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Original Studies |
Department of Obstetrics and Gynecology, University of Wisconsin (I.M.B.), Madison, Wisconsin 53715; the Department of Clinical Biochemistry, University of Edinburgh, Royal Infirmary of Edinburgh (J.I.M.), Edinburgh, Scotland EH3 9YW; and the Department of Obstetrics and Gynecology and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center (W.E.R.), Dallas, Texas 75235
Address all correspondence and requests for reprints to: Dr. Ian M. Bird, 7E Meriter Hospital Park, 202 South Park Street, Madison, Wisconsin 53715. E-mail: imbird{at}facstaff.wisc.edu
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Abstract |
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-hydroxylase cytochrome P450 (CYP17) or
cholesterol side-chain cleavage cytochrome P450 (CYP11A) mRNA.
Together, these findings were remarkably similar to those of our
previous studies regarding mechanisms regulating 3ßHSDII expression
and underline the existence of a subset of steroidogenic enzymes
regulated positively (CYP21 and 3ßHSDII) as opposed to negatively
(CYP17 and CYP11A) by the protein kinase C signaling pathway. The
additional finding of a small induction of CYP21 expression in response
to increased Ca2+, as previously reported for CYP17, but
not 3ßHSDII, expression, also demonstrates that the mechanisms of
control of CYP21 and 3ßHSDII are not identical. This latter finding
may also relate to how CYP21 as well as CYP17 expression continues in
the zona reticularis after adrenarche, whereas 3ßHSD expression
declines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (CYP17) in the zf and zr (2, 3),
3ß-hydroxysteroid dehydrogenase/isomerase (3ßHSD) in the zg and zf
(3, 4, 5), and 21-hydroxylase (CYP21) in the zg, zf, and zr (6, 7). It is
the different ratios of CYP17 vs. 3ßHSD expression in the
three zones that appear to determine the control of glucocorticoid
vs. C19 steroid secretion, primarily due to the pivotal
position of these enzymes in controlling the flux of pregnenolone into
the 
5- or 4-steroid metabolic pathways
(reviewed in Ref.8). Thus, although the expression of CYP17 is high in
both the zf and zr, the poor 3ßHSD expression after adrenarche in the
zr (5) ensures efficient conversion of pregnenolone to
17-hydroxypregnenolone and subsequent conversion to
dehydroepiandrosterone by 17,20-lyase action. Conversely, the high
level of 3ßHSD expression with CYP17 in the zf ensures the metabolism
of pregnenolone to the 4-steroid 17-hydroxyprogesterone,
which is a commitment to cortisol biosynthesis in the face of poor
17,20-lyase activity for 4-steroids. For this pathway to
be maximally active, however, once 5-pathway steroid
precursors have been successfully converted to 4-pathway
products (progesterone and 17-hydroxyprogesterone), it is important to
rapidly remove them because 3ßHSD will otherwise suffer product
inhibition (9). This is achieved by expression of 21-hydroxylase, which
has a low Km for its substrates, is normally
expressed at high levels in the human adrenal cortex, and so is not
normally rate limiting to glucocorticoid or mineralocorticoid
production (8). Partial or total deficiency of CYP21 expression,
however, results in increasing degrees of 5-pathway
activity in the zf with corresponding C19 steroid production excess,
cortisol deficiency, and adrenal hyperplasia from elevated circulating
ACTH. Near-complete deficiency can also result in inadequate
mineralocorticoid synthesis and associated salt-wasting disorders
(10).
Little is known of the mechanisms controlling zonal CYP21 expression in
the human. In this study we have focused on the roles for protein
kinase A and C, both alone and in combination, as well as the role of
Ca2+ in the control of CYP21 expression in the human
adrenocortical cell model H295R. Functionally, CYP21 expression would
be expected to follow that of 3ßHSD, yet zonal expression in the
human shows clear differences in the zr after adrenarche (5, 11). Our
findings show that, consistent with its physiological role of
withdrawing 4 products and so supporting efficient
3ßHSD type II (3ßHSDII) activity, CYP21 expression is largely
similar to that previously seen for 3ßHSDII expression in the human,
with both enzymes being strongly induced by protein kinase A and C,
alone or in combination. Furthermore, the induction of CYP21 and
3ßHSDII messenger ribonucleic acid (mRNA) required rapid protein
synthesis, but that of CYP17 and, indeed, CYP11A mRNA did not. An
increased expression of CYP21 was also seen, however, in response to
elevation of cellular Ca2+, a response not previously
observed for 3ßHSD in these cells (12). Thus, important additional
differences in the mechanism for control of CYP21 and 3ßHSD have been
newly identified that may relate to the differential expression of
these enzymes in the zr after adrenarche.
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Materials and Methods |
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H295R cells were initially obtained as NCI-H295 cells from the American Type Culture Collection (Rockville, MD) and then selected as described previously (13). Because of growth and culture differences between the original American Type Culture Collection cells and the selected subpopulation used for this study, these cells are designated H295R cells. Cells were maintained in a 1:1 mixture of DMEM and Ham’s F-12 medium (DMEM/F12 containing pyridoxine HCl, L-glutamine, and 15 mmol/L HEPES; Life Technologies, Gaithersburg, MD; catalogue no. 11331-014) supplemented with insulin (6.25 µg/mL), transferrin (6.25 µg/mL), selenium (6.25 ng/mL), and linoleic acid (5.35 µg/mL) added as 1% ITS Plus (Collaborative Research, Bedford, MA), 2% Ultroser G (Sepracore, France), 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; after 48 h, medium was removed, and cells were treated as indicated.
Northern analysis
To assess the effects of treatments on CYP21 mRNA, experiments were performed in medium containing 0.5% Ultroser G and antibiotics. Cells subcultured onto 100-mm dishes were maintained for 24 h in DMEM/F12 medium containing 0.5% Ultroser G and antibiotics. Medium was then renewed (10 mL/dish), and treatment was begun with the agents shown for a 20-h period unless otherwise indicated. Media were removed, and cells were lysed at 4 C into 1 mL RNAzol B solution (Cinna Biotecx, Houston, TX) before transfer to a microfuge tube. Phase separation was achieved by mixing with 0.15 mL CHCl3, incubation at 4 C for 5 min, and centrifugation (12,000 x 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 x 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 ethylenediamine tetraacetate, pH 7.0 (0.1 mL). After determination of recovery and purity by measuring 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 were stored at -70 C before analysis.
Samples of RNA (20 µg) 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 consistency between lanes. RNA was transferred to a Magna NT membrane (MSI, Westborough, MA) by pressure blotting (75 psi, 1 h; PossiBlot Pressure Blotter, Stratagene, La Jolla, CA) and was cross-linked under UV light. Prehybridization was carried out at 42 C overnight in a final buffer composition of 50% formamide, 5 x SSC, 1 x PE, and 50 µg/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-HCl (pH 7.5), 0.5% sodium pyrophosphate, 5% SDS, 1% polyvinylpyrrolidone, 1% Ficoll, 25 mm ethylenediamine tetraacetate, and 1% BSA]. Hybridizations were performed in the same buffer at 42 C for 16–24 h using antisense probes to CYP21 mRNA. The antisense probe was labeled with 32P by asymmetric PCR in the presence of [32P]deoxy (d)-CTP; (Amersham, Arlington Heights, IL) (14). The blots were then washed in 2 x SSC containing 0.1% SDS at room temperature for 15 min, and in 0.1 x SSC containing 0.1% SDS at room temperature twice for 30 min each time before direct radioimaging quantification of bound probe using a PhosphorImager (4-h exposure, GS250 PhosphorImager and Molecular Analyst V1.4 Software, Bio-Rad, Hercules, CA) and subsequent exposure to film (Hyperfilm, Amersham). Blots were then stripped by repeated washing in 0.1 x 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) mRNA using an antisense probe generated by asymmetric PCR (14) against bases 39–900 of the human complementary DNA (cDNA), and bound probe was quantified as described above. Binding of GAPDH probe per lane was then used to normalize data for CYP21 mRNA against minor variations in lane loading.
Northern analysis of the effects of cycloheximide on expression of steroid-metabolizing enzymes was performed exactly as described above, with cycloheximide (35 µmol/L) added 10 min before the addition of other agents. Northern analysis was performed using 25 µg RNA/lane as described, except the membranes were probed first for 3ßHSDII, CYP17, and then CYP11A mRNA using human cDNA antisense probes, exactly as previously described (14), and then stripped and reprobed for CYP21 mRNA, as described above. Because of the inhibitory action of cycloheximide on GAPDH expression, loading was normalized by probing for 28S subunit (below).
Probe preparation
Antisense probes were prepared by PCR in a 50-µL 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 was reduced to 5 µmol/L, and 50 µCi [32P]dCTP (3000 Ci/mmol; Amersham) were added. Template was added at 10 ng/kilobase, and labeling was performed through 40 cycles. Incorporation of label was routinely 60–75% by this procedure (14). Human CYP21 probe template was pc21/3c encoding a near-complete human cDNA (15), and the forward and reverse oligonucleotides were 5'-CTG CTG TGG AAC TGG TG-3' and 5'-TCG TGG TCT AGC TCC TC-3', respectively, encoding a probe of 861 bases. All antisense probes generated by asymmetric PCR were of similar specific activity (14).
28S ribosomal subunit probe was prepared by incubation of the
ligonucleotide 5'-AAACGATCAGAGTAGTGGTATTTCACCG-3' with
T4 polynucleotide kinase (Life Technologies) in the presence
of [
-32P]dATP (6000 Ci/mmol; Amersham).
Prehybridization and hybridization steps were performed exactly as
described above.
Analysis of 21-hydroxylase activity
To assess the effects of treatments on 21-hydroxylase activity,
cells were subcultured onto 12-well plates and maintained for 24 h
in DMEM/F12 medium containing 0.5% Ultroser G and antibiotics. Medium
was then renewed (1 mL/well), and treatment was begun with the agents
shown for a 48-h period. After treatment, cells were rinsed in DMEM/F12
medium and incubated for 2 h at 37 C with medium (1 mL) consisting
of DMEM/F12/0.01% BSA supplemented with 100,000 dpm/mL
[14C]17-hydroxyprogesterone (New England Nuclear-DuPont,
Boston, MA) and 20 µmol/L 17-hydroxyprogesterone. At the end of the
incubation, the medium was recovered and extracted into dichloromethane
(twice, 4 mL each time). Samples were then concentrated, applied to
thin layer chromatography plates (Keiselgel 60 F254, Merck/EM Science,
Gibbstown, NJ), and developed twice in chloroform-ethyl acetate
(90:10). 21-Hydroxylase activity was computed from the fractional
conversion of [14C]17-hydroxyprogesterone to
deoxycortisol plus cortisol, as identified against authentic standards.
Results were expressed as nanomoles per h/mg protein.
Protein determination
Cells were solubilized in Tris-HCl (50 mmol/L; pH 7.4), containing NaCl (150 mmol/L), SDS (1%), ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (5 mmol/L), MgCl2 (0.5 mmol/L), MnCl2 (0.5 mmol/L), and phenylmethylsulfonylfluoride (0.2 mmol/L), and stored frozen at -20 C. The protein content of samples was then determined by bicinchoninic acid protein assay, using the BCA assay kit (Pierce, Rockford, IL).
Statistical analysis
Statistical analysis of the data was accomplished using ANOVA, followed by Student-Newman-Keuls multiple comparison analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) revealed that forskolin induced a
progressive increase in CYP21 mRNA levels in H295R cells over a 36-h
period, whereas AII induced a smaller, biphasic rise to maximal levels
by 12 h and submaximal, but still elevated, levels by 24 h.
Treatment with K+ (14 mmol/L) also induced a time-dependent
rise in CYP21 mRNA levels, which was maximal at 1.5-fold by 12 h
of treatment (P < 0.05). Because of the small
magnitude of this response, we confirmed this increase by performing
further studies of dose dependency at 20 h of stimulation (Fig. 2). We found that K+ induced
a dose-dependent increase in CYP21 mRNA, achieving significance at
doses of 12 mmol/L or above.
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). The action of
forskolin alone on CYP21 expression was reproduced by treatment with
dibutyryl cAMP (dbcAMP), confirming the involvement of cAMP in this
response. The action of AII was greater than that of K+ or
the calcium channel agonist BAYK8644, suggesting that AII action was
not solely through the Ca2+ signaling pathway. The action
of AII was reproduced and indeed exceeded by the protein kinase C
activator, TPA, as has been previously shown for induction of 3ßHSDII
(12, 13, 16). The actions of forskolin alone were not significantly
different from those of TPA or AII alone, nor were they increased by
combined treatment with AII, suggesting no synergy of the effects of
protein kinase A and protein kinase C. This was confirmed by the
finding that the actions of forskolin and TPA in combination did not
exceed that of TPA alone.
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) agreed with those at the level of
CYP21 mRNA, with TPA inducing a significantly greater increase in
activity than forskolin, and the combined action of forskolin plus TPA
being significantly higher than that of forskolin, but similar to that
of TPA alone.
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). These findings were
also remarkably similar to the action of cycloheximide on induction of
3ßHSDII mRNA, but contrasted with the effects of these agonists on
CYP17 or CYP11A mRNA. Cycloheximide had no effect on the expression of
CYP17 or CYP11A mRNA in response to AII, forskolin, or dbcAMP.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that forskolin treatment induced a progressive increase in CYP21 mRNA levels over 36 h is consistent with the general stimulatory effect of the kinase A pathway on all steroid-metabolizing enzymes in H295R cells studied to date (12, 13, 16, 26, 27, 28). The finding that AII induced a smaller rise in CYP21 expression and that this was exceeded by treatment with TPA alone distinguishes the control of CYP21 expression from that of CYP17 or CYP11A expression and shows that CYP21 expression is more similar to that reported for 3ßHSDII in these cells (12, 13, 16). This is confirmed by the finding that TPA did not attenuate the effect of forskolin alone on CYP21 expression at the level of both mRNA and activity, as also previously reported for 3ßHSDII (12, 13, 16). Thus, the protein kinase C and A pathways did not act in opposition at the level of CYP21 expression, as has previously been shown for CYP17 and CYP11A expression in H295R cells (16).
The finding that treatment of H295R cells with K+ to stimulate the Ca2+ signaling pathways independently of protein kinase C activation induced a small time- and dose-dependent rise in CYP21 mRNA clearly indicates mechanistic differences in the control of expression of CYP21 and 3ßHSDII. In addition, as the magnitude of the response was smaller than that to AII, this suggests that AII could not be acting solely through Ca2+, consistent with the proposed additional involvement of protein kinase C in the AII response. The small magnitude of this CYP21 response, however, makes the physiological significance of K+-regulated CYP21 expression questionable compared to the previously described dramatic effect of K+ on CYP11B2 expression (26) or, to a lesser extent, on CYP17 expression (12).
The expression of some steroidogenic enzymes is dependent on the synthesis of short half-life trans-acting factors, and Staels et al. (27) previously showed CYP21 expression in response to protein kinase A activation to be cycloheximide sensitive in H295 cells. In view of the differential effects of K+ on CYP21 and 3ßHSDII expression, we also tested the effects of cycloheximide on the expression of CYP11A, CYP17, 3ßHSDII, and CYP21 in H295R cells. Our finding that cycloheximide selectively blocked the induction of both CYP21 and 3ßHSDII mRNA in response to AII, forskolin, and dbcAMP, but had no effect on CYP17 or CYP11A mRNA, is again consistent with the idea that expression of CYP21 largely follows that of 3ßHSDII in the human adrenal cortex. The small positive effect of K+ on CYP21 expression, but not on 3ßHSDII expression, however, indicates that the regulatory elements controlling CYP21 expression still differ from those controlling 3ßHSDII expression in H295R cells, even though both may involve common cycloheximide-sensitive factors. This finding may provide a clue as to why the expression of CYP21 as well as that of CYP17 can be maintained in the human zr after adrenarche (11), whereas expression of 3ßHSD declines (5).
In conclusion, we demonstrate that regulation of CYP21 expression in
H295R cells was remarkably similar to that previously reported for
3ßHSDII expression and underline the existence of a subset of
steroidogenic enzymes regulated positively (CYP21 and 3ßHSDII), as
opposed to negatively (CYP17 and CYP11A), by the protein kinase C
signaling pathway. The induction of CYP21 in response to both protein
kinase A and protein kinase C signaling pathways explains how adrenal
CYP21 expression is normally maintained at high levels in the zg and zf
in response to circulating ACTH and AII. The hormonal induction of
CYP21 and 3ßHSDII then effectively supports rapid and efficient
withdrawal of steroid substrate from the 5-pathway
toward aldosterone in the glomerulosa and cortisol in the fasciculata.
However, the finding that CYP21 expression, but not 3ßHSDII
expression, is increased in response to Ca2+ also
demonstrates that mechanistic differences still exist in control of the
expression of these two enzymes. Additional studies will be necessary
to determine exactly how these mechanistic differences may relate to
the differential expression of CYP21 and 3ßHSDII that occurs in the
zr after adrenarche.
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Footnotes |
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Received August 5, 1997.
Revised January 29, 1998.
Accepted February 5, 1998.
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References |
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