Actions of desacetyl-alpha-melanocyte-stimulating hormone on human adrenocortical cells

The responses of human adrenocortical cells to stimulation by ACTH(1-24), desacetyl-alpha-MSH, alpha-MSH and angiotensin II amide have been compared. Both desacetyl-alpha-MSH, thought to be the major form of the peptide in the human pituitary and in circulating plasma, and alpha-MSH caused a significant stimulation of aldosterone, corticosterone and cortisol secretion. Significant stimulation of the production of these steroids was obtained with desacetyl-alpha-MSH at a concentration of 1 nmol/l, while the response to alpha-MSH was considerably less sensitive, with a minimum effective concentration of 0.1 mumol/l. These values compared with minimum effective concentrations of 1 pmol/l for ACTH and 0.1 mumol/l for angiotensin II amide. Although cell types were not separated, it is possible to conclude that none of the peptides showed any specificity for the zona glomerulosa, and in each case the same minimum effective concentration of peptide was required for both aldosterone and cortisol secretion. Yields of steroid obtained under conditions of maximal stimulation by ACTH(1-24), alpha-MSH and desacetyl-alpha-MSH were at least three to five times the basal output of aldosterone, four to eight times that for corticosterone and more than seven to sixteen times that for cortisol. Angiotensin II amide was a relatively poor stimulant with maximal stimulation only 1.5 x basal. In these experiments the minimum effective concentration for desacetyl-alpha-MSH (1 nmol/l) was close to the circulating concentration of desacetyl-alpha-MSH (0.3 nmol/l) in man, and it is thus possible that this peptide may have a physiological role in the control of adrenocortical function.     

Corticotropin-releasing hormone stimulates steroidogenesis in cultured human adrenal cells

The effects of corticotropin releasing hormone (CRH) on steroid production by cultures of human fetal adrenal cells was investigated. We found that CRH, at concentrations that have been reported to exist in human fetal serum, stimulated dehydroepiandrosterone sulfate (DS) and cortisol production by cultured fetal zone and neocortical zone cells. A dose-dependent increase in secretion of both steroids was noted, with the cortisol pathway being preferentially enhanced by CRH at high concentrations. Pretreatment of adrenal cells for 3 days made them more responsive to ACTH stimulation and such effects were dose-dependent also. Inclusion of the antagonist, alpha-helical CRH (9-41) blocked CRH-induced stimulation of DS and cortisol over a broad dose range and also interfered with the augmentation of cortisol secretion noted after ACTH in CRH treated cells. CRH had no effects on adrenal cell proliferation or total cell protein. These studies are suggestive that CRH, either of systemic origin or else produced within the adrenal itself, has the potential to be a modulator of adrenal steroid production in the human.     

Aberrant expression of human luteinizing hormone receptor by adrenocortical cells is sufficient to provoke both hyperplasia and Cushing's syndrome features

CONTEXT: Aberrant expression of LH/human chorionic gonadotropin (hCG) receptor has been suggested in several cases of bilateral macronodular adrenal hyperplasia with Cushing's syndrome. The cortisol production is then directly controlled by endogenous secretion of LH/hCG. However, the direct involvement of this aberrant LH/hCG receptor expression in the development of the hyperplasia has not been demonstrated. Moreover in most cases, whenever investigated, the aberrant expression of LH/hCG receptor has been associated with the ectopic expression of other G protein-coupled receptors such as gastric inhibitory polypeptide, serotonin, or vasopressin receptors. OBJECTIVE: The aim of this study was to explore the action of LH/hCG receptor on the development of adrenal hyperplasia. RESULTS: The ectopic expression of this single nonmutated gene transduced into bovine adrenocortical cells was sufficient to induce not only the aberrant cortisol secretion but also hyperproliferation and benign transformation. The cells were transplanted beneath the kidney capsule of adrenalectomized immunodeficient mice. Only the cells expressing the LH/hCG receptor gene formed an enlarged tissue with a high proliferation rate. The tissue expressing LH/hCG receptor was responsible for elevated plasma cortisol and decreased plasma ACTH levels in transplanted mice. These animals displayed physiological changes similar to those of patients with Cushing's syndrome, including muscle atrophy, thin skin, spleen atrophy, and hyperglycemia. CONCLUSIONS: These results demonstrate that a single genetic event such as the inappropriate expression of the nonmutated LH/hCG receptor gene is sufficient to initiate the phenotypic changes that cause the development of a benign adrenocortical tumor.     

Effects of gonadotropin-releasing hormone and its agonists on prolactin secretion from normal and tumorous pituitary cells

Previous studies on the effect of gonadotropin-releasing hormone (LHRH) agonist on prolactin (PRL) secretion from normal and tumorous pituitary cells have not been conclusive as to the mechanism of action of these agonists. In this study the short-term administration of a LHRH agonist did not affect circulating PRL levels, but depleted the PRL content of the pituitary gland by 24, 49 and 73% after 2, 3 and 4 days, respectively, in normal female rats and by 75% after 4 days in normal male rats. This effect of the agonist could not be attributed to changes in the sex steroid environment: although plasma 17 beta-estradiol concentrations were significantly suppressed in female rats, circulating testosterone levels had not changed yet in the male rats. Interestingly, the pituitary luteinizing hormone (LH) content was depleted already from day 2 of LHRH agonist administration onwards, while the follicle-stimulating hormone (FSH) content of the pituitary glands had not changed even after 4 days. Culture studies with pituitary cells from normal adult male and female rats for 4-7 days did not reveal a direct effect of synthetic LHRH or an agonist on PRL release. Chronic systemic administration of a LHRH agonist greatly inhibited the growth of the transplantable PRL-secreting rat pituitary tumor 7315a in female rats, while circulating PRL levels were also suppressed. However, no direct effect of the LHRH agonist was observed on PRL release from a tumor cell clone, derived from the 7315a tumor, and no LHRH-binding sites were detectable on the tumor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticotropin-releasing hormone receptor type 1 and type 2 mediate differential effects on 15-hydroxy prostaglandin dehydrogenase expression in cultured human chorion trophoblasts

Throughout gestation, the chorion laeve controls the levels of biologically active prostaglandins (PGs) by its high level of nicotinamide adenine dinucleotide-dependent 15-hydroxy PG dehydrogenase (PGDH). In this study, we investigate the effects mediated by CRH receptors on the expression of PGDH in the chorion. We found that both CRHR1 and CRHR2 were localized in cultured chorion trophoblast cells, with CRH-R1alpha, R1beta, R1c, R1e, and R1f and CRHR2beta isoforms identified in these cells. To block the actions of endogenous CRH and its related peptides, cultured chorion trophoblasts were treated with an increasing concentration of alpha-helical CRH 9-41, the nonselective CRH receptor antagonist, which resulted in decreased mRNA and protein expression as well as the activity of PGDH. To investigate the individual role of CRHR1 and CRHR2, cell cultures were treated with the specific CRHR1 antagonist antalarmin and CRHR2 antagonist astressin2B, respectively. The results showed that antalarmin increased whereas astressin2B decreased mRNA and protein expression as well as the activity of PGDH in chorion cells. When the cells were treated with an exclusive CRHR2 agonist, urocortin II, elevated expression and activity of PGDH was exhibited. However, cells treated with either exogenous CRH or urocortin I showed significantly increased PGDH expression, and these effects could be blocked by astressin2B but not by antalarmin. We suggest that, in chorion trophoblast cells, CRHR1 and CRHR2 mediate divergent effects on PGDH expression, and this may provide a precise regulation of PGs levels from chorion to myometrium during pregnancy.     

Corticotropin-releasing hormone system in human adipose tissue

Mounting evidence exists for a role of the CRH system in energy balance, including a direct influence on human adipocytes, the regulation of adipose 11 beta-hydroxysteroid dehydrogenase type 1 activity, and cortisol formation. We characterized the expression of CRH receptors 1 and 2 and CRH-like peptides stresscopin and urocortin in human adipose tissue in comparison with other peripheral tissues, adrenal, and heart. The effect of CRH on CRH receptor and CRH-like peptide expression was analyzed in isolated human adipocytes using quantitative TaqMan PCR. CRH receptors were detectable in fat tissue at mRNA and protein levels. CRH-R2 expression in fat was comparable with its expression in the heart, the organ with the highest CRH-R2 expression known. CRH-R1:CRH-R2 ratio varied according to fat-depot type; whereas CRH-R1 expression was higher in sc fat than in visceral fat, the opposite was true for CRH-R2. Adipose tissue also expressed urocortin and stresscopin, the predominant ligands of peripheral CRH-R2. CRH down-regulated CRH-R1 and CRH-R2 mRNA expression in isolated adipocytes. These data, together with the recently published observation that CRH regulates adipocyte metabolism by down-regulating 11 beta-hydroxysteroid dehydrogenase, indicate that a CRH system exists within human adipose tissue. This system could be implicated in energy homeostasis and in mediating the anorexic effects of CRH at adipose level.     

Normal and tumorous human gonadotropic cells

Gonadotropic cells are scattered in the anterior and tuberal lobes and make up 10 to 20% of the anterior pituitary cells. Having a morphofunctional plasticity, they secrete FSH and LH, most often simultaneously. These hormones are stored together in the granulations. In addition, under the action of regulating factors, especially GnRH, the cells can secrete one hormone or the other, or even the alpha subunit. Gonadotropic adenomas range third in frequency among operated pituitary adenomas (12% in our series). The diagnosis is based on the presence of at least 5% of immunoreactive cells with specific antibodies to gonadotropic hormones. These adenomas are distributed into 3 major types: FSH-LH adenoma, the most frequent one, FSH adenoma and alpha-subunit adenoma. The LH adenoma and the beta FSH and beta LH adenomas are very rare. Tumoral gonadotropic cells lose their morphofunctional differentiation. They also lose, to an extent varying according to the cases, their control mechanisms as well as their capacity of synthesis and excretion of both subunits. The alpha subunit, the oldest one in ontogeny, remains the most often and longest-secreted substance. There is a continuum from the gonadotropic adenoma with high plasma gonadotropins levels to the non-functioning adenoma.     

Effects of angiotensin II and ACTH on normal and tumourous human adrenocortical cells

Isolated adrenocortical cells from 6 patients with a 'normal' zona fasciculata, 4 patients with a 'normal' zona glomerulosa, and tumour cells from 1 adrenocortical adenoma and 1 carcinoma were incubated with and without increasing concentrations of ACTH 1-24 (10(-13) M to 10(-9) M) or Asp1-Ile5-angiotensin II (10(-11) M to 10(-7) M). In 4/5 'normal' cases, cortisol was clearly stimulated by 10(-13) M ACTH. The maximum of the dose-response curve (5-fold stimulation) was reached at 10(-10) M ACTH. Angiotensin II (AII) started to stimulate 'normal' cells at 10(-11) M with a maximum (2-fold stimulation) at 10(-9) M. Aldosterone production by 'normal' cells was less markedly stimulated by ACTH and AII, although the threshold doses for both peptides were similar to those of the cortisol response curves. The cells of the adrenocortical adenoma from a patient with Cushing's syndrome produced large amounts of cortisol and small amounts of aldosterone, both steroids being clearly stimulated by ACTH and AII. The adrenocortical carcinoma cells produced small amounts of cortisol and no aldosterone. Cortisol production responded to ACTH, but not to AII. The results suggest that an activated renin-angiotensin system may stimulate the zona fasciculata, since 10(-11) M AII (= 10 pg AII/ml) is a normal plasma AII concentration on an unrestricted diet. Clinical evidence supporting this thesis is reviewed. However, cortisol production itself will rarely be increased by AII in vivo, since a down-regulation of ACTH would occur.     

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.     

FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells

FLT3 ligand is a hematopoietic growth factor that plays a key role in growth of primitive hematopoietic cells. FLT3 receptor mRNA is found in early hematopoietic progenitors and in human myeloid leukemia blasts. Much less is known about the surface expression of FLT3 receptor on human hematopoietic cells. Using human 125I-FLT3 ligand, we have identified and characterized surface FLT3 receptors on normal and malignant human hematopoietic cells and cell lines. Our results showed that surface display of FLT3 receptor was greatest in fresh myeloid leukemia blast cells and myeloid leukemia cell lines. Erythroleukemic and megakaryocytic leukemia cell lines (n = 5) bound little to no 125I- FLT3 ligand. Scatchard analysis of 125I-FLT3 ligand binding data shows that three myeloid leukemia cell lines, ML-1, AML-193, and HL-60, as well as normal human marrow mononuclear cells, exhibit high affinity FLT3 receptors. Crosslinking of 125I-FLT3 ligand to FLT3 receptors on the surface of ML-1 myeloid leukemia cells indicates that the FLT3 ligand. The rates of FLT3 ligand internalization and degradation were determined by binding 125I-FLT3 ligand to ML-1 cells and acid stripping to distinguish surface bound from internalized ligand. Internalized 125I-FLT3 ligand was detected within 5 minutes after binding to ML-1 cells. In addition, we evaluated the effect of FLT3 ligand on megakaryocytic colony growth and nuclear endoreduplication, alone or in the presence of thrombopoietin. FLT3 ligand did not promote colony forming unit megakaryocyte (CFU-Meg) colony growth or megakaryocyte nuclear maturation, nor did FLT3 ligand augment the effects of thrombopoietin on these measures of megakaryopoiesis. These data indicate that the FLT3 receptor shares several characteristics with the c-kit receptor including dimerization and rapid internalization. However, the more restricted cellular distribution of the FLT3 receptor may target the effects of FLT3 ligand to primitive hematopoietic cells and to myeloid and lymphoid progenitor cells, in contrast to the pleiotropic effects of the c-kit receptor ligand, stem cell factor.     

Corticotropin-releasing hormone stimulates NF-kappaB in human epidermal keratinocytes

Corticotropin-releasing hormone (CRH) has been shown to inhibit proliferation and modulate expression of inflammation markers in the epidermal cells. In the present study we report that CRH also stimulates nuclear factor-kappa B (NF-kappaB) activity. Incubation with CRH of human keratinocytes derived from primary cultures resulted in increased binding of DNA by NF-kappaB. CRH induced translocation of NF-kappaB subunit p65 from the cytoplasm to the nucleus and induced expression of kappaB-driven chloramphenicol acetyltransferase (CAT) reporter gene. NF-kappaB translocation was accompanied by degradation of the inhibitor of NF-kappaB alpha (IkappaB-alpha). Specificity of the CRH effect was demonstrated by the use of CRH-R antagonists antalarmin and alpha-helical CRH [9-41]. CRH-dependent stimulation of NF-kappaB activity is consistent with accumulated data about role of this neuropeptide in the regulation of local epidermal homeostasis.     

Differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells

In terms of regulation of gene expression, gonadotropin-releasing hormone receptor (GnRHR) found in extrapituitary tissues has been suggested to be different from that in the pituitary. In the present study, we examined the molecular basis of this difference using the pituitary alphaT3-1 and ovarian carcinoma OVCAR-3 cells. As a first step, the different expression levels of GnRHR mRNA in the pituitary and ovarian cells were investigated using semi-quantitative RT-PCR. Quantitative analysis showed that the expression level of hGnRHR is a nine-fold higher in primary pituitary tissues than the primary culture of ovarian carcinomas (PCO). In pituitary alphaT3-1 cells, the expression level of hGnRHR was ten-fold higher than ovarian carcinoma OVCAR-3 cells. The possibility of the differential use of various cell-specific promoters in different cells was addressed by transiently transfecting cells with 3'-deletion clones of human GnRHR promoter. Sequential deletion of the 5'-flanking region of the gene revealed the use of two putative promoters, designated PR1 (-771 to -557) and PR2 (-1351 to -1022), and a negative control region (-1022 to -771), in the pituitary and ovarian cells. The same promoters appeared to be utilized for driving the basal promoter activities in both alphaT3-1 and OVCAR-3 cells, suggesting that there is no cell-specific promoter usage for the human GnRHR gene. Alternatively, the involvement of different regulatory protein factors was investigated using electrophoretic gel mobility shift assays. When end-labeled PR1 was used as a probe, two unique shifted complexes were identified in OVCAR-3 cells when compared to alphaT3-1 cells. One unique protein-DNA complex was observed in alphaT3-1 cells compared to OVCAR-3 cells when incubated with end-labeled PR2 as a probe. These DNA-protein complexes appeared to be specific, as they competed with excess amount of unlabelled competitor PR1 and PR2, respectively. In summary, it is unlikely that the use of a cell-specific promoter contributes to the different characteristics of ovarian GnRHR. Our study demonstrates that one mechanism by which cell-specific expression of human GnRHR is achieved is through the binding of distinct and/or combinations of cell-specific regulatory factors to various promoter elements in the 5'-flanking region of the gene.     

Corticotropin-releasing hormone and its structurally related urocortin are synthesized and secreted by human mast cells

Stress activates the hypothalamic-pituitary-adrenal axis through CRH, leading to production of glucocorticoids that down-regulate immune responses. However, acute stress also has proinflammatory effects. We previously showed that restraint stress, as well as CRH and its structurally related urocortin (Ucn), could activate mast cells and trigger mast cell-dependent vascular permeability. Here we show for the first time that human cord blood-derived cultured mast cells (hCBMC) at 10 wk, but not at 2 wk, are immunocytochemically positive for CRH and Ucn; human leukemic mast cells are weakly positive for both peptides. The ability of these mast cells to synthesize CRH and Ucn was confirmed by showing mRNA expression with RT-PCR. hCBMC (8-14 wk) synthesize and store 1-10 ng/106 cells (10-20 microg/g) of both CRH and Ucn detected by ELISA of cell homogenates. Stimulation of IgE-sensitized hCBMC with anti-IgE results in secretion of most CRH and Ucn. These findings indicate that mast cells are not only the target, but also a potential source of CRH and Ucn that could have both autocrine and paracrine functions, especially in allergic inflammatory disorders exacerbated by stress.     

Gonadotropin-releasing hormone and its receptor in normal and malignant cells

Gonadotropin-releasing hormone (GnRH) is the hypothalamic factor that mediates reproductive competence. Intermittent GnRH secretion from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of the gonadotropins, LH and FSH. LH and FSH then stimulate sex steroid hormone synthesis and gametogenesis in the gonads to ensure reproductive competence. The pituitary requires pulsatile stimulation by GnRH to synthesize and release the gonadotropins LH and FSH. Clinically, native GnRH is used in a pump delivery system to create an episodic delivery pattern to restore hormonal defects in patients with hypogonadotropic hypogonadism. Agonists of GnRH are delivered in a continuous mode to turn off reproductive function by inhibiting gonadotropin production, thus lowering sex steroid production, resulting in medical castration. They have been used in endocrine disorders such as precocious puberty, endometriosis and leiomyomata, but are also studied extensively in hormone-dependent malignancies. The detection of GnRH and its receptor in other tissues, including the breast, ovary, endometrium, placenta and prostate suggested that GnRH agonists and antagonists may also have direct actions at peripheral targets. This paper reviews the current data concerning differential control of GnRH and GnRH receptor expression and signaling in the hypothalamic-pituitary axis and extrapituitary tissues. Using these data as a backdrop, we then review the literature about the action of GnRH in cancer cells, the utility of GnRH analogs in various malignancies and then update the research in novel therapies targeted to the GnRH receptor in cancer cells to promote anti-proliferative effects and control of tumor burden.     

Corticotropin-releasing hormone directly stimulates cortisol and the cortisol biosynthetic pathway in human fetal adrenal cells

Near term the human fetal adrenals (HFAs) initiate production of cortisol, which promotes organ maturation and acts to increase placental CRH biosynthesis. The objective of the present study was to determine whether CRH directly stimulates both cortisol production and expression of the steroidogenic enzymes in HFA-definitive zone cells. CRH stimulated the production of cortisol in a time- and dose-dependent manner, with an effective concentration of as low as 0.01 nm. In real-time RT-PCR experiments, CRH treatment increased the mRNA levels of steroidogenic acute regulatory protein and each of the enzymes needed to produce cortisol. CRH induced 3beta-hydroxysteroid dehydrogenase type II (HSD3B2) by 34-fold, 21-hydroxylase (CYP21) by 55-fold, and 11beta-hydroxylase by 41-fold. Induction of steroidogenic acute regulatory protein, cholesterol side chain cleavage (CYP11A), and 17alpha-hydroxylase (CYP17) mRNA by CRH was 6-, 4-, and 6-fold, respectively. We also demonstrated that submaximal concentrations of CRH (30 pm) and ACTH (30 pm) that are seen in fetal circulation were additive on cortisol biosynthesis and 3beta-hydroxysteroid dehydrogenase type II mRNA induction. We suggest that CRH may play an important role in the late gestational rise in cortisol secretion from the HFAs, which may serve to augment placental CRH production and therefore participate in the endocrine cascade that is involved in fetal organ maturation and potentially in the timing of human parturition.     

Expression of growth hormone-releasing hormone receptor splicing variants in human primary adrenocortical tumours

OBJECTIVE: Several splice variants (SVs) of GHRH receptor (GHRH-R) have been identified in various human cancers through which GHRH antagonists may exert their IGF-II-mediated antiproliferative action. Because the overexpression of the IGF-II gene is a frequent feature of adrenal carcinoma, we searched for the presence of GHRH-R SVs in these tumours. METHODS AND RESULTS: The expression of GHRH-R SVs was assessed by nested PCR in 45 human adrenocortical tumours. We have amplified 720-, 566- and 335-bp PCR products only in carcinomas. Their sequence revealed three open reading frames, corresponding to SV1, SV2 and SV4 of GHRH-R. SV2 was detected in five of 24 cancers examined, whereas the incidence of SV1 and SV4 was lower. Their simultaneous expression was observed in one carcinoma. No PCR products for SV3 or wild-type GHRH-R were found in carcinomas; mRNA for wild-type GHRH-R or SVs of GHRH-R were not observed either in adenomas or in normal adrenal or in NCI-H295R cells. Interestingly, all carcinomas which expressed SVs were also positive for the presence of GHRH mRNA. CONCLUSION: This is the first time that the expression of splice variants of GHRH-R has been demonstrated in human adrenal carcinoma. This study raises the possibility that splice variants might play a role in adrenal carcinogenesis and might offer the possibility for new therapeutic strategies at least in a subgroup of adrenal carcinomas.