Murine Adrenal Cortex Research Articles

Comparative Effect of ACTH and Related Peptides on Proliferation and Growth of Rat Adrenal Gland.

Pro-opiomelanocortin (POMC) is a polypeptide precursor known to yield biologically active peptides related to a range of functions. These active peptides include the adrenocorticotropic hormone (ACTH), which is essential for maintenance of adrenal growth and steroidogenesis, and the alpha-melanocyte stimulation hormone, which plays a key role in energy homeostasis. However, the role of the highly conserved N-terminal region of POMC peptide fragments has begun to be unraveled only recently. Here, we review the cascade of events involved in regulation of proliferation and growth of murine adrenal cortex triggered by ACTH and other POMC-derived peptides. Key findings regarding signaling pathways and modulation of genes and proteins required for the regulation of adrenal growth are summarized. We have outlined the known mechanisms as well as future challenges for research on the regulation of adrenal proliferation and growth triggered by these peptides.

Targeting the annexin 1-formyl peptide receptor 2/ALX pathway affords protection against bacterial LPS-induced pathologic changes in the murine adrenal cortex.

Hypothalamo-pituitary-adrenocortical dysfunction contributes to morbidity and mortality in a high proportion of patients with sepsis. Here, we provide new insights into the underlying adrenal pathology. Using a murine model of endotoxemia (LPS injection), we demonstrate that adrenal insufficiency is triggered early in the disease. LPS induced a local inflammatory response in the adrenal gland within 4 hours of administration, coupled with increased expression of mRNAs for annexin A1 (AnxA1) and the formyl peptide receptors [(Fprs) 1, 2, and 3], a loss of lipid droplets in cortical cells (index of availability of cholesterol, the substrate for steroidogenesis), and a failure to mount a steroidogenic response to ACTH. Deletion of AnxA1 or Fpr2/3 in mice prevented lipid droplet loss, but not leukocyte infiltration. LPS increased adrenal myeloid differentiation primary response gene 88 and TLR2 mRNA expression, but not lymphocyte antigen 96 or TLR4. By contrast, neutrophil depletion prevented leukocyte infiltration and increased AnxA1, Fpr1, and Fpr3 mRNAs but had no impact on lipid droplet loss. Our novel data demonstrate that AnxA1 and Fpr2 have a critical role in the manifestation of adrenal insufficiency in this model, through regulation of cholesterol ester storage, suggesting that pharmacologic interventions targeting the AnxA1/FPR/ALX pathway may provide a new approach for the maintenance of adrenal steroidogenesis in sepsis.

Endogenous biotin as a marker of adrenocortical cells with steroidogenic potential

Interpretation of adrenal cortex phenotypes is greatly facilitated by simultaneous examination of multiple markers at single cell resolution. However, the availability of multiple appropriate antibodies can be rate limiting, while their cognate antigens are often subject to variable accessibility. Specific markers not subject to these constraints thus have obvious utility. Here we report that endogenous biotin, when detected in fixed, frozen tissue sections using fluorescent streptavidin, is a specific marker of apparently all cells with steroidogenic potential in the murine adrenal cortex. While streptavidin stains presteroidogenic and mature cortical cells, it does not label the adrenal capsule, medulla or vascular endothelium. Developmental profiles reveal adrenal endogenous biotin labeling from E13.5 through adulthood. Comparisons with zonal markers, hypothalamic–pituitary–adrenal (HPA) axis-remodeled tissue, transgenic Shh-nLacZ or Gli1-nLacZ animals, and Shh mutant embryos further demonstrate the utility of this approach. Fluorescent streptavidin applied using a simple one-step staining protocol thus provides a potent counterstain for use in adrenal analyses.

Localization of Sonic hedgehog secreting and receiving cells in the developing and adult rat adrenal cortex

Sonic hedgehog signaling was recently demonstrated to play an important role in murine adrenal cortex development. The organization of the rat adrenal differs from that of the mouse, with the zona glomerulosa and zona fasciculata separated by an undifferentiated zone in the rat, but not in the mouse. In the present study we aimed to determine the mRNA expression patterns of Sonic hedgehog and the hedgehog signaling pathway components Patched-1 and Gli1 in the developing and adult rat adrenal. Sonic hedgehog expression was detected at the periphery of the cortex in cells lacking CYP11B1 and CYP11B2 expression, while signal-receiving cells were localized in the overlying capsule mesenchyme. Using combined in situ hybridization and immunohistochemistry we found that the cells expressing Sonic hedgehog lie between the CYP11B2 and CYP11B1 layers, and thus Sonic hedgehog expression defines one cell population of the undifferentiated zone.

CGMP‐dependent protein kinase II and aldosterone secretion

ACTH-stimulated aldosterone secretion can be inhibited by atrio-natriuretic peptide/cGMP. The mechanism behind this modulation has been reported to involve cGMP-dependent activation of phosphodiesterase 2 (PDE2) and hydrolysis of cAMP. Recently it was reported that activation of cGMP-dependent protein kinase II (cGKII) stimulated aldosterone secretion in rat zona glomerulosa cells. The zona glomerulosa of the murine adrenal cortex expresses cGKII and PDE2. We used mice with a homozygous inactivation of the cGKII gene to investigate in vivo the potential role of this kinase in aldosterone secretion. Basal plasma renin and aldosterone levels were similar in wild-type and cGKII(-/-) mice. In vivo injection of atrio-natriuretic peptide decreased ACTH-stimulated aldosterone secretion in wild-type mice, but had no effect in cGKII-deficient mice. These results support the view that cGKII modulates aldosterone secretion in the murine adrenal cortex.

Immunocytochemical demonstration of day/night changes of clock gene protein levels in the murine adrenal gland: differences between melatonin‐proficient (C3H) and melatonin‐deficient (C57BL) mice

The circadian system comprises several peripheral oscillators and a central rhythm generator that, in mammals, is located in the suprachiasmatic nucleus of the hypothalamus. Expression of clock genes is a characteristic feature of the central rhythm generator and the peripheral oscillators. With regard to the rhythmic production of glucocorticoids, the adrenal gland can be considered as peripheral oscillator, but little is known about clock gene expression in this tissue. Therefore, the present study investigates the levels of three clock gene proteins PER1, BMAL1 and CRY2 in the murine adrenal cortex and medulla at seven different time points of a 12-hr light/12-hr dark cycle. To determine a potential role of melatonin we compared the patterns of clock gene proteins in the adrenal gland of melatonin-proficient mice (C3H) with those of melatonin-deficient mice (C57BL). In C3H mice, both, the adrenal cortex and medulla displayed day/night variation in PER1-, CRY2- and BMAL1-protein levels. PER1 and CRY2 peaked in the middle of the light phase, whereas BMAL1 peaked in the dark phase. This pattern was also observed in the adrenal medulla of C57BL, but in the adrenal cortex of C57BL clock gene protein levels did not change with time and were consistently lower than in C3H mice. These results support the hypothesis that the adrenal gland is a peripheral oscillator and raise the possibility that melatonin may be involved in the control of clock gene protein levels in the adrenal cortex of mice.

Squamous cell carcinoma–related oncogene is highly expressed in developing, normal, and adenomatous adrenal tissue but not in aggressive adrenocortical carcinomas

Our previous work has demonstrated squamous cell carcinoma-related oncogene (SCCRO) expression in adult murine adrenocortical tissue. The aim of this study was to assess patterns of SCCRO expression in the embryonic murine adrenal gland, and in normal and neoplastic human adrenocortical tissues in order to determine its role as a marker of differentiation in adrenocortical development and neoplastic progression. Murine embryos were procured at developmental stages E8 to E18. A tissue microarray was constructed containing 38 normal, 39 adenomatous, and 87 carcinomatous human adrenocortical specimens. Immunohistochemistry for SCCRO was performed and its expression was graded in suitable tissues. SCCRO expression was detected in the murine adrenal cortex as early as E15 and persisted into the postnatal period. High-level SCCRO expression was identified in 94% of normal (32/34) and adenomatous (29/31) adrenocortical specimens but in only 63% (45/72) of adrenocortical carcinoma (ACC) specimens ( P = .001). Loss of SCCRO expression in primary ACC (13/34 (34%)) correlated with advanced stage ( P = .06), presence of M1 disease at presentation ( P = .03), and worse overall survival ( P = .006). SCCRO appears to be a marker of adrenocortical differentiation in both murine and human systems. SCCRO expression may be useful in distinguishing adrenocortical adenomas from ACC. Moreover, loss of SCCRO expression in primary ACC is associated with worse outcome and may be a marker of progressive dedifferentiation in these tumors.

Role of the Inhibin/Activin System and Luteinizing Hormone in Adrenocortical Tumorigenesis

Adrenal masses are one of the most common endocrine tumors diagnosed. Although most adrenal tumors are inactive adenomas, a considerable proportion is associated with hormonal hyperfunction and/or malignancy. The adrenocortical carcinoma (ACC) is a rare but highly malignant tumor. Most ACCs in adults are diagnosed in an advanced tumor stage limiting therapeutic options. Accordingly, despite some progress in diagnostic and therapeutic approaches, the overall survival rate of patients with ACC remains poor. However, the prerequisite for the development of new diagnostic tools and therapeutic options in the management of patients with ACC is the elucidation of the molecular pathogenesis of adrenal tumorigenesis. Although our understanding of adrenal tumor biology has increased substantially over the last decades, the regulation of many molecular pathways involved in adrenocortical growth and differentiation awaits further elucidation. Luteinizing hormone (LH) and activin have only recently emerged as hormones likely to play opposite roles in adrenocortical hormone secretion and cellular proliferation. Recent evidence from studies on human surgical tumor sample expression and detailed characterization of murine adrenal tumor models suggests stimulatory effects of LH on adrenocortical growth and function. On the contrary, activin, which plays a critical role as a paracrine and autocrine factor regulating cellular growth and differentiation, has been demonstrated to induce apoptosis and suppress proliferation in the human and murine adrenal cortex. In this review, we will summarize molecular and functional aspects of adrenal tumorigenesis and highlight some prospects for future clinical applications.

Irreversible Binding and Adrenocorticolytic Activity of the DDT Metabolite 3-Methylsulfonyl-DDE Examined in Tissue-Slice Culture

The persistent adrenocorticolytic DDT metabolite 3-methylsulfonyl-DDE (MeSO(2)-DDE) was originally identified in Baltic grey seals, a population suffering from adrenocortical hyperplasia. In mice, MeSO(2)-DDE induces mitochondrial degeneration and cellular necrosis in the adrenal zona fasciculata. In this study, we used precision-cut tissue slice culture to examine local CYP11B1-catalyzed irreversible binding of MeSO(2)-DDE in the murine adrenal cortex. We also examined effects on steroid hormone secretion, histology, and ultrastructure. As determined by microautoradiography, selective binding occurred in zona fasciculata of slices exposed to MeSO(2)-[(14)C]-DDE. Quantification of binding by phosphorautoradiography revealed a 3-fold reduction of binding in slices co-exposed to the CYP11B1 inhibitor metyrapone. As measured by HPLC, corticosterone and 11-deoxycorticosterone secretion to the medium increased linearly for at least 24 hr. Addition of the ACTH analog tetracosactide caused an 8-fold increase in corticosterone secretion. Addition of metyrapone reduced corticosterone secretion 4-fold. Exposure of slices to MeSO(2)-DDE (50 microM) reduced the rate of corticosterone secretion by 90% after 24 hr of incubation. As determined by electron microscopy, vacuolated mitochondria were present in zona fasciculata of slices exposed to MeSO(2)-DDE (50 microM) for 24 hr. Our findings show that all effects of MeSO(2)-DDE previously reported in vivo could be reproduced in adrenal slice culture ex vivo. This test system allows analysis of zone-specific irreversible binding and effects on steroid hormone secretion and target cell ultrastructure. We propose adrenal slice culture as a simple ex vivo test system with which to examine the adrenocorticolytic activity of xenobiotics in human and wild animal tissue.

Irreversible binding and adrenocorticolytic activity of the DDT metabolite 3-methylsulfonyl-DDE examined in tissue-slice culture.

The persistent adrenocorticolytic DDT metabolite 3-methylsulfonyl-DDE (MeSO(2)-DDE) was originally identified in Baltic grey seals, a population suffering from adrenocortical hyperplasia. In mice, MeSO(2)-DDE induces mitochondrial degeneration and cellular necrosis in the adrenal zona fasciculata. In this study, we used precision-cut tissue slice culture to examine local CYP11B1-catalyzed irreversible binding of MeSO(2)-DDE in the murine adrenal cortex. We also examined effects on steroid hormone secretion, histology, and ultrastructure. As determined by microautoradiography, selective binding occurred in zona fasciculata of slices exposed to MeSO(2)-[(14)C]-DDE. Quantification of binding by phosphorautoradiography revealed a 3-fold reduction of binding in slices co-exposed to the CYP11B1 inhibitor metyrapone. As measured by HPLC, corticosterone and 11-deoxycorticosterone secretion to the medium increased linearly for at least 24 hr. Addition of the ACTH analog tetracosactide caused an 8-fold increase in corticosterone secretion. Addition of metyrapone reduced corticosterone secretion 4-fold. Exposure of slices to MeSO(2)-DDE (50 microM) reduced the rate of corticosterone secretion by 90% after 24 hr of incubation. As determined by electron microscopy, vacuolated mitochondria were present in zona fasciculata of slices exposed to MeSO(2)-DDE (50 microM) for 24 hr. Our findings show that all effects of MeSO(2)-DDE previously reported in vivo could be reproduced in adrenal slice culture ex vivo. This test system allows analysis of zone-specific irreversible binding and effects on steroid hormone secretion and target cell ultrastructure. We propose adrenal slice culture as a simple ex vivo test system with which to examine the adrenocorticolytic activity of xenobiotics in human and wild animal tissue.

Multiple forms of cyclic nucleotide phosphodiesterase in a murine adrenal cortex cell line (Y-1)

Murine adrenal cortex tumor Y-1 cells contained both soluble and particulate forms of cyclic nucleotide phosphodiesterase (3′,5′-cyclic AMP 5′-nucleotide hydrolase, EC 3.1.4.17). The soluble forms of the enzyme comprised 80% of total cellular phosphodiesterase activity. The soluble enzyme(s) hydrolyzed both cyclic AMP and cyclic GMP, with apparent K m values of 125 and 30 μ m, respectively. Soluble cyclic AMP phosphodiesterase showed marked inhibition by the calcium chelator, ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid (EGTA), and the anticalmodulin drugs, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium. No alteration in soluble cyclic GMP phosphodiesterase activity was observed when cyclic AMP was added to the assay. Resolution of the soluble enzymatic activity by DEAE-cellulose chromatography in the presence of calcium showed two peaks of phosphodiesterase activity. Further purification of one of these peaks on DEAE-cellulose in the presence of EGTA yielded a phosphodiesterase activity peak that was stimulated fivefold by calmodulin. The particulate form of the enzyme hydrolyzed both cyclic AMP and cyclic GMP; the apparent K m values for these substrates were similar (90 and 100 μ m, respectively). Hydrolysis of cyclic GMP by the particulate enzyme was inhibited by cyclic AMP in a concentration-dependent manner with an apparent half-maximal inhibitory concentration of 100 μ m. The particulate form of phosphodiesterase was not inhibited by EGTA or anticalmodulin drugs.

Sterol metabolism VIII. Conversion of cholesterol 20α-hydroperoxide to 20α, 21- and 20 α,22 R-dihydroxycholesterol by adrenal cortex mitochondria

1. 1. Cholesterol 20α-hydroperoxide incubated with bovine and murine adrenal cortex mitochondrial enzyme preparations yields 20α, 22 R-dihydroxycholesterol and 20α, 21-dihydroxycholesterol, isolated as crystalline material, both sterols being identified by direct comparison of physical properties with authentic reference samples. 2. 2. A chemical synthesis of the previously undescribed 20α, 21-dihydroxycholesterol from 3β, 21-diacetoxypregn-5-en-20-one is described. 3. 3. The enzyme involved could be solubilized from acetone-dried mitochondria by sonic treatment, giving a specific actifity of 2–5 nmoles/min per mg protein (at 30°). Preparations of partially purified cytochrome P-450 derived from these mitochondrial extracts were also enriched in the sought enzyme. 4. 4. The transformations of cholesterol 20α-hydroperoxide to 20α, 21- and 20α, 22 R-dihydroxycholesterols do not occur with heat inactivated preparations nor with preparations subjected to prolonged sonication. 5. 5. The transformations do not require molecular oxygen or added NADPH. 6. 6. Decomposition of cholesterol 20α-hydroperoxide to pregnenolone, androst-5-ene-3β, 17β-diol, and androst-5-en-3β-ol is catalyzed slightly by protein, but at a much lower reaction velocity than the enzymic transformation, thus becoming noticeable only after prolonged incubations. 7. 7. A speculative proposal is made that the adrenal cortical cleavage of the isohexyl moiety of the cholesterol side-chain may proceed via 20α-hydroperoxide formation followed by enzymic rearrangement to 20α, 22 R-dihydroxycholesterol, which then undergoes bond cleavage between C-20 and C-22 to yield pregnenolone.

Adrenal cells in tissue culture. IV. Use of an inhibitor of steroid synthesis for the study of ACTH action.

This report describes the use of aminoglutethimide (AG), an inhibitor of steroid synthesis, for the study of some effects of ACTH on the metabolism of functional monolayer cultures of murine adrenal cortex tumors. At concentrations in the medium of 10 μg/ml or higher, the drug specifically blocks steroid biosynthesis from cholesterol. At concentrations over 50 μg/ml it is also a competitive inhibitor of 11β-hydroxylation. Its action is immediate and reversible. Concentrations in excess of 100 μg/ml have no adverse effect on cell growth, RNA and protein synthesis, glycolysis or cholesterol synthesis. Four effects of ACTH which we have previously described were investigated: the steroidogenic response to ACTH, the augmentation of steroidogenesis by prolonged stimulation with ACTH, the increased synthesis of cholesterol from acetate, and the stimulation of glycolytic activity. At concentrations over 10 μg/ml, both control and stimulated steroidogenic activity is blocked. At lower concentrations, the ratio of...