pG2 gene expression and its regulation in human adrenocortical and medullary tumors.

The cDNA clone pG2 was originally isolated from a human pheochromocytoma. The respective gene was found to be strongly expressed in normal adrenal zona glomerulosa and medulla, as well as in Conn's adenomas and pheochromocytomas. To shed more light on the expression and regulation of the pG2 gene, we investigated its expression in a wide variety of different adrenal neoplasms and cultured adrenal cells. Northern blot analysis was used to determine the steady state level of pG2 mRNA. Besides normal adrenals, Conn's adenomas and pheochromocytomas, we found abundant expression of pG2 mRNA in Cushing's, virilizing and nonfunctional adrenocortical adenomas and carcinomas, as well as in hyperplastic adrenals. The relative levels of pG2 mRNA in various adrenocortical tumors were not significantly different from those in normal adrenals and pheochromocytomas. In primary cultures of normal adrenal cells, treatment with adrenocorticotropin induced a 3- to 15-fold increase in the expression of pG2 mRNA (P<0.01), and this effect was reproduced by incubation with (Bu)2cAMP. In cultured pheochromocytoma cells, treatment with (Bu)2cAMP and a protein kinase inhibitor, staurosporine, increased pG2 mRNA accumulation (2- to 4-fold over the control level, P<0.01, and 3- to 8-fold, P<0.01, respectively). These results indicate that pG2 is widely expressed in normal and pathological adrenal tissues from both cortical and medullary origin, which eliminates its usefulness as a specific marker for zona glomerulosa or medullary adrenal tumors. Accumulation of pG2 mRNA is regulated by multiple differentiating factors through different pathways in primary cultures of normal adrenal and pheochromocytoma cells.     

The expression of inhibin/activin subunits in the human adrenal cortex and its tumours.

Inhibins and activins are dimeric proteins of the transforming growth factor-beta superfamily which have been shown to be expressed in the adrenal cortex. Recent studies have suggested a role for these peptides in the pathogenesis and/or function of adrenal tumours. To investigate further their physiological and pathological roles, we have documented immunoreactivity for inhibin alpha, betaA and betaB subunits in normal adult and fetal human adrenals, in hyperplastic adrenals and in adrenal tumours. In the normal and hyperplastic adult gland, diffuse immunopositivity was demonstrated for beta subunits, suggesting that activins (beta beta dimers) can be expressed in all zones. Inhibin alpha was limited to the zona reticularis and the innermost zona fasciculata in the normal gland, extending centripetally into the zona fasciculata in hyperplasia, supporting a role for ACTH in the regulation of expression, and suggesting that expression of inhibins (alpha beta dimers) is restricted. Immunopositivity for all three subunits was seen in both fetal and definitive zones of the fetal cortex, indicating that both inhibins and activins could be expressed in both. Immunopositivity for all three subunits was seen in most adrenocortical tumours. Loss of immunopositivity for inhibin alpha in a subgroup of carcinomas might indicate a role in tumour progression. The greater intensity of staining for inhibin alpha in tumours associated with Cushing's syndrome again suggests a link with cortisol production.     

p53 and Ki67 in adrenocortical tumors

The p53 tumor-supressor gene has been reported as the most frequent genetic abnormality seen in human malignancies. Here we studied immunohistochemically the expression of p53 in a large series of adrenocortical tumors. The proliferative activity was assessed by the expression of Ki67. Tumor material consisted of 60 adrenocortical adenomas and 27 adrenocortical carcinomas. A tumor was scored as positive for p53 if more than 10% of the cells showed nuclear staining. All adrenocortical adenomas were negative for p53 and the percentage of Ki67 positive cells was mostly 1-2% but never exceeded 5%. Hormonal activity did not reflect the proliferation index. Adrenocortical carcinomas, however, behaved differently depending on hormonal activity. 10/13 of non-functional , 0/3 Conn's, 3/7 Cushing's and 3/4 virilizing carcinomas were positive for p53. The proliferative activity was also higher in non-fuctional carcinomas compared with hormonally active tumors. Our data show that majority of adrenocortical carcinomas are positive for p53, whereas all adenomas are negative. Hormonal activity of carcinomas reflects both p53 status and proliferation index. Thus, immunohistochemical levels of p53 and Ki67 are higher in hormonally inactive adrenocortical carcinomas.     

Expression of activin/inhibin receptor and binding protein genes and regulation of activin/inhibin peptide secretion in human adrenocortical cells

Activins and inhibins are glycoprotein hormones produced mainly in gonads but also in other organs. They are believed to be important para/autocrine regulators of various cell functions. We investigated activin/inhibin receptor and binding protein gene expression and the regulation of activin/inhibin secretion in human adrenal cells. RT-PCR revealed inhibin/activin alpha-, betaA/B-subunit, follistatin, activin type I/II receptor, and inhibin receptor (betaglycan and inhibin-binding protein) mRNA expression in fetal and adult adrenals and cultured adrenocortical cells. Cultured cells secreted activin A and inhibin A/B as determined by specific ELISAs. ACTH stimulated inhibin A/B secretion in fetal (1.8- and 1.8-fold of control, respectively) and in adult cells (3.4- and 1.7-fold of control, respectively) without significant effect on activin A. 8-bromoadenosine cAMP (protein kinase A activator) increased activin A and inhibin A/B secretion in the human adrenocortical NCI-H295R cell line (32-, 17-, and 3-fold of control, respectively). 12-O-tetradecanoyl phorbol-13-acetate (protein kinase C activator) stimulated both activin A and inhibin A secretion (764- and 32-fold of control, respectively), and activin treatment increased inhibin B secretion in these cells (25-fold of control). In conclusion, human adrenocortical cells produce dimeric activins and inhibins. ACTH stimulates inhibin secretion and decreases activin/inhibin secretion ratio, probably via the protein kinase A signal transduction pathway. This, together with the adrenocortical activin/ inhibin receptor and binding protein expression, suggests a physiological role for activins and inhibins in the human adrenal gland.     

Evidence for a potential role for HDL as an important source of cholesterol in human adrenocortical tumors via the CLA-1 pathway.

CLA-1, a human homologue of rodent scavenger receptor class B1 (SR-B1), has been identified as a receptor for high density lipoprotein (HDL) and is highly expressed in the adrenal gland. Several studies have indicated that HDL might be a source of cholesterol for steroidogenesis in the adrenal gland. In this study, we show that ACTH and its second messenger cAMP stimulated CLA-1 protein expression in a human adrenocortical cell line. We also determined whether CLA-1 plays an important role in steroidogenesis by investigating CLA-1 expression levels in various adrenal tumors including the adenomas of Cushing's and Conn's syndrome. Western blot analysis showed that CLA-1 expression was much higher in the tumors of Cushing's syndrome than in non-tumor lesions of Conn's syndrome and pheochromocytoma. We were able to detect a strong CLA-1 signal in tumors of Conn's syndrome, too. On the other hand, much less CLA-1 expression was detected in Cushing's adenoma adjacent adrenal glands. The immunohistochemical analysis showed that CLA-1 was expressed in the outer region of the adrenal cortex mainly in plasma membranes of the cortical cells but not in the medulla. These findings demonstrated for the first time that ACTH increased CLA-1 protein in cultured human adrenocortical cells, and that cortisol- and aldosterone-secreting adenomas had high CLA-1 proteins in their cell surfaces.     

Expression of activin/inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis

Activins and inhibins are structurally related glycoprotein hormones modulating pituitary FSH secretion and gonadal steroidogenesis. Activins and inhibins are also produced in the adrenal cortex where their physiological role is poorly known. Hormonally active human adrenocortical tumors express and secrete inhibins, while in mice adrenal inhibins may function as tumor suppressors. To clarify the significance of adrenal activins and inhibins we investigated the localization of activin/inhibin signaling components in the adrenal gland, and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis.Activin receptor type II/IIB and IB, activin signal transduction proteins Smad2/3, and inhibin receptor betaglycan were expressed throughout the adrenal cortex, whereas Smad4 expression was seen mainly in the zona reticularis and the innermost zona fasciculata as evaluated by immunohistochemistry. Treatment of cultured adrenocortical carcinoma NCI-H295R cells with activin A inhibited steroidogenic acute regulatory protein and 17alpha-hydroxylase/17,20-lyase mRNA accumulation as evaluated by the Northern blot technique, and decreased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate secretion as determined by specific enzyme immunoassays. Activin A increased apoptosis as measured by a terminal deoxynucleotidyl transferase in situ apoptosis detection method. Inhibins had no effect on steroidogenesis or apoptosis.In summary, activin/inhibin signaling components are coexpressed in the zona reticularis and the innermost zona fasciculata indicating full signaling potential for adrenal activins and inhibins in these layers. Activin inhibits steroidogenic enzyme gene expression and steroid secretion, and increases apoptosis in human adrenocortical cells. Thus, the activin-inhibin system may have a significant role in the regulation of glucocorticoid and androgen production and apoptotic cell death in the human adrenal cortex.     

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

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

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

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

Role of local 11 beta-hydroxysteroid dehydrogenase type 2 expression in determining the phenotype of adrenal adenomas

It is not understood why some adrenal adenomas are nonfunctional and others with similar histopathology cause preclinical or overt Cushing's syndrome. Two isozymes of 11 beta-hydroxysteroid dehydrogenase, types 1 and 2 (HSD11B1 and HSD11B2), are known to modulate glucocorticoid levels in other tissues and might influence circulating levels of active and inactive glucocorticoids if they were expressed in adrenal adenomas. We determined levels of expression of these isozymes in normal adrenals and 61 adrenal adenomas by quantitative competitive RT-PCR and immunohistochemistry. There were no differences in HSD11B1 mRNA levels among adrenal tumor groups. HSD11B2 mRNA levels were high in nonfunctioning adenomas and preclinical Cushing's adenomas compared with levels in control adrenals or in adenomas causing overt Cushing's syndrome. HSD11B2 immunoreactivity was not detected in control adrenals, but was observed in more than half of these tumors. When nonfunctioning adenomas and those causing preclinical and overt Cushing's syndrome were considered as a single group, HSD11B2 mRNA levels were strongly correlated with the ratio of plasma cortisone to cortisol, and a simple model incorporating adrenal HSD11B2 expression and tumor size as variables could predict more than 50% of the interindividual variation in plasma cortisol levels (r(2) = 0.54; P < 0.0001). Adrenal HSD11B2 may regulate levels of active and inactive glucocorticoids in the systemic circulation under these conditions, presumably by acting in an autocrine or paracrine manner. Nonfunctioning adenomas and those causing preclinical and overt Cushing's syndrome may represent a continuum with clinical manifestations depending mainly on tumor size and HSD11B2 expression levels.     

Expression profiles of the glucose-dependent insulinotropic peptide receptor and LHCGR in sporadic adrenocortical tumors

Glucose-dependent insulinotropic peptide receptor (GIPR) and LHCGR are G-protein-coupled receptors with a wide tissue expression pattern. Aberrant expression of these receptors has rarely been demonstrated in adult sporadic adrenocortical tumors with a lack of data on pediatric tumors. We quantified the GIPR and LHCGR expression in a large cohort of 55 patients (25 children and 30 adults) with functioning and non-functioning sporadic adrenocortical tumors. Thirty-eight tumors were classified as adenomas whereas 17 were carcinomas. GIPR and LHCGR expression were analyzed by real-time PCR and normal human pancreatic and testicular tissue samples were used as positive controls. Mean expression values were determined by fold increase in comparison with a normal adrenal pool. GIPR mRNA levels were significantly higher in adrenocortical carcinomas than in adenomas from both pediatric and adult groups. LHCGR expression was similar in both carcinomas and adenomas from the pediatric group but significantly lower in carcinomas than in adenomas from the adult group (median 0.06 and 2.3 respectively, P<0.001). GIPR was detected by immunohistochemistry in both pediatric and adult tumors. Staining and real-time PCR results correlated positively only when GIPR mRNA levels were increased at least two-fold in comparison with normal adrenal expression levels. In conclusion, GIPR overexpression was observed in pediatric and adult adrenocortical tumors and very low levels of LHCGR expression were found in all adult adrenocortical carcinomas.     

Histopathology of the human adrenal cortex

The morphological features of the adult human adrenal cortex are described with particular respect to changes induced by alterations in function of the hypothalamo-pituitary axis. The occurrence of nodules in the normal and hyperplastic cortex (Cushing's and Conn's syndromes) is discussed in relation to the diagnostic problems that they still pose. Explanations based on the normal mechanisms of functional zonation in the cortex are provided for the different cell types which comprise cortical neoplasms associated with various syndromes of hypercorticalism (Cushing's, adrenogenital and Conn's syndromes), together with morphological and functional criteria to distinguish adenomas from carcinomas.     

Decreased expression of cyclic adenosine monophosphate-regulated aldose reductase (AKR1B1) is associated with malignancy in human sporadic adrenocortical tumors

The human aldose reductase, AKR1B1, participates in glucose metabolism and osmoregulation and is supposed to play a protective role against toxic aldehydes derived from lipid peroxidation and steroidogenesis that could affect cell growth/differentiation when accumulated. Adrenal gland is a major site of expression of AKR1B1, and we asked whether changes in its expression could be associated with adrenal disorders. Therefore, we examined AKR1B1 gene expression in human fetal adrenals, adrenocortical cell line, and tumors and compared the results with the expression of steroidogenic genes (StAR and CYP11A) and regulators of adrenal cortex development [steroidogenic factor-1 (SF-1) and dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1)]. Using specific antibodies, Northern blotting, and enzymatic assays, we present evidences that AKR1B1 detectable in 15-wk-old fetal glands is regulated by cAMP in NCI-H295 cells and thus that AKR1B1 is functionally related to the ACTH-responsive murine akr1b7/mvdp gene rather than to its direct ortholog, the mouse aldose reductase akr1b3 gene. Although low DAX1 expression in aldosterone-producing adenomas (n = 5) was confirmed (P < 0.05), no correlation was found between the expression of all other genes and the tumors endocrine activity. In contrast, relative abundance of AKR1B1 mRNA was decreased in adrenocortical carcinomas (n = 5; mean +/- sem, 0.95 +/- 0.2) when compared with adenomas (n = 12; 9.29 +/- 3.05; P < 0.001). Most (seven of eight) adrenocortical carcinomas (19.0 +/- 5.4) had very low relative AKR1B1 protein levels when compared with benign tumors (cortisol-producing adenomas, n = 5, 63.0 +/- 9.8; nonfunctional adenomas, n = 5, 58.0 +/- 10.4; aldosterone-producing adenomas, n = 4, 65.3 +/- 7.7; P < 0.001), Cushing's hyperplasia (n = 5, 54.6 +/- 5.3; P < 0.01), or normal adrenals (n = 4; 37.1 +/- 5.3; P < 0.001). These properties provide the first evidence that expression of cAMP-regulated AKR1B1 is decreased in adrenocortical cancer. This might take part in adrenal tumorigenesis and could be investigated as a marker of malignancy for the diagnosis of adrenal tumors.