Type beta transforming growth factor is a potent modulator of differentiated adrenocortical cell functions

Whereas TGF-beta exhibited no detectable effect on DNA synthesis, it was found to exert a striking inhibitory effect on the steroidogenic activities of bovine adrenocortical cells in culture. Basal, as well as ACTH- and angiotensin II- activated adrenocortical cortisol productions were inhibited in a time and dose-dependent manner following TGF-beta treatment. Half-maximum inhibition of ACTH- and AII-activated steroidogenesis was observed with TGF-beta concentrations of 0.40 and 0.12 ng/ml, respectively. This effect was half maximal after 6 hours of cell exposure to optimally effective TGF-beta concentrations (1 ng/ml) and reached a plateau after 12-15 hours, resulting in an average 60% inhibition in the steroidogenic response to ACTH and 90% in the case of AII. Supply of different exogenous steroid substrates to support steroidogenesis in adrenocortical cells pointed to a marked loss in steroid-17 alpha hydroxylase activity as a major alteration following TGF-beta treatment. TGF-beta thus appears as a potent modulator of differentiated adrenocortical cell functions in vitro; in this regard it may play a significant role in the development and the regulation of adrenal cortex in vivo.     

Morphologic change and elevation of cortisol secretion in cultured human normal adrenocortical cells caused by mutant p21K-ras protein

In our previous study on the tumorigenesis of human functional adrenal tumors, we observed a high frequency of K-ras point mutations in clinical specimens. Furthermore, we cloned the mutated K-ras gene from the tumors and inserted it into vectors to transfect normal bovine adrenocortical cells to express the mutated K-ras gene. The mRNA level of steroidogenic enzymes such as cholesterol sidechain cleavage enzyme (P450SCC), 17alpha-hydroxylase/17,20-lyase (P450c17), and 3beta-hydroxysteroid dehydrogenase (3betaHSD) in the mutant K-ras stably transfected cells were elevated. Cultured normal adrenocortical cells from donors and patients with adrenocortical tumors were then transfected with mutant K-ras expression plasmids constructed from human adrenal tumors. Stable transfectants grew faster than normal cells. Additionally, morphologic change was observed in the transfected cells. Moreover, when the synthesis of hormones was analyzed, the mRNA of P450SCC, P450C17, and 3betaHSD was found to have increased, and the level of cortisol was 18 to 25 times that in control cells. The increased steroid hormone production in mutant K-ras-transfected cells was reversed by lovastatin, a pharmacologic inhibitor of p21ras function. These results, combined with previous reports of steroidogenic K-ras in bovine adrenocortical cells, suggest that the K-ras oncogene is involved in steroidogenesis in human adrenocortical cells.     

The enhancement of pregnenolone production as the main mechanism of the prolonged stimulatory effect of ACTH on cortisol production by guinea-pig adrenocortical cells

The prolonged stimulatory influence of corticotropin (ACTH) on the adrenocortical steroidogenic response to ACTH was studied in guinea-pig adrenocortical cells harvested from control and ACTH-treated animals (ACTH1-24, 50 micrograms s.c. twice daily on the day preceding the in vitro experiment). The maximal capacity to produce cortisol in response to ACTH (by 10(5) cells and 2 h incubation) was increased from 341.8 +/- 36.3 ng (control group) to 663.3 +/- 37.6 ng for cells obtained from guinea-pigs treated in vivo with ACTH. In the presence of trilostane, added to the cells in order to block the conversion of pregnenolone to cortisol, the net maximal output of pregnenolone and 17-hydroxypregnenolone in response to ACTH was significantly increased in adrenocortical cells from ACTH-treated animals (449.5 +/- 35.8 ng pregnenolone and 85.7 +/- 10.5 ng 17-hydroxypregnenolone vs 269.1 +/- 36.3 ng pregnenolone and 43.7 +/- 8.51 ng 17-hydroxypregnenolone for cells from control guinea-pigs). It appeared therefore that the total production of pregnenolone (as estimated by the sum of pregnenolone and 17-hydroxypregnenolone produced by the cells incubated with trilostane) nearly reached the level of the maximal production of cortisol in response to ACTH and was also significantly enhanced for cells from ACTH-treated animals (532.2 +/- 38.4 ng vs 312.8 +/- 40.0 ng for cells from control group). By contrast, no effect was documented on 17 alpha-hydroxylase activity since 17 alpha-hydroxylation index was similar for both types of adrenocortical cells (16.3 +/- 2.05% for ACTH-treated animals and 14.2 +/- 2.83% for control group). It was concluded therefore that the prolonged stimulatory influence of ACTH on pregnenolone production is the main mechanism of the enhancement of cortisol synthesis by guinea-pig adrenocortical cells previously stimulated by ACTH.     

Purification and properties of steroid 17 alpha-hydroxylase from calf testis

Steroid 17 alpha-hydroxylase has emerged as a key enzyme in steroidogenic cells: (i) it represents the branch point between the 17-deoxy (mineralo) and the 17-hydroxy (gluco) corticosteroid pathways in the adrenal cortex; (ii) the corresponding specific cytochrome (P-450(17 alpha] is highly dependent upon hormonal regulation; and (iii) the enzyme also catalyzes the steroid 17-20 lyase reaction, leading to the major androgens in the testis. As a prerequisite to the study of its regulation in intact cell, 17 alpha-hydroxylase was purified from calf testis microsomal preparations. Following five chromatographic steps, the enzyme was obtained as an apparently homogeneous protein of Mr = 57 kDa upon gel electrophoresis. The procedure yielded a recovery of about 10% as judged by cytochrome P-450 assay. Whereas 17 alpha-hydroxylase specific activity was about 30-fold enriched during the purification, that of the C17-20 lyase was increased by about 6-fold, strongly suggesting that its organelle environment may modulate the enzymatic activity. The purified enzyme yielded a 20 N-terminal amino-acid sequence showing a complete homology with that of its adrenal counterpart and a polyclonal antibody raised against our preparation revealed a 57 kDa protein band in bovine adrenocortical microsomal extracts, upon immunoblotting experiments. It was thus concluded that bovine 17 alpha-hydroxylase activity is supported by highly similar if not identical enzymatic proteins in both testis and adrenal cortex tissues. The purified P-450(17 alpha) preparation is now being used in reconstitution experiments which suggest that microsomal components may contribute to a different expression of the enzyme specificity in its native testis or adrenocortical intracellular environment, respectively.     

Adenovirus-delivered DKK3/WNT4 and Steroidogenesis in Primary Cultures of Adrenocortical Cells.

The Wnt family molecules Dickkopf-3 (DKK3) and WNT4 are present at higher concentrations in the zona glomerulosa than in the rest of the adrenal cortex. In order to study direct effects of these proteins on adrenocortical cell function, we created adenoviruses encoding human DKK3 and WNT4. When added to cultured human adrenocortical cells, DKK3 inhibited aldosterone and cortisol biosynthesis, either alone or together with cyclic AMP. WNT4 increased steroidogenesis when added alone but decreased it in the presence of cyclic AMP. A control adenovirus encoding GFP had no effect. RNA was prepared from cultured cells and was assayed by real-time PCR. CYP11A1 (cholesterol side-chain cleavage enzyme), HSD3B2 (3beta-hydroxysteroid dehydrogenase type II), CYP17 (17alpha-hydroxylase), CYP21 (21-hydroxylase) and CYP11B1 (11beta-hydroxylase) mRNAs were all increased by cyclic AMP, whereas CYP11B2 (aldosterone synthase) was unaffected. DKK3 decreased cyclic AMP-stimulated CYP17. WNT4 increased both CYP17 and CYP21 in the absence of cyclic AMP. Both DKK3 and WNT4 increased the level of CYP11B2. These data show that these Wnt signaling molecules have multiple actions on steroidogenesis in adrenocortical cells, including effects on overall steroidogenesis (aldosterone and cortisol biosynthesis) and distinct effects on steroidogenic enzyme mRNA levels. The co-localization of DKK3 and WNT4 in the glomerulosa and their stimulation of CYP11B2 imply an action on glomerulosa-specific function.     

Regulation of ferredoxin gene in steroidogenic and nonsteroidogenic cells

Ferredoxin is an electron transport intermediate for all the mitochondrial cytochromes P450. It is especially abundant in steroidogenic organs where it functions in steroid biosynthesis. The regulation of ferredoxin gene expression was studied in both steroidogenic and nonsteroidogenic cell lines. In steroidogenic cell line Y1, the expression of ferredoxin was stimulated by cAMP and repressed slightly by angiotensin II and phorbol ester PMA. These drugs exhibited the same effect on the basal promoter of the ferredoxin gene, which includes one TATA box and an SP1 site. In human adrenocortical cell line H295, the stimulation of the ferredoxin gene by cAMP was blocked by cycloheximide, as observed in bovine adrenocortical cell culture. In nonsteroidogenic cell lines such as HeLa and COS-1, the stimulation of ferredoxin gene expression by cAMP was not observed, although basal expression was strong. Transfection studies showed that the ferredoxin promoter could not be stimulated by cAMP in nonsteroidogenic cells. Therefore the steroidogenic cell-specific regulation and the general expression pattern appears to be a property unique to the ferredoxin gene.     

Significantly increased cortisol secretion in normal adrenocortical cells transfected with K-ras mutants derived from human functional adrenocortical tumors

Our previous study showed that the mutation hotspots of the K-ras proto-oncogene in human functional adrenocortical tumors are in codons 15, 16, 18, and 31, thus differing from the sites in other tumors. In addition, analyzing the K-Ras protein by a recombinant DNA technique showed that the activity of endogenic GTPase and the GTPase-activating protein (GAP)-binding ability were significantly decreased in patients with these tumors. The aim of this study was to understand whether those K-ras mutants, which were found only in human adrenocortical tumors, play an important role in these tumors. Thus, the mutant K-ras cDNA was constructed with mammalian expression vectors and transfected into normal adrenocortical cells. The amount of cortisol secreted by the transfected cells was 20 to 30 times that of normal cells. Furthermore, Northern blot analysis revealed that the expression of the three steroidogenesis-related genes P450(scc) (cholesterol side-chain cleavage enzyme), P450(C17) (17alpha-hydroxylase/17, 20-lyase), and P450(C21) (steroid 21-hydroxylase) gene increased in the transfected cells. The K-ras oncogene significantly increases cortisol secretion by normal adrenocortical cells.     

Induction of 17 alpha-hydroxylase (cytochrome P-450(17)alpha) activity by adrenocorticotropin in bovine adrenocortical cells maintained in monolayer culture

Using bovine adrenocortical cells in monolayer culture it has been shown that treatment with adrenocorticotropin (ACTH) causes a dramatic increase in 17 alpha-hydroxylase activity. In postmitochondrial supernatant fractions (PMS) prepared from cells maintained in culture, there was a 15-fold increase in 17 alpha-hydroxylase activity 36 h following initiation of ACTH treatment compared with the activity measured in PMS prepared from control cells. In the continued presence of ACTH, 17 alpha-hydroxylase activity declined; however, even after 60 h of exposure to ACTH, 17 alpha-hydroxylase activity was eight times higher than that present in control cells. The dramatic increase in 17 alpha-hydroxylase activity provides an explanation for the previously observed phenomenon that following initiation of ACTH treatment of bovine adrenocortical cells in monolayer culture there is a shift in the pattern of corticosteroid secretion from approximately equal amounts of cortisol and corticosterone to almost exclusively cortisol. Thus, the modulation of 17 alpha-hydroxylase activity by ACTH action appears to serve a key regulatory role in the pattern of corticosteroid production. Soluble cytosolic factors apparently do not participate in the regulation of 17 alpha-hydroxylase activity in the bovine adrenal cortex. Increases in the magnitude of substrate-induced absorbance changes are indicative that the increase in 17 alpha-hydroxylase activity is due, at least in part, to an elevation of cytochrome P-450(17)alpha synthesis.     

LH action in the Leydig cell: modulation by angiotensin II and corticotropin releasing hormone, and regulation of P450(17) alpha mRNA

Luteinizing hormone is the major regulator of Leydig cell differentiation and steroidogenic function. A number of hormones produced by the Leydig cell (e.g. estrogen, angiotensin, CRF, vasopressin) and the tubular compartment (inhibin, TGF beta), can influence both acute and long-term actions of LH. Conversely, hormones produced in the Leydig cells modulate tubular function (e.g. androgen, beta-endorphin, oxytocin). The LH stimulatory event can be negatively influenced by the action of angiotensin II through the guanyl nucleotide inhibitory unit of adenylate cyclase. We have recently discovered an action of corticotrophin releasing hormone through specific high-affinity low-capacity receptors in the Leydig cells which involves a pertussis toxin insensitive guanyl nucleotide regulatory unit with interaction between signalling pathways and resulting inhibition of LH induced cAMP generation and consequently of steroidogenesis. In contrast to other tissues the CRF receptor in the Leydig cells did not couple to Gs. CRF action is exerted through direct or indirect action of protein kinase C, at the level of the catalytic subunit of adenylate cyclase. Physiological increases in endogenous LH cause positive regulation of membrane receptors and steroidogenesis, while major elevations in circulating gonadotropin can induce down-regulation of LH receptors and desensitization of steroid responses in the adult cell. Gonadotropin-induced desensitization in adult rat tests include an estrogen mediated steroidogenic lesion of the microsomal enzymes 17 alpha-hydroxylase/17,20-desmolase. For further understanding of the regulation of this key enzyme of the androgen pathway the rat P450(17) alpha cDNA was cloned and sequenced. This cDNA expressed in COS-1 cells 17 alpha-hydroxylase/17,20-desmolase activities. From the deduced amino acid sequence, two transmembrane regions were identified, a signal peptide for insertion in the ER, and a 2nd transmembrane region separated from the first by 122 amino acids. The carboxy terminal non-transmembrane region possesses 4 hydrophobic clefts, of which cleft II would contain the putative steroid binding site for both hydroxylase and lyase activities. The rat cDNA was employed to evaluate the hormonal regulation of mRNA levels in adult and fetal Leydig cells. Low dose hCG treatment caused an early increase in mRNA levels followed by a return to control values at later times, while with higher desensitizing doses the initial increase in mRNA was followed by a marked reduction in mRNA at 24 h and a small recovery at 48 h. Fetal rat Leydig cells treated with E2 showed a 70% decrease in P450 mRNA levels, and testosterone production closely followed the changes in mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of phosphatidylinositol turnover by acetylcholine, angiotensin II and ACTH in bovine adrenal fasciculata cells

The effect of acetylcholine, angiotensin II and adrenocorticotropin (ACTH) on phosphatidylinositol (PI) metabolism was examined using bovine adrenocortical fasciculata cell suspensions. The three agents, which acutely stimulate glucocorticoid production by these cells, were all able to increase [32P]Pi incorporation into cellular PI. However, whereas the relative steroidogenic potency (at maximally active concentrations) was ACTH greater than or equal to angiotensin II greater than acetylcholine, the effect on PI labeling was in the order angiotensin II greater than acetylcholine greater than ACTH. The dose-response curves for steroidogenesis and that for PI labeling were superimposable in the case of angiotensin II (ED50 = 1 X 10(-8) M) and of acetylcholine (ED50 = 5 X 10(-7) M), while the two responses were dissociated under graded ACTH challenge. Both steroidogenic response and increased PI labeling elicited by angiotensin II and acetylcholine were respectively inhibited by (Sar1-Ala8)-angiotensin II and muscarinic antagonists. Time-course study showed that in the case of angiotensin II and acetylcholine, the sequence of events was: increased phosphatidic acid labeling, increased PI labeling, activated steroidogenesis. By sharp contrast, under ACTH stimulation, increased steroidogenesis was detected well before activation of PI metabolism. These data suggest that in bovine adrenocortical fasciculata cell, steroidogenesis may be activated by two different pathways. The first one would act mainly through cyclic AMP-dependent intracellular events and is usually accepted in the mechanism of action of ACTH. The other, cyclic AMP-independent pathway, as in the case of angiotensin II and acetylcholine actions, may involve phospholipid-mediated intracellular processes.     

Regulation of steroid hormone biosynthesis enzymes and organic anion transporters by forskolin and DHEA-S treatment in adrenocortical cells

Several important physiological functions are regulated by cortisol. Previously, we demonstrated the involvement of human organic anion transporter 3 (hOAT3) in cortisol release. In the present study, we investigated the influence of dehydroepiandrosterone sulfate (DHEA-S) and estrone sulfate on cortisol release in a human adrenocortical cell line (NCI-H295R) compared with forskolin stimulation. Additionally, we examined the impact of forskolin and DHEA-S on the expression of key enzymes in steroid biosynthesis and expression of hOAT3 and -4 in NCI-H295R cells. The cortisol release was increased 10-fold after 24-h incubation with DHEA-S, but incubation with estrone sulfate did not show any significant change in cortisol release. When cells were incubated with DHEA-S in the presence of forskolin, an additive influence of DHEA-S stimulation of cortisol was recorded over forskolin alone. The 24-h stimulation of NCI-H295R cells with forskolin increased the expression of steroidogenic acute regulatory protein (StAR), CYP17, CYP21A2, and CYP11A1, whereas only StAR mRNA expression was increased significantly by incubation with DHEA-S. Immunofluorescence analyses revealed strongly elevated expression of hOAT3 by forskolin as well as by DHEA-S stimulation. We conclude that the increased cortisol release of adrenocortical cells by DHEA-S and forskolin stimulation is probably due to high expression of the key enzymes of steroid biosynthesis and hOAT3.     

Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries.

To test the hypothesis that the hyperandrogenemia associated with polycystic ovary syndrome (PCOS) results from an intrinsic abnormality in ovarian theca cell steroidogenesis, we examined steroid hormone production, steroidogenic enzyme activity, and mRNA expression in normal and PCOS theca cells propagated in long-term culture. Progesterone (P4), 17alpha-hydroxyprogesterone (17OHP4), and testosterone (T) production per cell were markedly increased in PCOS theca cell cultures. Moreover, basal and forskolin-stimulated pregnenolone, P4, and dehydroepiandrosterone metabolism were increased dramatically in PCOS theca cells. PCOS theca cells were capable of substantial metabolism of precursors into T, reflecting expression of an androgenic 17beta-hydroxysteroid dehydrogenase. Forskolin-stimulated cholesterol side chain cleavage enzyme (CYP11A) and 17alpha-hydroxylase/17,20-desmolase (CYP17) expression were augmented in PCOS theca cells compared with normal cells, whereas no differences were found in steroidogenic acute regulatory protein mRNA expression. Collectively, these observations establish that increased CYP11A and CYP17 mRNA expression, as well as increased CYP17, 3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase enzyme activity per theca cell, and consequently increased production of P4, 17OHP4, and T, are stable properties of PCOS theca cells. These findings are consistent with the notion that there is an intrinsic alteration in the steroidogenic activity of PCOS thecal cells that encompasses multiple steps in the biosynthetic pathway.     

Corticotropin-induced changes in enzymatic activities of the post-pregnenolone steroidogenic pathway in rabbit adrenocortical cells

In an attempt to delineate the effect of corticotropin (ACTH) on post-pregnenolone steroidogenesis, the activity of enzymatic systems operative in conversion of pregnenolone into glucocorticoids and androgens was studied in adrenocortical cells from control rabbits and from animals treated with ACTH for 12 days (ACTH 1-24, 200 micrograms s.c. daily). The cells from ACTH-treated rabbits exhibited an increased overall steroidogenic capacity and produced much more cortisol (P less than 0.0005) as well as other 17-hydroxylated steroids as a result of increased activity of 17 alpha-hydroxylase; corticosterone generation was concomitantly reduced. The increased conversion of pregnenolone or progesterone into androgens, as a result of previous treatment with ACTH, provides additional evidence for an effect of ACTH on 17 alpha-hydroxylase activity. A stimulatory effect of ACTH on 11 beta-hydroxylase was also evidenced by these cells, since conversion of 11-deoxycortisol into cortisol was enhanced (P less than 0.005). The increased production of androgens from 17-hydroxylated precursors by cells from ACTH-treated rabbits suggests that ACTH also exerts a prolonged stimulatory effect on 17,20-lyase. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was apparently not influenced by chronic treatment with ACTH, judged from unchanged conversion of dehydroepiandrosterone into androstenedione. The activity of 11 beta-dehydrogenase was likewise unchanged in these conditions.     

Enhanced 17 alpha-hydroxylation of pregnenolone and increased androgen production by rabbit adrenocortical cells stimulated chronically with corticotropin

The postulated chronic stimulatory effect of corticotropin (ACTH) on pregnenolone production and on 17 alpha-hydroxylase activity was evaluated on adrenocortical cells obtained from control and chronically ACTH-treated rabbits. The cells were incubated with various concentrations of ACTH added alone or together with trilostane, so as to inhibit further conversion of pregnenolone and dehydroepiandrosterone. The maximal steroidogenic effect of ACTH (determined in the absence of trilostane) was increased 2-fold in adrenocortical cells from ACTH-treated animals; furthermore, cortisol production was increased whereas that of corticosterone decreased. While the generation of pregnenolone was of comparable magnitude for cells from both experimental groups, chronic in vivo treatment with ACTH was followed by a 40-fold enhancement in 17-hydroxypregnenolone production. Concomitantly, maximal DHEA production documented in the presence of ACTH and trilostane was enhanced more than 200-fold, from 0.45 +/- 0.20 pmol in control rabbits to 147 +/- 67 pmol in cells from ACTH-treated animals. The corresponding values of DHEA-sulphate production were 0.86 +/- 0.12 and 432 +/- 334 pmol, respectively. Thus, a prolonged stimulatory effect of ACTH on rabbit adrenocortical cells consists in an enhancement of the capacity to generate pregnenolone, and to convert this compound into 17-hydroxylated steroids.     

Differentiation of human adipose tissue–derived mesenchymal stromal cells into steroidogenic cells by adenovirus-mediated overexpression of NR5A1 and implantation into adrenal insufficient mice

Background aimsCell therapy for adrenal insufficiency is a potential method for physiological glucocorticoid and mineralocorticoid replacement. We have previously shown that mouse mesenchymal stromal cells (MSCs) differentiated into steroidogenic cells by the viral vector–mediated overexpression of nuclear receptor subfamily 5 group A member 1 (NR5A1), an essential regulator of steroidogenesis, and their implantation extended the survival of bilateral adrenalectomized (bADX) mice.MethodsIn this study, we examined the capability of NR5A1-induced steroidogenic cells prepared from human adipose tissue-derived MSCs (MSC [AT]) and the therapeutic effect of the implantation of human NR5A1-induced steroidogenic cells into immunodeficient bADX mice.ResultsHuman NR5A1-induced steroidogenic cells secreted adrenal and gonadal steroids and exhibited responsiveness to adrenocorticotropic hormone and angiotensin II in vitro. In vivo, the survival time of bADX mice implanted with NR5A1-induced steroidogenic cells was significantly prolonged compared with that of bADX mice implanted with control MSC (AT). Serum cortisol levels, which indicate hormone secretion from the graft, were detected in bADX mice implanted with steroidogenic cells.ConclusionsThis is the first report to demonstrate steroid replacement by the implantation of steroid-producing cells derived from human MSC (AT). These results indicate the potential of human MSC (AT) to be a source of steroid hormone-producing cells.