Adrenocortical Cells In Primary Culture Research Articles

Melanocortin 2 Receptor Antagonists in Canine Cushing’s Disease: In Vitro Studies

Melanocortin 2 receptor antagonists in canine Cushing’s disease: in vitro studiesCushing’s disease (CD), caused by an ACTH-secreting pituitary adenoma, is one of the most common endocrinopathies in dogs. The current medical treatment options involve adrenocortical steroid synthesis inhibitors, but a selective targeted approach to block ACTH receptor at its receptor would be much more attractive. The objective of this study was to preclinically investigate the effect of MC2R antagonists on adrenocortical hormone production, cell viability, and mRNA expression of steroidogenic enzymes in canine primary adrenocortical cell cultures from adrenal glands of healthy dogs. Three different MC2R antagonists were used: CRN.1, CRN.2, and CRN.4. Canine primary adrenocortical cell cultures (n = 8) were incubated with 50 nM ACTH for 24h, to mimic CD. Thereafter, 10 nM (IC50) and 2 μM (maximal concentration) of CRN.1, CRN.2, and CRN.4 were added. The two concentrations were established based on preliminary studies. After 24 hours of incubation, adrenocortical hormone concentrations were measured in the culture medium using liquid chromatography-mass spectrometry. RNA was isolated from the cells using the RNeasy Microkit (Qiagen) for subsequent real-time quantitative PCR analysis. Cell viability was assessed after 24 hours of incubation using alamarBlue™ Cell Viability Reagent. All CRN compounds effectively inhibited cortisol concentrations, while leaving aldosterone concentrations unaffected. In incubations with a maximal concentration of the three compounds, cortisol concentration decreased to undetectable levels. The mRNA expression levels of steroidogenic enzymes StAR, CYP11A1, CYP17A1, HSD3B2, CYP21, and CYP11B were significantly inhibited in most conditions when compared to the ACTH-stimulated control. The mRNA expression of melanocortin 2 receptor accessory protein (MRAP) was suppressed as well. Cell viability was not affected by CNR.1 or CNR.4, but was slightly inhibited by CRN.2. In summary, canine adrenocortical cell culture is a useful model system for drug testing. Incubation with MC2R antagonists demonstrated the potential of CNR.1 and CNR.4 as new treatment options for CD. Future in vivo studies in dogs with spontaneous CD are indicated.

Abiraterone Acetate for Cushing Syndrome: Study in a Canine Primary Adrenocortical Cell Culture Model.

Abiraterone acetate (AA) is a potent inhibitor of steroidogenic enzyme 17α-hydroxylase/17,20-lyase (CYP17A1). AA is approved for the treatment of prostate cancer but could also be used to treat patients with Cushing syndrome (CS). Similar to humans, canine glucocorticoid synthesis requires CYP17A1, providing a useful animal model. The objective of this study was to preclinically investigate the effect of AA on adrenocortical hormone production, cell viability, and mRNA expression of steroidogenic enzymes in canine primary adrenocortical cell cultures (n = 9) from the adrenal glands of nine healthy dogs. The cells were incubated with AA (0.125 nM to 10 μM) for 72 hours under basal conditions and with 100 nM ACTH(1-24). Adrenocortical hormone concentrations were measured in culture medium using liquid chromatography-mass spectrometry, RNA was isolated from cells for subsequent real-time quantitative PCR analysis, and cell viability was assessed with an alamarBlue™ assay. AA reduced cortisol (IC50, 21.4 ± 4.6 nM) without affecting aldosterone under basal and ACTH-stimulated conditions. AA increased progesterone under basal and ACTH-stimulated conditions but reduced corticosterone under basal conditions, suggesting concurrent inhibition of 21-hydroxylation. AA did not affect the mRNA expression of steroidogenic enzymes and did not inhibit cell viability. In summary, primary canine adrenocortical cell culture is a useful model system for drug testing. For the treatment of CS, AA may to be superior to other steroidogenesis inhibitors due to its low toxicity. For future in vivo studies, dogs with endogenous CS may provide a useful animal model.

Melanocortin 2 receptor antagonists in canine pituitary-dependent hypercortisolism: in vitro studies

Canine hypercortisolism is most often caused by an ACTH-secreting pituitary adenoma (pituitary-dependent hypercortisolism; PDH). An interesting target for a selective medical treatment of PDH would be the receptor for ACTH: the melanocortin 2 receptor (MC2R). In this study we investigated whether two peptide compounds, BIM-22776 (#776) and BIM-22A299 (#299), are effective MC2R antagonists in vitro. Their effects on cortisol production and mRNA expression of steroidogenic enzymes, MC2R and melanocortin 2 receptor accessory protein (MRAP) were evaluated in primary adrenocortical cell cultures (n = 8) of normal canine adrenal glands. Cortisol production stimulated by 50 nM ACTH was dose-dependently inhibited by #299 (inhibition 90.7 ± 2.3% at 5 μM) and by #776 (inhibition 38.0 ± 5.2% at 5 μM). The ACTH-stimulated mRNA expression of steroidogenic enzymes, MC2R and MRAP was significantly inhibited by both compounds, but most potently by #299. These results indicate that canine primary cell culture is a valuable in vitro system to test MC2R antagonists, and that these compounds, but especially #299, are effective MC2R antagonists in vitro. To determine its efficacy in vivo, further studies are warranted. Antagonism of the MC2R is a promising potential treatment approach in canine PDH.

Nampt (Visfatin) Influence on Proliferative Activity of Normal Rat Adrenocortical Cells and Human Adrenal Corticocarcinoma Nci-H295r Cells

Abstract Nampt (Nicotinamidephosphoribosyltransferase - also known as visfatin/PBEF) is the enzyme that regulates the NAD+ level, therefore influencing many metabolic pathways within the cells. As circulating cytokine, extracellular Nampt (eNampt) exerts pro-inflammatory, pro-chemotactic, pro-angiogenic and insulin-like effects; however the mechanism of eNampt action is still unclear.Earlier studies have shown that eNampt exerts a stimulating effect on the proliferation of many cancer cell lines. However, the effect of this cytokine on cell proliferation in primary culture is little known. Therefore, the aim of the study was to analyse the influence of eNampt on the proliferation of rat adrenocortical cells in primary culture and to investigate similar influence of eNampt on the line H295R of human adrenal corticocarcinoma cells. Proliferation of the examined cells was assessed using the RTCA (Real Time Cell Analyzer) method. The obtained results indicate that eNampt stimulates the proliferation of H295R cells, but does not change the proliferation of cultured rat adrenocortical cells. In primary culture of rat adrenocortical cells, Fk866 (specific Nampt inhibitor) does not modify the rate of proliferation of tested cells. In H295R cells the addition of Fk866 alone inhibits proliferative activity and stimulates apoptosis. Fk866 also inhibits the stimulating effect of eNampt on H295R cell proliferation.

Steroidogenic factor-1 inverse agonists as a treatment option for canine hypercortisolism: in vitro study

Hypercortisolism is one of the most commonly diagnosed endocrinopathies in dogs, and new targeted medical treatment options are desirable. Steroidogenic factor-1 (SF-1), an orphan nuclear hormone receptor, is a key regulator of adrenal steroidogenesis, development, and growth. In pituitary-dependent hypercortisolism (PDH), high plasma ACTH concentrations increase the transcriptional activity of SF-1. In adrenal-dependent hypercortisolism, SF-1 expression is significantly greater in dogs with recurrence after adrenalectomy than in those without recurrence. Inhibition of SF-1 could therefore be an interesting treatment option in canine spontaneous hypercortisolism. We determined the effects of 3 SF-1 inverse agonists, compounds IsoQ A, #31, and #32, on cortisol production, on the messenger RNA (mRNA) expression of steroidogenic enzymes and SFs, and on cell viability, in primary adrenocortical cell cultures of 8 normal adrenal glands and of 3 cortisol-secreting adrenocortical tumors (ATs). To mimic PDH, the normal adrenocortical cell cultures were stimulated with ACTH. The results show that only compound #31 inhibited cortisol production and SF-1 target gene expression in non–ACTH-stimulated and ACTH-stimulated normal adrenocortical cells but did not affect cell viability. In the AT cell cultures, the effects of #31 on cortisol production and target gene expression were variable, possibly caused by a difference in the SF-1 mRNA expressions of the primary tumors. In conclusion, inhibition of SF-1 activity shows much promise as a future treatment for canine hypercortisolism.

Abrogation of TLR4 and CD14 Expression and Signaling in Human Adrenocortical Tumors

Abstract Context: Adrenocortical carcinoma (ACC) is a rare tumor with poor prognosis. The expression of innate immunity receptor Toll-like receptor (TLR)-4 was recently reported in various human tumors, and TLR4 was shown to regulate tumor immune escape processes, proliferation, and resistance to chemotherapeutical agents. Objective: The aim of this study was to investigate TLR4 expression, signaling, and function in the process of tumorigenesis in the human adrenal cortex. Measurements and Main Results: Real-time PCR analysis of human ACC (n = 8), adenoma (n = 8), and ACC cell lines (SW13, NCI-H295R, and HAC15) revealed a significant down-regulation of TLR4, MD2, and CD14 mRNA compared with normal human adrenal cortex and adrenocortical cells in primary culture. Furthermore, immunohistochemistry revealed an abrogation of TLR4 and CD14 expression in ACC but not adenoma tissues. Western blot analysis of MAPK, AKT, activator protein-1, and nuclear factor-κB signaling revealed that the ACC cell lines are unresponsive to lipopolysaccharide action. Restoration of TLR4 signaling by stable transfection of TLR4-CD14 plasmid into NCI-H295R cells sensitized them to lipopolysaccharide incubation as shown by nuclear factor-κB activation and decreased cell viability and induced apoptosis in these cells. Conclusion: Our results demonstrate a significant reduction in the expression of TLR4 and CD14 and an inactivation of TLR4 signaling in ACCs. Furthermore, our data show that reintroduction of TLR4 expression in ACCs may provide a novel therapeutic strategy for adrenal cancer.

Abrogation of TLR4 and CD14 Expression and Signaling in Human Adrenocortical Tumors

Adrenocortical carcinoma (ACC) is a rare tumor with poor prognosis. The expression of innate immunity receptor Toll-like receptor 4 (TLR4) was recently reported in various human tumors, and TLR4 was shown to regulate tumor immune escape processes, proliferation, and resistance to chemotherapeutical agents. The aim of this study was to investigate TLR4 expression, signaling, and function in the process of tumorigenesis in the human adrenal cortex. Real-time PCR analysis of human ACC (n=8), adenoma (n=8), and ACC cell lines (SW13, NCI-H295R, and HAC15) revealed a significant down-regulation of TLR4, MD2 (myeloid differentiation protein-2), and cluster of differentiation 14 (CD14) mRNA compared with normal human adrenal cortex and adrenocortical cells in primary culture. Furthermore, immunohistochemistry revealed an abrogation of TLR4 and CD14 expression in ACC but not adenoma tissues. Western blot analysis of MAPK, AKT, activator protein-1, and nuclear factor-κB signaling revealed that the ACC cell lines are unresponsive to lipopolysaccharide action. Restoration of TLR4 signaling by stable transfection of TLR4-CD14 plasmid into NCI-H295R cells sensitized them to lipopolysaccharide incubation as shown by nuclear factor-κB activation and decreased cell viability and induced apoptosis in these cells. Our results demonstrate a significant reduction in the expression of TLR4 and CD14 and an inactivation of TLR4 signaling in ACCs. Furthermore, our data show that reintroduction of TLR4 expression in ACCs may provide a novel therapeutic strategy for adrenal cancer.

The Divergent Effect of Insulin-Like Growth Factor Binding Protein (IGFBP) - 1 on IGF-induced Steroidogenesis in Bovine Adrenocortical Cells is not due to its Phosphorylation Status

In previous studies we have shown that insulin-like growth factor (IGF)-II stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I. The steroidogenic effect of both, IGF-I and IGF-II, is mediated through the IGF-I receptor and modified by locally produced IGF binding proteins. Further studies demonstrate that bovine adrenocortical cells do synthesize IGFBP-1 to -4 and that their secretion is regulated differentially by IGFs and ACTH. Since IGFBP-1 selectively is induced 3- to 4- fold by ACTH, the aim of the following studies was to evaluate the effect of IGFBP-1 on IGF-induced cortisol secretion from bovine adrenocortical cells in primary culture. Coincubation of bovine adrenocortical cells with purified IGFBP-1 (30 nM) induced a time--and dose-dependent potentiating effect (38+/-2%) on IGF-I-stimulated (6.5 nM) cortisol-secretion, wheras the IGF-II induced steroidogenic effect was inhibited (40+/-3%) by IGFBP-1. Similar results were found when bovine adrenocortical cells were preincubated with IGFBP-1 before stimulation experiments with IGFs were performed. In order to evaluate the influence of different phosphorylation states of IGFBP-1 on the steroidogenic effect of IGF-I and IGF-II and on the affinity to IGFs, a highly phosphorylated (pIGFBP-1) and a dephosphorylated isoform (dIGFBP-1) of IGFBP-1 have been separated by anion exchange chromatography for further incubation experiments and binding studies. No different effects on IGF-I or IGF-II-induced steroidogenesis were observed when a highly phosphorylated IGFBP-1 isoform was used, compared to the dephosphorylated IGFBP-1 isoforms. In binding studies IGFBP-1 did show high affinity binding for IGF-I with a calculated association constant (K (a)) of 2,17 x 10 (9) M (-1). Similar association constants were calculated for dIGFBP-1 and pIGFBP-1 (1,93 x 10 (9) M (-1) and 2,67 x 10 (9) M (-1) respectively) and no difference in binding capacity was observed when IGF-II was used as ligand. IN CONCLUSION, these results demonstrate that in bovine adrenocortical cells IGFBP-1 time- and dose-dependently inhibits the steroidogenic effect of IGF-II and stimulates the effect of IGF-I. The observed modulatory effect of IGFBP-1 is independent of the mode of incubation and its phosphorylation status. The previously observed stronger steroidogenic potency of IGF-II in bovine adrenocortical cells therefore can not be explained by an interaction with IGFBP-1. The mechanisms by which IGFBP-1 divergently regulates the steroidogenic effect of IGF-I and IGF-II remain unclear at present and further investigation is necessary to elucidate the mechanisms by which IGFBP-1 modulates IGF action in the adult adrenal gland.

Stimulation of the pituitary–adrenal axis and of adrenocortical steroidogenesis ex vivo by administration of di-2-ethylhexyl phthalate to prepubertal male rats

Phthalate esters exert deleterious effects on testicular physiology and, consequently, on reproduction and fertility. However, little is presently known concerning potential adverse effects of these environmental pollutants on the hormonal functions of the adrenal gland. Therefore, we have investigated the effects of administering to rats of different developmental ages di-2-ethylhexyl phthalate (DEHP) on the hypothalamic-pituitary-adrenal axis in vivo, as well as on adrenocortical steroidogenesis ex vivo. Oral exposure to DEHP once daily for 4 days elevated the serum levels of ACTH and corticosterone in rats 20 and 40 days of age, but not in adult, 60-day-old animals. Furthermore, primary cultures of adrenocortical cells isolated from 20- and 40-day-old rats treated with DEHP exhibited an enhanced capacity to produce corticosterone in response to ACTH, dibutyryl cAMP, and 22R-hydroxycholesterol, as well as increased ACTH-stimulated transport of endogenous cholesterol into mitochondria. Neither DEHP nor its major metabolite mono-2-ethylhexyl phthalate altered steroidogenesis in cultures of adrenocortical cells isolated from untreated rats. These findings demonstrate that in male rats, DEHP exerts an age-dependent influence on the pituitary-adrenocortical axis in vivo and adrenocortical steroidogenesis ex vivo. Such perturbation may be of pathological significance in connection with disorders of the hormonal stress response, especially in very young human beings.

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

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

The N-Terminal Neurotensin Fragment, NT1–11, Inhibits Cortisol Secretion by Human Adrenocortical Cells

Neurotensin (NT) modulates corticosteroid secretion from the mammalian adrenal gland. The objective of this study was to investigate the possible involvement of NT in the control of cortisol secretion in the human adrenal gland. In vitro studies were conducted on cultured human adrenocortical cells. This study was conducted in a university research laboratory. Adrenal explants from patients undergoing expanded nephrectomy for kidney cancer were studied. Cortisol secretion from cultured adrenocortical cells was measured. NT1-11, the N-terminal fragment of NT, dose-dependently inhibited basal and ACTH-stimulated cortisol production by human adrenocortical cells in primary culture. In contrast, NT had no influence on cortisol output at concentrations up to 10(-6) m. HPLC and RT-PCR analyses failed to detect any significant amounts of NT and NT mRNA, respectively, in adrenal extracts. Molecular and pharmacological studies were performed to determine the type of NT receptor involved in the corticostatic effect of NT1-11. RT-PCR analysis revealed the expression of NT receptor type (NTR) 3 mRNA but not NTR1 and NTR2 mRNAs in the human adrenal tissue. However, the pharmacological profile of the adrenal NT1-11 receptor was different from that of NTR3, indicating that this receptor type is not involved in the action of NT1-11 on corticosteroidogenesis. Our results indicate that NT1-11 may act as an endocrine factor to inhibit cortisol secretion through activation of a receptor distinct from the classical NTR1, NTR2, and NTR3.

Effects of met-enkephalin on cell proliferation in different models of adrenocortical-cell growth

Met-enkephalin (met-Enk) is an opioid peptide that acts via three main subtypes of receptors referred to as mu (mu)-, delta (delta)- and zeta (zeta)-receptor. While the first two receptor subtypes mediate the classic opioid effects of met-Enk, zeta-receptors are reported to be involved in the non-opioid actions of the peptide, i.e. the inhibitory effect on the cell growth. Despite the fact that met-Enk is known to regulate the function of the hypothalamic-pituitary-adrenal axis acting on both its central and peripheral branches, none is known on the effects of met-Enk on adrenal growth. Hence, we have investigated the effects of met-Enk and its receptor agonists and antagonists on cell proliferation in three different models of rat adrenal growth: i) immature adrenal cortex, ii) regenerating adrenal cortex and iii) primary cultures of adrenocortical cells. In in vivo experiments, rats were given subcutaneous injections of 1 nmol/100 g of the peptides 28, 16 and 4 h before the sacrifice, and proliferative activity was assessed by counting the number of metaphase-arrested cells (after vincristine administration). In in vitro studies, cultured adrenocortical cells were exposed for 48 h to the peptides at a concentration of 10(-6) M, and proliferative activity was measured by the EZ4U method. The blockade of mu- and delta-receptors raised proliferative activity in immature adrenals and decreased it in regenerating glands, and the effects were reversed by mu- and delta-receptor agonists. Naltrexone-induced blockade of all met-Enk receptor subtypes decreased proliferative activity in immature adrenal and raised it in regenerating glands. The exposure to either mu- or delta-receptor agonists and antagonists evoked doubtful or no effects on the proliferative activity of cultured adrenocortical cells. In contrast, met-Enk exerted a marked antiproliferogenic effect that was reversed by naltrexone. Taken together, these findings allow us to draw the following conclusions: i) mu- and delta-receptor activation inhibits the growth of immature adrenals, stimulates adrenal regeneration, and does not affect proliferation of cultured adrenocortical cells; ii) zeta-receptors mediate the growth inhibitory effect of met-Enk on both regenerating adrenals and cultured adrenocortical cells, but unexpectedly their activation stimulates the growth of immature gland; and iii) the effects of mu- and delta-receptor activation in in vivo experiments are probably mediated by extra-adrenal indirect mechanisms.

Triiodothyronin (T3) time and dose dependently stimulates cortisol secretion from bovine adrenocortical cells in primary culture

Triiodothyronin (T3) time and dose dependently stimulates cortisol secretion from bovine adrenocortical cells in primary culture

Role of adrenocorticotropic hormone in the development and maintenance of the adrenal cortical vasculature.

The adrenal cortex is a highly vascularized endocrine tissue. A dense network of blood capillaries centripetally irrigates the adrenal gland, allowing every endocrine cell to be in contact with an endothelial cell. The pituitary hormone ACTH controls the coordinated development of the vasculature and the endocrine tissue mass. This suggests that paracrine secretions between steroidogenic adrenocytes and capillary endothelial cells participate in the control of adrenocortical homeostasis. Besides its effect on the vascular tone of arteries, ACTH induces the expression of the angiogenic cytokine VEGF-A (vascular endothelial growth factor-A) in primary cultures of adrenocortical cells. This growth factor is a specific mitogen for endothelial cells and is likely to mediate the hormonal control of adrenocortical vascularization through a paracrine mechanism. The newly discovered angiogenic factor EG-VEGF (endocrine-gland-derived vascular endothelial growth factor), the expression of which is restricted to endocrine glands and which is preferentially mitogenic for endocrine tissue-derived endothelial cells, is another candidate mediator of great potential interest.

Adenoviral vectors can impair adrenocortical steroidogenesis: Clinical implications for natural infections and gene therapy

Recombinant adenoviral vectors are effective in transferring foreign genes to a variety of cells and tissue types, both in vitro and in vivo. However, during the gene transfer, they may alter the principal function and local environment of transfected cells. Increasing evidence exists for a selective adrenotropism of adenovirus during infections and gene transfer. Therefore, using bovine adrenocortical cells in primary culture, we analyzed the influence of different adenoviral deletion mutants on cell morphology and physiology. Transfection of cells with an E1/E3-deleted adenoviral vector, engineered to express a modified form of the Aequorea victoria green fluorescent protein, was highly efficient, as documented by fluorescent microscopy. Ultrastructural analysis, however, demonstrated nuclear fragmentation and mitochondrial alterations in addition to intranuclear viral particles. Basal secretion of 17-OH-progesterone, 11-deoxycortisol, and cortisol was significantly increased by E1/E3-deleted vectors; yet, the corticotropin-stimulated release of these steroids was decreased. Interestingly, neither purified viral capsids nor E3/E4-deleted adenoviral mutants altered basal and stimulated steroidogenesis of adrenocortical cells. An intact adrenal response is crucial for adaptation to stress and survival. Therefore, the implications of our findings need to be considered in patients with adenoviral infections and those undergoing clinical studies using adenoviral gene transfer. At the same time, the high level of transfection in adrenocortical cells might make appropriately modified adenoviral vectors suitable for gene therapy of adrenocortical carcinomas with poor prognosis.

Identification and characterization of insulin-like growth factor (IGF)-binding protein expression and secretion by adult human adrenocortical cells: differential regulation by IGFs and adrenocorticotropin.

In previous studies we have shown that IGF-II stimulates basal as well as ACTH-induced cortisol secretion from adult human adrenocortical cells more potently than IGF-I, and that both IGFs predominantly stimulate androgen biosynthesis. The steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we identified and characterized IGFBP synthesis in normal adult human adrenocortical cells in primary culture, and investigated the effect of ACTH and recombinant human IGF-I and -II on the regulation of IGFBP expression and secretion. Using RT-PCR, we identified the mRNA of all six high-affinity IGFBPs, in both adrenocortical tissue and monolayer cell cultures of adrenocortical cells. Using Western ligand and immunoblotting and two-dimensional Western ligand blotting we confirmed the secretion of IGFBP-1, -2, -3, -4 and -5 by adrenocortical cells in primary culture. The quantification of IGFBPs indicated that IGFBP-3 accounts for almost half the binding activity in conditioned medium of unstimulated cells (47%), followed by IGFBP-4 (20%), IGFBP-5 (15%), IGFBP-2 (12%) and IGFBP-1 (6%). After treatment with ACTH, the abundance of IGFBP-1 was upregulated significantly 2.6-fold, while IGFBP-3 was induced only slightly (1.3-fold). IGFBP-2, -4 and -5 remained unchanged. In contrast, IGF-I and -II (6.5 nM) predominantly induced the abundance of IGFBP-5 (2- and 1.6-fold respectively) and IGFBP-3 (2- and 1.7-fold respectively), while IGFBP-1, -2 and -4 were unaltered. The induction of IGFBP-1 and -5 by ACTH and IGFs, respectively, was paralleled by an increase in the amount of IGFBP-1 and -5 mRNA in these cells. In conclusion, all six high-affinity IGFBPs are expressed in the adult human adrenal gland, and the presence of at least five high-affinity IGFBPs has been demonstrated in conditioned medium of adult human adrenocortical cells. Furthermore, the expression and secretion of IGFBP-1 is upregulated by ACTH, whereas IGFBP-5 is induced by IGF-I and -II. Together with earlier findings, these results suggest that IGFBPs play an important modulatory role in the regulation of the differentiated adrenocortical function.

Interleukin‐3 and Interleukin‐6 Stimulate Bovine Adrenal Cortisol Secretion Through Different Pathways

Several interleukins have been reported to play a major role in the regulation of steroid secretion at all three levels of the hypothalamic-pituitary-adrenal axis. The objective of this study was to investigate the effect of interleukin-3 (IL-3) and interleukin-6 (IL-6) on cortisol secretion of bovine adrenocortical cells in primary culture under serum-free conditions. Both IL-3 and IL-6 stimulated basal cortisol secretion dose-dependently to a similar extent at a similar time course. After incubation with IL-3 or IL-6 at concentrations of 100 microg/l, a maximum 4.1-fold increase of the cortisol secretion was reached after 12 h (P<0.01). Coincubation of IL-3 and IL-6 (100 microg/l) revealed no significant synergism. To elucidate a possible involvement of arachidonic acid metabolites in the signal transduction, we coincubated IL-3 or IL-6 together with the cyclo-oxygenase inhibitor indometacin or the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Coincubation with indometacin completely abolished the stimulatory effect of IL-6 but had no effect on IL-3 stimulated cortisol secretion. In contrast, specific inhibition of the lipoxygenase system by nordihydroguaiaretic acid blocked IL-3 stimulated steroidogenesis while the effect of IL-6 was not affected. Neither IL-3 nor IL-6 altered cAMP levels significantly, whereas ACTH significantly induced cAMP levels in parallel to its steroidogenic effect. In conclusion, our data indicate that IL-3 and IL-6 stimulate the steroid secretion of bovine adrenocortical cells to a similar extent and with a similar time course. However, the effects of IL-3 and IL-6 are mediated through different, cAMP-independent pathways. While the stimulatory effect of IL-3 seems to be dependent on the lipoxygenase pathway, the effect of IL-6 on adrenocortical cortisol secretion is mediated through the cyclo-oxygenase pathway.

Expression of Three Negative Regulators of CYP17 Gene Transcription in Adrenocortical Cells

The expression of negative regulators of CYP17 gene expression: DAX-1, COUP-TF and N-CoR was investigated in bovine adrenocortical cells in primary culture. The cells were incubated for 6 hours in a defined medium, containing ACTH (10 nM) or forskolin (25 μM). Total RNA was isolated, the concentration of CYP17 gene transcript was determined by hybridization analysis, and polyA RNA was reverse-transcribed to obtain cDNA. Fragments of CYP17, DAX-1, COUP-TF, N-CoR and β-actin cDNA were amplified by PCR and the amplified cDNAs were analyzed by agarose gel electrophoresis followed by densitometry.It was found that DAX-1 and COUP-TF mRNA levels were unaffected by treatment with ACTH or forskolin. On the contrary, both compounds enhanced the level of mRNA encoding N-CoR. Under the same conditions, ACTH or forskolin substantially stimulated CYP17 mRNA accumulation.Our results suggest that in the adrenocortical cells in culture, the expression of DAX1 and COUP-TF is constitutive, whereas N-CoR expression might be inducible.

Adrenal Tumorigenesis Targeted by the Corticotropin-Regulated Promoter of the Aldo-Keto Reductase AKR1B7 Gene in Transgenic Mice

Studies of ACTH functions in adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y1 cells. In order to obtain alternative cell lines with a more differentiated zona fasciculata (ZF) phenotype we used targeted tumorigenesis strategy. We have generated transgenic mice expressing the SV40 T antigen under the control of the ACTH-dependent promoter for the AKR1B7/MVDP gene (aldo-keto reductase 1B7/mouse vas deferens protein), which encodes an enzyme responsible for detoxifying isocaproaldehyde, the product of side-chain cleavage of cholesterol generated by steroidogenesis. Our previous data indicated that in the mouse adrenal, AKR1B7 expression was restricted to the ZF and that a 0.5-kb promoter region was able to target specific adrenal expression in transgenic mice. In situ hybridization analyses indicate that AKR1B7 expression during fetal and post-natal periods paralleled the onset of glucocorticoid synthesis and the development of ZF. In transgenic mice. ACTH control and developmental programming of the CAT gene driven by the 0.5-kb promoter followed endogenous gene regulation. Then transgenic mice harboring the 0.5-kb/SV40 T antigen construct were generated and two founders out of three developed adrenal tumors. Cells derived from the tumor of founder 1 (ATC1) were grown in presence of forskolin to maintain ACTH receptor expression and were tested for ACTH responsiveness by immunocychemistry and northern blot analyses. Even after several passages, the ACTH induced AKR1B7 and P450c11β mRNAs accumulations were similar to that observed in mouse primary adrenocortical cell cultures. Our findings suggest that ATC1 cells have conserved essential features of ZF cells. In order to achieve complete characterization of these cells further analyses are currently performed to investigate their steroidogenic activity.

Relationship of p21WAF1/CIP1/SDI1 to cell proliferation in primary cultures of adrenocortical cells

p21(WAF1/CIP1/SDI1) was originally described as a protein expressed at high levels in senescent human fibroblasts. We have studied the expression of p21 in adrenocortical cells, p21 is not expressed under most circumstances in the intact adrenal gland in vivo, except when the gland is damaged. When human and bovine adrenocortical cells are isolated and placed in both short-term and long-term culture, p21 levels are much higher. These levels did not show a large increase when the cells senesce after long-term proliferation. Thus, these observations raise the question of whether the elevated p21 in primary cultures of adrenocortical cells is caused by damage or whether p21 is elevated because the cells are dividing rather than quiescent, because it has been reported that p21 levels peak in G1 and G2 in dividing cells. In the present experiments on bovine and human adrenocortical cells in primary culture, labeling techniques that correlated nuclear p21 with measures of cell proliferation (bromodeoxyuridine incorporation and nuclear Ki-67 antigen) supported the hypothesis that p21 is associated with cell division and not with damage. This is consistent with recent data showing that, when adrenocortical cells are transplanted into immunodeficient mice, p21 is associated with healthy dividing cells in the transplant, p21 is not a unique marker for senescence, and more studies are required both to clarify its role in cell biology and to determine molecular features which characterize the senescent state of cells both in vitro and in vivo.