Steroidogenic acute regulatory protein mRNA expression in adrenal tumours

The rate limiting step in steroidogenesis is cholesterol transport through the outer to the inner mitochondrial membrane and the cytochrome P450 side chain cleavage (P450scc) complex. The protein factor responsible for this transport, and as such necessary for regulating the acute production of steroids, has been identified and named the steroidogenic acute regulatory protein (StAR). We investigated the expression of StAR in functional and non-functional adrenal neoplasms and compared the expression with that of P450scc. Poly A RNA was extracted from normal adrenal glands (NAG, n=5), aldosterone producing adenomas (APA, n=4), cortisol producing adenomas (CPA, n=5), adrenocortical carcinomas (ACC, n=6) and non-functional adenomas (NFA, n=3), electrophoresed through a 1% agarose gel, blotted and hybridised with a PCR-generated cDNA labelled with [(32)P]CTP. The blots were stripped and re-hybridised with a P450scc cDNA and a mouse beta-actin probe. Compared with P450scc, StAR mRNA expression showed little variability in the magnitude of expression and did not correlate with the endocrine profiles (NAG: StAR 100+/-16%, P450scc 100+/-14%; APA: StAR 80+/-3%, P450scc 94+/-13%; CPA: StAR 71+/-10%, P450scc 109+/-15%; NFA: StAR 64+/-9.5%, P450scc 18+/-5%; means+/-s.e.m.). ACC expressed low levels of both genes probably as a result of dedifferentiation (StAR 29+/-9%, P450scc 46+/-18%). Incubation of the NCI-h295 tumour cell line with 10nmol ACTH and 10micromol forskolin induced an increase in the abundance of StAR and P450scc mRNA, demonstrating gene regulation by the cAMP protein kinase A pathway. Furthermore, we incubated the NCI-h295 tumour cell line with the adrenostatic compounds, aminoglutethimide and metyrapone. We could not detect an effect on the expression of StAR mRNA, whereas the expression of P450scc mRNA was significantly reduced. We conclude that, in contrast to P450scc, StAR seems to be evenly expressed in adrenocortical adenomas. Therefore, the endocrine activity of a given tumour cannot be explained by the abundance of StAR expression. In ACC, both StAR and P450scc expression is low, explaining the relatively inefficient steroid production of these tumours.     

Angiotensin II type 1 receptor and ACTH receptor expression in human adrenocortical neoplasms

OBJECTIVE: Type 1 angiotensin II (Ang II) receptors transduce most of the known actions of Ang II, including steroidogenesis and trophic actions on the adrenal cortex. We investigated the type 1 Ang II receptor expression in adrenocortical tissues to define its regulation in adrenocortical neoplasms and to compare its expression with that of the ACTH receptor (ACTH-R). PATIENTS AND MEASUREMENTS: Poly A RNA was extracted from tumour tissue and electrophoresed through a 1.0% agarose gel, blotted and hybridized with alpha32P-CTP labelled PCR generated type 1 Ang II receptor cDNA probe. Receptor autoradiography was performed on slices from normal adrenals and tumour tissue by incubation with 125I-Sar1, Ile8-Ang II with and without pretreatment with cold Ang II or with the selective type 1 receptor antagonist losartan. RESULTS: Ang II type 1 receptor mRNA was high in cortisol producing (CPA; n = 5) and aldosterone producing (APA; n = 4) adenomas (normal adrenals 100 +/- 12% vs. 180 +/- 16% in CPA and 154 +/- 26% in APA, mean +/- SEM), but was low in nonfunctioning adenomas (NFA; n = 2; 2 +/- 1%). ACTH receptor mRNA followed a similar pattern (CPA 178 +/- 17, APA 196 +/- 30, NFA 0%, carcinomas 56 +/- 11%) with a good correlation between Ang II type 1 receptor and ACTH-R mRNA of r = 0.692, P = 0.0019. Receptor autoradiography in normal adrenals demonstrated Ang II type 1 receptors predominantly in the zona glomerulosa. In tumour tissue, mainly type 1 receptor expression was found confirming the Northern blot data. CONCLUSIONS: Angiotensin II type 1 receptor and ACTH receptor expression seems to be correlated with the functional status of adrenocortical tumours, suggesting regulation by similar factors. The predominant receptor expressed in adrenocortical tumours is the Angiotensin II type 1 receptor whereas type 2 receptor expression is minimal.     

Markedly increased expression of cytochrome P-450 17 alpha-hydroxylase (P-450c17) mRNA in adrenocortical adenomas from patients with Cushing's syndrome.

We studied the contents of cortisol (F) and dehydroepiandrosterone (DHEA) and the expression of mRNA of cytochrome P-450 for side-chain cleavage (P-450scc), 17 alpha-hydroxylase (P-450c17), 21 alpha-hydroxylase (P-450c21) and 11 beta-hydroxylase (P-450c11) in adrenocortical adenomas from three patients with Cushing's syndrome. The F content was significantly higher in adrenocortical adenomas than in normal adrenal glands, while the DHEA level was similar to that in normal adrenal glands. The adrenal adenomas showed a markedly higher level of P-450c17 mRNA, and a slightly but not significantly increased level of P-450c21 mRNA, compared with normal adrenal glands. The expression of P-450scc and P-450c11 mRNA in the adenomas was similar to that in normal adrenal glands. These results suggest that the overproduction of cortisol in adrenocortical adenomas associated with Cushing's syndrome results from an increased expression of P-450c17 and P-450c21 mRNA.     

Expression of cytochrome P-450 mRNAs in steroidogenesis of adrenocortical adenomas from patients with primary aldosteronism

We studied the contents of aldosterone and cortisol (F) and the expression of mRNA of cytochrome P-450 for side-chain cleavage (P-450scc), 17 alpha-hydroxylase (P-450c17), 21-hydroxylase (P-450c21) and 11 beta-hydroxylase (P-450c11) in adrenocortical adenomas from three patients with primary aldosteronism. The aldosterone content was significantly higher in adrenocortical adenomas than in normal adrenal glands, while F content in adenomas was similar to the level in normal adrenal glands. The aldosterone-producing adenomas showed a markedly higher level of P-450c11 mRNA, a slightly but not significantly increased level of P-450c21 mRNA and a significantly decreased level of P-450c17 mRNA, compared with those in normal adrenal glands. The expression of P-450scc mRNA in adenomas was similar to the level in normal adrenal glands. These results suggested that the renin-independent overproduction of aldosterone in adrenocortical adenomas from the patients with primary aldosteronism results from increasing expression of the mRNA for P-450c11 and decreasing expression of the mRNA for P-450c17.     

Frequent mutations of beta-catenin gene in sporadic secreting adrenocortical adenomas

Objective Molecular alterations remain largely unknown in most sporadic adrenocortical tumours and hyperplasias. In our previous work, we demonstrated the differential expression of several Wnt/β‐catenin signalling‐related genes implicated in ACTH‐independent macronodular adrenal hyperplasias (AIMAH). To better understand the role of Wnt/β‐catenin signalling in adrenocortical tumours, we performed mutational analysis of the β‐catenin gene. Methods We studied 53 human adrenocortical samples (33 adenomas, 4 carcinomas, 13 AIMAH, 3 ACTH‐dependent adrenal hyperplasias) and the human adrenocortical cancer cell line NCI‐H295R. All samples were screened for somatic mutations in exons 3 and 5 of the β‐catenin gene. Eleven and six samples were analysed for β‐catenin protein expression by Western blotting and immunohistochemistry, respectively. Results No mutations were detected in adrenocortical carcinomas, AIMAH and ACTH‐dependent hyperplasias. Genetic alterations were found in 5 (15%) out of 33 adenomas: three cortisol‐secreting adenomas, one aldosterone‐secreting adenoma and one nonfunctional adenoma. Two‐point mutations occurred at serine residues of codons 37 and 45 (S37C and S45F). The remaining three tumours contained deletions of 6, 55 and 271 bp. H295R cells carry an activating S45P mutation. Western blot analysis of samples with 55‐ and 271‐bp deletions showed an additional shorter and more intense band representing an accumulation of the mutated form of β‐catenin protein. In addition, cytoplasmic and/or nuclear accumulation of β‐catenin was observed in mutated adenomas by immunohistochemistry. Conclusions Activating mutations of exon 3 of the β‐catenin gene are frequent in adrenocortical adenomas, and further characterization of the Wnt/β‐catenin signalling pathway should lead to a better understanding of adrenal tumourigenesis.     

Adrenaline stimulates cholesterol side-chain cleavage cytochrome P450 mRNA accumulation in bovine adrenocortical cells.

The effect of adrenaline on the accumulation of mRNA encoding cholesterol side-chain cleavage cytochrome P450 (P450scc) and cortisol secretion was studied in bovine adrenocortical cells in primary culture. Treatment of cultured cells with adrenaline resulted in a 2-fold increase in mRNA encoding P-450scc, as revealed by Northern blot analysis. Under these conditions the maximal stimulation with ACTH resulted in a 6-fold accumulation of mRNA encoding P450scc. The effect of adrenaline on the expression of P450scc was abolished by the beta-blocker propranolol, while propranolol had no effect on ACTH-induced P450scc mRNA accumulation. Adrenaline stimulated the secretion of cortisol in a dose-dependent manner with a median effective dose of 0.5 mumol/l. The adrenaline-stimulated cortisol secretion amounted to 42% of the effect of ACTH (0.1 nmol/l). Upon adrenaline treatment, cAMP concentration in the culture medium increased about 50-fold over the basal value. It is concluded that the stimulatory action of adrenaline upon cortisol formation requires beta-adrenergic receptors and is due, at least in part, to a cAMP-mediated increases in the accumulation of mRNA encoding P450scc.     

Alteration in the expression of genes for cholesterol side-chain cleavage enzyme and 21-hydroxylase by hypophysectomy and ACTH administration in the rat adrenal

The changes in steady-state levels of mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and steroid 21-hydroxylase cytochrome P-450 (P-450c21) caused by hypophysectomy and ACTH treatment were determined in rat adrenals. Hypophysectomy caused marked decreases in adrenal weight and total RNA per gland. Administration of ACTH resulted in increases in adrenal weight and total RNA. A significant correlation between the amount of RNA and adrenal weight was observed. Both P-450scc and P-450c21 mRNAs were decreased by hypophysectomy and increased by ACTH treatment. P-450scc mRNA decreased to 20% and P-450c21 mRNA to 76% of control values 1 day after hypophysectomy. ACTH caused a significant increase in P-450scc mRNA after 3 h. However, a significant increase in P-450c21 mRNA was observed 12 h after administration of ACTH. These results are concordant with previous studies in vitro utilizing cultured adrenocortical cells. Moreover, the induction of steady-state levels of P-450scc mRNA was faster than that observed by other investigators in studies in vitro. These results may indicate that integrity of the adrenal gland in vivo is important for the action of ACTH.     

Identification of adrenocorticotropin receptor messenger ribonucleic acid in the human pituitary and its loss of expression in pituitary adenomas

The ACTH receptor (ACTH-R) is the second member of the melanocortin (MC-2) receptor family that includes five seven-transmembrane G protein-coupled receptors and has been shown to be predominantly expressed in the adrenal cortex. It has been postulated that ACTH may regulate its own secretion through ultra-short-loop feedback within the pituitary. ACTH-secreting adenomas are characterized by resistance to glucocorticoid feedback, and they may have dysregulated ACTH feedback. We therefore investigated the ACTH-R in normal and adenomatous human pituitary tissue. We report here the identification of ACTH-R mRNA in the human pituitary gland, which was confirmed by direct sequencing. We studied the expression of the ACTH-R in 23 normal pituitary specimens and 53 pituitary adenomas (22 ACTH-secreting, nine GH-secreting, eight prolactin-secreting, one TSH-secreting, one FSH-secreting, 10 nonfunctioning, and two silent corticotroph adenomas), using the sensitive technique of real-time quantitative PCR. Contamination of ACTH-secreting adenomas and nonfunctioning pituitary adenomas with nonadenomatous tissue was excluded by lack of Pit-1 expression. ACTH-R mRNA was detected in all normal pituitary specimens, and in situ hybridization colocalized expression to ACTH staining cells only. However, ACTH-R mRNA levels were undetectable in 16 of 22 ACTH-secreting tumors and in both silent corticotroph tumors. Diagnostic preoperative plasma ACTH levels were significantly lower in the ACTH-R positive ACTH-secreting tumors, compared with those who were ACTH-R negative (P = 0.0006). Direct sequencing of the coding region of the ACTH-R in cDNA from three ACTH-secreting tumors positively expressing the receptor showed no mutations, as did sequencing of genomic DNA in three receptor negative ACTH-secreting tumors and the two silent corticotrophs. These results provide further evidence compatible with an ACTH feedback loop in the pituitary and suggest that loss of expression of the ACTH-R in corticotroph adenomas of patients with Cushing's disease may play a role in the resistance to feedback of the pituitary-adrenal axis seen in these patients.     

Leptin interferes with adrenocorticotropin/3',5'-cyclic adenosine monophosphate (cAMP) signaling, possibly through a Janus kinase 2-phosphatidylinositol 3-kinase/Akt-phosphodiesterase 3-cAMP pathway, to down-regulate cholesterol side-chain cleavage cytochrome P450 enzyme in human adrenocortical NCI-H295 cell line

CONTEXT: Obesity has adverse effects on adrenocortical functions. Adipocyte-derived leptin, a biomarker molecule of obesity, may directly control adrenal steroidogenesis via an unclear mechanism. OBJECTIVE: We studied the mechanism underlying leptin action on adrenal steroidogenesis in human adrenocortical NCI-H295 tumor cell line. METHODS: Levels of progesterone, cortisol, and cAMP were determined by ELISA. Western blotting was used to detect protein amounts of P450 side-chain cleavage (P450scc), Janus kinase 2 (JAK2), Akt, and their phosphorylated forms. The mRNA expressions of P450scc and leptin receptors were measured by RT-PCR and real-time PCR. P450scc promoter activity was analyzed with a luciferase reporter system. RESULTS: Cholera toxin mimicked ACTH action by increasing adrenal cAMP levels and steroid secretion. Leptin did not affect basal release but significantly inhibited ACTH/cholera toxin-induced steroid secretion. The concomitant inhibitions by leptin on cholera toxin-induced protein and ACTH/cholera toxin-induced mRNA expression of P450scc were confirmed. Leptin inhibited ACTH/cholera toxin-induced CYP11A1 promoter activity via a known cAMP-responsive region located between -1.7 and -1.5 kb. Leptin activated phosphorylations of JAK2 and Akt. Inhibitory effects of leptin on ACTH/cholera toxin-induced cAMP levels, CYP11A1 promoter activity, and steroid secretion were blunted by either inhibitor of JAK2 (AG490) or phosphatidylinositol 3-kinase/Akt (wortmannin) as well as inhibitors of cAMP-degrading phosphodiesterases (PDEs), including nonspecific 3-isobutyl-1-methylxanthine and PDE3-specific SKF94836. Leptin failed to affect the inductions of CYP11A1 promoter activity and steroid secretion by PDE-nonhydrolyzable N(6)-monobutyryl-cAMP. CONCLUSIONS: Leptin interferes with ACTH/cAMP signaling, possibly through a cAMP-degrading mechanism involving activation of JAK2, phosphatidylinositol 3-kinase, and PDE3, to down-regulate P450scc expression and consequent adrenal steroidogenesis.     

Expression of inhibin alpha in adrenocortical tumours reflects the hormonal status of the neoplasm

Inhibins are gonadal glycoprotein hormones whose main endocrine function is to inhibit pituitary FSH secretion. In addition to testes and ovaries, other steroid-producing organs are sites of inhibin alpha subunit expression. To study the role of inhibins in human adrenal gland, we screened a panel of 150 adrenals (10 normal adrenals, 25 adrenocortical hyperplasias, 65 adrenocortical adenomas, 30 adrenocortical carcinomas and 20 phaeochromocytomas) for inhibin alpha expression. mRNA levels of inhibin alpha subunit were studied in 57 samples and all tissues were stained immunohistochemically with an inhibin alpha subunit-specific antibody. Inhibin alpha mRNA was detected in all adrenocortical tissues. Virilizing adenomas possessed a 10-fold higher median inhibin alpha mRNA expression than did normal adrenals. Bilaterally and nodularly hyperplastic adrenals and other than virilizing adrenocortical tumours had their median inhibin alpha mRNA levels close to those of normal adrenals. Immunohistochemically, inhibin alpha subunit was detectable in all normal and hyperplastic adrenals, as well as in 73% of the adrenocortical tumours. However, the percentage of inhibin alpha-positive cells varied greatly in different tumour types. The median percentage of positive cells was 10 in non-functional and Conn's adenomas, 30 in Cushing's adenomas and 75 in virilizing adenomas. In malignant adrenocortical tumours the median percentage of inhibin alpha-immunopositive cells was 20 in non-functional carcinomas, 30 in Conn's carcinomas, 65 in Cushing's carcinomas and 75 in virilizing carcinomas. All phaeochromocytomas were negative for inhibin alpha subunit both at the mRNA level and immunohistochemically. Our data show that inhibin alpha subunit is highly expressed in both normal and neoplastic androgen-producing adrenocortical cells, with less expression in cortisol-producing and hardly any in aldosterone-producing cells. This suggests a specific role for inhibins in the regulation of adrenal androgen production. We did not find any significant difference in inhibin alpha expression between benign and malignant adrenocortical tumours. Thus inhibin alpha gene does not seem to have a tumour suppressor role in human adrenal cortex.     

Food restriction enhances endogenous and corticotropin-induced plasma elevations of free but not total corticosterone throughout life in rats

Chronic food restriction (FR), which retards many aging processes, enhances the endogenous diurnal peak of plasma total corticosterone (B) in young rats. Although the FR-dependent enhancement of total B disappears in aged rats, increased levels of the bioavailable fraction, free B, appear to be maintained. In young rats, we previously found that the FR-induced increase in the diurnal peak of total B is associated with increased adrenal response to corticotropin, also known as adrenocorticotropic hormone (ACTH). Here we show that the FR-enhanced adrenal response of total B to ACTH disappears with age but that the enhanced response of free B is maintained. We measured the endogenous diurnal peak and the response to ACTH of total and free B in 10-, 16-, and 22-month-old ad-libitum fed and FR male Fischer 344 rats in the afternoon, when plasma B peaks. At 10 and 16 months, FR rats showed enhanced total plasma B responses to ACTH relative to ad-libitum fed rats, but not at 22 months. By contrast, the response of free B to ACTH was enhanced by FR at all ages. The effect of FR on patterns of endogenous total and free diurnal B in these three age groups paralleled the ACTH-response data. The enhanced adrenocortical response of FR rats to ACTH does not reflect an increased expression of ACTH-receptor (ACTH-R) mRNA, because ACTH-R mRNA/microg adrenal RNA and ACTH-R mRNA/mg adrenal weight did not differ between ad-libitum fed and FR rats at any age.     

Expression of the novel adrenocorticotropin-responsive gene selective Alzheimer's disease indicator-1 in the normal adrenal cortex and in adrenocortical adenomas and carcinomas

Selective Alzheimer's disease indicator-1 (seladin-1) is a novel gene with antiapoptotic activity that is down-regulated in vulnerable brain regions in Alzheimer's disease. This gene encodes 3-beta-hydroxysterol Delta-24-reductase (DHCR24), which converts desmosterol into cholesterol. In the adrenal cortex, increased expression of seladin-1/DHCR24, which appears to be modulated by ACTH, has been recently reported in cortisol-secreting adenomas, compared with the adjacent atrophic tissue. In our study, we measured the expression level of seladin-1/DHCR24 in cortisol- (n = 18) and aldosterone-secreting (n = 16) adrenocortical adenomas, in carcinomas (n = 17), and in normal adrenal glands (n = 8) by quantitative real-time RT-PCR. The amount of seladin-1/DHCR24 mRNA was significantly reduced in carcinomas (total RNA, 2.5 +/- 0.8 pg/ micro g) compared with the other groups (P < 0.01). Western blot analysis confirmed the mRNA results. Similarly, in adrenal malignancies, significantly reduced levels of expression of the ACTH receptor gene were found. In the adrenal cancer cell line H295R and in primary cultures from adrenocortical cells, ACTH (1 nM) and forskolin (10 micro M) effectively increased seladin-1/DHCR24 expression, confirming that seladin-1/DHCR24 is modulated by the ACTH/cAMP-driven pathway. In summary, this is the first demonstration that seladin-1/DHCR24 expression is reduced in adrenal cancer, suggesting that it might be viewed as a new potential marker of adrenal malignancies.     

Thrombospondin expression in aldosterone-producing adenomas.

Thrombospondin (TSP) 1 and 2 are extracellular matrix proteins that appear to play a role in cell adhesion and cell migration. It has been demonstrated that the pattern of TSP expression is shifted from TSP1 to TSP2 under adrenocorticotrophic hormone treatment in bovine adrenocortical cells. We investigated the expression in human adrenal tissues by Northern blot analysis and correlated these data with the expression of the adrenocorticotrophic hormone-receptor (ACTH-R). All adrenal tissues (control adrenals, nonfunctional adenomas and ACTH-dependent aldosterone-producing adenomas (APA)) expressed both TSP1 and TSP2 mRNAs. Compared to control adrenals (TSP1 and TSP2 expression = 100 +/- 12%, respectively), TSP1 expression was negatively (51 +/- 10%, p < 0.01) and TSP2 expression was positively (289 +/- 36%, p < 0.01) regulated in APA. No significant differences in TSP1 and TSP2 expressions were found between control adrenals and nonfunctional adenomas. In APA, TSP1 (r = -0.86, p<0.01) and TSP2 (r = 0.88, p < 0.01) expressions correlated closely with the expression of ACTH-R. These results suggest that ACTH activity plays an important role in regulating the expression of TSPs in human adrenal tissues. We speculate that the shift of expression observed in APA may be associated with the phenotype of the tumors.     

ACTH induces up-regulation of ACTH receptor mRNA in mouse and human adrenocortical cell lines

Corticotropin (ACTH) binds to specific receptors in the adrenal cortex and thereby regulates glucocorticoid and mineralocorticoid production. The number of ACTH binding sites on adrenocortical cells is increased by exposure of cells to activators of the cAMP pathway. The mechanism responsible for the increase in ACTH binding sites is not known. We therefore studied the levels of ACTH-R mRNA in mouse Y-l and human NCI-H295 (H295) adrenocortical carcinoma cell lines. ACTH induced an increase in mouse ACTH-R mRNA in Y-l cells that was time and dose dependent, increasing 6-fold over basal levels following exposure to 10⁻⁸ M ACTH for 19–24 h. The amount of human ACTH-R mRNA in H295 cells increased 2–4-fold following a 24 h exposure to 10⁻⁸ M ACTH, 1 mM dbcAMP, or 10⁻⁵ M Forskolin. Treatment of H295 cells with angiotensin II (A-II) was found to dramatically increase the level of ACTH-R mRNA. These data indicate that regulation of ACTH-R mRNA levels is at least one mechanism by which ACTH and A-II elevate the number of ACTH binding sites in the adrenocortical cell.     

Hormonal regulation of messenger ribonucleic acids for P450scc (cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase) in cultured human fetal adrenal cells

ACTH has acute and long term effects on adrenal steroidogenesis by week 14 of fetal life. We used human fetal adrenal cells to investigate the long term effect of physiological doses of ACTH on mRNAs for P450scc (the cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase). Monolayer cultures of 18- to 24-week gestation fetal zone adrenal cells were maintained in the presence and absence of 10(-9) or 10(-8) M ACTH for up to 12 days. As assessed by RNA dot blots probed with cloned homologous human cDNAs, ACTH increased P450scc and P450c17 mRNAs 4- and 9-fold, respectively, over control values on day 7 of culture. ACTH-mediated stimulation was slightly less on day 12 of culture. The ACTH-mediated accumulation of those mRNAs were time dependent. When cells were exposed to a single 10(-8)-M dose of ACTH, the amount of P450scc and P450c17 mRNA was increased by 24 h, reaching a maximum at 48 h and diminishing by 72 h. When cells were maintained in 10(-8) M ACTH continuously, mRNA for both enzymes accumulated in a similar pattern, reaching a peak at 48 h but remaining at nearly maximal values thereafter, up to 96 h. Dibutyryl cAMP (10(-3) M) mimicked these stimulatory actions of ACTH, although its effect was greater at 24 h and more stable up to 96 h. Angiotensin II (1-100 ng/mL) and hCG (1-100 ng/mL) had no effect on accumulation of P450scc and P450c17 mRNAs. The production of both dehydroepiandrosterone sulfate and cortisol also was stimulated by ACTH, suggesting that the increased mRNAs were translated into active enzymes. These results indicate that ACTH induces human fetal adrenal cells to accumulate mRNAs for both P450scc and P450c17; this effect of ACTH is probably mediated by cAMP. Chronic 96-h stimulation of human fetal adrenal cells did not diminish their responsiveness to ACTH. Together with our earlier studies of the human fetal adrenal, these data indicate that fetal adrenal tissue does not exhibit the desensitization to trophic hormone stimulation characteristic of adult tissue.