Adrenal Expression Research Articles

Differentially expressed genes in adrenal gland of H22 liver cancer mice with different syndromes and in different stages

To reveal the characteristics of gene expression in adrenal gland of H22 tumor mice with typical syndromes and in different liver cancer stages. By the quantitative four diagnosis and syndrome differentiation methods and GeneChip Mouse Exon 1.0 ST Array, we observed adrenal gland gene expression in H22 tumor mice with pathogenic factor-toxin predominance syndrome and qi deficiency syndrome in the earlier stage, yang-qi deficiency syndrome in the intermediate stage, and qi-yin-yang deficiency syndrome in the advanced stage. Genes highly expressed and remarkably different were analyzed in this study. A total of seventy-three up-regulated coincident genes and twenty-six down-regulated coincident genes in different stages were investigated in the study. Up-regulated coincident genes included Hp, C3, Anxa1, Procr, C2, Il4ra, Cd14, Ptprc, Cd52, C4b, Eno3, Xdh, Gpx3, and so on. Down-regulated coincident genes included nervous system function-related genes such as Plp1, Mbp, Aldh1a1, Cck, Atn1, genes associated with electrolyte metabolism such as Aldh1a1 and Slc22a17, genes related to signal transduction such as Cxcr4, Spag5 and Stmn3, etc, and genes related to transcriptional control and protein biosynthesis such as Hspa1a, Dnajb1, Thra, Hhex and so on. With the development of the tumorigenesis, the symptoms and signs and differentially expressed genes in adrenal gland of H22 tumor mice can be measured. Up-regulated and down-regulated coincident genes may be the features of H22 tumor mice different from those of normal mice.

Estrogen Reduces Aldosterone, Upregulates Adrenal Angiotensin II AT2 Receptors and Normalizes Adrenomedullary Fra-2 in Ovariectomized Rats

We studied the effect of ovariectomy and estrogen replacement on expression of adrenal angiotensin II AT₁ and AT₂ receptors, aldosterone content, catecholamine synthesis, and the transcription factor Fos-related antigen 2 (Fra-2). Ovariectomy increased AT₁ receptor expression in the adrenal zona glomerulosa and medulla, and decreased adrenomedullary catecholamine content and Fra-2 expression when compared to intact female rats. In the zona glomerulosa, estrogen replacement normalized AT₁ receptor expression, decreased AT_1B receptor mRNA, and increased AT₂ receptor expression and mRNA. Estrogen treatment decreased adrenal aldosterone content. In the adrenal medulla, the effects of estrogen replacement were: normalized AT₁ receptor expression, increased AT₂ receptor expression, AT₂ receptor mRNA, and tyrosine hydroxylase mRNA, and normalized Fra-2 expression and catecholamine content. We demonstrate that the constitutive adrenal expression of AT₁ receptors, catecholamine synthesis and Fra-2 expression are partially under the control of reproductive hormones. Our results suggest that estrogen treatment decreases aldosterone production through AT₁ receptor downregulation and AT₂ receptor upregulation. AT₂ receptor upregulation and modulation of Fra-2 expression may participate in the estrogen-dependent normalization of adrenomedullary catecholamine synthesis in ovariectomized rats. The AT₂ receptor upregulation and the decrease in AT₁ receptor function and in the production of the fluid-retentive, pro-inflammatory hormone aldosterone partially explain the protective effects of estrogen therapy.

Effect of dietary polyunsaturated fatty acids on the expression of peroxisomal ABC transporters

Peroxisomal ABC transporters encoded by the ABCD genes are thought to participate in the import of specific fatty acids in the peroxisomal matrix. ABCD1 deficiency is associated with X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder which is characterized by the accumulation of saturated very-long-chain fatty acids (VLCFA). ABCD2 (the closest homolog of ABCD1) and ABCD3 have been shown to have partial functional redundancy with ABCD1; only when overexpressed, they can compensate for VLCFA accumulation. Other lipids, for instance polyunsaturated fatty acids (PUFA), should be possible candidate substrates for the ABCD2 and ABCD3 gene products, ALDRP and PMP70 respectively. Moreover, PUFA, which are known regulators of gene expression, could therefore represent potent inducers of the ABCD genes. To test this hypothesis, littermates of n-3-deficient rats were subjected to an n-3-deficient diet or equilibrated diets containing ALA (α-linolenic acid, 18:3 n-3) as unique source of n-3 fatty acids or ALA plus DHA (docosahexaenoic acid, 22:6 n-3) at two different doses. We analyzed the expression of peroxisomal ABC transporters and of the peroxisomal acyl-CoA oxidase gene 1 ( Acox1) in adrenals, brain and liver. Whatever the diet, we did not observe any difference in gene expression in adrenals and brain. However, the hepatic expression level of Abcd2 and Abcd3 genes was found to be significantly higher in the n-3-deficient rats than in the rats fed the ALA diet or the DHA supplemented diets. This was accompanied by important changes in hepatic fatty acid composition. In summary, the hepatic expression of Abcd2 and Abcd3 but not of Abcd1 and Abcd4 appears to be highly sensitive towards dietary PUFA. This difference could be linked to the substrate specificity of the peroxisomal ABC transporters and a specific involvement of Abcd2 and Abcd3 in PUFA metabolism.

Farnesoid X receptor regulation of adrenal cell steroidogenic enzyme expression

Farnesoid X receptor (FXR, NR1H4), a member of the nuclear receptor superfamily, is expressed in a number of tissues including the liver, ovary and adrenal. In the liver, FXR regulates cholesterol and glucose homeostasis, but its function in the ovary and adrenal remains unknown. Herein, we tested the hypothesis that FXR regulates expression of the enzymes involved in steroid hormone biosynthesis. Human adrenocortical cells were transfected with FXR expression vectors and treated with the FXR ligand GW4064; followed by microarray analysis. Among the adrenal FXR target genes were the well established liver FXR target, OSTα as well as the steroid‐metabolizing enzyme, 3β‐hydroxysteroid dehydrogenase type II (HSD3B2). HSD3B2 promoter construct studies and quantitative real‐time RTPCR showed that FXR stimulates HSD3B2 transcriptional activity and mRNA levels. Sequential deletions and mutational analysis of the HSD3B2 promoter defined an inverted hormone response element repeat between −137 and −124 that was necessary for FXR‐induced reporter activity. Chromatin immunoprecipitation assay demonstrated the ability of FXR to bind this response element. Since HSD3B2 is critical for the production of both adrenal and ovarian steroid hormones, these results indicate a new and important role of FXR in the regulation of steroidogenesis in the ovary and adrenal.

Metastin Stimulates Aldosterone Synthesis in Human Adrenal Cells

Kisspeptins, including metastin, are encoded by the KiSS-1 gene and play an important role in regulating the hypothalamic gonadotropin-releasing hormone (GnRH) system via G protein-coupled receptor 54 (GPR54, also called KiSS-1R). Normally, metastin (also called Kp-54) levels are quite low, except during pregnancy, when levels increase 1000-fold over those found in men and nonpregnant women. However, the potential hormonal role of metastin in the fetal and maternal circulation is unknown. In this study, the authors examine the levels of GPR54 mRNA expression in human adult and fetal adrenals using quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). In addition, they examine the effects of metastin on steroidogenesis and steroidogenic enzyme mRNA levels in fetal adrenal cells and in the H295R adrenocortical cell line using enzyme immunoassay and RT-PCR techniques. The authors demonstrate that GPR54 mRNA is significantly higher (50-fold) in human fetal adrenals than in adult adrenals. Immunohistochemical studies have demonstrated that the GPR54 protein is predominantly expressed in the neocortex of human fetal adrenals in the third trimester. Metastin increases aldosterone production (approximately 2-fold) in both fetal neocortex adrenal cells and H295R adrenal cells, with a maximal increase seen at 100 nM. In addition, metastin increased angiotensin II (Ang II)-stimulated aldosterone production by approximately 1.5-fold. Metastin also increased the ability of the H295R cells to metabolize exogenously added pregnenolone to aldosterone but had no effect on the expression of aldosterone synthase (CYP11B2). These results suggest that the high fetal/maternal levels of metastin seen during pregnancy may affect adrenal production of aldosterone.

6A-4 Impact of in vitro embryo culture on fetal adrenal growth and steroidogenic gene expression in the sheep in late gestation

6A-4 Impact of in vitro embryo culture on fetal adrenal growth and steroidogenic gene expression in the sheep in late gestation

P1-72 Adrenal gene expression in sheep is altered by maternal dexamethasone treatment in early gestation

P1-72 Adrenal gene expression in sheep is altered by maternal dexamethasone treatment in early gestation

Vasopressin-Responsive ACTH-Independent Macronodular Adrenal Hyperplasia Causing Cushing's Syndrome

ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a rare cause of endogenous Cushing's syndrome (CS). The diagnosis is suggested by bilateral adrenal nodular enlargement on conventional imaging and the demonstration of ACTH-independent hypercortisolism. Cortisol secretion in AIMAH can be regulated by the aberrant adrenal expression of receptors for gastric inhibitory polypeptide, vasopressin, catecholamines, luteinizing hormone/human chorionic gonadotropin and serotonin. We report on a 47-year-old man with CS due to AIMAH. The cortisol levels were increased after vasopressin stimulation, suggesting aberrant expression of vaso-pressin receptor in the adrenal glands. Total adrenalectomy is considered a standard therapy for AIMAH.

Upregulation of sodium-dependent vitamin C transporter 2 expression in adrenals increases norepinephrine production and aggravates hyperlipidemia in mice with streptozotocin-induced diabetes

The hyperglycemia and hyperoxidation that characterize diabetes lead to reduced vitamin C (l-ascorbic acid, AA) levels in diabetic humans and animals. We examined the possibility that diabetes-induced low plasma AA levels impair AA distribution to various tissues and that these changes are closely related to the development of diabetic complications. AA levels were markedly decreased in the plasma and increased in the adrenals of mice with streptozotocin (STZ)-induced diabetes. Consistently with these results, in [1−14C]AA accumulation assays, the efficiency of [1−14C]AA accumulation was significantly higher in the adrenals (which had the greatest ability to accumulate [1−14C]AA) of diabetic mice than in those of controls. Expression of sodium-dependent vitamin C transporter (SVCT)-2, a transporter of AA, was upregulated in diabetic adrenals. Furthermore, increased AA incorporation into the diabetic adrenals by SVCT-2 led to increased plasma norepinephrine, triglyceride and free fatty acid levels in mice with STZ-induced diabetes. Therefore, oversupplementation with AA could be deleterious in diabetic patients, because overexpression of adrenal SVCT-2 in diabetes could lead to excessive AA uptake, thus enhancing norepinephrine production and exacerbating some diabetic complications. Interestingly, however, treatment with AA dose-dependently abolished the increased expression of adrenal SVCT-2 and normalized the abovementioned plasma parameters in diabetic mice. These results suggest SVCT-2-mediated increases in AA uptake by the adrenals followed by excessive production of plasma norepinephrine may play a pivotal role in the development of diabetic complications.

Gender and gonadal status differences in zona reticularis expression in marmoset monkey adrenals: Cytochrome b5 localization with respect to cytochrome P450 17,20-lyase activity

Neonatal marmosets express an adrenal fetal zone comparable to humans. While adult males fail to express a functional ZR, with barely detectable blood DHEA levels, females produce higher levels of DHEA than males in adulthood. We investigated the presence of a putative functional ZR in adult female marmosets. In contrast to males, immunohistochemical analysis showed the ZR marker cytochrome b5 was elevated in the innermost zone in cycling females (compared to testis-intact males), further elevated in the adrenals from anovulatory females, and substantially elevated and continuous in ovariectomized females. As a functional test in vivo, following overnight dexamethasone treatment, cycling and anovulatory females showed higher levels of DHEA relative to males, but DHEA failed to increase in response to ACTH. In direct contrast, while ovariectomized females exhibited lower initial DHEA levels, clear increases were detectable after ACTH administration (p<0.05), suggesting an adrenal origin. The apparent differences in cytochrome b5 expression between groups were also further verified by Western blotting of adrenal microsomes, and compared to 17,20-lyase activity; the two parameters were positively correlated (p<0.01) across multiple treatment groups. We conclude that the cycling female marmoset expresses a rudimentary ZR with at least a capacity for DHEA production that becomes significantly ACTH-responsive after anovulation. Expression of cytochrome b5 in this region may be directly or indirectly controlled by gonadal function, and is, at least in part, a critical determinant in the development of an adrenal ZR that is more defined and significantly ACTH-responsive.

Impact of Periconceptional Undernutrition on Adrenal Growth and Adrenal Insulin-Like Growth Factor and Steroidogenic Enzyme Expression in the Sheep Fetus during Early Pregnancy

Periconceptional undernutrition (PCUN) results in an earlier prepartum activation of the pituitary-adrenal axis in twin compared with singleton fetuses. We have tested the hypotheses that the functional development of the fetal sheep adrenal is delayed in twins compared with singletons in early gestation and that PCUN accelerates adrenal growth and increases the expression of intraadrenal IGF-I and -II and cytochrome P450 17-hydroxylase (CYP17) as early as 55 d gestation. We have investigated the effect of PCUN in the ewe (restricted at 70% of control allowance, n=21; control, n=24) from at least 45 d before mating until d 7 after mating on maternal cortisol and progesterone concentrations, fetal adrenal weight, adrenal IGF-I, IGF-I receptor (IGF-IR), IGF-II, IGF-IIR, and CYP17 mRNA expression and placental 11beta-hydroxysteroid dehydrogenase-1 and -2 mRNA and protein expression at d 53-56 pregnancy. The relative weight of the fetal adrenal and adrenal IGF-I, IGF-IR, IGF-II, IGF-IIR, and CYP17 mRNA expression were lower in twin compared with singleton fetuses. In singleton fetuses of PCUN ewes, there was a loss of the relationship between adrenal IGF-II/IGF-IIR expression and either adrenal weight or CYP17 mRNA, which was present in controls. Similarly in twin fetuses, PCUN resulted in the loss of the relationships between adrenal weight and IGF-I expression and between adrenal CYP17 and IGF-II expression, which were present in controls. Our findings suggest that differences in the timing of the prepartum activation of the fetal adrenal in twins and singletons have their origins in early gestation and highlight the importance of the interaction between the periconceptional environment and embryo number in setting the growth trajectory of the fetal adrenal.

Expression and Externalization of Annexin 1 in the Adrenal Gland: Structure and Function of the Adrenal Gland in Annexin 1-Null Mutant Mice

Annexin 1 (ANXA1) is a member of the annexin family of phospholipid- and calcium-binding proteins with a well demonstrated role in early delayed (30 min to 3 h) inhibitory feedback of glucocorticoids in the hypothalamus and pituitary gland. This study used adrenal gland tissue from ANXA1-null transgenic mice, in which a beta-galactosidase (beta-Gal) reporter gene was controlled by the ANXA1 promoter, and wild-type control mice to explore the potential role of ANXA1 in adrenal function. RT-PCR and Western blotting revealed strong expression of ANXA1 mRNA and protein in the adrenal gland. Immunofluorescence labeling of ANXA1 in wild-type and beta-Gal expression in ANXA1-null adrenals localized intense staining in the outer perimeter cell layers. Immunogold electron microscopy identified cytoplasmic and nuclear ANXA1 labeling in outer cortical cells and capsular cells. Exposure of adrenal segments in vitro to dexamethasone (0.1 mum, 3 h) caused an increase in the amount of ANXA1 in the intracellular compartment and attached to the surface of the cells. The N-terminal peptide ANXA1(Ac2-26) inhibited corticosterone release. Corticosterone release was significantly greater from ANXA1-null adrenal cells compared with wild type in response to ACTH (10 pm to 5 nm). In contrast, basal and ACTH-stimulated aldosterone release from ANXA1-null adrenal cells was not different from wild type. Morphometry studies demonstrated that ANXA1 null adrenal glands were smaller than wild-type, and the cortical/medullary area ratio was significantly reduced. These results suggest ANXA1 is a regulator of adrenocortical size and corticosterone secretion.

Pre-B-Cell Transcription Factor 1 and Steroidogenic Factor 1 Synergistically Regulate Adrenocortical Growth and Steroidogenesis

A variety of transcription factors including Wilms tumor gene (Wt-1), steroidogenic factor 1 (Sf-1), dosage-sensitive sex reversal, adrenal hypoplasia congenita on the X-chromosome, Gene 1 (Dax-1), and pre-B-cell transcription factor 1 (Pbx1) have been defined as necessary for regular adrenocortical development. However, the role of Pbx1 for adrenal growth and function in the adult organism together with the molecular relationship between Pbx1 and these other transcription factors have not been characterized. We demonstrate that Pbx haploinsufficiency (Pbx1(+/-)) in mice is accompanied by a significant lower adrenal weight in adult animals compared with wild-type controls. Accordingly, baseline proliferating cell nuclear antigen levels are lower in Pbx1(+/-) mice, and unilateral adrenalectomy results in impaired contralateral compensatory adrenal growth, indicating a lower proliferative potential in the context of Pbx1 haploinsufficiency. In accordance with the key role of IGFs in adrenocortical proliferation and development, real-time RT-PCR demonstrates significant lower expression levels of the IGF-I receptor, and up-regulation of IGF binding protein-2. Functionally, Pbx1(+/-) mice display a blunted corticosterone response after ACTH stimulation coincident with lower adrenal expression of the ACTH receptor (melanocortin 2 receptor, Mc2-r). Mechanistically, in vitro studies reveal that Pbx1 and Sf-1 synergistically stimulates Mc2-r promoter activity. Moreover, Sf-1 directly activates the Pbx1 promoter activity in vitro and in vivo. Taken together, these studies provide evidence for a role of Pbx1 in the maintenance of a functional adrenal cortex mediated by synergistic actions of Pbx1 and Sf-1 in the transcriptional regulation of the critical effector of adrenocortical differentiation, the ACTH receptor.

Shared Long-Range Regulatory Elements Coordinate Expression of a Gene Cluster Encoding Nicotinic Receptor Heteromeric Subtypes

The nicotinic acetylcholine receptor (nAChR) beta4/alpha3/alpha5 gene cluster encodes several heteromeric transmitter receptor subtypes that are essential for cholinergic synaptic transmission in adrenal gland, autonomic ganglia, pineal gland, and several nuclei in the central nervous system. However, the transcriptional mechanisms coordinating expression of these subunit genes in different cell populations are unknown. Here, we used transgenic methods to investigate long-range transcriptional control of the cluster. A 132-kb P1-derived artificial chromosome (PAC) encoding the rat cluster recapitulated the neurally- and endocrine-restricted expression patterns of the endogenous beta4/alpha3/alpha5 genes. Mutation of ETS factor binding sites in an enhancer, beta43', embedded in the beta4 3'-untranslated exon resulted in greatly diminished beta4, alpha3, and alpha5 expression in adrenal gland and to a lesser extent in the superior cervical ganglion (SCG) but not in other tissues. Phylogenetic sequence analyses revealed several conserved noncoding regions (CNRs) upstream of beta4 and alpha5. Deletion of one of them (CNR4) located 20 kb upstream of beta4 resulted in a dramatic decrease in beta4 and alpha3 expression in the pineal gland and SCG. CNR4 was sufficient to direct LacZ transgene expression to SCG neurons, which express the endogenous beta4alpha3alpha5 subunits, and pineal cells, which express the endogenous beta4alpha3 combination. Finally, CNR4 was able to direct transgene expression to major sites of expression of the endogenous cluster in the brain. Together, our findings support a model in which cell type-specific shared long-range regulatory elements are required for coordinate expression of clustered nAChR genes.

Adrenal expression of orexin receptor subtypes is differentially regulated in experimental streptozotocin induced type-1 diabetes

Orexins (hypocretins) are involved in the regulation of energy homeostasis and sleeping behavior. Orexins were also implicated in the regulation of neuroendocrine and autonomic functions. Recent data show the expression of orexin receptors within the hypothalamic–pituitary–adrenal (HPA) axis and suggest specific actions of orexins at the pituitary and adrenal glands. To further evaluate the role of orexin in the HPA axis, we investigated the mRNA expression of prepro-orexin (PPO) and orexin receptors within the HPA axis of streptozotocin-injected (STZ) rats showing type-1 like diabetes. PPO, as well as OX 1 and OX 2 receptor levels were analyzed by quantitative real-time PCR (qPCR). STZ rats were characterized by decreased body weight, plasma insulin, and leptin levels and by increased plasma glucose. Hypothalamic PPO mRNA levels were significantly reduced in STZ compared to non-diabetic control rats. No differences were found in the mRNA levels of hypothalamic or pituitary OX 1 and OX 2 receptors between control and STZ rats. In adrenals, OX 1 receptor mRNA levels were significantly elevated in STZ rats while OX 2 receptors were significantly reduced. Our results imply distinct functions of adrenal orexin receptor subtypes during type-1 like diabetes.

Whole Genome Expression Profiling of Glucose-Dependent Insulinotropic Peptide (GIP)- and Adrenocorticotropin-Dependent Adrenal Hyperplasias Reveals Novel Targets for the Study of GIP-Dependent Cushing’s Syndrome

The mechanisms responsible for the ectopic adrenal expression of glucose-dependent insulinotropic peptide (GIP) receptor (GIPR) in GIP-dependent Cushing's syndrome (CS) are unknown. Chronic adrenal stimulation by ACTH in Cushing's disease or GIP in GIP-dependent ACTH-independent macronodular adrenal hyperplasia both lead to the induction of genes implicated in adrenal proliferation and steroidogenesis. The objective of the study was to identify genes differentially expressed specifically in GIP-dependent CS that could be implicated in the ectopic expression of GIPR. We used the Affymetrix U133 plus 2.0 microarray oligochips to compare the whole genome expression profile of adrenal tissues from five cases of GIP-dependent bilateral ACTH-independent macronodular adrenal hyperplasia with CS, one case of GIP-dependent unilateral adenoma with CS, five cases of ACTH-dependent hyperplasias, and a pool of adrenals from 62 normal individuals. After data normalization and statistical filtering, 723 genes with differential expression were identified, including 461 genes or sequences with a known functional implication, classified in eight dominant functional classes. Specific findings include repression of perilipin, the overexpression of 13 G protein-coupled receptors, and the potential involvement of Rho-GTPases. We also isolated 94 probe sets potentially linked to the formation of GIP-dependent nodules adjacent to the diffuse hyperplasia. These included probe sets related to the linker histone H1 and repression of RXRa and CCND2. The expression profiles for eight genes were confirmed by real-time RT-PCR. This study identified an extensive series of potentially novel target candidate genes that could be implicated in the molecular mechanisms of ectopic expression of the GIPR as well as in the multistep progression of GIP-dependent CS.

Increases in preproenkephalin mRNA levels in the Syrian hamster: The influence of glucocorticoids is dependent on age and tissue

In adult hamsters, basal proenkephalin (Penk) gene expression in adrenals is independent of glucocorticoids and glucocorticoid receptor blockade, by RU 486, increases striatal preproenkephalin (PPenk) mRNA levels. However, glucocorticoids maintain both basal and induced Penk gene expression in rat adrenal (medulla) and striatum. This suggests species and tissue-specific differences in Penk gene regulation. Since studies show temporal coordination in Penk gene expression in developing hamster adrenal and striatum, we tested the hypothesis that increasing PPenk mRNA levels are dependent, while basal levels are independent of glucocorticoids in developing hamsters. To facilitate this study, we examined the influence of glucocorticoids on the temporal increases in developing hamster PPenk mRNA observed in adrenals between postnatal days 0 and 4 and in striatum between postnatal days 12 and 48. PPenk mRNA levels were determined in hamster pups after treatment with increasing doses of metyrapone (an 11β hydroxylase inhibitor) or with the glucocorticoid receptor antagonist RU 486 ± metyrapone between postnatal days 2 and 4. Levels were also determined 36 days after hypophysectomy at age 16–17 days. Although plasma glucocorticoid levels and/or the influence from glucocorticoids were reduced, only developmental increases in PPenk mRNA are influenced by glucocorticoids in hamster adrenals, while basal adrenal mRNA levels are unchanged. However, pituitary influence on striatal PPenk mRNA levels appears complex and may involve steroid and/or non-steroid factors. These results suggest that glucocorticoids regulate hamster Penk gene expression via a mechanism that varies with age and tissue and functions during the induction of the Penk gene and not to maintain basal gene expression. Possible mechanisms and species variation are discussed.

Impact of Fetal Glucocorticoid Exposure on Gene Expression Profiles in the Adult Adrenal Gland

An adverse in-utero environment increases the risk of development of adult diseases such as hypertension, diabetes and the metabolic syndrome. Although elevated circulating fetal levels of glucocorticoids are critically involved in programming events of adult diseases, the genetic basis is not completely understood. The aim of this study was to examine the impact of elevated fetal glucocorticoid levels on changes in gene expression in the adult adrenal gland via a genome-wide approach. The adrenal gland was selected for analysis since it secretes hormones that can directly influence cardiovascular, endocrine and metabolic functions. Total mRNA from adrenal glands was isolated from 5 wk old male CD1 mice exposed prenatally (GD14-21) via maternal injections of the synthetic glucocorticoid dexamethasone (DEX; 100 ug/kg/day i.p.) or saline (control). To probe changes in neuroendocrine transcriptome induced in fetal programming, total mRNA was hybridized to Affymetrix GeneChip Mouse Genome 430v2.0 Arrays (~39K genes). Data was analyzed using a model-based expression algorithm (dCHIP) and filtered (>1.5 fold change in gene expression; statistical significance, p<0.05). Results indicate that only 0.7% of genes (290 genes) were differentially expressed in adrenal glands of mice prenatally exposed to DEX. Widespread alterations in gene expression in diverse systems were observed, including catecholamine biosynthesis (DDC, PNMT), stress proteins (HSP27, HSP68, HSP70) and oxidative stress. These global changes induced by elevated fetal glucocorticoids suggest altered adrenal function may contribute to the pathogenesis of fetal programming of adult diseases.

Elevated Expression of Luteinizing Hormone Receptor in Aldosterone-Producing Adenomas

The mechanisms driving steroid production in aldosterone-producing adenomas (APAs) are poorly defined. However, previous studies have shown that steroid production in some cortisol-producing adenomas is regulated by aberrant expression of G protein-coupled receptors. Aberrant adrenal expression of LH receptors has been shown to cause Cushing's syndrome, but the role of LH receptors in Conn's disease (hyperaldosteronism) has not been studied. The objective of the study was to determine whether APAs express elevated LH receptor, compared with normal adrenal (NA). Pools of RNA from NA and APAs were hybridized to oligonucleotide microarrays. Data were confirmed using real-time RT-PCR analysis of RNA derived from NA (n = 20) and APAs (n = 18). Aldosterone synthase transcription was studied in H295R adrenocortical cells transfected with an LH receptor expression construct and reporter constructs prepared from CYP11B2 5'-flanking DNA. The patient population consisted of 20 normal control adrenals and 18 adenomas from patients with APAs. Regulation of CYP11B2 gene expression by aberrant LH receptor expression in aldosterone-producing adrenal adenoma was measured. LH/choriogonadotropin receptor gene and CYP11B2 are indicated as having greater than 25-fold expression in one pool of APA mRNA samples over NA using microarray analysis. Real-time RT-PCR analyses indicated that one APA sample (APA-LH receptor) exhibited more than 2400-fold elevation in LH receptor expression over NA. Examination of LH receptor mRNA levels in 18 independent APA samples indicated elevated expression in nine samples when compared with NA. In H295R cells transfected with LH receptor, LH treatment caused a concentration-dependent increase in CYP11B2 reporter activity. LH receptor expression is elevated in many APAs, which makes LH a potential cause of the excessive production of aldosterone in a subset of these adrenal tumors.

Expression profile of serotonin4 (5-HT4) receptors in adrenocortical aldosterone-producing adenomas

We aimed to investigate the expression profile of serotonin4 (5-HT4) receptors in adrenocortical aldosterone-producing adenoma (APA) tissues in comparison with normal adrenal cortex. Total 5-HT4 receptor mRNAs were quantified by real-time quantitative polymerase chain reaction (PCR) assay, and the mRNAs encoding the 5-HT4 receptor isoforms were characterized by reverse transcription (RT)-PCR in seven normal adrenal cortices and 11 APA tissues. The distribution of 5-HT4 receptor mRNAs was investigated by in situ hybridization in both normal adrenal and APA tissues, and the presence of 5-HT in APA tissues was studied by immunohistochemistry. Real-time PCR analysis revealed that 5-HT4 receptor mRNA expression was 4.7-47 times higher in APA tissues than in normal glands. In situ hybridization studies showed that 5-HT4 receptor mRNAs were expressed in both zona glomerulosa and zona fasciculata/reticularis of the normal cortex and in groups of APA steroidogenic cells disseminated in the tumor tissues. Characterization of 5-HT4 receptor splice variants by RT-PCR revealed different profiles of expression in APAs versus normal adrenals. Isoforms (a) and (b) were not expressed in any APA but were present in the majority of normal adrenocortical tissues. Conversely, isoform (d) was expressed in 5/11 APAs but only in 1/7 adrenals. Immunohistochemical studies revealed the presence of 5-HT-immunoreactivity in both mast cells and clusters of steroidogenic cells in APA tissues. Our results show overexpression and different splicing of the 5-HT4 receptor in APA tissues in comparison with normal adrenocortical tissue. They also demonstrate the presence of 5-HT in both mast cells and tumor steroidogenic cells, providing evidence for a possible autocrine/paracrine activation of aldosterone secretion within adenoma tissues.