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.

[Construction of transgenic mice with specific Cre recombinase expression in the zona fasciculata in adrenal cortex].

The adrenal gland is an important endocrine organ of human body. CYP11B1 gene was specifically expressed in the zona fasciculata in adrenal cortex. In order to better study the function of genes specifically expressed in the zona fasciculata in adrenal cortex, the mice with Cre recombinase specifically expressed in the zona fasciculata in adrenal cortex were constructed. It was then confirmed that CYP11B1 was specifically expressed in adrenal glands. Then, using CRISPR/Cas9 technique, CYP11B1-2A-GfpCre recombinant vector was constructed and subsequently injected into the fertilized eggs of mice. It was confirmed that the Cre gene was mainly expressed in the zona fasciculata in adrenal cortex of CYP11B1Cre mice by using mTmG and LacZ staining. The CYP11B1Cre mice were then mated with cystathionine γ-lyase (CTH)f/f mice, thereby generating CTHf/f/CYP11B1Cre mice. It was also confirmed that CTH gene in the zona fasciculata in adrenal cortex was specifically knocked out in these mice. These results suggest that transgenic mice with specific Cre recombinase expression in the zona fasciculata in adrenal cortex were constructed successfully. This animal model can be a powerful tool for the study of the function of genes expressed in the zona fasciculata in adrenal cortex.

Role of Ad4-binding protein/steroidogenic factor 1 in regulating NADPH production in adrenocortical Y-1 cells.

Ad4-binding protein/steroidogenic factor 1 (Ad4BP/SF-1), a member of the nuclear receptor superfamily, is expressed in steroidogenic cells and regulates all steroidogenic gene expression. We recently employed mRNA and chromatin immunoprecipitation sequence (ChIP-seq) to demonstrate that Ad4BP/SF-1 directly regulates the expression of nearly all glycolytic genes. The pentose phosphate pathway (PPP) contributes to the production of nicotinamide adenine dinucleotide phosphate (NADPH). Although the expression of PPP genes and intracellular NADPH were decreased by Ad4BP/SF-1 knockdown, these genes were not the direct targets of Ad4BP/SF-1. This study therefore investigates whether Ad4BP/SF-1 directly regulates genes implicated in NADPH production. Examination of previously published data sets of mRNA sequence (mRNA-seq) and ChIP-seq strongly suggested a possibility that other NADPH-producing genes, such as malic enzyme 1 (Me1) and methylenetetrahydrofolate dehydrogenase 2 (Mthfd2), are the direct targets of Ad4BP/SF-1. Reporter gene assays and determination of intracellular NADPH concentration supported the notion that Ad4BP/SF-1 regulates NADPH production by regulating these genes. NADPH is required for macromolecule synthesis of compounds such as steroids, and for detoxification of reactive oxygen species. When synthesizing steroid hormones, steroidogenic cells consume NADPH through enzymatic reactions mediated by steroidogenic P450s. NADPH is also consumed through elimination of reactive oxygen species produced as the byproducts of the P450 reactions. Overall, Ad4BP/SF-1 potentially maintains the intracellular NADPH level through cooperative regulation of genes involved in the biological processes for consumption and supply.

Suppressed Sex Hormone Biosynthesis by Alkylresorcinols: A Possible Link to Chemoprevention.

Alkylresorcinols (ARs, 5-n-alkylresorcinols) are amphiphilic phenolic lipids in whole grain rye and wheat, with a long odd-numbered carbon chain. A preventive effect of whole grain diet on sex hormone-dependent cancers has been recognized, but the active component(s) or mechanisms are not known. We have investigated the effects of the ARs C15:0, C19:0, and C21:0, individually and in combination, on steroid hormone production by using the human adrenocortical cell line H295R. Decreased synthesis of dehydroepiandrosterone (DHEA), testosterone, and estradiol was demonstrated at low concentrations of C15:0 and C19:0. There were no indications of additive effects on steroid secretion from the combined treatment with equimolar concentrations of the three ARs. Gene expressions of CYP21A2, HSD3B2, and CYP19A1 were downregulated and CYP11A1 was upregulated by the ARs. The results on gene expression could not explain the effects on steroidogenesis, which may be due to direct effects on enzyme activities, such as inhibition of CYP17A1. Our results demonstrate suppressed synthesis of testosterone and estradiol by ARs suggesting a novel mechanism for ARs in the chemoprevention of prostate and breast cancer.

Histomorphometrical and electron microscopic study of adrenocorticocytes following surgically induced extrahepatic biliary obstruction in adult female albino rats.

Cholestasis, which is a component of many liver diseases, is often associated with symptoms that resemble clinical adrenal insufficiency. This work aimed to study the histomorphometrical and electron microscopic structure of adrenocorticocytes after surgical induction of bile duct resection (BDR) in adult female albino rats. Sixty rats were randomly divided into control, BDR and sham-operated groups. Six weeks after surgery, the blood serum of the rats was examined biochemically, and the suprarenal cortexes were prepared for histological, morphometrical and statistical studies. The BDR group showed a highly significant increase in bilirubin and serum alkaline phosphatase levels, whereas aldosterone and cortisol levels were highly significantly decreased. The area percentages of positive immunoreactions for P53, cyclooxygenase II (COX-II) and inducible nitric oxide synthase (INOS) revealed highly significant increases in the BDR group. Electron microscopic examination of the BDR group showed marked cytoplasmic vacuolations, large lipid droplets, swollen mitochondria and many small dark nuclei in the adrenocorticocytes. The zona fasciculata had heterogeneously electron-dense mitochondria and dilated smooth endoplasmic reticulum. Some of the zona reticularis cells contained lipofuscin pigments. The surgical induction of BDR produced deleterious effects on the structure and function of the adrenocorticocytes. A long-term study using different animal species is recommended for further examination.

[Hormone-sensitive lipase/cholesteryl esterase from the adrenal cortex - structure, regulation and role in steroid hormone synthesis]. / Hormonozalezna lipaza/esteraza cholesterolowa z kory nadnerczy - struktura, regulacja i rola w syntezie hormonów steroidowych.

Hormone-sensitive lipase/cholesteryl esterase (HSL), encoded by LIPE gene, plays a very important role in the metabolism of acylglycerols in the adipose tissue and cholesteryl esters in the adrenal cortex, gonads and placenta. Isoforms of this enzyme supply fatty acids, important energy substrates, and free cholesterol required for steroid hormone synthesis. Recent discoveries on hormonal regulation of HSL synthesis with special emphasis given to the regulation of LIPE gene expression, its tissue-specific promoters and activation of the gene products by phosphorylation, as well as the role of HSL in the metabolism of cholesteryl esters were reviewed. In the concluding remarks, the gaps in our knowledge of the metabolism of acylglyceroles and cholesteryl esters, as well as the possibility of effects, synergic with HSL, influencing metabolism of these compounds were discussed.

Effect of ions of potassium and lithium on NO synthase expression in the human adrenal cortex.

The expression of endothelial and inducible NO synthase in the human adrenal glands was studied under a change in the concentration of K(+), which plays a regulatory role in aldosterone secretion. K(+) ions stimulated the expression of both isoforms of NO synthase in the human adrenal cortex. A stimulatory effect of K(+) on NO synthase is probably related to activation of the calmodulin system and potassium-induced translocation of protein kinase C. Lithium produced n inhibitory effect on both isoforms of NO synthase, which suggests that protein kinase C serves a major regulator of expression in the human adrenal glands.

The relevance of chemical interactions with CYP17 enzyme activity: assessment using a novel in vitro assay.

The steroidogenic cytochrome P450 17 (CYP17) enzyme produces dehydroepiandrosterone (DHEA), which is the most abundant circulating endogenous sex steroid precursor. DHEA plays a key role in e.g. sexual functioning and development. To date, no rapid screening assay for effects on CYP17 is available. In this study, a novel assay using porcine adrenal cortex microsomes (PACMs) was described. Effects of twenty-eight suggested endocrine disrupting compounds (EDCs) on CYP17 activity were compared with effects in the US EPA validated H295R (human adrenocorticocarcinoma cell line) steroidogenesis assay. In the PACM assay DHEA production was higher compared with the H295R assay (4.4 versus 2.2nmol/h/mg protein). To determine the additional value of a CYP17 assay, all compounds were also tested for interaction with CYP19 (aromatase) using human placental microsomes (HPMs) and H295R cells. 62.5% of the compounds showed enzyme inhibition in at least one of the microsomal assays. Only the cAMP inducer forskolin induced CYP17 activity, while CYP19 was induced by four test compounds in the H295R assay. These effects remained unnoticed in the PACM and HPM assays. Diethylstilbestrol and tetrabromobisphenol A inhibited CYP17 but not CYP19 activity, indicating different mechanisms for the inhibition of these enzymes. From our results it becomes apparent that CYP17 can be a target for EDCs and that this interaction differs from interactions with CYP19. Our data strongly suggest that research attention should focus on validating a specific assay for CYP17 activity, such as the PACM assay, that can be included in the EDC screening battery.

Androgen synthesis in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

A hallmark of severe congenital adrenal hyperplasia due to 21-hydroxylase deficiency is pre- and postnatal virilization. The most characteristic biochemical abnormality is the elevation of 17α-hydroxyprogesterone, which is metabolized to the most potent androgen receptor agonist dihydrotestosterone. 17α-Hydroxyprogesterone can be metabolized to dihydrotestosterone via 4-androstenedione through the classical Δ4-pathway or via 17α-hydroxypregnenolone and dehydroepiandrosterone through the classical Δ5-pathway, as well as through an alternative route, called the 'backdoor pathway', that bypasses dehydroepiandrosterone, 4-androstenedione, and testosterone as intermediates. This review article will summarize recent advances in the understanding of the activities of androgen synthesis pathways in patients with 21-hydroxylase deficiency obtained by urinary steroid metabolomics based on gas chromatography-mass spectrometry. Compared with healthy controls, the relative activities of the backdoor and Δ4-pathways increase in patients with congenital adrenal hyperplasia during neonatal age and infancy, whereas the activity of the Δ5-pathway remains unchanged. Thereafter, the activity of the Δ5-pathway dominates, whereas a decreasing 5α-reductase activity leads to a diminished role of the backdoor pathway for androgenic steroid production. Beside the backdoor pathway, the Δ4-pathway seems to be responsible for increased androgen generation in patients with 21-hydroxylase deficiency before the onset of adrenarche, whereas the Δ5-pathway might contribute to the increased androgen formation in those patients only after the onset of adrenarche.

Control of CYP11B2/CYP11B1 expression ratio and consequences for the zonation of the adrenal cortex.

Access of corticotropin to glucocorticoid synthesis in adrenocortical cells is provided by the expression of the ACTH receptor (MC2R). Activation of the MC2R increases stimulatory G-protein, adenylyl cyclase, and protein kinase A (PKA) activities. Furthermore, PKA phosphorylates transcription factors that have a stimulating effect on glucocorticoid synthesis. Sensitivity of adrenocortical cells to renin/angiotensin-2 is conferred by the expression of the inhibitory G-protein-linked angiotensin-2 type 1 receptor (AT1R) that additionally associates to the phospholipase C-activating G-protein q. The AT1R is connected to the adrenal potassium sensory system and regulates calcium influx as well as phospholipase C-ß (PLC-ß) and thus calmodulin kinase-dependent transcription of steroidogenic enzymes. While AT1R signaling suppresses the influence of corticotropin on the generation of cyclic adenosine monophosphate, the expression of the AT1R and its associated enzyme activities are under the control of glucocorticoids. Thus, dominance of one of the two signaling pathways is dependent on two factors: the extracellular concentration of their ligands and the products of their signaling pathways. These findings are in favor of the hypothesis that the centripetal blood flow through the adrenal gland builds up a glucocorticoid gradient creating a morphogenetic field along which adrenal cortical cells adopt different functional states, leading to the typical zonation of the adrenal cortex.

ACAT-selective and nonselective DGAT1 inhibition: adrenocortical effects--a cross-species comparison.

Acyl-coenzyme A: cholesterol O-Acyltransferase (ACAT) and Acyl-coenzyme A: diacylglycerol O-acyltransferase (DGAT) enzymes play important roles in synthesizing neutral lipids, and inhibitors of these enzymes have been investigated as potential treatments for diabetes and other metabolic diseases. Administration of a Acyl-coenzyme A: diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor with very limited cellular selectivity over ACAT resulted in significant adrenocortical degenerative changes in dogs. These changes included macrosteatotic vacuolation associated with adrenocyte cell death in the zonae glomerulosa and fasciculata and minimal to substantial mixed inflammatory cell infiltration and were similar to those described previously for some ACAT inhibitors in dogs. In the mouse, similar but only transient adrenocortical degenerative changes were seen as well as a distinctive nondegenerative reduction in cortical fine vacuolation. In the marmoset, only the distinctive nondegenerative reduction in cortical fine vacuolation was observed, suggesting that the dog, followed by the mouse, is the most sensitive species for cortical degeneration. Biochemical analysis of adrenal cholesterol and cholesteryl ester indicated that the distinctive reduction in cortical fine vacuolation correlated with a significant reduction in cholesteryl ester in the mouse and marmoset, whereas no significant reduction in cholestryl ester, but an increase in free cholesterol was observed in dogs. Administration of a DGAT1 inhibitor with markedly improved selectivity over ACAT to the marmoset and the mouse resulted in no adrenal pathology at exposures sufficient to cause substantial DGAT1 but not ACAT inhibition, thereby implicating ACAT rather than DGAT1 inhibition as the probable cause of the observed adrenal changes. Recognizing that the distinctive nondegenerative reduction in cortical fine vacuolation in the mouse could be used as a histopathological biomarker for an in vivo model of the more severe changes observed in dogs, the mouse has subsequently been used as a model to select DGAT1 inhibitors free of adrenocortical toxicity.

Adiponectin (15-36) stimulates steroidogenic acute regulatory (StAR) protein expression and cortisol production in human adrenocortical cells: role of AMPK and MAPK kinase pathways.

Adiponectin is an abundantly circulating adipokine, orchestrating its effects through two 7-transmembrane receptors (AdipoR1 and AdipoR2). Steroidogenesis is regulated by a variety of neuropeptides and adipokines. Earlier studies have reported adipokine mediated steroid production. A key rate-limiting step in steroidogenesis is cholesterol transportation across the mitochondrial membrane by steroidogenic acute regulatory protein (StAR). Several signalling pathways regulate StAR expression. The actions of adiponectin and its role in human adrenocortical steroid biosynthesis are not fully understood. The aim of this study was to investigate the effects of adiponectin on StAR protein expression, steroidogenic genes, and cortisol production and to dissect the signalling cascades involved in the activation of StAR expression. Using qRT-PCR, Western blot analysis and ELISA, we have demonstrated that stimulation of human adrenocortical H295R cells with adiponectin results in increased cortisol secretion. This effect is accompanied by increased expression of key steroidogenic pathway genes including StAR protein expression via ERK1/2 and AMPK-dependent pathways. This has implications for our understanding of adiponectin receptor activation and peripheral steroidogenesis. Finally, our study aims to emphasise the key role of adipokines in the integration of metabolic activity and energy balance partly via the regulation of adrenal steroid production. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Very-low-density lipoprotein mediates transcriptional regulation of aldosterone synthase in human adrenocortical cells through multiple signaling pathways.

Diabetic dyslipidemia is characterized by increased circulatory very-low-density lipoprotein (VLDL) levels. Aldosterone, apart from its role in fluid and electrolyte homeostasis, has also been implicated in insulin resistance and myocardial fibrosis. The impact of VLDL as a potential risk factor for aldosterone-mediated cardiovascular injury in diabetes mellitus, however, remains to be investigated. We have therefore studied native and modified VLDL-mediated steroidogenesis and its underlying molecular mechanisms in human adrenocortical carcinoma cells, NCI H295R. Native VLDL (natVLDL), isolated from healthy volunteers, was subjected to in vitro modification with glucose (200 mmol/l) or sodium hypochlorite (1.5 mmol/l) for preparation of glycoxidized and oxidized VLDL, respectively. VLDL treatment induced steroidogenesis in both a concentration- and time-dependent manner. Native and glycoxidized VLDL (50 µg/ml) were almost two-fold more potent in adrenocortical aldosterone release than angiotensin II (100 nmol/l). These forms of VLDL significantly augmented transcriptional regulation of aldosterone synthase (Cyp11B2), partially through scavenger receptor class B type I, as evident from the effect of BLT-1. In contrast to glycoxidized VLDL, oxidized VLDL significantly attenuated the stimulatory effect of natVLDL on adrenocortical hormone synthesis. Moreover, treatment with specific pharmacological inhibitors (H89, U0126, AG490) provided supporting evidence that VLDL, irrespective of modification, presumably recruited PKA, ERK1/2 and Jak-2 for steroid hormone release through modulation of Cyp11B2 mRNA level. In conclusion, this study demonstrates a novel insight into intracellular mechanism of VLDL-mediated aldosterone synthesis through transcriptional regulation of steroidogenic acute regulatory protein (StAR) and Cyp11B2 expression in human adrenocortical carcinoma cell line.

Comparison of aldosterone production among human adrenocortical cell lines.

Several human adrenocortical cell lines have been used as model systems for aldosterone production. However, these cell lines have not been directly compared with each other. Human adrenal cell lines SW13, CAR47, the NCI-H295 and its sub-strains and sub-clones were compared with regard to aldosterone production and aldosterone synthase (CYP11B2) expression. Culture media was collected 48 h after incubation, aldosterone secretion was measured and the data were normalized to the amount of cell protein. RNA was isolated for microarray analysis and quantitative RT-PCR (qPCR). The cell lines with the highest aldosterone production were further tested with regard to angiotensin II (Ang II) stimulation. Neither aldosterone nor CYP11B2 transcript were detected in SW13 or CAR47 cells. The aldosterone production by the NCI-H295, H295A, H295R-S1, H295R-S2, H295R-S3, HAC13, HAC15 and HAC50 were 119, 1, 6, 826, 18, 139, 412, and 1 334 (pmol/mg protein/48 h), respectively. H295A and H295R-S1 expressed less CYP11B2 than the commonly used H295R-S3 cells; while NCI-H295, H295R-S2, HAC13, HAC15 and HAC50 expressed 24-, 14-, 3-, 10-, and 35-fold higher CYP11B2 compared with the H295R-S3 cells. When treated with Ang II, NCI-H295, H295R-S2, HAC13, HAC15 and HAC50 showed significantly higher aldosterone production than the basal level (p<0.05). A comparison of the available human adrenal cell lines indicates that the H295R-S2 and the clonal cell lines, HAC13, HAC15 and HAC50 produced the highest levels of aldosterone and responded well to Ang II.

Mechanism of intermolecular interactions of microsomal cytochrome P450s CYP17 and CYP21 involved in steroid hormone biosynthesis.

Protein-protein interactions play a significant role in regulation of functional activity of cytochrome P450s. The aim of the present study was to elucidate the molecular interactions between steroidogenic enzymes CYP17 and CYP21 localized in endoplasmic reticulum membranes of adrenal cortex and involved in biosynthesis of corticosteroid hormones. In the present work, we demonstrate for the first time the direct interaction at molecular level between highly purified human recombinant cytochrome P450s in a mixed reconstituted system. The dependence of the interaction between CYP17 and CYP21 on concentration of the redox-partner - NADPH-cytochrome P450 reductase - is demonstrated, and it is shown that electrostatic interactions play a crucial role in the interaction between CYP17 and CYP21.

The high-affinity cAMP-specific phosphodiesterase 8B controls steroidogenesis in the mouse adrenal gland.

The functions of the phosphodiesterase 8B (PDE8) family of phosphodiesterases have been largely unexplored because of the unavailability of selective pharmacological inhibitors. Here, we report a novel function of PDE8B as a major regulator of adrenal steroidogenesis using a genetically ablated PDE8B mouse model as well as cell lines treated with either a new PDE8-selective inhibitor or a short hairpin RNA (shRNA) construct against PDE8B. We demonstrate that PDE8B is highly enriched in mouse adrenal fasciculata cells, and show that PDE8B knockout mice have elevated urinary corticosterone as a result of adrenal hypersensitivity toward adrenocorticotropin. Likewise, ablation of PDE8B mRNA transcripts by an shRNA construct potentiates steroidogenesis in the commonly used Y-1 adrenal cell line. We also observed that the PDE8-selective inhibitor (PF-04957325) potentiates adrenocorticotropin stimulation of steroidogenesis by increasing cAMP-dependent protein kinase activity in both primary isolated adrenocortical cells and Y-1 cells. It is noteworthy that PDE8s have their greatest control under low adrenocorticotropin-stimulated conditions, whereas other higher K(m) PDE(s) modulate steroidogenesis more effectively when cells are fully stimulated. Finally, both genetic ablation of PDE8B and long-term pharmacological inhibition of PDE8s cause increased expression of steroidogenic enzymes. We conclude that PDE8B is a major regulator of one or more pools of cAMP that promote steroidogenesis via both short- and long-term mechanisms. These findings further suggest PDE8B as a potential therapeutic target for the treatment of several different adrenal diseases.

The expression of ghrelin O-acyltransferase (GOAT) in human tissues.

Ghrelin is a circulating growth hormone-releasing and appetite-inducing brain-gut peptide. It needs to be acylated on its serine-3 with octanoate for its endocrine actions. The acyl-transferase that catalyses ghrelin octanoylation has recently been identified and named as GOAT (ghrelin O-acyltransferase); GOAT enzyme is coded by the MBOAT4 gene. This study aimed to investigate GOAT expression in the human. The distribution of GOAT mRNA expression was studied in various human tissues using classical and real-time reverse transcription and polymerase chain reaction. GOAT expression was found in all tissues studied (stomach, adrenal cortex, breast, right and left colon, duodenum, jejunum, ileum, fat, Fallopian tube, gallbladder, lymph node, lymphocyte cell line, kidney, liver, lung, muscle, myocardium, pituitary, oesophagus, pancreas, ovary, placenta, prostate, testis, spleen and thyroid). The widespread expression of GOAT corresponds to the widespread distribution of ghrelin expression. GOAT expression was high in stomach and gut, the major ghrelin-secreting tissues, and in the pituitary, in which ghrelin is known to show autocrine and paracrine effects. Identification of GOAT expression in various tissues support the concept that in addition to the important endocrine effect of acylated ghrelin, the paracrine effects of locally synthetised and acylated ghrelin may be important.

A phosphodiesterase 11 (Pde11a) knockout mouse expressed functional but reduced Pde11a: Phenotype and impact on adrenocortical function.

Phosphodiesterases catalyze the hydrolysis of cyclic nucleotides and maintain physiologic levels of intracellular concentrations of cyclic adenosine and guanosine mono-phosphate (cAMP and cGMP, respectively). Increased cAMP signaling has been associated with adrenocortical tumors and Cushing syndrome. Genetic defects in phosphodiesterase 11A (PDE11A) may lead to increased cAMP signaling and have been found to predispose to the development of adrenocortical, prostate, and testicular tumors. A previously reported Pde11a knockout (Pde11a-/-) mouse line was studied and found to express PDE11A mRNA and protein still, albeit at reduced levels; functional studies in various tissues showed increased cAMP levels and reduced PDE11A activity. Since patients with PDE11A defects and Cushing syndrome have PDE11A haploinsufficiency, it was particularly pertinent to study this hypomorphic mouse line. Indeed, Pde11a-/- mice failed to suppress corticosterone secretion in response to low dose dexamethasone, and in addition exhibited adrenal subcapsular hyperplasia with predominant fetal-like features in the inner adrenal cortex, mimicking other mouse models of increased cAMP signaling in the adrenal cortex. We conclude that a previously reported Pde11a-/- mouse showed continuing expression and function of PDE11A in most tissues. Nevertheless, Pde11a partial inactivation in mice led to an adrenocortical phenotype that was consistent with what we see in patients with PDE11A haploinsufficiency.

The cAMP-responsive element binding protein (CREB) regulates the expression of acid ceramidase (ASAH1) in H295R human adrenocortical cells.

Acid ceramidase (encoded by ASAH1) is a lipid hydrolase that catalyzes the conversion of ceramide (cer) into sphingosine (SPH) and a free fatty acid. Adrenocortical steroidogenesis is regulated by the trophic peptide hormone adrenocorticotropin (ACTH), which induces the expression of steroidogenic genes in the human adrenal cortex primarily via a cAMP/protein kinase A (PKA)-dependent pathway. ACTH also stimulates sphingolipid metabolism in H295R adrenocortical cells leading to changes in steroidogenic gene expression. Based on our previous data identifying SPH as an antagonist for the nuclear receptor steroidogenic factor 1 (SF-1) and the role of ACTH-stimulated changes in sphingolipid metabolism on steroidogenic gene transcription, the aim of the current study was to determine the role of ACTH signaling in regulating the expression of the ASAH1 gene in H295R cells. We show that activation of the ACTH signaling pathway induces ASAH1 gene expression by stimulating the binding of the cAMP-responsive element binding protein (CREB) to multiple regions of the ASAH1 promoter. CREB binding promotes the recruitment of the coactivators CREB binding protein (CBP) and p300 to the CREB-responsive regions of the promoter. Consistent with transcriptional activation, we show that cAMP signaling increases the trimethylation of Lys 4 on histone H3 (H3K4) along the ASAH1 promoter. Finally, RNA interference (RNAi) experiments demonstrate that CREB is indispensable for cAMP-induced ASAH1 transcription. These data identify the ACTH/cAMP signaling pathway and CREB as transcriptional regulators of the ASAH1 gene in the human adrenal cortex.

Optimizing the conditions of a multiple reaction monitoring assay for membrane proteins: quantification of cytochrome P450 11A1 and adrenodoxin reductase in bovine adrenal cortex and retina.

Approximately 30% of naturally occurring proteins are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is traditionally understudied due to limitations of the available analytical tools. To facilitate the analysis of membrane proteins, the analytical methods for their soluble counterparts must be optimized or modified. Multiple reaction monitoring (MRM) assays have proven successful for the absolute quantification of proteins and for profiling protein modifications in cell lysates and human plasma/serum but have found little application in the analysis of membrane proteins. We report on the optimization of sample preparation conditions for the quantification of two membrane proteins, cytochrome P450 11A1 (CYP11A1) and adrenodoxin reductase (AdR). These conditions can be used for the analysis of other membrane proteins. We have demonstrated that membrane proteins that are tightly associated with the membrane, such as CYP11A1, can be quantified in the total tissue membrane pellet obtained after high-speed centrifugation, whereas proteins that are weakly associated with the membrane, such as AdR, must be quantified in the whole tissue homogenate. We have compared quantifications of CYP11A1 using two different detergents, RapiGest SP and sodium cholate, and two different trypsins, sequencing grade modified trypsin and trypsin, type IX-S from porcine pancreas. The measured concentrations in these experiments were similar and encouraged the use of either combination of detergent/trypsin for quantification of other membrane proteins. Overall, the CYP11A1 and AdR quantified in this work ranged from 110 pmol to 10 fmol per milligram of tissue protein.