6, P < 0.05), a finding in agreement with our previous observations at the level of 17a-hydroxylase activity (22) and with a progressive time- dependent decline in P450c17 mRNA reported by Staels et al. (23). However, the attenuative action of AII on the forskolin- induced increase in P450c17 mRNA was reproduced, and indeed exceeded, by TPA with a 76% reduction in message level compared with treatment with forskolin alone. This finding is again in agreement with our findings at the level of activity when TPA is used in combination with dbcAMP (24). Smaller but otherwise similar findings were observed for the effects of these factors on P450scc mRNA, with the exception that TPA treatment alone had no significant effect on basal levels of P450scc mRNA. Once again, this lack of effect of 20-h treatment with TPA on basal P450scc mRNA levels in H295 cells is in agreement with that of Staels et al.
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(23), although these authors also reported a transient fall in message from 3-12 h after treatment.
Treatment with AII also promoted a significant increase in mRNA for 30-HSD (2.2-fold), which, consistent with our activity data shown above, was less than that in response to forskolin (3.2-fold). Cotreatment of cells with AII and forskolin, however, had little further effect on 38-HSD mRNA levels relative to the effect of forskolin, in contrast with the additivity observed by activity assay. This would suggest that, unlike P450c17 expression, changes in 3ß- HSD activity may also be regulated by processes in ad- dition to changes in transcription or mRNA level. Treat- ment with TPA alone had a still greater effect (5.7-fold) on induction of 30-HSD mRNA than AII or, indeed, forskolin alone, and treatment with TPA and forskolin combined increased 36-HSD mRNA to levels significantly above that for treatment with forskolin alone. However, although the
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effect of forskolin and TPA combined did not exceed that in response to treatment with TPA alone, the larger effect of TPA treatment alone may have maximally stimulated 3ß-HSD mRNA levels, and so, resulted in a lack of addi- tivity in combination with forskolin. These observations at the level of mRNA are also in agreement with our recent findings at the level of 36-HSD protein as determined by Western analysis (24). Although our probe for Northern analysis encodes a region of type II 33-HSD that is ho- mologous to type I, and the molecular sizes of the mRNA in each case are similar, we have also shown previously by Western analysis that the isoform expressed in H295R cells demonstrates an apparent mol wt of 44K characteristic of the type II isoform and not the higher weight of 45K shown by the type I isoform. Furthermore, there was no change in mol wt of the immunodetectable protein on hormone treatment with forskolin or TPA alone or in combination (24).
Discussion
We have previously described the H295R cell as a pluri- potent adrenocortical cell on the basis that prolonged (48 h) treatment of H295R cells with forskolin or AII alone pro- motes a markedly different steroid secretory profile in each case (16, 17, 20). Our findings confirm and extend these observations, showing that treatment with forskolin pro- moted expression of 17a-hydroxylase together with a marked increase in cortisol and C19 steroids while having a less pronounced effect on aldosterone production. In con- trast, treatment with AII had a greater effect on aldosterone production but a lesser effect on cortisol production and no effect on DHEA. We have previously shown that, in part, this predominant effect of AII on aldosterone production is sup- ported by increased expression of aldosterone synthase in H295R cells (20, 25) and fetal bovine adrenocortical cells (26). We have also shown here, however, that the stimulatory effect of AII on cortisol production is accompanied by a corresponding increase in 17-hydroxylase expression. Al- though the effect of AII on 17a-hydroxylase expression is much less than that seen for forskolin, this small increase in 17a-hydroxylase activity may still have an effect on steroid production because of the low basal activity for 17a-hydrox- ylase in these cells. In contrast, whereas both AII and fors- kolin promoted similar increases in 3B-HSD expression, the relative magnitudes were, however, small because of high basal activity. Thus, the overall effect of AII treatment was a sufficient increase in the 17«-hydroxylase/3ß-HSD activity ratio to allow a small increase in steroid flux into cortisol synthesis, even in the face of increased aldosterone synthase expression and aldosterone synthesis.
Treatment with forskolin alone promoted large increases in cortisol and DHEA production, but a comparatively small increase in aldosterone production, a result consistent with our previous findings (17, 20). Cotreatment with increasing doses of AII resulted in further enhancement of aldosterone production but marked attenuation of cortisol and C19 ste- roid synthesis seen in response to forskolin alone. At the level of activity, the large forskolin-induced increase in 17x-hy- droxylase activity was also severely attenuated by AII co- treatment in a dose-dependent manner, and comparable changes were seen for both P450c17 and P450scc mRNA. In contrast, combined treatment with forskolin and AII resulted in significantly higher 38-HSD activity than treatment with forskolin alone, but once again, the relative increase was small compared with the initially high basal activity. As such, the ability of AII to markedly attenuate forskolin induction of 17a-hydroxylase with comparatively little effect on 38- HSD activity would result in a drop in the 17a-hydroxylase/ 38-HSD activity ratio compared with that induced by fors- kolin alone, and this explains the changes in steroidogenesis observed. DHEA production is entirely dependent on both the 17a hydroxylation and the less efficient 17,20 lyase ac- tivity of P450c17, whereas cortisol production only requires 17a-hydroxylation. Thus, All attenuation of forskolin-in- duced 17a-hydroxylase activity will have a more detrimental effect on DHEA synthesis than on cortisol synthesis. In con- trast, aldosterone production occurs independent of 17a- hydroxylation. Indeed, 17@-hydroxylation effectively re-
moves pregnenolone from the aldosterone synthetic pathway. AII attenuation of forskolin-induced 17a-hydrox- ylase expression, therefore, will ensure commitment of more pregnenolone to the mineralocorticoid synthetic pathway.
Our studies also provide further insight into the mecha- nism through which AII mediates these effects in H295R cells, and it seems that multiple signaling pathways are in- volved. In contrast to our previous findings in bovine ad- renocortical cells, TPA did not reproduce the effects of AII alone on induction of P450c17 but, instead, reduced basal expression at the level of mRNA and activity by almost 50% (see also 22). Thus, the increased expression of P450c17 in response to AII alone does not seem to be mediated directly or indirectly by protein kinase C in human adrenocortical cells. We recently have shown that treatment of H295R cells with K+ significantly increases expression of P450c17, and to a lesser extent, P450scc (22). Although the effect is not re- produced by TPA, K+ does promote a marked increase in [Ca2+]], and the effect on P450c17 expression is reproduced by the Ca2+ channel agonist BAYK8644 and inhibited by the corresponding antagonist, nifedipine. However, K+ does not alter expression of 30-HSD, which is known to be induced by both the kinase C and kinase A pathways in these cells (22). Thus, in the H295R cell, the Ca2+ signaling pathway may increase expression of P450c17 directly, rather than through an indirect action on cAMP. In contrast, AII attenuation of forskolin-induced P450c17 expression is reproduced by TPA, suggesting this inhibitory action of AII is mediated via pro- tein kinase C.
The mechanism by which AII mediates its positive effects of 3ß-HSD expression seems to differ from that by which All increases P450c17 expression. We show here that, although not mediated through elevation of [Ca2+]; (22), the action of AII alone on 38-HSD expression could be reproduced and even exceeded by substitution with TPA. Although cotreat- ment with the combination of forskolin and AII did not alter the level of 30-HSD mRNA compared with the effects of forskolin alone, the combined effect of TPA with forskolin was greater than either agent alone. Furthermore, increases in 3B-HSD activity by forskolin and AII were additive. Thus, it seems that AII mediates its effects through a kinase C pathway to induce 38-HSD both in the presence and absence of activators of the kinase A pathway but that other post transcription events may clearly be involved in the regula- tion of activity.
Our findings concerning the small positive effects of AII alone on P450c17 expression disagree with those on fetal adrenocortical cell cultures (15) but concur with the more recent studies on adult human adrenocortical cell cultures (14). Our results also confirm that AII can attenuate the more potent stimulatory effect of activators of the protein kinase A pathway on P450c17 expression and that this attenuative effect of AII could be reproduced by TPA (12, 13). Although this attenuative effect of AII was not observed in adult hu- man adrenocortical cell cultures (14), the authors did confirm that TPA could attenuate the effect of ACTH on P450c17 expression. Our findings concerning the effects of AII alone, or in combination with forskolin, on expression of 30-HSD are in agreement with those observed in fetal and adult human adrenocortical cell cultures (12-14), but additivity
| H295R Cell treatment | ||||
|---|---|---|---|---|
| Basal | AII | Forskolin | Forskolin/AII | |
| P450c17 expression Stimulatory pathway | None detected | Ca2+ None detected | cAMP/kinase A None detected | cAMP/kinase A |
| Inhibitory pathway | DG/kinase C | |||
| Final activity | + | ++ | ||
| 36-HSD expression | ||||
| Stimulatory pathway | DG/kinase C | cAMP/kinase A | cAMP/kinase A, DG/kinase C | |
| Inhibitory pathway | None detected | None detected | None detected | |
| Final activity | ||||
| Final P450c17/38-HSD ratio P450c17:38-HSD | 0 | Low | High | Intermediate |
| Final steroid products | ||||
| Aldosterone | + | |||
| Cortisol | + | |||
| DHEA | 0 | 0 | + | |
was only observed for the effects of AII and forskolin at the level of activity in H295R cells. However, all studies agree on the ability of TPA to reproduce the effects of AII on 38-HSD expression.
In conclusion, our findings, summarized in Table 1, confirm that All alone could weakly stimulate cortisol, as well as aldo- sterone production. The changes in steroid production were associated with increased expression of P450c17, as well as 3B-HSD, resulting in an overall increase in the 17a-hydroxy- lase/3B-HSD activity ratio. Only induction of 3B-HSD, how- ever, was mediated through a protein kinase C-dependent pathway. In the light of recent findings concerning the actions of K+ on P450c17 expression (22), it seems that the mechanism by which AII promotes P450c17 expression is an elevation of [Ca2+] ;. As described previously, forskolin treatment alone in- duced an increase in expression of both P450c17 and 3ß-HSD and a concomitant increase in the 17a-hydroxylase/38-HSD activity ratio, causing predominantly 17a-hydroxylated steroid products. We have shown here that combined treatment with AII in the presence of forskolin modified the effect of forskolin on H295R cells to enhance aldosterone production while sup- pressing both C19 steroid and cortisol production. This was accompanied by a markedly attenuated expression of P450c17 seen in response to forskolin alone but only a marginal accom- panying increase in levels of 3ß-HSD expression over the effect of forskolin alone. Thus, cotreatment with forskolin plus AII resulted in a reduction in the 17@-hydroxylase/3ß-HSD activity ratio achieved in response to forskolin alone. These effects of AII that attenuate forskolin induction of P450c17 or further increase forskolin induction 38-HSD were both apparently achieved through a kinase C-dependent mechanism. Our findings con- cerning 36-HSD expression seem to contrast with those for bovine and ovine adrenocortical cells (4, 10, 11) where we found that AII weakly attenuated the forskolin induction of 30-HSD. However, combined with the far greater attenuative effect on P450c17 expression in all three species, the end result is similar, i.e. a reduction of the 17a-hydroxylase/36-HSD activity ratio and suppression of C19 steroid and cortisol synthesis but an enhancement of mineralocorticoid production. Thus, in con- trast to recent findings in primary cultures of human andre- nocortical cells (14), our results support the concept that similar mechanisms control the ratio of 17@-hydroxylase and 30-HSD
expression in H295R cells as previously reported in ovine and bovine adrenocortical cell studies and that control of this ratio is a powerful influence in determining flux of newly formed pregnenolone into the aldosterone, cortisol, and C19 steroid biosynthetic pathways. The reasons for the differences between H295R cells and primary cultures of human adrenocortical cells may relate to the timing of studies performed in primary culture because basal P450c17 expression was still declining from that in freshly isolated cells at the time of treatment (14), but this will clearly require further investigation.
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