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Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
Address all correspondence and requests for reprints to: Jean-Guy LeHoux, University of Sherbrooke, Department of Biochemistry, Faculty of Medicine, Sherbrooke, Québec J1H 5N4, Canada.
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Abstract |
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In conclusion, we showed that ACTH acutely increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that posttranslational modifications of the StAR precursor occurred during the early stimulatory phase and before the apparent translation of the newly formed mRNA. The rapid induction of protooncogenes suggests their participation in the action of ACTH to stimulate steroidogenesis. Under chronic stimulation by ACTH, adrenals were hypertrophied, and the expression of many steroidogenic enzymes was modified, particularly the level of StAR protein was increased in the ZG and ZFR, confirming the importance of this protein in the control of steroidogenesis in a situation similar to that of Cushing’s syndrome.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among steroidogenic enzymes, 3ß-hydroxysteroid dehydrogenase
(3ßHSD) is responsible for the transformation of 
5 to
4 steroids, and cytochrome P450 21-hydroxylase (P450C21)
is responsible for the catalysis of progesterone to yield
deoxycorticosterone (1). In most mammalian species studied to date,
deoxycorticosterone has been shown to be transformed into aldosterone
in the adrenal zona glomerulosa (ZG) by cytochrome P450 aldosterone
synthase (P450aldo), which possesses both 11ß-hydroxylase and
18-methyloxidase activities (1). In the zonae fasciculata and
reticularis (ZFR), cytochrome P450 11ß-hydroxylase (P450C11)
catalyzes the transformation of deoxycorticosterone to corticosterone
and that of 11-deoxycortisol to cortisol.
The regulation of steroidogenesis by ACTH is mediated by cAMP, which produces acute effects occurring within minutes and chronic effects that require hours before being discernible (1). In a preliminary report (13) we have also shown that administration of ACTH to rats produced, within a few minutes, increases in the messenger RNA (mRNA) levels of adrenal c-jun, c-fos, junB, and fosB, suggesting that these protooncogenes might play a role in the acute stimulation by ACTH.
The purpose of this study was to examine the short-term (acute) and long-term (chronic) effects of ACTH administration on temporal changes occurring in the expression of rat adrenal P450s, 3ßHSD, StAR protein, and protooncogenes of the Jun and Fos family. Dexamethasone was also administered to determine the effect of the inhibition of ACTH secretion on the expression of the above-mentioned adrenal components.
We found that acute stimulation by ACTH rapidly changed the expression of the genes of the jun/fos family in the ZG and the ZFR. Under such conditions, the expression of steroidogenic enzyme proteins was little affected, with the exception of StAR; the level of StAR mRNA was increased, followed by a delayed increase in the level of its protein. Our results indicate that during the first hour after ACTH stimulation, StAR protein does not accumulate in the mitochondrion and, therefore, could act from the external surface of the organelle. Under chronic stimulation by ACTH, the levels of StAR protein in both ZG and ZFR were elevated, showing the importance of this protein in the control of steroidogenesis in a situation that mimics Cushing’s syndrome. Dexamethasone treatment perturbed the expression of many steroidogenic enzymes, thus confirming the importance of ACTH in maintaining the functional status of the adrenal cortex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and treatments
Two-month-old male Long-Evans rats were purchased from Charles
River (St. Constant, Canada). Rats received a single treatment of ACTH
consisting of 4 U Acthar (for rapid action) and 2 U Acthar Gel/250 g
BW, or two daily injections of Synacthen Depot (7.5 U/250 g BW) for a
sustained action. Dexamethasone acetate (400 µg/250 g BW) was
injected once daily. Controls received vehicle only. Animals were
killed by decapitation (in accordance with the ethical standards of the
institutional review committee) at different times after the first
injection, as specified in Results. Blood was collected, and
adrenals were removed. The zona glomerulosa was separated from the
zonae fasciculata and reticularis containing the medulla by the method
of Giroud et al. (14).
RNA extraction from tissues and Northern blotting analysis
Total RNA from rat adrenal ZG and ZFR was extracted using the
Tri-Reagent protocol (Molecular Research Center, Cincinnati, OH). RNA
(15 µg) samples were denatured with glyoxal (15), and then
fractionated by electrophoresis on a diethylpyrocarbonate-treated 1%
agarose gel in 0.01 M phosphate buffer, pH 7.0. The
fractionated RNA was transferred to positively charged nylon membranes
(Boehringer Mannheim, Mannheim, Germany), which were then hybridized
for 16 h at 42 C with the following 
-32P-labeled
probes: 1) bovine P450scc and P450C21 complementary DNAs (cDNAs)
obtained from Dr. M. R. Waterman (Vanderbilt University,
Nashville, TN); 2) rat 3ßHSD cDNA (16); 3) hamster StAR cDNA (17); 4)
hamster P450C11 cDNA (18); 5) oligonucleotide sequence specific to the
rat P450aldo (19); 6) v-fos from Oncor (Gaithersburg, MD);
7) c-jun, junB, and fosB cDNAs
obtained from the American Type Culture Collection (Rockville, MD); 8)
and bovine adrenal angiotensin II receptor type 1 (AT1)
cDNA (20). All blots were also analyzed with an 18S cDNA probe
(American Type Culture Collection), and the results obtained were used
to standardize quantities of mRNA. Autoradiograms were observed by
exposing the blots to Kodak X-Omat RP films (Eastman Kodak, Rochester,
NY) with Cronex Lightning plus enhancing screens (DuPont Cronex,
Wilmington, DE). The intensity of bands on the films was determined
using an LKB 2222–020 Ultroscan XL laser densitometer (Pharmacia
Canada, Baie d’Urfe, Canada). In some experiments radioactivity was
detected using an optical imager (PhosphorImager SF, Molecular
Dynamics, Sunnyvale, CA).
Immunoblotting
Homogenates of rat adrenal ZG and ZFR (15) were analyzed by
immunoblotting as previously described (21). Tissues were homogenized
in 50 mM Tris (pH 7.4), 0.25 M sucrose, 5
mM EDTA, 1 mM phenylmethylsulfonylfluoride, 0.1
mM leupeptin, 30 mM iodoacetamide, and 0.125
µM aprotinin using a Potter-Elvehjem homogenizer with a
loosely fitting Teflon pestle. A portion of the homogenate was
centrifuged for 10 min at 900 x g, and the supernatant
was then centrifuged for 15 min at 9500 x g.
Mitochondrial pellet was resuspended in homogenization buffer. Cholate
and SDS were added to homogenate and mitochondrial fractions to final
concentrations of 1% and 0.1%, respectively. Preparations were frozen
in liquid nitrogen, thawed twice, and solubilized in Laemmli sample
buffer (22). They were passed through a 26-gauge needle, then boiled
for 5 min, and finally centrifuged at 12,000 x g for 2
min. Soluble proteins, 50 µg of homogenate and mitochondrial
preparations, were electrophoresed on 10% polyacrylamide gel in the
presence of 0.1% SDS (SDS-PAGE) and analyzed by immunoblotting as
previously described (21, 23) using a rabbit polyclonal antimouse StAR
peptide antibody provided by Dr. D. M. Stocco (Department of Cell
Biology and Biochemistry, Texas Tech University, Health Sciences
Center, Lubbock, TX). Antibodies used to analyze c-jun and
c-fos were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Other antibodies against other steroidogenic enzymes were
the same as those used in a previous work (23). Immunoreactive proteins
were detected using ECL light-emitting reagents (Amersham
International, Aylesbury, UK). Autoradiograms were observed by exposing
the blots to Kodak X-Omat RP films. The intensity of bands on the films
was determined using the above-mentioned laser densitometer.
Steroid analysis
Corticosterone and aldosterone analyses were performed as
previously described (15).
Statistical analysis
Differences between mean mRNA or protein levels were analyzed by
ANOVA followed by Dunnett’s test, using the SigmaStat program for
Windows (Jandel Corp., San Rafael, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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, ACTH
administration to rats provoked increases in the levels of StAR mRNA in
adrenal ZG and ZFR; changes in the level of StAR mRNA bands were
observed as early as 30 min after treatment; levels were maximally
elevated between 1 and 3 h, with increases of 220% (for the
1.6-kb band) and 370% (for the 3.5-kb band) in the ZG and of 300%
(for the 1.6-kb band) and 350% (for the 3.5-kb band) in the ZFR.
Although ACTH administration always induced a rapid increase in the
adrenal level of StAR mRNA, the order of magnitude varied from one
experiment to another (data not shown). Dexamethasone had no effect on
the adrenal StAR mRNA level 3 h after its administration.
Furthermore, the coadministration of dexamethasone with ACTH did not
block the increases induced by ACTH (results not shown).
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) after ACTH administration, there was a
delay before an increase in the StAR protein level occurred. This is
well illustrated in Fig. 2 and Table 2a, which show that the level of StAR
protein was unchanged at 1 h and was increased only 3–5 h after
ACTH treatment in homogenates of both ZG and ZFR preparations. We also
determined whether StAR protein accumulated more rapidly in
mitochondria than in homogenates. Figure 2 and Table 2a show that
mitochondrial preparations of both zones were enriched in StAR protein
at the same time as for homogenate preparations, but not before.
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and Table 2).
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shows results confirming
our preliminary findings from a first series of experiments (13) that
the mRNA levels of c-jun, c-fos, junB,
and fosB were all increased within 30 min after ACTH
administration in both adrenal ZG and ZFR. These high levels were not
sustained, as they were already diminished after 3 h and returned
to near or below basal values at 5 h. Similar results were found
in a third series of experiments (results not shown). The
coadministration of dexamethasone with ACTH did not prevent the action
of the hormone to increase the levels of the protooncogenes. Western
blotting analysis revealed that the ZG and ZFR contents in
c-jun and c-fos were increased 1 h after
ACTH administration (Table 2b and Fig. 3).
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). When ACTH was administered for 36 h, and animals were
killed 24 h later, the levels of plasma corticosterone and
aldosterone in these latter groups were decreased to near control
values.
Long-term effect of ACTH on StAR mRNA and protein
Compared with controls, the twice daily administration of the long
acting ACTH preparation resulted in levels of StAR mRNA that were
elevated at 12 h in the ZG (310% for the 1.6-kb band, and 290%
for the 3.5-kb band) and in the ZFR (410% for the 1.6-kb band, and
490% for the 3.5-kb band); these levels remained elevated at 24 and
36 h after the first injection (Fig. 5). When rats were injected for 36 h
and killed 24 h after the last injection, the levels of StAR mRNA
had returned to or near basal values. In another series of experiments
(Fig. 6 and Table 3), we
found that after 9 days of ACTH treatment, StAR mRNA levels remained
elevated in the ZG (210% for the 1.6-kb band and 270% for the 3.5-kb
band) and the ZFR (870% for the 1.6-kb band and 570% for the 3.5-kb
band). After 5 days of dexamethasone treatment, the StAR mRNA levels
did not change significantly in the ZG (Table 3) and were elevated in
the ZFR (450% for the 1.6-kb band and 300% for the 3.5-kb band).
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and Table 4). No significant changes occurred in adrenal StAR
protein content under dexamethasone treatment for 5 days.
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and Table 3 show that ACTH administration to rats for 9
days resulted in a decrease in the c-jun mRNA level in the
ZG. A faint junB mRNA band was detected in the ZG and the
ZFR, this did not appear to vary during chronic ACTH treatment (results
not shown). c-fos and fosB mRNAs were not
detected. During dexamethasone treatment for 5 days, junB
mRNA was significantly increased by 400% in the ZFR (results not
shown). The level of c-jun mRNA did not change during
dexamethasone treatment in the ZG and ZFR. Western analysis shows that
the level of c-Jun protein was increased by 190% in the ZG and
remained unchanged in the ZFR (Fig. 7 and Table 4).
Long-term effect of ACTH on steroidogenic enzyme mRNAs
Nine days of ACTH administration provoked changes in the levels of
some adrenal steroidogenic enzyme mRNAs in both ZG and ZFR (Fig. 6 and
Table 3). In the ZG, a decrease in the mRNA levels of 3ßHSD was
observed, with no significant changes in those of P450scc and P450C21.
The level of AT1 receptor mRNA was decreased by 90% in the
ZG, similarly to that of P450aldo mRNA (70%). In the ZFR, the mRNA of
P450scc was significantly increased (260%), whereas those of P450C11,
P450C21, and 3ßHSD were not significantly changed.
When endogenous ACTH secretion was inhibited by daily injections of
dexamethasone for 5 days, and the rats were killed 24 h after the
last injection, the level of P450scc mRNA in the ZG was decreased by
76%, whereas the levels of P450C21, P450aldo, and 3ßHSD were similar
to control values (Table 3 and Fig. 6). The level of AT1
receptor mRNA was not significantly changed in the ZG by dexamethasone
treatment. In the ZFR, the levels of P450scc, P450C21, and P450C11 mRNA
were decreased by 90%, 60%, and 98%, respectively, whereas that of
3ßHSD mRNA was slightly increased (120%). These results clearly show
that inhibition of ACTH secretion affected the levels of many
steroidogenic enzyme mRNAs in both ZG and ZFR, and therefore, they
suggest an important role for ACTH in maintaining these adrenal zones
in a functional state.
Western blotting analysis revealed that, when expressed on a milligram
of protein basis, the content of P450scc protein was increased in the
ZG and was decreased in the ZFR after 9 days of ACTH treatment (Fig. 7, and Table 4). The content of P450C11 protein was not significantly
changed in the ZFR after 9 days of ACTH treatment (data not shown). The
content of P450C21 protein was decreased in the ZG and the ZFR. No
significant changes occurred in the 3ßHSD protein of either
zones.
Dexamethasone treatment for 5 days resulted in significant decreases in the level of P450C21 in the ZG and no changes in the ZFR. The levels of P450scc and 3ßHSD were not changed in either zone by dexamethasone treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Acute ACTH stimulation provoked a rapid increase in the levels of plasma corticosteroids and StAR mRNA within 0.5–1 h. The newly transcribed StAR mRNA was apparently not immediately translated into protein, as the ZG and ZFR StAR protein contents did not differ between preparations from 1-h treated and control animals. There was thus a delay between the increase in the levels of StAR mRNA and StAR protein. Clark et al. (24), using an in vitro model, found that progesterone production, StAR mRNA, and StAR protein expression were concomitant in MA-10 cells stimulated by (Bu)2cAMP after a lag period of 30 min. Clark et al. (24) also found a spatial and temporal relationship between StAR protein expression and the capacity to produce steroid hormones in vivo, although they did not determine whether StAR protein synthesis occurred concomitantly or was slightly retarded compared with the expression of StAR mRNA. In our in vivo study, the delay observed between the increase in the level of StAR mRNA and that in its protein might be tentatively explained by the fact that the rat adrenal cortex cells, under acute stimulation, might be using existing StAR protein precursor for their immediate function before apparently translating newly transcribed StAR mRNA; this situation might be different in unstimulated MA-10 cells because they have an extremely low basal level of StAR protein (25). Supporting our findings, in response to tropic hormones, precursor StAR protein undergoes several posttranslational modifications to yield mature 30-kDa proteins (6, 26, 27, 28, 29, 30). Moreover, previous studies showed that actinomycin D did not inhibit hormonal stimulation of corticosterone production when administered to rats or added in vitro to rat adrenal quarters or slices (31, 32, 33), suggesting that transcription may not be required for the hormonally induced acute steroidogenic response.
Surprisingly, the StAR protein content of mitochondria increased at the same time as that of homogenates. This indicates that during the first hour after ACTH administration, although steroidogenesis was maximally stimulated, StAR protein did not accumulate in the mitochondria. Hence, it can be hypothesized that it is not necessary for StAR protein to enter the mitochondria to increase the transport of cholesterol leading to increased steroidogenesis. This hypothesis is in agreement with results reported by Arakane et al. (34), which found that deletion of up to 62 amino acid residues of StAR protein from the N-terminus did not affect its steroidogenic-enhancing activity, but prevented its importation into mitochondria. Arakane’s results and the fact that StAR protein was not accumulated into mitochondria when steroidogenesis was maximally stimulated imply that StAR protein acts on the external surface of the mitochondria during the acute enhanced steroidogenesis phase.
Although ACTH had a short-term enhancing effect on the level of StAR protein, only small changes occurred in the P450scc protein level in the ZFR within 3 h, with a small decrease at 5 h and a small increase at 3 h for the ZG. A similar situation was reported in the hamster, where ACTH administration did not affect the adrenal P450scc protein content (23). In rat ovaries treated in vivo with hCG, Sandhoff and McLean (35) reported that in contrast to the dramatic increase in the expression of StAR mRNA, P450scc mRNA levels remained unchanged in response to hormonal stimulation; this agrees with our findings. Also in agreement with results reported for hamster adrenal, acute ACTH stimulation did not greatly affect the levels of P450C21, P450C11, and 3ßHSD proteins in the rat adrenal. Taken together, these results indicate that corticosteroidogenesis is not controlled by changes in the levels of adrenal P450 and 3ßHSD enzymes under acute ACTH stimulation.
As recently reviewed (1), there is good evidence that the members of the jun/fos oncogene family play a role in the mechanism of action of ACTH in the adrenal. Indeed, a circadian variation in the expression of c-fos was found in rat adrenal ZFR using immunocytochemistry (36). Also the level of c-fos was rapidly increased after ACTH administration in the adrenal cortex of intact (37) and hypophysectomized rats (38). Viard et al. (39) also reported rapid increases in the levels of junB, c-fos and c-jun mRNA after the addition of ACTH to incubation medium of bovine and ovine adrenal cells in culture. In agreement with the studies mentioned above, in this work we found that administration of ACTH to rats induced a rapid increase in the level of c-fos mRNA and in other early response genes, c-jun, junB, and fosB; furthermore, these mRNAs were translated into proteins, as increases in adrenal ZG and ZFR contents in c-jun and c-fos proteins were observed as early as 1 h after ACTH administration. As previously mentioned, the level of StAR mRNA began to increase in the adrenal ZG and ZFR as early as 30 min after ACTH treatment and continued to increase up to 3–5 h, whereas the mRNA levels of protooncogenes c-jun, c-fos, and fosB were already maximally increased at 0.5–1 h and those for fosB were maximally increased at 1–3 h, then decreased thereafter to near control values by 3–5 h. Taken together, these results suggest that under physiological conditions, early response genes of the jun/fos family may participate in the induction of StAR expression.
Chronic stimulation by ACTH changed the level of StAR protein, but in contrast to the acute stimulation, the long-term treatment also changed the levels of other steroidogenic enzymes in rat adrenals. Under such conditions, the levels of StAR mRNA were significantly elevated in the ZG (210–270%) and ZFR (450–870%), and the levels of StAR protein (expressed on a milligram of protein basis) were also significantly elevated in the ZG (150%) and ZFR (160%), indicating the importance of StAR protein in maintaining a high level of steroidogenesis in a situation similar to that which prevails in Cushing’s syndrome. In agreement with our results, ACTH administration to hypophysectomized rats resulted in an increase in adrenal StAR mRNA 24 h after treatment (40). In H295R adrenocortical cells, (Bu)2cAMP induced StAR protein expression (41).
There was not always a correlation between the levels of mRNA and protein of steroidogenic enzymes. In fact, there was no correlation between the levels of mRNA and protein for P450scc in adrenals of rats who had received ACTH for 9 days. Indeed, in the ZG, the level of P450scc mRNA was not changed, whereas its protein level was significantly increased by 150%; in the ZFR, the level of P450scc mRNA was increased to 260%, whereas its protein level was decreased to 56%. P450scc mRNA was significantly increased in whole adrenals of rats treated with ACTH for 3 days (42). Sander et al. (43) found no changes in the level of P450scc mRNA in adrenals of Sprague-Dawley rats after 8 days of ACTH treatment. In vitro, ACTH stimulation of adrenocortical cells increased P450scc mRNA accumulation (44). In NCI-H295R cells, (Bu)2cAMP led to an increase in the level of P450scc mRNA at 12 h (45) and 20 h after treatment (46).
There was also no correlation between the levels of P450C21 mRNA and
P450C21 protein in the ZG and ZFR. Indeed, we found no changes in mRNA
levels, whereas the protein level was decreased during chronic ACTH
treatment, as seen in Tables 3 and 4. In vitro,
pharmacological doses of ACTH increased bovine adrenal P450C21 protein,
whereas P450C21 mRNA increased only slightly (47).
In the adrenal ZFR of rats treated with ACTH for 9 days, the levels of P450C11 mRNA and protein were not significantly changed. These results are in agreement with those of Sander et al. (43), who reported that ACTH treatment of rats for 8 days did not stimulate P450C11 mRNA expression. However, Engeland et al. (48) reported that P450C11 increased in ACTH-treated rats between 1–4 days in the inner ZFR. Our results are in agreement with those of a previous study that reported that the level of P450C11 mRNA was not affected by a high plasma ACTH level induced by 3 days of 4-aminopyrazolopyrimidine treatment (49). Furthermore, ACTH administration for 24 h did not change the level of P450C11 mRNA or P450C11 protein in hamsters (23). In vitro in cultured bovine adrenocortical cells, ACTH increased the concentration of P450C11 transcripts (50).
In the case of 3ßHSD, a decrease in mRNA was observed in the ZG but not in the ZFR, whereas no change in the protein level was found in either zone after chronic ACTH treatment. In situ hybridization studies performed on adrenals of rats treated with ACTH for 1–4 days showed that 3ßHSD did not increase until 4 days (48). In vitro in NCI-H295R cells (51), protein kinase A signaling pathway activators enhanced, over a 48-h treatment period, the level of 3ßHSD mRNA.
In this study, the P450aldo mRNA level in the rat adrenal ZG was decreased to 30% of the control value, showing that chronic ACTH administration also affected another rate-limiting enzyme of aldosterone formation. These results are in agreement with those of Sander et al. (43), which reported that 8 days of ACTH treatment rendered P450aldo mRNA undetectable in rat adrenals, and also with the results of Aguilera et al. (52), which reported that in isolated adrenal ZG cells from chronically stressed rats, P450aldo mRNA levels and binding of 125I-[Sar1,Ile8]angiotensin II were significantly reduced. We reported that chronic ACTH administration to rats for 9 days resulted in a significant decrease in the capacity of adrenal ZG cells to bind angiotensin II (53). In agreement with our data, activation of the protein kinase A pathway in NCI-H295R cells rapidly decreased the level of AT1 receptor mRNA, and this was paralleled by a loss of both AT1 receptor binding and phosphoinositidase C response (54). The 90% decrease in AT1 receptor mRNA level found in the ZG after chronic ACTH administration suggests that this change may be responsible for the decrease in the level of P450aldo mRNA. The bovine AT1 receptor cDNA probe used recognizes both rat AT1A and AT1B receptors, and as the latter is the principal species in the rat adrenal (55), it is likely that the observed changes are due to changes in the level of AT1B receptors.
In agreement with previous reports, we found that plasma corticosterone and aldosterone levels were increased during chronic ACTH stimulation for 9 days (53). A discrepancy thus seems to exist between the low adrenal contents of P450aldo and AT1 receptor mRNA and the high plasma aldosterone level; at present we cannot explain this discrepancy. We can only speculate that 1) the antialdosterone antibody used may also have cross-reacted with unknown metabolites formed during chronic ACTH stimulation; and 2) it is possible that adrenal hypertrophy by itself might be responsible for the observed high plasma aldosterone level. Indeed, the rat adrenal increases in size during chronic ACTH administration (43, 53). The consequence of this hypertrophy is that the adrenal content of some steroidogenic enzymes might be considerably increased. For example, this is effectively the case for P450scc and P450C11. Indeed, when expressed per gland, P450scc protein content was significantly increased by 400% and 360% in the ZG and ZFR, respectively, and P450C11 protein content was significantly increased by 500% and 690% in the ZG and ZFR, respectively; hence, it is possible that the hypertrophy of the gland might have contributed to the formation of high concentrations of aldosterone precursors. As mentioned above, there was not always a correlation between changes in the level of mRNA and protein during chronic ACTH stimulation. This could be the consequence of a rapid turnover of steroidogenic enzyme mRNA and protein under such stressful conditions. A rapid turnover could also tentatively explain discrepancies found between results from different reports. Taken together, these results thus show profound perturbations in adrenal steroidogenic pathways in a situation similar to that of Cushing’s syndrome, in which the adrenal is also under chronic ACTH stimulation.
When rats were injected twice daily to maintain a high plasma ACTH level, c-fos and fosB mRNAs were not detectable after 9 days of treatment. However, although decreased, c-jun and junB mRNAs were still detectable under such conditions; moreover, c-jun protein, although decreased, was still detectable in the ZG and ZFR after 9 days of ACTH treatment. These results suggest a basic role for protooncogenes of the jun family in maintaining the integrity and function of the adrenal cortex. More work will be needed to delineate the function of protooncogenes in relation to the expression of StAR protein during chronic stimulation by ACTH.
Dexamethasone treatment for 5 days, to decrease ACTH secretion, decreased the level of circulating corticosterone and aldosterone and the levels of P450scc mRNA and P450C21 protein in the ZG. In the ZFR, the mRNA levels of P450scc, P450C21, and P450C11 were significantly decreased, whereas the levels of their respective proteins were not significantly changed by dexamethasone treatment. These results demonstrate the need for the presence of ACTH to maintain corticosteroidogenesis in a functional status. However, during dexamethasone treatment, the level of StAR mRNA was increased in the ZFR, but that of its protein remained unchanged. At present we cannot explain these differences; however, these results indicate that during low levels of circulating ACTH, the adrenals conserved a normal amount of StAR protein. Under such conditions, the level of c-jun mRNA was not different from control values in the ZFR. Although the relationship between protooncogenes of the Jun family and StAR protein in the ZFR has yet to be established, results obtained in this work open the way to further studies to clarify this point.
In conclusion, we have shown that acute stimulation by ACTH acts to increase the level of StAR mRNA, followed by a delay, by an increase in the level of StAR protein in the rat adrenal; this suggests that posttranscriptional modifications of the precursor of StAR protein occur before the translation of newly formed StAR mRNA. Our results suggest that during the first hour after ACTH stimulation, StAR protein acts on the external membrane of mitochondria. The rapid induction of protooncogenes of the jun/fos family also suggests their early participation in the action of ACTH to stimulate steroidogenesis.
Chronic stimulation by ACTH provoked the enlargement of the adrenal cortex and changes in the mRNA and protein levels of many steroidogenic enzymes of the ZG and ZFR. Under such conditions, the levels of StAR protein in both zones were elevated, showing the importance of this protein in the control of steroidogenesis during chronic ACTH stimulation, a situation that mimics Cushing’s syndrome.
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Acknowledgments |
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Footnotes |
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Received January 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5–4-isomerase expression in rat and characterization
of the testis isoform. Mol Cell Endocrinol 80:21–31[CrossRef][Medline]
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J Clin Endocrinol Metab 65:170–175[Abstract]
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h after
treatment. Analyses were performed on 15 µg of total RNA using
c-jun, c-fos, jun B, and fos B cDNAs as
probes.
