Evaluation of posttranslational processing of proopiomelanocortin in the banded houndshark pituitary by combined cDNA cloning and mass spectrometry

Proopiomelanocortin (POMC) is cleaved into small peptides, such as adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormones (MSHs), and β-endorphin (β-END), by tissue-specific posttranslational processing in the corticotrophs of the pars distalis (PD) and melanotrophs of the neurointermediate lobe (NIL) of the pituitary. We examined the posttranslational processing of POMC in the pituitary of the banded houndshark Triakis scyllium by molecular cloning and subsequent mass spectrometric identification of the POMC-derived peptides in the pituitary extracts. One-fifth of the randomly selected clones from a Triakis pituitary cDNA library contained a cDNA encoding for POMC. Triakis prePOMC contained 4 MSHs and a single β-END, as has been observed in case of other cartilaginous fish POMCs. These predicted hormonal segments were flanked by basic amino acid residues, which are the cleavage sites for the processing enzymes, i.e., protein convertases. Mass spectrometry was performed using PD (including most parts of the rostral and proximal PD) and NIL extracts to detect mass values corresponding to the POMC-derived peptides. Consequently, ACTH, β-END, and the joining peptide (JP) were detected in the PD extract, while MSHs, processed β-END, and some other POMC-derived peptides were identified in the NIL extract; however, neither acetylated α-MSH nor acetylated β-END was detected in the latter. These tissue-specific POMC processing patterns are similar to those of the other vertebrate pituitaries; however, the absence of acetylated peptides suggests the lack of an acetylation system in the melanotrophs in the NIL of the Triakis pituitary.     

Role of proopiomelanocortin-derived peptides and their receptors in the osteoarticular system: from basic to translational research

Proopiomelanocortin (POMC)-derived peptides such as melanocortins and β-endorphin (β-ED) exert their pleiotropic effects via binding to melanocortin receptors (MCR) and opioid receptors (OR). There is now compelling evidence for the existence of a functional POMC system within the osteoarticular system. Accordingly, distinct cell types of the synovial tissue and bone have been identified to generate POMC-derived peptides like β-ED, ACTH, or α-MSH. MCR subtypes, especially MC1R, MC2R (the ACTH receptor), MC3R, and MC4R, but also the μ-OR and δ-OR, have been detected in various cells of the synovium, cartilage, and bone. The respective ligands of these POMC-derived peptide receptors mediate an increasing number of newly recognized biological effects in the osteoarticular system. These include bone mineralization and longitudinal growth, cell proliferation and differentiation, extracellular matrix synthesis, osteoprotection, and immunomodulation. Importantly, bone formation is also regulated by the central melanocortin system via a complex hormonal interplay with other organs and tissues involved in energy metabolism. Among the POMC-derived peptides examined in cell culture systems from osteoarticular tissue and in animal models of experimentally induced arthritis, α-MSH, ACTH, and MC3R-specific agonists appear to have the most promising antiinflammatory actions. The effects of these melanocortin peptides may be exploited in future for the treatment of patients with inflammatory and degenerative joint diseases.     

Proopiomelanocortin and energy balance: insights from human and murine genetics

Proopiomelanocortin (POMC) undergoes extensive and tissue-specific posttranslational processing to yield a range of biologically active peptides. Historically, the most clearly defined roles of these peptides are in the control of adrenal steroidogenesis by corticotroph-derived ACTH and skin pigmentation by alphaMSH. However, a rapidly expanding body of work has established that POMC-derived peptides synthesized in neurons of the hypothalamus play a central role in the control of energy homeostasis. We review how inherited abnormalities in POMC synthesis and processing and defects in the action of POMC-derived peptides in both humans and mice have helped shape our current understanding of the importance of the melanocortin system in human energy balance.     

Proopiomelanocortin-derived peptides in rat cerebrospinal fluid and hypothalamic extracts: evidence that secretion is regulated with respect to energy balance

Regulation of proopiomelanocortin (POMC) is an important means of controlling the central melanocortin system. It has never been established whether the spectrum of POMC-derived peptides synthesized and secreted from the hypothalamus is altered in response to changes in energy homeostasis in vivo. To monitor secretion, we analyzed peptide content of rat cerebrospinal fluid. Strikingly, both the POMC precursor and ACTH were readily detected. Moreover, levels of both were lower in samples from obese Zucker rats (fa/fa) vs. lean Zucker rats (+/+, fa/+) and from fasted vs. fed rats, whereas alpha MSH could not be detected. POMC levels were also decreased in hypothalamic extracts from obese and fasted animals. In contrast, despite being the most predominant peptide in extracts, alpha MSH levels were not significantly changed in any of the rat models. The ratio of precursor to derived peptides in cerebrospinal fluid was significantly higher in obese vs. lean and fed vs. fasted rats, indicating that secretion of POMC-derived peptides is differentially down-regulated during negative energy balance. In contrast to peptide analysis, we found that POMC gene expression was not significantly decreased in fasted rat hypothalami. We conclude that regulation of peptide secretion is an important mechanism by which the POMC system is controlled.     

Agouti-related protein (83-132) is a competitive antagonist at the human melanocortin-4 receptor: no evidence for differential interactions with pro-opiomelanocortin-derived ligands

Interactions between pro-opiomelanocortin (POMC)-derived peptides, agouti-related protein (AGRP) and the melanocortin-4 receptor (MC4-R) are central to energy homeostasis. In this study we have undertaken comprehensive pharmacological analysis of these interactions using a CHOK1 cell line stably transfected with human MC4-R. Our main objectives were (1) to compare the relative affinities and potencies of POMC-derived peptides endogenously secreted within the hypothalamus, (2) to investigate the potency of AGRP(83-132) antagonism with respect to each POMC-derived peptide and (3) to determine whether AGRP(83-132) and POMC-derived peptides act allosterically or orthosterically. We have found that beta melanocyte-stimulating hormone (betaMSH), desacetyl alpha MSH (da-alphaMSH) and adrenocorticotrophic hormone all have very similar affinities and potencies at the MC4-R compared with the presumed natural ligand, alphaMSH. Moreover, even MSH precursors, such as beta lipotrophic hormone, showed significant binding and functional activity. Therefore, many POMC-derived peptides could have important roles in appetite regulation and it seems unlikely that alphaMSH is the sole physiological ligand. We have shown that AGRP(83-132) acts as a competitive antagonist. There was no significant difference in the potency of inhibition by AGRP(83-132) or agouti(87-132) at the MC4-R, regardless of which POMC peptide was used as an agonist. Furthermore, we have found that AGRP(83-132) has no effect on the dissociation kinetics of radiolabelled Nle4,D-Phe7 MSH from the MC4-R, indicating an absence of allosteric effects. This provides strong pharmacological evidence that AGRP(83-132) acts orthosterically at the MC4-R to inhibit Gs-coupled accumulation of intracellular cAMP.     

Corticotropin-releasing hormone and oxytocin stimulate the release of placental proopiomelanocortin peptides

Human placenta contains the POMC-derived peptides ACTH, alpha MSH, and beta-endorphin, and CRH. High concentrations of immunoreactive (IR) CRH have been recently demonstrated in maternal plasma during pregnancy. To determine if the human placenta secretes CRH and POMC-derived peptides, we developed an in vitro human placental fragment perifusion system. The perifused tissue released IR-CRH and POMC-derived peptides at a constant rate for at least 5 h. The mean IR-CRH concentration in the effluent (under basal conditions) was 158 +/- 26 (+/- SD) pg (34.5 +/- 5.8 fmol)/5-min fraction.g tissue. IR-alpha MSH, IR-beta-endorphin, and IR-ACTH were also released into the perifusion medium; the mean concentration of alpha MSH released was 24.6 +/- 8 pg (14.8 +/- 4.8 fmol)/fraction.g, that of ACTH was 2.9 +/- 1.9 pg (0.65 +/- 0.43 fmol)/fraction.g, and that of beta-endorphin was 12.9 +/- 6 pg (3.8 +/- 1.7 fmol)/fraction.g. We examined the effects of human CRH, oxytocin, vasopressin, and dexamethasone on placental POMC peptide secretion. Five-minute pulses of 10(-8) or 10(-6) mol/L human CRH or oxytocin produced an immediate and dose-dependent increase in all POMC peptides in the effluent. A 5-min pulse of 10(-8) or 10(-6) mol/L vasopressin had no effect. A continuous 4-h exposure to 10(-6) mol/L dexamethasone had no effect on either basal IR-CRH or POMC-derived peptide or their KCl-induced release. In conclusion, we found that 1) human placenta releases IR-CRH and POMC-derived peptides in vitro; this phenomenon seems to be independent of glucocorticoid control; 2) placental CRH may have a paracrine effect on placental POMC peptide release in addition to its possible action on maternal pituitary hormone release; and 3) oxytocin, but not vasopressin, stimulates placental POMC peptide release.     

Expression of pro-opiomelanocortin-like gene in the testis and epididymis.

Adrenocorticotropin (ACTH), beta-endorphin, and the melanocyte-stimulating hormones (MSHs), which are products of a common precursor, pro-opiomelanocortin (POMC), are present in a variety of tissues other than pituitary. The recent detection of immunoreactive POMC-derived peptides in the male reproductive tract raised the possibility that these hormones might regulate reproductive function. To determine whether the low concentrations of POMC-derived peptides in the male reproductive tract are synthesized locally and are not contaminants from blood, we have demonstrated POMC-like gene expression in both testis and epididymis. The identification of cells in testis capable of synthesizing POMC mRNA was established by showing the presence of this mRNA in mouse Leydig cell lines (TM3 and I10A). The hybridizing species of POMC-like mRNA in the testis, epididymis, and Leydig cell lines (TM3 and I10A) were approximately 150 bases shorter than those in the pituitary or hypothalamus but were similar in size to that in the amygdaloid nucleus of rat brain. The concentration of POMC-like mRNA in the testis is almost as high as that in the hypothalamus. This finding is quite unexpected because the concentrations of POMC-derived peptides in the testis were 2-3 orders of magnitude lower than those in the hypothalamus. The demonstration of a POMC-like gene expression in male reproductive tissues suggests that POMC-derived peptides are synthesized in Leydig cells and epididymis. These observations are consistent with the postulate that POMC-derived peptides may exert paracrine and/or autocrine effects in these organs.     

The effects of proopiomelanocortin deficiency on murine adrenal development and responsiveness to adrenocorticotropin

The mature adrenal cortex is dependent upon proopiomelanocortin (POMC)-derived peptides for the maintenance of its size, structure, and endocrine function. Recent studies in mice genetically deficient in POMC have suggested that early exposure to POMC-derived peptides might also be necessary for the development of a functionally competent adrenal. We examined adrenal morphology and function in an independent line of mice lacking all POMC-derived peptides (Pomc-/-). Adrenal glands were found in all mice, although the glands of Pomc-/- mice had markedly reduced weight compared with control animals (0.5 +/- 0.1 vs. 2.1 +/- 0.1 mg, respectively; P < 0.05) and had disrupted cortical architecture. In Pomc-/- mice, plasma corticosterone was undetectable, and plasma aldosterone was significantly reduced compared with wild-type mice (498 +/- 88 vs. 1845 +/- 168 nmol/liter, respectively; P < 0.001). Heterozygous mice (Pomc+/-) had smaller adrenal glands with significantly lower levels of corticosterone both basally and in response to CRH and ACTH than wild-type mice, indicating that two functional copies of the Pomc gene are necessary to support the fully normal function of the hypothalamic-pituitary-adrenal axis. Three-month-old Pomc-/- mice were treated for 10 d with a highly specific ACTH analog. This treatment restored adrenal weight, cortical morphology, and plasma corticosterone to the levels seen in wild-type littermates. In conclusion, murine adrenal glands can develop without exposure to endogenous POMC-derived peptides during fetal and neonatal life. Although such glands are atrophic and hypofunctional, exposure to ACTH alone can restore their size, morphology, and corticosterone secretion.     

A role for the endogenous opioid beta-endorphin in energy homeostasis

Proopiomelanocortin (POMC) neurons in the hypothalamus are direct targets of the adipostatic hormone leptin and contribute to energy homeostasis by integrating peripheral and central information. The melanocortin and beta-endorphin neuropeptides are processed from POMC and putatively coreleased at axon terminals. Melanocortins have been shown by a combination of pharmacological and genetic methods to have inhibitory effects on appetite and body weight. In contrast, pharmacological studies have generally indicated that opioids stimulate food intake. Here we report that male mice engineered to selectively lack beta-endorphin, but that retained normal melanocortin signaling, were hyperphagic and obese. Furthermore, beta-endorphin mutant and wild-type mice had identical orexigenic responses to exogenous opioids and identical anorectic responses to the nonselective opioid antagonist naloxone, implicating an alternative endogenous opioid tone to beta-endorphin that physiologically stimulates feeding. These genetic data indicate that beta-endorphin is required for normal regulation of feeding, but, in contrast to earlier reports suggesting opposing actions of beta-endorphin and melanocortins on appetite, our results suggest a more complementary interaction between the endogenously released POMC-derived peptides in the regulation of energy homeostasis.     

PP/PYY cells from endocrine pancreas of the scincid lizard Eumeces inexpectatus synthesize ACTH- and alpha-MSH-like molecules

The endocrine pancreas of the scincid lizard Eumeces inexpectatus secretes four major hormones, insulin, glucagon, somatostatin, and pancreatic polypeptide (PP); in addition, other peptides and neuropeptides, often colocalized in one of the principal cell types (A, B, D, and PP), were detected by light and ultrastructural immunocytochemistry. In particular, the pancreas is rich in peptide tyrosine tyrosine (PYY), ACTH, and alpha-MSH immunoreactivity. When single- and double-immunolabeled serial sections were compared for immunostaining for PP, PYY, ACTH, and alpha-MSH, there was broad coincidence with PP, termed PP/PYY, cells in view of the extensive colocalization of these two peptides. Furthermore, ultrastructural morphometric studies revealed similar secretory granules for PP immunoreactive (ir) and ACTH ir cells, while the endocrine cells express pro-opiomelanocortin (POMC) mRNA, indicating an active, extrapituitary synthesis of the POMC-derived peptides in these cells. In conclusion, the presence of POMC-derived peptides in the endocrine pancreatic cells suggests that they may regulate insulin secretion.     

On the potential role of proopiomelanocortin in skin physiology and pathology

Pro-opiomelanocortin (POMC) is the precursor of ACTH, α-MSH and β-endorphin, neuropeptides with multiple regulatory functions. Both the pituitary gland and peripheral tissues such as mammalian skin are capable of generating POMC-derived neuropeptides. Mammalian skin is also a target for POMC products; their possible roles in skin physiology and pathology are discussed in this communication.     

Effects of loperamide and other opioid-related substances on the transcriptional regulation of the rat pro-opiomelanocortin gene in AtT20 cells

Although opioid peptides are involved in the regulation of the hypothalamic-pituitary-adrenal axis, their role in pro-opiomelanocortin (POMC) gene expression at the pituitary level is not known. We therefore examined the effects of opioid receptor agonists, including recently discovered endogenous opioid peptides, on POMC gene expression using the AtT20PL cell line, a subclone of AtT20 in which the rat POMC 5'-promoter-luciferase fusion gene was stably incorporated. The endogenous mu-opioid receptor agonists endomorphin 1 and 2 had no effect on either basal or corticotropin-stimulating-hormone-induced POMC expression. This was also the case with the delta-agonist BUBUC, the kappa-agonist U50488H and the orphan receptor agonist orphanin FQ. In contrast, the synthetic mu-agonist loperamide significantly inhibited basal and yet enhanced cAMP-induced POMC expression. The inhibitory effect of loperamide was mimicked by the calmodulin antagonist W7 and antagonized by the calcium channel blocker nifedipine, whereas neither the inhibitory nor the enhancing effect of loperamide was influenced by the opioid antagonist naloxone. These results suggest that the synthetic mu-agonist loperamide has a modulatory effect on the 5'-promoter activity of the POMC gene. This effect does not seem to be mediated through the classical mu-opioid receptor but rather in part through a calcium/calmodulin-related mechanism.     

Ontogenesis of proopiomelanocortin gene expression and regulation in the rat pituitary intermediate lobe

During ontogenesis, proopiomelanocortin (POMC) mRNA appears in the pituitary intermediate lobe (IL) at embryonic day 16 (E16), rather later than in the hypothalamic arcuate nucleus (E13) or the pituitary anterior lobe (E15). POMC mRNA onset in the IL correlates with the appearance of POMC-derived peptides detected by immunocytochemistry (ICC), indicating that there is probably no time lag between POMC mRNA translation. Subsequently, while the IL lobular organization developed progressively, the number of in situ hybridization-(ISH) and ICC-positive cells increased until after birth. During postnatal development, coinciding with innervation of the IL, the POMC mRNA level in the lobe, measured by quantitative ISH, increased about 4-fold to reach the adult value at weaning. The effects of acute or chronic postnatal treatment with a dopamine antagonist (haloperidol) or a dopamine agonist (bromocriptine) show that the physiological dopaminergic inhibitory control of POMC gene expression operates as early as postnatal day 5. The subsequent increase in mRNA levels despite the inhibitory innervation raises the question of the existence of some unknown positive regulation active during postnatal development.