Estudio de estabilidad de las concentraciones de cortisol, 25-hidroxivitamina D y corticotropina (ACTH) / Study of the stability of Cortisol, 25 OH vitamin D and adrenocorticotrophic hormone (ACTH) in plasma

Objetivo. Evaluar la estabilidad de las concentraciones de cortisol y 25-hidroxivitamina D (25OH-vit D) en suero y de corticotropina (ACTH) en plasma en función del tiempo de almacenamiento en las condiciones habituales de trabajo para estas muestras y comparar dos protocolos diferentes de centrifugación. Material y método. Se obtuvieron muestras de 25 voluntarios sanos (5 hombres y 20 mujeres) para el estudio de cortisol y 25OH-vit D. Para el estudio de ACTH se utilizaron muestras de 15 de estos voluntarios sanos. Las muestras se procesaron en el analizador Liaison® DiaSorin en tiempos comprendidos entre 90 minutos y 7 días desde la toma de la muestra hasta la obtención del resultado. Resultados. El cortisol y 25OH-vit D se mantuvieron estables a lo largo de todo el estudio en las condiciones especificadas. La ACTH se mantuvo estable hasta 8 horas a 4°C. En ninguna de las tres magnitudes se observaron diferencias significativas entre los resultados obtenidos en muestras centrifugadas tras su obtención o inmediatamente antes de su procesamiento hasta 6 horas después de la obtención de la muestra. Conclusiones. Las condiciones habituales de manejo de las muestras, 8 horas a temperatura ambiente con o sin centrifugación previa y posteriormente almacenadas a 4°C durante una semana, no comprometen la estabilidad en el caso de cortisol y 25OH-vit D. En el caso de ACTH es preciso congelar las muestras tras su obtención si no van a ser procesadas en un tiempo inferior a 8 horas (AU)

Objective. To asses the stability of cortisol, and 25 (OH)-Vitamin D concentrations in serum, and adrenocorticotrophic hormone (ACTH) in plasma samples, according to the storage time under normal working conditions for these samples, and to compare two different centrifugation protocols. Material and method. The samples were obtained from 25 healthy volunteers (5 men and 20 women) for the study of cortisol and 25 OH-vit D. For the ACTH only 15 of the 25 samples were used. The samples were processed in the Liaison® DiaSorin analyser at times between 90minutes and 7 days from the time the sample was taken until the result was obtained. Results. Cortisol and 25 OH-Vit D remained stable during the time of the study under the specified conditions. ACTH remained stable only until the 8th hour at 4° C. There were no significant differences in relation to the time of centrifugation. Conclusions. The regular management conditions of the samples, 8hours at room temperature with or without previous centrifugation and later storage at 4° C for a week does not jeopardize the stability of cortisol and 25 OH-vit D. In the case of ACTH the samples have to be frozen after they are obtained if they are not going to be processed in less than 8 hours (AU)

Leptin alters adrenal responsiveness by decreasing expression of ACTH-R, StAR, and P450c21 in hypoxemic fetal sheep.

The late gestation increase in adrenal responsiveness to adrenocorticotropin (ACTH) is dependent upon the upregulation of the ACTH receptor (ACTH-R), steroidogenic acute regulatory protein (StAR), and steroidogenic enzymes in the fetal adrenal. Long-term hypoxia decreases the expression of these and adrenal responsiveness to ACTH in vivo. Leptin, an adipocyte-derived hormone which attenuates the peripartum increase in fetal plasma cortisol is elevated in hypoxic fetuses. Therefore, we hypothesized that increases in plasma leptin will inhibit the expression of the ACTH-R, StAR, and steroidogenic enzymes and attenuate adrenal responsiveness in hypoxic fetuses. Spontaneously hypoxemic fetal sheep (132 days of gestation, PO(2) ≈ 15 mm Hg) were infused with recombinant human leptin (n = 8) or saline (n = 7) for 96 hours. An ACTH challenge was performed at 72 hours of infusion to assess adrenal responsiveness. Plasma cortisol and ACTH were measured daily and adrenals were collected after 96 hours infusion for messenger RNA (mRNA) and protein expression measurement. Plasma cortisol concentrations were lower in leptin- compared with saline-infused fetuses (14.8 ± 3.2 vs 42.3 ± 9.6 ng/mL, P < .05), as was the cortisol:ACTH ratio (0.9 ± 0.074 vs 46 ± 1.49, P < .05). Increases in cortisol concentrations were blunted in the leptin-treated group after ACTH(1-24) challenge (F = 12.2, P < .0001). Adrenal ACTH-R, StAR, and P450c21 expression levels were reduced in leptin-treated fetuses (P < .05), whereas the expression of Ob-Ra and Ob-Rb leptin receptor isoforms remained unchanged. Our results indicate that leptin blunts adrenal responsiveness in the late gestation hypoxemic fetus, and this effect appears mediated by decreased adrenal ACTH-R, StAR, and P450c21 expression.

Endotoxin tolerance of adrenal gland: attenuation of corticosterone production in response to lipopolysaccharide and adrenocorticotropic hormone.

OBJECTIVES: Reversible adrenal insufficiency frequently has been diagnosed in critically ill patients with sepsis who have either low basal cortisol levels or low cortisol responses to adrenocorticotropic hormone (ACTH) stimulation. It is generally accepted that a phenomenon called "endotoxin tolerance" contributes to immunosuppression during sepsis. The present study was to investigate whether endotoxin tolerance occurs in the adrenal gland, leading to hyporesponsiveness of adrenal gland during sepsis. DESIGN: Controlled laboratory experiment. SETTING: University research laboratory. SUBJECTS: Sprague-Dawley male rats 200-250 g and primary isolated adrenal fasciculata-reticularis cells. INTERVENTIONS: Rats received intra-arterial injection of purified lipopolysaccharide (0.5 mg/kg) through indwelling femoral arterial catheters, and 24 hrs later the adrenocortical sensitivity to exogenous ACTH (10 ng/kg) was detected. Primary fasciculata-reticularis cells were pretreated with lipopolysaccharide at 0.1-100 ng/mL or with ACTH at 0.01-10 ng/mL and then challenged, in fresh media, with 1 µg/mL lipopolysaccharide or 10 ng/mL ACTH. MEASUREMENTS AND MAIN RESULTS: Toll-like receptor 4 was expressed in adrenal gland and primary fasciculata-reticularis cells. Plasma corticosterone response to ACTH was decreased in rats receiving preinjection of lipopolysaccharide. Lipopolysaccharide pretreatment caused a significant decrease in corticosterone production in response to subsequent ACTH and lipopolysaccharide stimulation in primary fasciculata-reticularis cells. Lipopolysaccharide pretreatment inhibited ACTH- and lipopolysaccharide-induced expression of steroid metabolizing enzymes. Lipopolysaccharide significantly decreased Toll-like receptor 4 and ACTH receptor expression. CONCLUSIONS: Pre-exposure to lipopolysaccharide resulted in hyporesponsiveness to ACTH stimulation in rats. In vitro, lipopolysaccharide pretreatment impaired corticosterone production of fasciculata-reticularis cells in response to ACTH and lipopolysaccharide, which was associated with decreased expression of synthetic enzymes required for corticosterone production. Our results indicate that endotoxin tolerance of adrenal gland is one of the mechanisms for adrenocortical insufficiency during sepsis.

Developmental changes in adrenocorticotrophin (ACTH)-induced expression of ACTH receptor and steroid acute regulatory protein mRNA in ovine fetal adrenal cells.

OBJECTIVES: Adrenocorticotrophin (ACTH) plays an important role in mediating the increase in cortisol output in the late gestation sheep fetus. At the adrenal itself, heightened expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) appear to be important parallel changes. This study examined how ACTH affects ACTH-R and StAR mRNA expression, and cortisol production in adrenocortical cells isolated from fetuses of varying gestational age (dGA). We hypothesized that the ability of ACTH to stimulate its receptor and StAR mRNA expression would be greater close to term than earlier in development. METHODS: Adrenals were obtained from fetuses (100-105, 120, or 135-139 dGA), and the cortical cells were dispersed. After 3 days of culture, cells were stimulated with ACTH(1-24), and the cells and medium were collected at different time points (0, 3, 6, 9, 12, and 24 hours) for measurement of cortisol and ACTH-R and StAR mRNA. RESULTS: Cortisol secretion was increased after ACTH treatment in all three age cohorts. Cells from the 135-139 dGA group secreted the most cortisol, followed by the 100-105 and then the 120 dGA groups (P <.05). ACTH-R mRNA levels before and after ACTH were higher in the late compared to both earlier groups. StAR mRNA levels before and after ACTH were higher in the 100-105 and 135 than in the 120 dGA group. The time to peak ACTH-R mRNA response was age-dependent, with the 100-105 dGA cells taking longer to attain maximum levels. Maximal StAR mRNA levels were not age-related. CONCLUSION: The data suggest that ACTH-R and StAR are indeed key mediators of fetal adrenocortical responsiveness, and that ACTH is able to up-regulate responsiveness, and hence cortisol production, by increasing their expression.

Evidence for direct actions of melanocortin peptides on bone metabolism.

Expression of melanocortin-4 receptor (MC4R) mRNA in developing rat limb buds, teeth, and skull bone first indicated a possible role for MC4R in bone metabolism. We therefore investigated whether MC4R mRNA was expressed in the rat osteosarcoma UMR106.06 cell line and in primary rat osteoblast cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot analysis, and ribonuclease protection assay (RPA) were used to demonstrate MC4R mRNA expression in UMR106.06 and primary osteoblast cells. MC4R mRNA was found to be localized to the periosteum of mouse bone using in situ hybridization. We also used RT-PCR and rat specific MC2R and MC5R oligonucleotides to amplify the correct size DNA fragments for these melanocortin receptors from rat primary osteoblasts. In conclusion, melanocortin receptor expression in mouse periosteum and rat osteoblasts suggests a direct role for POMC derived peptides in bone development and bone metabolism.

Steroidogenic factor 1 differentially regulates basal and inducible steroidogenic gene expression and steroid synthesis in human adrenocortical H295R cells.

The significance of steroidogenic factor 1 (SF-1) in adrenal steroidogenesis was studied using adrenocortical cell lines transformed with a dominant negative mutant of SF-1. Constitutive expression of the mutant did not only impair the activity of endogenous SF-1 but also diminish its own expression, suggesting that SF-1 was under autoregulation. Inhibition of the endogenous SF-1 activity significantly reduced basal and inducible transcription of CYP17, CYP21B and CYP11B1, but exhibited little effects on StAR and CYP11A1 expression. Stimulating the transformed cells with potassium and cAMP freed CYP11B2 from the mutant-caused transcriptional inhibition, whereas the transformation abolished induction of CYP17 by both stimulants. Consistent with the transcriptional changes of steroidogenic genes, basal and inducible synthesis of cortisol and androgens drastically declined in the transformed cell lines. The relief of CYP11B2 repression following the potassium and cAMP stimulation removed the restraint the mutant exerted on aldosterone synthesis, and resulted in aldosterone overproduction in the stimulated transformed cells. SF-1 also plays a role in regulating the adrenocorticotrophic hormone (ACTH) responsiveness of the adrenocortical cells. Inhibition of SF-1 activity significantly decreased basal expression of ACTH receptor and its induction by potassium and cAMP.

Transgenic mice expressing green fluorescent protein under the control of the melanocortin-4 receptor promoter.

The melanocortin-4 receptor (MC4-R) is an important regulator of energy homeostasis, and evidence suggests that MC4-R-expressing neurons are downstream targets of leptin action. MC4-Rs are broadly expressed in the CNS, and the distribution of MC4-R mRNA has been analyzed most extensively in the rat. However, relatively little is known concerning chemical profiles of MC4-R-expressing neurons. The extent to which central melanocortins act presynaptically or postsynaptically on MC4-Rs is also unknown. To address these issues, we have generated a transgenic mouse line expressing green fluorescent protein (GFP) under the control of the MC4-R promoter, using a modified bacterial artificial chromosome. We have confirmed that the CNS distribution of GFP-producing cells is identical to that of MC4-R mRNA in wild-type mice and that nearly all GFP-producing cells coexpress MC4-R mRNA. For example, cells coexpressing GFP and MC4-R mRNA were distributed in the paraventricular hypothalamic nucleus (PVH) and the dorsal motor nucleus of the vagus (DMV). MC4-R promotor-driven GFP expression was found in PVH cells producing thyrotropin-releasing hormone and in cholinergic DMV cells. Finally, we have observed that a synthetic MC3/4-R agonist, MT-II, depolarizes some GFP-expressing cells, suggesting that MC4-Rs function postsynaptically in some instances and may function presynaptically in others. These studies extend our knowledge of the distribution and function of the MC4-R. The transgenic mouse line should be useful for future studies on the role of melanocortin signaling in regulating feeding behavior and autonomic homeostasis.

Adrenocorticotropic hormone (MC-2) receptor mRNA is expressed in rat sympathetic ganglia and up-regulated by stress.

Stress triggered cardiovascular disorders are associated with elevated activity of the sympathetic nervous system, the major source of elevated plasma norepinephrine levels. Our previous studies revealed that administration of adrenocorticotropic hormone (ACTH) increases the gene expression of norepinephrine biosynthetic enzymes and several neuropeptides in rat sympathetic ganglia as much as stress. Here, we examine whether an ACTH-responsive receptor is expressed in rat superior cervical (SCG) and stellate ganglia (StG). Using reverse transcriptase-polymerase chain reaction (RT-PCR) we found expression of MC-2 receptor mRNA in these ganglia. Identical DNA fragments were amplified with mRNA from SCG, StG or from adrenal cortex. Sequencing revealed extensive homology to published sequences of mouse and human MC-2 receptor. Real time PCR was used to quantitate MC-2 receptor mRNA levels in the SCG under basal conditions and following immobilization stress. Immobilization stress triggered a large increase in MC-2 receptor mRNA in SCG. The results provide the first evidence that rat sympathetic ganglia express MC-2 receptor gene and are a target tissue for the peripheral actions of ACTH in response to stress.

Molecular and behavioral interactions between central melanocortins and cocaine.

Behavioral and molecular studies have established a link between drugs of abuse and the central melanocortin system, particularly the melanocortin 4 receptor (MC4-R). The present study expands this line of investigation to characterization of the neurochemical and behavioral interactions between MC4-R and the psychomotor stimulant, cocaine. The results demonstrate that repeated, but not acute, cocaine administration up-regulates MC4-R mRNA expression in the striatum and hippocampus, but not cerebral cortex. Pharmacological studies indicate that the up-regulation of MC4-R expression occurs via dopamine D1 and D2 receptor-dependent mechanisms. The D1/D2 antagonist haloperidol and the D2-selective antagonist eticlopride mimic the effect of cocaine on MC4-R expression. In addition, coadministration of a D1-selective antagonist, SCH 23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine], completely blocks the up-regulation of MC4 mRNA by cocaine, demonstrating that D1 receptor activation is necessary for this response. Moreover, the results demonstrate that cocaine treatment increases behavioral responses (grooming and locomotor activity) to infusions of a melanocortin agonist, indicating that up-regulation of MC4-R expression results in functional consequences. These data further support a role for the melanocortin-MC4-R neuropeptide system in the biochemical and behavioral effects of cocaine.

Maternal undernutrition throughout pregnancy increases adrenocorticotrophin receptor and steroidogenic acute regulatory protein gene expression in the adrenal gland of twin fetal sheep during late gestation.

We have previously demonstrated that maternal undernutrition during either the 'periconceptional' (i.e. from 60 days (d) before until 7 d after mating) or 'gestational' periods (i.e. from 8 d after mating until the end of pregnancy) have differential effects on the subsequent development of the hypothalamo-pituitary-adrenocortical (HPA) axis and on adrenal growth and steroidogenesis in the sheep fetus during late gestation (term=147+/-3 d gestation). The specific mechanisms by which periconceptional or gestational undernutrition result in activation of the fetal HPA axis in late gestation are unclear. We have therefore investigated the impact of maternal nutrient restriction imposed either during the periconceptional period, or between 8 and 147 d gestation on the expression of specific genes in the fetal pituitary and adrenal which regulate adrenal steroidogenesis in late gestation. Ewes were maintained on either a Control (C) or Restricted (R, 70% of C) diet from 60 d before until 7 d after mating (periconceptional period) and then maintained on either a Control or Restricted diet from 8 d after mating for the remainder of pregnancy (gestational period). Four nutritional treatment groups were therefore generated (C-C, C-R, R-R and R-C). Whilst periconceptional undernutrition (R-R and R-C groups) resulted in higher fetal plasma adrenocorticotrophic hormone (ACTH) at 135-146 d gestation, there was no change in the relative level of expression of the ACTH receptor (MC2R), steroidogenic acute regulatory protein (StAR) or steroidogenic enzyme mRNAs in the fetal adrenal in late gestation. Exposure to gestational undernutrition (R-R and C-R groups), however, resulted in a stimulation in the relative level of expression of MC2R mRNA (P=0.001) and StAR mRNA (P=0.007) in the fetal adrenal during late gestation. This study provides new insights into the potential mechanisms by which alterations of the nutrient environment of the fetus at different stages of gestation may result in differential activation of the fetal HPA axis.

Skin colour and skin cancer - MC1R, the genetic link.

Pigmentary traits such as red hair, fair skin, lack of tanning ability and propensity to freckle (the RHC phenotype) have been identified as genetic risk factors for both melanoma and non-melanocytic skin cancers when combined with the environmental risk factor of high ultraviolet light exposure. The human melanocortin-1 receptor (MC1R) is a key determinant of the pigmentation process and can account in large part for the diverse range of variation in human pigmentation phenotypes and skin phototypes. The coding sequence is highly polymorphic in human populations, with several of these variant forms of the receptor now known to be associated with the RHC phenotype. We have examined variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins with defined pigmentation characteristics. Variant allele frequencies have also been determined in several case-control studies of sporadic melanoma, basal cell carcinoma and squamous cell carcinoma, and in familial melanoma kindreds collected within Australia. These studies have shown that three RHC alleles - Arg151Cys, Arg160Trp and Asp294His - were associated with increased risk in all forms of skin cancer and with penetrance and age of onset in familial melanoma in mutation carriers. There is a significant RHC allele heterozygote carrier effect on skin phototype and skin cancer risk, which indicates that variant alleles do not behave in a strictly recessive manner. Ultimately, the genetic and chemical assessment of melanin synthesis rather than skin colour will be the best indicator for skin cancer risk, and such genetic association studies combined with functional analysis of variant alleles should provide the link to understanding skin phototypes.

Involvement of microphthalmia-associated transcription factor (MITF) in expression of human melanocortin-1 receptor (MC1R).

Analysis of the nucleotide sequence of human melanocortin-1 receptor (MC1R) promoter indicated that an E-box (CANNTG) is present immediately upstream of the transcriptional initiation site. The presence of the CATGTG motif suggests that MC1R gene expression may be regulated by a basic helix-loop-helix-leucine-zipper (bHLH-LZ) type transcription factor. The microphthalmia-associated transcription factor (MITF), which belongs to the family of bHLH-LZ type transcription factors, regulates the transcription of melanogenesis-related enzyme genes such as the tyrosinase and TRP-1 genes. We investigated whether MITF regulates human MC1R gene expression through the same transcriptional mechanism as tyrosinase and TRP-1 genes in melanocytes. For this purpose, the effect of co-expression of cDNA encoding MITF on MC1R promoter activity in NIH/3T3 cells was studied. MC1R promoter activity was induced to the extent of approximately 5-fold in the presence of MITF. In addition, electrophoretic mobility shift assay indicated that nuclear extracts of human SK-Mel-2 cells contain a protein that binds specifically to the MC1R promoter region containing the CATGTG motif. These results suggested that MITF regulates not only the expression of enzymes involved in melanin synthesis, but also the expression of a receptor which plays an essential role in melanocyte functions.

One melanocortin 4 and two melanocortin 5 receptors from zebrafish show remarkable conservation in structure and pharmacology.

We report the cloning, genome mapping, functional expression, pharmacology and anatomical distribution of three melanocortin (MC) receptors from zebrafish (z). Phylogenetic analysis showed with high bootstrap support that these genes represent one MC4 receptor and two MC5 receptors. Chromosomal mapping showed conserved synteny between regions containing zMC4 and human (h) MC4 receptors, whereas the two zMC5 receptor genes map on chromosome segments in which the zebrafish has several genes with two orthologues of a single mammalian gene. It is likely that the two copies of zMC5 receptors arose through a separate duplication in the teleost lineage. The zMC4, zMC5a, and zMC5b receptors share 70-71% overall amino acid identity with the respective human orthologues and over 90% in three TM regions believed to be most important for ligand binding. All three zebrafish receptors also show pharmacological properties remarkably similar to their human orthologues, with similar affinities and the same potency order, when expressed and characterized in radioligand binding assay for the natural MSH) peptides alpha-, beta-, and gamma-MSH. Stimulation of transfected mammalian cells with alpha-MSH caused a dose-dependent increase in intracellular cAMP levels for all three zebrafish receptors. All three genes were expressed in the brain, eye, ovaries and gastrointestinal tract, whereas the zMC5b receptor was also found in the heart, as determined by RT-PCR. Our studies, which represent the first characterization of MC receptors in a nonamniote species, indicate that the MC receptor subtypes arose very early in vertebrate evolution. Important pharmacological and functional properties, as well as gene structure and syntenic relationships have been highly conserved over a period of more than 400 million years implying that these receptors participate in vital physiological functions.

Steroidogenic factor-1 is essential for compensatory adrenal growth following unilateral adrenalectomy.

While the orphan nuclear receptor steroidogenic factor-1 (SF-1) has been shown to function as an induction factor to define adrenocortical cell lineage, it remains unclear whether SF-1 plays an additional role as a growth promoting agent in the adult adrenal cortex. The proliferative potential of the adrenal cortex in adult SF-1(+/-) mice was examined using the model of compensatory adrenal growth following unilateral adrenalectomy (uADX). While the right adrenal gland of wild-type (wt) mice grew significantly after uADX, the adrenal of SF-1(+/-) mice exhibited a blunted, statistically nonsignificant weight increase. Accordingly, a profound increase in the proliferation marker proliferating cell nuclear antigen could be detected only in wt mice after uADX but not in the SF-1(+/-) mice. The proposed key regulator in adrenal compensatory growth, the recently cloned adrenal secretory serine protease was up-regulated in the remaining adrenal of wt mice, whereas this increase was blunted in SF-1(+/-) mice. While no differences in preadipocyte factor-1, the presumed marker of primitive adrenocortical cells, were detectable in the adrenals of wt and SF-1(+/-) mice, an increase in the ACTH receptor as well as agouti-related protein was observed only in wt animals but not in the SF-1(+/-) mice following uADX. Taken together, these results reflect a primary inability of adrenal cortical cells of SF-1(+/-) mice to undergo compensatory adrenal growth in response to uADX.

Variable expression of the transcription factors cAMP response element-binding protein and inducible cAMP early repressor in the normal adrenal cortex and in adrenocortical adenomas and carcinomas.

The molecular mechanisms leading to adrenocortical tumorigenesis have been only partially elucidated so far. Because the pituitary hormone ACTH, via activation of the cAMP pathway, regulates both cell proliferation/differentiation and steroid synthesis in the adrenal cortex, in this study we focused on the cAMP-dependent transcription factors cAMP responsive element modulator (CREM) and cAMP responsive element binding protein (CREB). We studied CREM and CREB expression by RT-PCR in human normal adrenal cortex (n = 3), adrenocortical adenomas (n = 8), and carcinomas (n = 8). We found transcripts corresponding to the isoforms alpha, beta, gamma, and tau2 of the CREM gene in all of the normal adrenal tissues, in the adenomas, and in seven of eight carcinomas. On the other hand, mRNA for the inducible cAMP early repressor isoforms, which derive from an internal promoter of CREM gene, was detected in the normal adrenal and in seven of eight adenomas, but in only three of eight carcinomas. Similarly, CREB transcripts were readily detectable in all normal adrenals and adenomas, whereas they were not found in four of eight adrenal carcinomas. To further characterize the carcinomas, telomerase activity and the expression of the ACTH receptor gene were determined. Telomerase activity in the carcinomas resulted in levels significantly higher than in the adenomas, whereas the levels of ACTH receptor mRNA were lower in the carcinomas. No correlation was found in the carcinomas between the levels of the ACTH receptor transcript and the loss of expression of CREB/inducible cAMP early repressor, suggesting that this alteration is not secondary to an upstream disregulation at the receptor level. In conclusion, our results suggest that an alteration in cAMP signaling may be associated with malignancies of the adrenal cortex.

alpha-Melanocyte stimulating hormone acts as a selective inducer of secretory functions in human mast cells.

In the present study, we have investigated the pro-opiomelanocortin (POMC)-derived neuropeptide alpha-MSH for its ability to modulate activation of human mast cells. The in vitro ability of purified human skin mast cells to secrete various types of mast cell mediators was monitored in response to alpha-MSH at the mRNA and at the protein level. Picomolar concentrations of alpha-MSH induced a dose-dependent release of histamine from isolated human skin mast cells and from skin punch biopsies. However, no effect of alpha-MSH was seen regarding the expression of IL-1, IL-6, IL-8, TGF-beta, and TNF-alpha. Melanocortin receptor MC-1 was identified at the transcriptional level by RT-PCR analysis but not at the protein level, whereas, in leukemic human mast cells (HMC-1), the mRNAs and the proteins for the MC-1 and MC-5 receptor were identified. These results suggest that alpha-MSH may selectively induce acute inflammatory effects via secretion of histamine.

The melanocortin 5 receptor is expressed in human sebaceous glands and rat preputial cells.

Melanocortins regulate pigmentation, adrenal hormone secretion, immune functions, lipid metabolism, and feeding behaviors in rodents. These peptides include adrenocorticotrophic hormone, melanocyte stimulating hormone, beta-lipotrophin, and the endorphins. Lipid metabolism in sebaceous glands and preputial glands of rodents is regulated by alpha-melanocyte stimulating hormone, the major agonist for melanocortin receptors. Five melanocortin receptor subtypes have been identified that differ in their tissue localization and affinities for melanocortin ligands. Targeted disruption of the melanocortin 5 receptor in transgenic mice results in widespread dysfunction of exocrine glands, including a marked decrease in sebum production. A role for melanocortins in the modulation of human sebum production has not been established. The goal of this study is to determine which melanocortin receptors are expressed in human sebaceous glands. Messenger RNA was isolated from human sebaceous glands and the reverse transcriptase polymerase chain reaction was performed using primers specific for each of the melanocortin receptor subtypes. Transcripts were detected for the melanocortin 5 receptor. A polyclonal chicken antihuman antibody to the melanocortin 5 receptor localized to sebaceous glands, eccrine glands, hair follicles, and epidermis in human skin, rat skin, cultured human sebocytes, and rat preputial cells. Presence of the melanocortin 5 receptor protein in human sebaceous glands and rat preputial glands was further verified by Western blotting. These data support further investigation of the role of melanocortins in the regulation of human sebum production and support the use of the rat preputial system as an experimental model in sebaceous gland physiology.

Regulation by adrenocorticotropin (ACTH), angiotensin II, transforming growth factor-beta, and insulin-like growth factor I of bovine adrenal cell steroidogenic capacity and expression of ACTH receptor, steroidogenic acute regulatory protein, cytochrome P450c17, and 3beta-hydroxysteroid dehydrogenase.

The purpose of this study was to evaluate the time-course effect of a 36-h treatment with ACTH (10(-8) M), transforming growth factor-beta1 (TGFbeta1; 10(-10) M), angiotensin II (AngII; 10 (-7) M), and insulin-like growth factor I (IGF-I; 10(-8) M) on the steroidogenic capacity of bovine adrenocortical cells (BAC) and on messenger RNA (mRNA) levels of ACTH receptor, cytochrome P450c17, 3beta-hydroxysteroid dehydrogenase (3betaHSD), steroidogenic acute regulatory protein (StAR), and StAR protein. ACTH and IGF-I enhanced, in a time-dependent manner, the acute 2-h ACTH-induced cortisol production, whereas TGFbeta 1 and AngII markedly reduced it. ACTH, IGF-I, and AngII increased ACTH receptor mRNA, but the opposite was observed after TGFbeta1 treatment. ACTH and IGF-I increased P450c17 and 3betaHSD mRNAs, whereas AngII and TGFbeta1 had the opposite effects. However, the effects of the four peptides on ACTH-induced cortisol production appeared before any significant alterations of the mRNA levels occurred. The most marked and rapid effect of the four peptides was on StAR mRNA. The stimulatory effect of ACTH was seen within 1.5 h, peaked at 4-6 h, and declined thereafter, but at the end of the 36-h pretreatment, the levels of StAR mRNA and protein were higher than those in control cells. IGF-I also enhanced StAR mRNA levels within 1.5 h, and these levels remained fairly constant. The effects of AngII on StAR mRNA expression were biphasic, with a peak within 1.5-3 h, followed by a rapid decline to almost undetectable levels of both mRNA and protein. TGFbeta1 had no significant effect during the first 3 h, but thereafter StAR mRNA declined, and at the end of the experiment the StAR mRNA and protein were almost undetectable. Similar results were observed when cells were treated with ACTH plus TGFbeta1. A 2-h acute ACTH stimulation at the end of the 36-h pretreatment caused a higher increase in StAR mRNA and protein in ACTH- or IGF-I-pretreated cells than in control cells, which, in turn, had higher levels than cells pretreated with TGFbeta1, ACTH plus TGFbeta1, or AngII. These results and the fact that the stimulatory (IGF-I) or inhibitory (AngII and TGFbeta1) effects on ACTH-induced cortisol production were more pronounced than those on the ability of cells to transform pregnenolone into cortisol strongly suggest that regulation of StAR expression is one of the main factors, but not the only one, involved in the positive (IGF-I) or negative (TGFbeta1 and AngII) regulation of BAC for ACTH steroidogenic responsiveness. A high correlation between steady state mRNA level and acute ACTH-induced cortisol production favors this conclusion.

Involvement of mi-transcription factor in expression of alpha-melanocyte-stimulating hormone receptor in cultured mast cells of mice.

The microphthalmia (mi) locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (MITF). We have reported that expression of several genes was impaired in cultured mast cells (CMCs) of mi/mi mice due to a defective transactivation ability of mutant MITF (mi-MITF). We also found that mi/mi CMCs did not express a receptor (MC1R) for alpha-melanocyte-stimulating hormone. The overexpression of the wild-type (+/+) MITF but not mi-MITF normalized the expression of the MC1R in mi/mi CMCs, indicating the involvement of +-MITF in the MC1R gene expression. Next, we analyzed the promoter region of the MC1R gene by the transient cotransfection assay. The luciferase construct under the control of the MC1R promoter and the cDNA-encoding +-MITF or mi-MITF were cotransfected into NIH/3T3 fibroblasts. The cotransfection of +-MITF but not mi-MITF increased the luciferase activity. There were five CANNTG motifs recognized by bHLH-Zip-type transcription factors in the cloned promoter region. We found +-MITF bound two of five CANNTG motifs, and both motifs were essential for the transactivation of the MC1R gene by +-MITF. These results indicated that +-MITF directly transactivated the MC1R gene through these two motifs.

Upregulation of mRNA for the melanocortin-1 receptor but not for melanogenic proteins in macrophage x melanoma fusion hybrids exhibiting increased melanogenic and metastatic potential.

Fusion of mouse peritoneal macrophages or human blood monocytes with weakly metastatic mouse Cloudman S91 melanoma cells resulted in hybrids with enhanced metastatic potential (Rachkovsky et al., 1998. Clin. Exp. Metastasis, 16: 299-312). With few exceptions, such hybrids also showed increased basal- and MSH-induced pigmentation, at least in part through increased N-glycosylation of melanogenic proteins (Sodi et al., 1998. Pigment Cell Res., 11: 299-309). Here we report analyses regarding expression of the melanocyte-stimulating hormone (MSH) receptor (melanocortin-1 receptor, MC1-R) and the melanogenic proteins, tyrosinase (E.C. 1.14.18.1), tyrosinase-related protein 1 (TRP-1), and the tyrosinase-related protein 2 (TRP-2, E.C. 5.3.2.3), by a panel of cell lines consisting of parental Cloudman S91 melanoma cells, macrophages from DBA/2J mice, artificially derived macrophage x melanoma hybrids of high and low metastatic potential, and a naturally occurring highly metastatic hybrid between a Cloudman S91 tumor cell and a DBA/2J tumor-infiltrating cell. We show that incubation of cells with MSH/isobutylmethylxanthine (IBMX) resulted in strong melanogenic and morphologic responses in high metastatic hybrids compared to parental cells and the low metastatic hybrid, and that high metastatic hybrids exhibit increased mRNA expression for MC1-R accompanied by increased 125I-alphaMSH binding. Although tyrosinase activity and the protein level for tyrosinase and TRP-2, but not for TRP-1, were increased in the high metastatic hybrids versus the other cells, no significant changes in mRNA either for tyrosinase or for TRPs were observed in them. Furthermore, unlike tyrosinase, the abundance and gel mobility pattern of TRP-2 did not correlate with changes in activity in all hybrids and parental melanoma cells. The results suggest that although the activity MC1-R and tyrosinase correlate with enhanced basal as well as MSH-induced melanogenesis in metastatic/melanotic hybrids, their expression is differentially regulated, i.e., regulation of MC1-R while at transcriptional level, the TRPs are primarily regulated via post-transcriptional mechanisms in high metastatic hybrids.