Steroidogenic differentiation and PKA signaling are programmed by histone methyltransferase EZH2 in the adrenal cortex.

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.

Phospholipase D activity underlies very-low-density lipoprotein (VLDL)-induced aldosterone production in adrenal glomerulosa cells.

Aldosterone is the mineralocorticoid responsible for sodium retention, thus increased blood volume and pressure. Excessive production of aldosterone results in high blood pressure as well as renal disease, stroke, and visual loss via both direct effects and effects on blood pressure. Weight gain is often associated with increased blood pressure, but it remains unclear how obesity increases blood pressure. Obese patients typically have higher lipoprotein levels; moreover, some studies have suggested that aldosterone levels are also elevated and represent a link between obesity and hypertension. Very-low-density lipoprotein (VLDL) functions to transport triglycerides from the liver to peripheral tissues. Although previous studies have demonstrated that VLDL can stimulate aldosterone production, the mechanisms underlying this effect are largely unclear. Here we show for the first time that phospholipase D (PLD) is involved in VLDL-induced aldosterone production in both a human adrenocortical cell line (HAC15) and primary cultures of bovine zona glomerulosa cells. Our data also reveal that PLD mediates steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2) expression via increasing the phosphorylation (activation) of their regulatory transcription factors. Finally, by using selective PLD inhibitors, our studies suggest that both PLD1 and PLD2 isoforms play an important role in VLDL-induced aldosterone production.

Protein kinase C and Src family kinases mediate angiotensin II-induced protein kinase D activation and acute aldosterone production.

Recent evidence has shown a role for the serine/threonine protein kinase D (PKD) in the regulation of acute aldosterone secretion upon angiotensin II (AngII) stimulation. However, the mechanism by which AngII activates PKD remains unclear. In this study, using both pharmacological and molecular approaches, we demonstrate that AngII-induced PKD activation is mediated by protein kinase C (PKC) and Src family kinases in primary bovine adrenal glomerulosa cells and leads to increased aldosterone production. The pan PKC inhibitor Ro 31-8220 and the Src family kinase inhibitors PP2 and Src-1 inhibited both PKD activation and acute aldosterone production. Additionally, like the dominant-negative serine-738/742-to-alanine PKD mutant that cannot be phosphorylated by PKC, the dominant-negative tyrosine-463-to-phenylalanine PKD mutant, which is not phosphorylatable by the Src/Abl pathway, inhibited acute AngII-induced aldosterone production. Taken together, our results demonstrate that AngII activates PKD via a mechanism involving Src family kinases and PKC, to underlie increased aldosterone production.

Chronic hyperaldosteronism in cryptochrome-null mice induces high-salt- and blood pressure-independent kidney damage in mice.

Although aldosterone has an essential role in controlling electrolyte and body fluid homeostasis, aldosterone also exerts certain pathological effects on the kidney. Several previous studies have attempted to examine these deleterious effects. However, the majority of these studies were performed using various injury models, including high-salt treatment and/or mineralocorticoid administration, by which the kidney changes observed were not only due to aldosterone but also due to prior injury caused by salt and hypertension. In the present study, we investigated aldosterone's pathological effect on the kidney using a mouse model with a high level of endogenous aldosterone. We used cryptochrome-null (Cry 1, 2 DKO) mice characterized by high aldosterone levels and low plasma renin activity and observed that even under normal salt exposure conditions, these mice showed increased albumin excretion and kidney tubular injury, decreased nephrin expression and increased reactive oxygen species production in the absence of hypertension. Exposure to high salt levels exacerbated the kidney damage observed in these mice. Moreover, we noted that decreasing blood pressure without blocking aldosterone action did not provide beneficial effects to the kidney in high-salt-treated Cry 1, 2 DKO mice. Thus, our findings support the hypothesis that aldosterone has deleterious effects on the kidney independent of high-salt exposure and high blood pressure.

A role for phospholipase D in angiotensin II-induced protein kinase D activation in adrenal glomerulosa cell models.

The mineralocorticoid aldosterone plays an important role in regulating blood pressure, with excess causing hypertension and exacerbating cardiovascular disease. Previous studies have indicated a role for both phospholipase D (PLD) and protein kinase D (PKD) in angiotensin II (AngII)-regulated aldosterone production in adrenal glomerulosa cells. Therefore, the relationship between AngII-activated PLD and PKD was determined in two glomerulosa cell models, primary bovine zona glomerulosa (ZG) and HAC15 human adrenocortical carcinoma cells, using two inhibitors, 1-butanol and the reported PLD inhibitor, fluoro-2-indolyl des-chlorohalopemide (FIPI). FIPI was first confirmed to decrease PLD activation in response to AngII in the two glomerulosa cell models. Subsequently, it was shown that both 1-butanol and FIPI inhibited AngII-elicited PKD activation and aldosterone production. These results indicate that PKD is downstream of PLD and suggest that PKD is one of the mechanisms through which PLD promotes aldosterone production in response to AngII in adrenal glomerulosa cells.

Adrenocortical zonation in humans under normal and pathological conditions.

CONTEXT: Aldosterone synthase (CYP11B2) and steroid 11 beta-hydroxylase (CYP11B1) catalyze the terminal steps for aldosterone and cortisol syntheses, respectively, thereby determining the functional differentiation of human adrenocortical cells. Little is known, however, about how the cells expressing the enzymes are actually distributed in the adrenals under normal and pathological conditions. OBJECTIVE: The objective of the study was to determine the localization of CYP11B2 and -B1 in human adrenal specimens by using developed antibodies capable of distinguishing the two enzymes from each other. RESULTS: Under normal conditions, CYP11B2 was sporadically detected in the zona glomerulosa, whereas CYP11B1 was entirely detected in the zonae fasciculata-reticularis. Adrenocortical cells lacking both enzymes were observed in the outer cortical regions. In addition to conventional zonation, we found a variegated zonation consisting of a subcapsular cell cluster expressing CYP11B2, which we termed aldosterone-producing cell cluster, and a CYP11B1-expressing area. Aldosterone-producing adenomas differed in cell populations expressing CYP11B2 from one another, whereas CYP11B1-expressing and double-negative cells were also intermingled. Adenomas from patients with Cushing's syndrome expressed CYP11B1 entirely but not CYP11B2, resulting in atrophic nontumor glands. The nontumor portions of both types of adenomas bore frequently one or more aldosterone-producing cell clusters, which sustained CYP11B2 expression markedly under the conditions of the suppressed renin-angiotensin system. CONCLUSION: Immunohistochemistry of the human normal adrenal cortex for CYP11B2 and CYP11B1 revealed a variegated zonation with cell clusters constitutively expressing CYP11B2. This technique may provide a pathological confirmatory diagnosis of adrenocortical adenomas.

Salt-sensitive hypertension in circadian clock-deficient Cry-null mice involves dysregulated adrenal Hsd3b6.

Malfunction of the circadian clock has been linked to the pathogenesis of a variety of diseases. We show that mice lacking the core clock components Cryptochrome-1 (Cry1) and Cryptochrome-2 (Cry2) (Cry-null mice) show salt-sensitive hypertension due to abnormally high synthesis of the mineralocorticoid aldosterone by the adrenal gland. An extensive search for the underlying cause led us to identify type VI 3beta-hydroxyl-steroid dehydrogenase (Hsd3b6) as a new hypertension risk factor in mice. Hsd3b6 is expressed exclusively in aldosterone-producing cells and is under transcriptional control of the circadian clock. In Cry-null mice, Hsd3b6 messenger RNA and protein levels are constitutively high, leading to a marked increase in 3beta-hydroxysteroid dehydrogenase-isomerase (3beta-HSD) enzymatic activity and, as a consequence, enhanced aldosterone production. These data place Hsd3b6 in a pivotal position through which circadian clock malfunction is coupled to the development of hypertension. Translation of these findings to humans will require clinical examination of human HSD3B1 gene, which we found to be functionally similar to mouse Hsd3b6.

Angiotensin II-activated protein kinase D mediates acute aldosterone secretion.

Dysregulation of the renin-angiotensin II (AngII)-aldosterone system can contribute to cardiovascular disease, such that an understanding of this system is critical. Diacylglycerol-sensitive serine/threonine protein kinase D (PKD) is activated by AngII in several systems, including the human adrenocortical carcinoma cell line NCI H295R, where this enzyme enhances chronic (24h) AngII-evoked aldosterone secretion. However, the role of PKD in acute AngII-elicited aldosterone secretion has not been previously examined. In primary cultures of bovine adrenal glomerulosa cells, which secrete detectable quantities of aldosterone in response to secretagogues within minutes, PKD was activated in response to AngII, but not an elevated potassium concentration or adrenocorticotrophic hormone. This activation was time- and dose-dependent and occurred through the AT1, but not the AT2, receptor. Adenovirus-mediated overexpression of constitutively active PKD resulted in enhanced AngII-induced aldosterone secretion; whereas overexpression of a dominant-negative PKD construct decreased AngII-stimulated aldosterone secretion. Thus, we demonstrate for the first time that PKD mediates acute AngII-induced aldosterone secretion.

The role of calcium influx pathways in phospholipase D activation in bovine adrenal glomerulosa cells.

The steroid hormone aldosterone maintains sodium homeostasis and is therefore important in the control of blood volume and pressure. Angiotensin II (AngII) and elevated extracellular potassium concentrations ([K(+)](e)), the prime physiologic regulators of aldosterone secretion from adrenal glomerulosa cells, activate phospholipase D (PLD) in these cells. The role of Ca(2+) in the activation by these agents is unknown, although nitrendipine, a voltage-dependent Ca(2+) channel antagonist, does not inhibit AngII-elicited PLD activation, despite the fact that this compound blocked elevated [K(+)](e)-stimulated PLD activity. PLD activation triggered by AngII was also unaffected by the T-type calcium channel inhibitor nickel. Nevertheless, Ca(2+) influx was required for AngII-induced PLD activation in both primary cultures of bovine adrenal glomerulosa cells and a glomerulosa cell model, the NCI H295R adrenocortical carcinoma cell line. The involvement of store-operated Ca(2+) (SOC) influx and Ca(2+) release-activated Ca(2+) (CRAC) influx pathways in PLD activation was investigated using thapsigargin, an endoplasmic reticulum Ca(2+) pump inhibitor that empties the store to induce SOC influx, and the SOC inhibitor YM-58483 (BTP2), as well as a CRAC inhibitor, tyrphostin A9. In bovine glomerulosa cells, tyrphostin A9 inhibited AngII-induced PLD activation without affecting elevated [K(+)](e)-stimulated enzyme activity. On the other hand, differences were observed between the bovine adrenal glomerulosa and H295R cells in the involvement of Ca(2+) influx pathways in PLD activation, with the involvement of the SOC pathway suggested in the H295R cells. In summary, our results indicate that Ca(2+) entry only through certain Ca(2+) influx pathways is linked to PLD activation.

Characterization and phospholipase D mediation of the angiotensin II priming response in adrenal glomerulosa cells.

Bovine adrenal glomerulosa cells are primed by an initial treatment with angiotensin II (AngII) to respond with enhanced secretion to a second exposure to AngII or agents that increase calcium influx. We hypothesized that the mechanism of priming involves a persistent increase in diacylglycerol (DAG) generated via sustained activity of phospholipase D (PLD). In this report, we sought to define the time frame of this priming response as well as determine its mechanism using assays of aldosterone secretion, PLD activation, and radiolabeled diacylglycerol levels. We found that in primary cultures priming was observed for up to 50 min after AngII washout, suggesting that the priming window is protracted in these cultures relative to freshly isolated cells. The phorbol ester, phorbol 12,13-dibutyrate (PDBu), was used to investigate the role of sustained PLD activation in the persistent DAG and priming responses. PDBu was able to both prime glomerulosa cells to respond with enhanced secretion to AngII and elicit a persistent increase in DAG after PDBu washout. This persistent increase in DAG levels with an initial exposure to PDBu or AngII was not the result of maintained PLD activity after agent removal because PLD activation returned to basal levels by 30 min after washout. Finally, inhibition of PLD signaling during the initial AngII treatment inhibited the subsequent response to AngII or another agent that increases calcium influx. Thus, our results suggest that persistent DAG resulting from PLD signaling mediates the priming response to AngII or PDBu.

Mechanisms of endothelin-1-induced MAP kinase activation in adrenal glomerulosa cells.

G protein-coupled receptors (GPCRs) such as angiotensin II, bradykinin and endothelin-1 (ET-1) are critically involved in the regulation of adrenal function, including aldosterone production from zona glomerulosa cells. Whereas, substantial data are available on the signaling mechanisms of ET-1 in cardiovascular tissues, such information in adrenal glomerulosa cells is lacking. Bovine adrenal glomerulosa (BAG) cells express receptors for endothelin-1 (ET-1) and their stimulation caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), extracellularly regulated signal kinases (ERK1/2), and their dependent proteins, p90 ribosomal S6 kinase (RSK-1) and CREB. ET-1 elicited these responses predominantly through activation of a G(i)-linked cascade with a minor contribution from the G(q)/PKC pathway. Whereas, selective inhibition of EGF-R kinase with AG1478 caused complete inhibition of EGF-induced ERK/RSK-1/CREB activation, it caused only partial reduction (30-40%) of such ET-1-induced responses. Consistent with this, inhibition of matrix metalloproteinases (MMPs) with GM6001 reduced ERK1/2 activation by ET-1, consistent with partial involvement of the MMP-dependent EGF-R activation in this cascade. Activation of ERK/RSK-1/CREB by both ET-1 and EGF was abolished by inhibition of Src, indicating its central role in ET-1 signaling in BAG cells. Moreover, the signaling characteristics of ET-1 in cultured BAG cells closely resembled those observed in clonal adrenocortical H295R cells. The ET-1-induced proliferation of BAG and H295 R cells was much smaller than that induced by Ang II or FGF. These data demonstrate that ET-1 causes ERK/RSK-1/CREB phosphorylation predominantly through activation of G(i) and Src, with a minor contribution from MMP-dependent EGF-R transactivation.

Regulation of aldosterone production from zona glomerulosa cells by ANG II and cAMP: evidence for PKA-independent activation of CaMK by cAMP.

Aldosterone production in zona glomerulosa (ZG) cells of adrenal glands is regulated by various extracellular stimuli (K(+), ANG II, ACTH) that all converge on two major intracellular signaling pathways: an increase in cAMP production and calcium (Ca(2+)) mobilization. However, molecular events downstream of the increase in intracellular cAMP and Ca(2+) content are controversial and far from being completely resolved. Here, we found that Ca(2+)/calmodulin-dependent protein kinases (CaMKs) play a predominant role in the regulation of aldosterone production stimulated by ANG II, ACTH, and cAMP. The specific CaMK inhibitor KN93 strongly reduced ANG II-, ACTH-, and cAMP-stimulated aldosterone production. In in vitro kinase assays and intact cells, we could show that cAMP-induced activation of CaMK, using the adenylate cyclase activator forskolin or the cAMP-analog Sp-5,6-DCI-cBIMPS (cBIMPS), was not mediated by PKA. Activation of the recently identified cAMP target protein Epac (exchange protein directly activated by cAMP) by 8-pCPT-2'-O-Me-cAMP had no effect on CaMK activity and aldosterone production. Furthermore, we provide evidence that cAMP effects in ZG cells do not involve Ca(2+) or MAPK signaling. Our results suggest that ZG cells, in addition to PKA and Epac/Rap proteins, contain other as yet unidentified cAMP mediator(s) involved in regulating CaMK activity and aldosterone secretion.

Ascorbate stimulates monooxygenase-dependent steroidogenesis in adrenal zona glomerulosa.

It is well known that ascorbic acid (Asc) is highly concentrated in the adrenal gland, but its function in the gland is not thoroughly elucidated. We therefore examined the possibility that Asc participates in steroidogenic monooxygenase systems of the adrenal cortex with the aid of the regenerating system including outer mitochondrial membrane cytochrome b (OMb). When Asc availability was limited in rat mutants unable to synthesize Asc, the increase in plasma aldosterone concentration under Na-deficiency was suppressed without effect on plasma corticosterone concentration. Aldosterone formation in the isolated mitochondrial fraction of the zona glomerulosa (zG) of the adrenal cortex was stimulated by the addition of Asc and NADH, while corticosterone formation was not. Consistently zG showed a high level of Asc regeneration activity and was rich in OMb among adrenocortical zones. Taken together, the enhanced aldosterone formation that is catalyzed by one of the steroidogenic monooxygenases, P450aldo, may be supported by Asc with its regenerating system.

Mechanisms of extracellularly regulated kinases 1/2 activation in adrenal glomerulosa cells by lysophosphatidic acid and epidermal growth factor.

The regulation of adrenal function, including aldosterone production from adrenal glomerulosa cells, is dependent on a variety of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In many cell types, GPCR-mediated MAPK activation is mediated through transactivation of RTKs, in particular the epidermal growth factor (EGF) receptor (EGF-R). However, the extent to which this cross-communication between GPCRs and RTKs is operative in the adrenal glomerulosa has not been defined. Bovine adrenal glomerulosa cells express receptors for lysophosphatidic acid (LPA) and EGF. In cultured bovine adrenal glomerulosa cells, LPA, which is predominantly coupled to Gi and partially to Gq/protein kinase C alpha and epsilon, caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), EGF-R, protein kinase B/Akt, extracellularly regulated signal kinases 1/2, and their dependent protein, p90 ribosomal S6 kinase. Overexpression of dominant negative mutants of Ras or EGF-R, and selective inhibition of EGF-R kinase with AG1478, significantly reduced LPA-induced ERK1/2 phosphorylation. However, this was not impaired by inhibition of matrix metalloproteinase (MMP) and heparin-binding EGF. LPA-induced ERK1/2 activation occurs predominantly through EGF-R transactivation by Gi/Src and partly through activation of protein kinase C, which acts downstream of EGF-R and Ras. In contrast, LPA-induced phosphorylation of Shc and ERK1/2 in clonal hepatocytes (C9 cells) was primarily mediated through MMP-dependent transactivation of the EGF-R. These observations in adrenal glomerulosa and hepatic cells demonstrate that LPA phosphorylates ERK1/2 through EGF-R transactivation in a MMP-dependent or -independent manner in individual target cells. This reflects the ability of GPCRs expressed in cell lines and neoplastic cells to utilize distinct signaling pathways that can elicit altered responses compared with those of native tissues.

Zone-specific cell proliferation during adrenocortical regeneration after enucleation in rats.

A quantitative analysis of zone-specific proliferation was done to determine the recovery of adrenal cortical zonation during regeneration after enucleation. Adult male rats underwent adrenal enucleation [unilateral enucleation (ULE)] or sham surgery, both accompanied by contralateral adrenalectomy. At 2, 5, 10, and 28 days, blood and adrenals were collected to assess functional recovery. Adrenal sections were immunostained for Ki67 (proliferation), cytochrome P-450 aldosterone synthase (P-450aldo, glomerulosa), and cytochrome P-450 11beta-hydroxylase (P-45011beta, fasciculata). Unbiased stereology was used to count proliferating glomerulosa and fasciculata cells. Recovery of fasciculata secretory function occurred by 28 days as reflected by plasma ACTH and corticosterone, whereas glomerulosa function reflected by plasma aldosterone remained low at 28 days. At 5 days, ULE adrenals showed increased Ki67+ cells in the glomerulosa and inner fasciculata, whereas at 10 and 28 days increased proliferation was restricted to the outer fasciculata. These data show that enucleation results in transient elevations in glomerulosa and inner fasciculata cell proliferation followed by a delayed increase in the outer fasciculata. To assess adrenal growth in enucleated adrenals previously suppressed by the presence of an intact adrenal, rats underwent ULE and sham surgery; after 4 wk, the intact adrenal was removed and enucleated adrenals were collected at 2, 5, and 10 days. Overall, proliferation was delayed in this model, but at 5 days, Ki67+ cells increased in the outer fasciculata, whereas by 10 days, increased proliferation occurred in the outer and inner fasciculata. The key novel finding of increased proliferation in the inner fasciculata suggests that the delayed growth of the enucleated adrenal results in part from a regenerative response.

Real-time monitoring of the PDE2 activity of live cells: hormone-stimulated cAMP hydrolysis is faster than hormone-stimulated cAMP synthesis.

Cyclic nucleotide phosphodiesterases (PDEs) are the enzymes that catalyze the hydrolysis of cAMP and cGMP, thereby restricting the activity of these second messengers in cells. A unique ability to shape gradients of cyclic nucleotides and compartmentalize their signaling implies a high potency and a rapid action of PDEs. However, it has not been demonstrated how fast PDEs can hydrolyze cAMP in a living system. Here we perform a real-time monitoring of PDE2 activity in aldosterone-producing adrenal cells using a recently developed genetically encoded, fluorescent cAMP sensor, which reveals enormously rapid kinetics of cAMP degradation. Activation of PDE2 results in a rapid decrease of intracellular cAMP from high micromolar to the sub-micromolar range within a few seconds. Moreover, the kinetics of atrial natriuretic peptide-stimulated PDE2 activity (measured as decline of cAMP) are much faster than the speed of ACTH and isoprenaline-induced cAMP-synthesis (measured as cAMP accumulation) in the cells, revealing high catalytic activity and fast action of PDEs in regulating cAMP signaling in a physiological system.

Effects of nitric oxide on aldosterone synthesis and nitric oxide synthase activity in glomerulosa cells from bovine adrenal gland.

This study investigated the effects of two NO-releasing agents, diethylenetriamine-NO (deta-NO) and sodium nitroprusside (SNP), on basal, ACTH-, and angiotensin II (AngII)-stimulated aldosterone production in glomerulosa cells from bovine adrenal gland. NO donors inhibited basal and ACTH- or AngII-stimulated aldosterone synthesis in a concentration-dependent manner. Deta-NO and SNP also provoked a concentration-dependent stimulation of cGMP production. However, cGMP was not responsible for the inhibition of aldosterone secretion, because a cGMP analog did not reproduce the inhibitory effect. Moreover, soluble guanylyl cyclase or protein kinase G inhibitors did not revert the inhibitory effect of NO on aldosterone production. NO donors did not modify ACTH-stimulated cAMP production or AngII-stimulated PLC activity stimulation, but inhibited 22[R] hydroxycholesterol- or pregnenolone-stimulated aldosteronogenesis. NO can be synthesized in bovine glomerulosa cells because nitrite production was determined and characterization of NOS activity was also performed. Nitrite accumulation was not modified in the presence of ACTH, AngII, or other factors used to induce iNOS. NOS activity that showed a Michaelis-Menten kinetic was NADPH- and calcium-dependent and was inhibited by two competitive inhibitors, L-NAME and L-NMMA. These results show that NO inhibits aldosterone production in glomerulosa cells acting on P450scc and other P450-dependent steroidogenic enzymes, and these cells display NOS activity suggesting that NO can be produced by constitutive NOS isozymes.

A role for the NGFI-B family in adrenal zonation and adrenocortical disease.

The three zones of the human adrenal cortex are functionally distinct with the glomerulosa producing aldosterone, the fasciculata producing cortisol, and the reticularis producing DHEA/DHEAS. This functional zonation is largely due to the zone-specific expression of steroidogenic enzymes. Recent evidence suggests a role for the NGFI-B family of orphan nuclear receptors (particularly NURR1 and NGFI-B) in the zone-specific expression of two key steroidogenic enzymes, aldosterone synthase (CYP11B2) and 3beta-hydroxysteroid dehydrogenase (HSD3B2). Herein we discuss the evidence that suggests a role for NURR1 (NR4A2) in the expression of CYP11B2 in the glomerulosa as well as in the dysregulation of CYP11B2 gene expression as is seen in aldosterone-producing adenoma (APA), a major cause of endocrine hypertension. NURR1 appears to be important for CYP11B2 transcription and is found at higher levels in glomerulosa and in APA. Its expression in adrenal cells is also readily increased by angiotensin II treatment. HSD3B2 is a steroid-metabolizing enzyme that is essential for adrenal production of mineralocorticoids and glucocorticoids. Thus, HSD3B2 is expressed at high levels in the glomerulosa and fasciculata where these steroids are produced but at low levels in the adrenal reticularis, which produces mainly DHEA. We recently demonstrated that NGFI-B (nur77 or NR4A1) plays an important role in the regulation of HSD3B2 transcription and may play an important role in the functional zonation of the adrenal gland. Immunohistochemistry confirmed that, within adult and fetal adrenal gland, NGFI-B expression paralleled expression of HSD3B2. Transient transfections demonstrated that NGFI-B family members enhanced HSD3B2 reporter activity but had no effect on a 17alpha-hydroxylase (CYP17) promoter construct. Taken together these results suggest that the NGFI-B family of transcription factors plays a role in establishing the functional zonation of the human adrenal by regulating CYP11B2 and HSD3B2 gene transcription.

Angiotensin II activates cholesterol ester hydrolase in bovine adrenal glomerulosa cells through phosphorylation mediated by p42/p44 mitogen-activated protein kinase.

In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) occurs via activation of the Ca2+ messenger system, increased expression of the steroidogenic acute regulatory protein, and enhanced transfer of cholesterol to the inner mitochondrial membrane. We examined here whether Ang II affects the activity of cholesterol ester hydrolase (CEH), also named hormone-sensitive lipase, the enzyme recruiting cholesterol from intracellular pools, in bovine adrenal glomerulosa cells. In bovine adrenal tissue, CEH activity was detected with characteristics similar to those reported in other tissues (Michaelis constant = 46.3 +/- 6.7 microM, n = 3; maximal velocity = 1 nmol/mg.min). This activity was significantly enhanced in isolated bovine glomerulosa cells challenged for 2 h with 10 nM Ang II (to 149 +/- 11% of controls, n = 3). Similarly, 25 microM forskolin raised CEH activity to 151 +/- 5% of controls (n = 3). This increase in activity of CEH was not due to an increase in the amount of enzyme protein but was associated with an increased phosphorylation of the enzyme to 337 +/- 33% of controls (n = 9, P < 0.0001). Potassium ion (K+) and forskolin also stimulated [32P]orthophosphate incorporation, although to a lesser extent (to 157 +/- 18% and 186 +/- 25% of controls, respectively). On SDS-PAGE, the majority of this radioactivity was associated with a species of 172 kDa, corresponding to a CEH dimer. Both Ang II-induced CEH phosphorylation and pregnenolone production were significantly reduced (to 47 +/- 6% and 50 +/- 8% of controls with Ang II alone, respectively) in the presence of PD098059, an inhibitor of p42/p44 MAPK. Indeed, Ang II challenge led to a rapid 32P incorporation into p42/p44 MAPK. These results demonstrate that, in addition to its known effects on intramitochondrial cholesterol transfer, Ang II also promotes aldosterone biosynthesis by rapidly increasing cholesterol supply to the outer mitochondrial membrane.

Interferon-inducible genes in the rat adrenal gland and vascular smooth muscle cells.

Chronic stimulation of the renin-angiotensin system results in increased zona glomerulosa cells and in cells expressing the final enzyme in the synthesis of aldosterone, the cytochrome P-450 aldosterone synthase. The genes activated during adrenal remodeling are not well defined. We have reported that the expression of interferon-inducible genes, 9-27, 1-8D and 1-8U in H295R cells is stimulated by A-II. The 9-27 gene is expressed mainly in leukocytes and is associated with cell proliferation. In this study, we searched for similar genes in a rat zona glomerulosa cDNA library, and examined the regulation of the expression of these genes. We found the Rat8 gene, which has been reported to be similar to human interferon-inducible genes, as well as two similar genes, No. 10 (1096 bp), and No. 16 (630 bp). Rat8 gene and No. 16 were mainly expressed in zona glomerulosa. The product of No. 10 is thought to be a secreted protein, unlike those of 8 and 16, and its expression in the adrenal was weak in comparison. The control of the expression of rat8 or No. 16 genes differs depending on the tissue. Expression in A10 cells (derived from rat embryo thoracic aorta) was not stimulated by A-II, nor was it influenced by salt intake in the adrenal gland, but it was reduced in vascular smooth muscle cells (VSMC) of rats on a low sodium diet. These results show that genes similar to the human 1-8 gene family are expressed in rat adrenal glomerulosa cells and VSMC, but their expression is not regulated by A-II. The function of these genes in VSMC and adrenal is unknown.