Processing Of Proopiomelanocortin Research Articles

An Analysis of the Proopiomelanocortin Systems in the Pituitary of the Squamate Reptile Lacerta galloti

Acid extracts of the pars intermedia of the squamate reptile Lacerta galloti were screened for immunoreactive forms of proopiomelanocortin (POMC)-related end products following Sephadex G-50 column chromatography, α-MSH-sized end products were detected with a Val-NH 2, C-terminal-specific RIA, and β-endorphin-sized end products were detected with a separate C-terminal-directed RIA. Five peaks of α-MSH-related immunoreactivity were isolated following fractionation by reversed-phase HPLC. Based on a comparison of the reversed-phase HPLC properties and the net positive charges (pH 2.75) of the Lacerta forms of α-MSH to those of the mammalian forms of α-MSH and Anolis carolinensis ACTH(I-13)NH 2, it appears that the N-acetylation of α-MSH is a major post-translational processing event in the pars intermedia of L. galloti. Although multiple forms of β-endorphin were detected in the pars intermedia of L. galloti following cation-exchange chromatography, the low levels of N-acetylated β-endorphin detected with an N -acetyl-specific β-endorphin RIA indicate that the N-acetylation of β-endorphin in this species is a minor post-translational processing event. This pattern of POMC processing in the pars intermedia of L. galloti is similar to the processing events observed for the turtle Pseudemys scripta , but distinct from the processing events observed in the squamate reptile A. Carolinensis.

The effect of time of day on levels of hypothalamic proopiomelanocortin primary transcript, processing intermediate and messenger ribonucleic acid in proestrous and estrous rats.

Several lines of evidence from different laboratories suggest that hypothalamic beta-endorphinergic activity decreases around the time of initiation of the LH surge and may increase on estrus to extinguish the expression of the daily neuronal signal for the surge. In several hormone systems, factors that stimulate or suppress hormone release also stimulate or repress transcription of the hormone gene and translation of the messenger RNA encoding the hormone. Therefore, information about neurohormone activity may be inferred from data on changes in the levels of RNA species encoding these neurohormones. We used a solution hybridization/RNase protection assay to test the hypotheses that 1) the abundance of primary transcript of the hypothalamic POMC gene decreases at the time of initiation of the proestrous LH surge and 2) levels of POMC primary transcript (and by inference, levels of beta-endorphin neuronal activity and secretion) increase on estrus. 96 rats exhibiting at least two consecutive 4-day estrous cycles were killed at either 0600 or 1300 h on proestrus and estrus. Dissections of the medial basal hypothalamus were pooled into 4 samples at each time-point (6 rats per sample) and RNA was extracted from nuclear and cytoplasmic fractions separately. We measured levels of POMC primary transcript, processing intermediate and fully spliced mRNA in the nuclear fractions and POMC mRNA in cytoplasmic fractions. Compared to 0600 h, levels of POMC primary transcript decreased significantly during the afternoons of both proestrus and estrus (P < 0.05). Levels of nuclear processing intermediate RNA and cytoplasmic mRNA followed the same trend but the afternoon declines did not reach statistical significance. We conclude from these data that the afternoon decline in POMC gene expression is not unique to the day of proestrus and we speculate that an afternoon decline in beta-endorphinergic neuronal activity may instead be a component of the daily signal for the LH surge.

Effects of a chronic inflammatory stress on levels of pro-opiomelanocortin-derived peptides in the rat spleen and thymus

Adjuvant-induced arthritis (AA) in specific strains of rats is an immunologically mediated inflammatory disease which is also characterised by activation of the endocrine system. To further investigate the effects of AA on processing of the pro-opiomelanocortin (POMC) precursor in rat immune tissues, we utilised radioimmunoassays for adrenocorticotropin (ACTH), β-endorphin and α-melanocyte-stimulating hormone (α-MSH) to measure these peptides in the spleen and thymus. 14 days following adjuvant injection, spleen levels of ACTH were elevated in the AA group (4.47 ± 1.04 ng/g tissue, n = 9) compared to controls 2.42±0.4 ng/g) and exacerbation of the disease by removal of circulating glucocorticoids through bilateral adrenalectomy (ADX) resulted in further elevation of spleen ACTH (5.11 ± 1.22 ng/g). β-Endorphin levels in both the AA (10.60 ± 1.61 ng/g) and AA/ADX (13.37 ± 2.36 ng/g) groups were higher than controls 5.57 ± 0.65 ng/g). Conversely, α-MSH spleen levels were decreased in the AA (2.89 ± 0.22 ng/g) and AA/ADX (2.22 ± 0.33 ng/g) groups compared to controls (4.62 ± 0.45 ng/g) and were also decreased following adrenalectomy. In the thymus, ACTH levels were elevated in the AA group (8.95 ± 1.41 ng/g) compared to controls (5.79 ± 0.63 ng/g), and the same pattern wasevident for thymic α-MSH (0.64 ± 0.08 ng/g in AA animals compared to control levels of 0.35 ± 0.03 ng/g). Following G50 gel filtration, ACTH and β-endorphin immunoreactivities (ir) were present in both spleen and thymus as two peaks, one which eluted near the void volume and one which eluted in a lower molecular mass position than the standards. However, ir-ACTH and ir-β-endorphin were present in multiple peaks in AA spleen extracts, indicating extensive processing. These data are the first evidence that ir-ACTH and ir-β-endorphin tissue content and POMC processing in the spleen and thymus can be influenced by a chronic stress.

Post-translational processing of proopiomelanocortin (POMC) in mouse pituitary melanotroph tumors induced by a POMC-simian virus 40 large T antigen transgene.

Mice harboring a transgene composed of proopiomelanocortin (POMC) gene promoter sequences (nucleotides -706 to +64) ligated to the simian virus (SV) 40 early gene encoding large T antigen developed large POMC-expressing pituitary tumors. Histologically the tumors arose from the intermediate lobe, contained nuclear SV40 T antigen and POMC peptides, but did not express other pituitary hormones. POMC processing in the pituitary tumors was indistinguishable from normal mouse intermediate lobe melanotrophs and was characterized by high proportions of acetylated and carboxyl-terminal shortened beta-endorphins, and amino-terminal acetylated alpha-melanocyte-stimulating hormone, and virtually no adrenocorticotropic hormone (ACTH)(1-39), beta-lipotropin, or POMC. The tumors contained abundant levels of mRNA for the prohormone convertase PC2 and undetectable levels of PC1. Normal mouse neurointermediate lobe also has a high ratio of PC2/PC1 expression that is distinct from the relative abundance of PC1 in anterior lobe and AtT-20 corticotroph cells. In contrast, extracts from tumors transplanted subcutaneously in nude mice contained predominantly nonacetylated forms of beta-endorphin(1-31) and -(1-27), very little ACTH(1-39), almost no corticotropin-like intermediate peptide or alpha-melanocyte-stimulating hormone, and higher proportions of intact POMC. Surprisingly, despite the less efficient proteolytic cleavage, a transplanted tumor expressed both PC1 and PC2. These studies are the first biochemical documentation of a melanotroph pituitary tumor in a rodent species and provide a new model for the investigation of pituitary oncogenesis and the molecular basis of tissue-specific prohormone post-translational processing.

Proopiomelanocortin-derived peptides and mRNA are expressed in rat heart.

Proopiomelanocortin (POMC) is posttranslationally processed to a-MSH, p-endorphin, and a variety of other peptides, not only in the pituitary and brain, but in peripheral tissues as well. P-Endorphin and its immediate precursor p-lipotropin (P-LPH) have recently been identified in cardiac tissue' suggesting that the POMC gene is expressed in heart, although neither the cellular localization nor the posttranslational processing of cardiac POMC peptides has been fully characterized. The localization of p-endorphin, as well as other opioid peptides, in cardiac tissue is consistent with extensive evidence that opiates produce direct effects on cardiac function, reducing cardiac output, coronary pressure, and heart rate.2s3 ACTH, a-MSH, and related ACTH fragments also exhibit potent, albeit opposing, cardioregulatory activities, increasing heart rate and ~ont rac t i l i ty ,~ ,~ although their biosynthesis in cardiac tissue has not, as yet, been demonstrated. In the present study, we further characterized POMC processing in rat heart and also showed, using in situ hybridization, that POMC mRNA is localized within cardiac ventricular muscle cells. Initial gel filtration high-performance liquid chromatography (HPLC) analysis revealed that immunoreactive ACTH and a-MSH are both present in rat heart extracts; however, ACTH levels are quite low relative to a-MSH concentrations (s5%), suggesting that ACTH is almost entirely converted to a-MSH. Reverse phase HPLC further demonstrated that the principal a-MSH-related peptides in heart are a-MSH and N,O-diacetyl-a-MSH; des-acetyl-a-MSH is a relatively minor constituent (FIGURE l). Similarly, sequential gel filtration and cation exchange HPLC analysis indicated that p-LPH is almost entirely converted to p-endorphin and that P-endorphin-1-3 1 is further modified through both N-acetylation and C-terminal proteolysis (FIGURE 1). N-Acetyl-P-endorphin-( 1-31) is the predomi-

Characterization of pro-opiomelanocortin processing in heterologous neuronal cells that express PC2 mRNA

We have investigated processing of monkey pro-opiomelanocortin (POMC) following transfection into heterologous neuronal Neuro 2A (N2A) cells. In several separately transfected stable cell lines (termed N2A POMC2 -like ; n = 4), POMC was processed to βE only, by direct cleavage from the precursor. Thus, these cell lines did not produce βE in the orderly manner observed in the pituitary, that is, via the intermediate peptide βLPH. Analysis of one representative N2A POMC2 cell line revealed that the extent of processing to βE appeared to be negatively correlated with precursor expression level, suggesting that the processing enzyme(s) in these cells was present in limiting amounts. Northern analysis of PC1 and PC2, two recently cloned processing enzymes, showed that N2A POMC2 cells expressed low levels of PC2 mRNA, but no detectable PC1 mRNA. These data suggest that (1) the order of processing observed in the pituitary is not exclusively determined by tertiary folding of the precursor, but rather by the complement of processing enzymes in a particular cell, and (2) if PC2 is responsible for POMC processing in N2A POMC2 cells, this enzyme, expressed in limiting amounts, appeared to show selectivity for the βE amino terminal processing site.

Differential proopiomelanocortin processing in the rhesus monkey intermediate pituitary.

Differential proopiomelanocortin processing in the rhesus monkey intermediate pituitary.

Foetal endocrine maturation.

In domestic ruminants such as the sheep, birth is effected through sequential maturation of the foetal hypothalamic-pituitary-adrenal (HPA) axis, leading to the increased output of cortisol. Factors regulating foetal pituitary adrenocorticotrophin (ACTH) secretion have been delineated, and these include corticotrophin releasing hormone (CRH), arginine vasopressin, prostaglandin (PG) E2 and endogenous opioids. The pre-partum increase in foetal plasma ACTH is associated with a rise in pro-opiomelanocortin (POMC) mRNA in the foetal pars distalis, and with an altered pattern of POMC post-translational processing. Foetal adrenal activation results from an increase in ACTH receptors and enhanced coupling through the Gs protein to adenylate cyclase, and increased expression of key steroidogenic enzymes including P450c17. Cortisol modulates the mechanism by which ACTH activates foetal adrenal function, through specific glucocorticoid receptors (GR) in the foetal adrenal cortex. Although the numbers of GR change with gestation, the relative abundance of GR mRNA does not, pointing to post-translational regulatory mechanisms. Cortisol also stimulates an increase in the concentration of its own high affinity binding protein (corticosteroid binding globulin; CBG) in the foetal circulation, apparently by increasing CBG gene expression in the foetal liver, and by altering the extent of foetal CBG glycosylation in a manner that would be expected to decrease the metabolic clearance of this glycoprotein. Clear evidence for placental CRH and ACTH production is lacking in sheep, but PGE2, produced in increasing amounts by the placenta during late pregnancy, may augment the drive to HPA maturation. Aspects of the maturational pathway of cortisol biosynthesis have been described in other species, including the horse, and some comparison is made with the more detailed information currently available from species such as the sheep.

Proopiomelanocortin processing and its role in immunomodulation

Proopiomelanocortin processing and its role in immunomodulation

Plasma immunoreactive joining peptide in man: a new marker of proopiomelanocortin processing and corticotroph function.

Joining Peptide (JP) is a 30 amino acid fragment separating the N-terminal peptide and ACTH within their common polypeptide precursor POMC. Using antibody Jamie directed against the C-terminal amidated residue Glu-NH2 we studied the molecular weight forms and the variations of plasma immunoreactive (IR)-JP in man under various physiological, pharmacological, and pathological conditions. In 21 plasma samples from patients with ACTH hypersecretory syndromes from pituitary and nonpituitary tumors, IR-JP had the same elution pattern on Sephadex G-75 showing a predominant, if not single, peak corresponding to a mol wt of 7000 as expected for a JP-homodimer. Normal male volunteers at 0800 h had plasma IR-JP values ranging from undetectable (< 6 pmol/L) to 28 pmol/L; all values were suppressed by the overnight 1 mg dexamethasone test. Plasma IR-JP had circadian variations and responded to the metyrapone test in a manner strictly similar to that of ACTH and lipotropins (LPHs). One hundred and fifteen plasma samples covering a large range of pathological ACTH values (from 10(0) to 10(4) pmol/L) were also assayed by the JP and LPH RIAs. All three immunoreactivities strongly correlated with each other with a molar ratio close to 1:1. Discrepancies were observed in two situations where both IR-JP and IR-LPH were much higher than ACTH: they occurred in some patients with the ectopic ACTH syndrome and in all patients with chronic renal failure; they are explained by the further degradation of ACTH into corticotropin-like intermediary lobe peptide in the first case, by the prolonged plasma half-life of JP and LPH, compared to that of ACTH, in the second case. These data show that JP is a normal end-product of POMC processing in man which circulates in blood mainly as a homodimer. It provides yet another marker of the overall corticotroph function and may be used to unravel abnormal POMC processing in some nonpituitary tumors.

Plasma immunoreactive joining peptide in man: a new marker of proopiomelanocortin processing and corticotroph function

Plasma immunoreactive joining peptide in man: a new marker of proopiomelanocortin processing and corticotroph function

β-Endorphin processing and cellular origins in rat spinal cord

While enkephalin and dynorphin peptides have been well characterized in the spinal cord, the cellular localization of β-endorphin (βE) and the processing of pro-opiomelanocortin (POMC) to βE and other non-opioid peptides in the cord have not been extensively investigated. Other investigators have characterized the various βE forms present in rat spinal cord regions. Previous studies have also suggested that spinal POMC content is entirely derived from supraspinal sources. However, high proportions of βE precursors present in spinal cord sieving profiles led us to suspect the presence of POMC cell bodies intrinsic to the cord. In this study, we performed thoracic spinal cord lesions on a group of animals and demonstrated the persistence of about one-third of control levels of βE immunoreactivity (βE-IR) below the level of the lesions. We also characterized POMC processing in various regions of the spinal cord both before and after lesioning. These data suggested that there may be intrinsic POMC/endorphinergic neuronal systems in the spinal cord.

Unrestrained production of proopiomelanocortin (POMC) and its peptide fragments by pituitary corticotroph adenomas in Cushing's disease

The hallmark of ACTH oversecretion in Cushing's disease is its partial resistance to the normal suppressive effect of glucocorticoids. Because ACTH secretion by the pituitary tumor is not normally restrained ACTH is overproduced with subsequent chronic hypercortisolism. Since peripheral tissues have retained their normal sensitivity to the action of cortisol they appropriately develop the features of Cushing's disease. The question of whether a collection of corticotroph cells, eventually arranged in an adenomatous-like fashion, is a primary pituitary event or is corticotropin-releasing factor driven has had no response so far. Clonal composition of such lesions has been determined by X chromosome inactivation using DNA probes which detect multiallelic polymorphism in females. A monoclonal pattern is found in all macroadenomas. ACTH is co-secreted with other peptide fragments derived from their common polypeptide precursor, proopiomelanocortin (POMC). As a rule POMC processing in pituitary tumors is qualitatively unaltered: plasma values of the N-terminal fragment, the joining peptide, the β-and γ-lipotropins, and β-endorphin all are valid alternate markers of the tumor activity. Tumor POMC peptides including ACTH and its phosphorylated form usually show no peculiar or unexpected molecular forms in contrast with what is often found when POMC expression occurs in a non-pituitary tumor.

Isolation and structural characterization of peptides related to α- and γ-melanocyte-stimulating hormone (MSH) from the frog brain

Peptides that are derived from the processing of proopiomelanocortin were isolated in pure form from the brain of the frog Rana ridibunda. The primary structure of the most abundant of those peptides was established as: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. This amino acid sequence is identical to that of mammalian and frog pituitary α-melanocyte-stimulating hormone (MSH) and the peptide co-eluted with synthetic desacetyl α-MSH, indicating that it is COOH-terminally α-amidated. A second component, which exhibited a shorter retention time, co-eluted with the glycine-extended form of desacetyl α-MSH [ACTH(1–14)]. The primary structure of the third peptide isolated in pure form from the brain extract was established as: Lys-Tyr-Val-Met-Ser-His-Phe-Arg-Trp-Asn-Lys-Phe-NH 2. This sequence corresponds to Lys-γ 1-MSH as predicted from the nucleotide sequence of frog proopiomelanocortin. The presence of substantial amounts of desacetyl α-MSH and Lys-γ 1-MSH in the frog brain supports the concept that, in amphibia, melanotropins may act as neurotransmitters and/or neuromodulators as well as hormonal peptides.

A certain step of proteolytic processing of proopiomelanocortin occurs during the transition between two distinct stages of secretory granule maturation in rat anterior pituitary corticotrophs

A certain step of proteolytic processing of proopiomelanocortin occurs during the transition between two distinct stages of secretory granule maturation in rat anterior pituitary corticotrophs

A certain step of proteolytic processing of proopiomelanocortin occurs during the transition between two distinct stages of secretory granule maturation in rat anterior pituitary corticotrophs.

To obtain antibodies specific for precursor POMC and its cleaved peptides, we prepared synthetic peptides corresponding to the cleavage site (ST-1) between ACTH and beta-lipotropic hormone moieties of POMC and to amidated joining peptide (JP; ST-3) and generated their polyclonal antibodies in rabbits. The anti-ST-1 serum immunoprecipitated only POMC in the extract of AtT-20 cells. In addition to a similar immunoprecipitation examination, an immunoabsorption test showed that anti-ST-3 serum recognizes cleaved peptides, 16K amino-terminal peptide, and JP, but not POMC precursor. Double immunolabeling with gold particles of different sizes for the precursor POMC and its cleaved peptides revealed that the former was localized in electron-dense secretory granules, whereas the latter was found in the electron-lucent secretory granules in the same ultrathin cryosections. Colocalization of POMC and its cleaved peptides in the same secretory granules was rarely observed. Only POMC immunolabeling was detected in the Golgi cisternae. These results suggest that a certain step of proteolytic processing of POMC occurs in the secretory granules during the transition between the two distinct stages of secretory granule maturation in the rat anterior pituitary corticotroph.

Coordinate regulation of mRNA levels of pro-opiomelanocortin and the candidate processing enzymes PC2 and PC3, but not furin, in rat pituitary intermediate lobe

Pro-opiomelanocortin is a multivalent hormone precursor which is processed at pairs of basic residues in a tissue-specific manner to release biologically active peptides. We have examined the message levels of three candidate pro-opiomelanocortin processing enzymes in the intermediate lobe of the rat pituitary following treatment with a dopamine receptor agonist and antagonist which are known to regulate pro-opiomelanocortin mRNA levels. Message levels for PC2 and PC3 but not furin were coordinately regulated with pro-opiomelanocortin transcripts supporting a role for PC2 and PC3 in the maturation of the pro-opiomelanocortin precursor in the rat pituitary intermediate lobe.

Expression and processing of mouse proopiomelanocortin in bovine adrenal chromaffin cells. A model system to study tissue-specific prohormone processing

Many neuroendocrine precursor proteins, such as proopiomelanocortin (POMC), are cleaved in a tissue specific manner at distinct pairs of basic amino acids. Elucidating the specificity of the prohormone endoprotease(s) is essential to understanding cleavage specificity. However, isolation of these enzymes has been difficult, due to the inability to distinguish authentic maturation enzyme from the many other trypsin-like activities present in tissue homogenates. Recently, a "signature" of the insulin cell endoprotease(s) was defined in vivo by assessing the processing of a series of mutant cleavage sites in a model prohormone, mouse POMC (mPOMC) (Thorne, B. A., and Thomas, G. (1990) J. Biol. Chem. 265, 8436-8443. To investigate mechanisms of tissue-specific processing, we sought to identify the endoprotease signature of a cell having a processing phenotype distinct from insulinoma cells. In this report, the cleavage site specificity of the endoprotease(s) expressed in bovine adrenal chromaffin cells is examined. High levels of mPOMC (1.6 pmol/10(6) cells) were expressed in these cells using a vaccinia virus vector, and the precursor was targeted to the regulated secretory pathway. Analysis of POMC-derived peptides revealed that chromaffin cells processed the prohormone to a set of peptides highly similar to anterior pituitary corticotrophs, including adrenocorticotropin hormone (ACTH) and beta-lipotropin, gamma-lipotropin, and beta-endorphin. This processing contrasted with the pattern of cleavage site utilization in Rin m5F insulinoma cells, which more closely resembled that of the intermediate pituitary melanotrophs. However, the processing preference for the sequences of pairs of basic amino acids (as tested using the entire series of mutant cleavage sites; -LysArg- (native), -ArgArg-, -ArgLys-, -LysLys-, -HisArg-, -MetArg- at the ACTH/beta-lipotropin junction and -LysLys- (native), -LysArg-, -ArgArg-, -ArgLys- in beta-endorphin) was the same in both insulinoma and adrenal chromaffin cells, suggesting recognition and cleavage by similar enzymes in both cell types. The cell-specific processing of mPOMC may thus result from expression of a common core set of processing enzymes and factors unique to each cell type affecting the enzyme accessibility to precursor cleavage sites.

Processing of Proopiomelanocortin (POMC) Approached by Immunoelectron Microscopy Pre-Embedding Method

Immunoelectron microscopy pre-embedding method was applied to study the processing of proopiomelanocortin (POMC). Premature pituitary cells and cancer cells showed POMC derived peptides in perinuclear spaces (PNS), rough endoplasmic reticula (RER) and in a few secretory granules (SG). These cells demonstrated immature processing of POMC and suggested constitutive pathway. Cultured mouse fibroblasts transfected with human POMC gene was a good model for this system. Mature pituitary cells and pituitary adenomas showed POMC derived peptides predominantly in SG which correlated with authentic processing of POMC. Cultured mouse AtT 20 cells transfected with human POMC gene showed similar secretory pathway and processing. These results emphasized that SG is mandatory for proper processing of POMC. SG is probably essential for the regulated system of secretion.

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.