Processing Of Proopiomelanocortin Research Articles

Nur77 gene expression levels were involved in different ACTH-secretion autonomy between Cushing's disease and subclinical Cushing's disease.

Cushing's disease (CD) and subclinical Cushing's disease (subCD) are both diseases caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas. However, ACTH autonomy in subCD is weaker than in CD and there are no Cushingoid features in subCD. The differences of molecular mechanisms in ACTH autonomy between CD and subCD have not yet been reported. Therefore, we aimed to investigate the differences in molecular mechanisms of ACTH-secretion autonomy between CD and subCD. The study included 23 patients [7 CD, 6 subCD, and 10 non-functioning pituitary tumors (NFTs)] who underwent transsphenoidal surgery at the Osaka University Hospital between December 2009 and October 2013. Using quantitative real-time PCR, various ACTH-related gene expressions in tumor tissues from CD, subCD, and NFT were measured such as pro-opiomelanocortin (POMC), POMC transcription factor (Tpit, Pitx1, NeuroD1, and Nur77), POMC peptide processing enzymes (prohormone convertase: PC1/3 and PC2), and ACTH secretion-related factors (corticotropin-releasing hormone receptor 1: CRHR1 and glucocorticoid receptor α: GRα). Only Nur77 mRNA levels were significantly higher in CD than in subCD. Furthermore, we stained 6 CD and 6 subCD with anti-Nur77 antibody. All tumor samples from CD had Nur77 protein positive cells. On the other hand, Nur77 protein was expressed in only one tumor sample from subCD. This sample showed high expression of Nur77 mRNA. Nur77 is an important to regulate POMC transcription and negative-feedback by glucocorticoids. Nur77 gene expression levels might involve different autonomy of ACTH production between CD and subCD.

Melanin production through novel processing of proopiomelanocortin in the extracellular compartment of the auricular skin of C57BL/6 mice after UV-irradiation.

The production of melanin is regulated by α-melanocyte-stimulating hormone (α-MSH), which is produced from proopiomelanocortin (POMC). Keratinocytes release POMC along with lower levels of α-MSH and ACTH. To clarify the mechanism of melanogenesis after ultraviolet (UV)-irradiation, this study focused on the expression of POMC and POMC-derived peptides after UV-irradiation. Western blot analysis and immunoassays indicated that both POMC and α-MSH-like immunoreactivity (α-MSH-LI) increased after UV-irradiation. However, other POMC-derived products were very low. In hypophysectomized mice, α-MSH-LI increased to the same level as in control mice after UV-irradiation. Structural analysis revealed that the major α-MSH-LI product was ACTH(1–8). Furthermore, ACTH(1–8) competed with [125I]-α-MSH for receptor binding and increased melanin production via a melanocortin-1 receptor. These results suggested that melanin was produced through ACTH(1–8) after UV-irradiation. Trypsin-like enzymatic activity, which is responsible for POMC activation, increased after UV-irradiation and was identified as tryptase. In mast cell-deficient mice, which do not produce tryptase, α-MSH-LI levels were unchanged after UV-irradiation. The present study demonstrates the production of ACTH(1–8) from POMC by tryptase, which is a novel peptide-processing mechanism in the extracellular compartment of the skin.

Modulation of proopiomelanocortin gene expression by ethanol in mouse anterior pituitary corticotrope tumor cell AtT20

In humans, alcoholism is associated with a decrease in basal ACTH and cortisol levels, and blunted pituitary ACTH responses to administered corticotropin-releasing hormone (CRH) during active drinking and after long-term abstinence. Preclinical studies indicate that a persistent decrease in pituitary activation after chronic exposure to ethanol is due to a direct effect of ethanol on the corticotrope of the anterior pituitary. The present studies were undertaken to determine if ethanol has effects on proopiomelanocortin (POMC) gene transcription activity in mouse anterior pituitary corticotrope tumor cell AtT20. We measured the levels of the POMC primary nuclear RNA transcript (PT), processing intermediate, and mature mRNA in the nucleus and the levels of the POMC mRNA in the cytoplasm after treatment of AtT20 cells with 5–15mM concentrations of ethanol. After 15mM ethanol for 60 to 120min, the POMC PT levels were significantly decreased. This decreased POMC gene transcription activity was coupled with a significant reduction of the POMC cytoplasmic mRNA levels. After ethanol for 4h, however, both the decreases were no longer observed. After 8h, a decrease in the ACTH secretion in the medium was found. We further investigated if CRH or glutamate modulates the effects of ethanol on the POMC gene transcription activity. CRH at 10nM after 60min increased the POMC PT levels, and 15mM ethanol attenuated the effect of CRH on the nuclear transcription activity. Glutamate receptor proteins, including NMDA receptor subtype NR1 (but not NR2A or NR2B) and GluR2, were identified by Western immunoblot analysis in AtT20 cells, with similar sizes to those in mouse hypothalamus. The inhibitory effect of 60min ethanol at 5 to 15mM on the POMC PT levels was attenuated by 50μML-glutamate. Together, our data showed that: (1) ethanol treatment in intoxicate doses significantly inhibited POMC gene transcription activity in a dose- and time-dependent manner in AtT20 cells, and (2) the POMC gene transcription activity in response to CRH or glutamate was altered by ethanol. Our results suggest that ethanol has an inhibitory effect on the POMC gene transcription activity in the anterior pituitary corticotrope, which may contribute to the persistent decrease in pituitary activation after chronic ethanol exposure.

Identification of Pathways Associated With Endothelial Dysfunction in Obesity

Obesity has become an increasingly serious health problem and a widespread social focus. It is associated with many diseases, including cardiovascular disease through the induction of endothelial dysfunction. The aim of this study was to analyze the endothelial genes that become dysfunctional in obesity and then describe the most significant signal pathways using a systematic bioinformatics approach. Two sets of genes were recovered from the PubMed database. One set was constrained to vascular endothelial dysfunction (VED), and the other set was strictly constrained to both obesity and VED. These two gene sets were mapped to the pathway databases in GeneGo and DAVID to identify pathways associated with endothelial dysfunction in obesity.One hundred and fifty-nine genes were collected in the first set of VED genes. An additional 64 genes associated with obesity also played a role in endothelial dysfunction. Two major pathways (Proopiomelanocortin (POMC) processing and angiotensin system maturation in protein folding and maturation) associated with obesity and endothelial dysfunction were explored. This paper studied obesity at the systems biology level and identified important pathways associated with this disorder. These pathways could serve as targets for treatment and drug discovery in the future.

Chapter Five - Constitutive Activity in Melanocortin-4 Receptor: Biased Signaling of Inverse Agonists

The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis, including both energy intake and energy expenditure. It mediates the actions of a number of hormones on energy balance. The endogenous ligands for MC4R include peptide agonists derived from processing of proopiomelanocortin and the antagonist Agouti-related peptide (AgRP). Wild-type MC4R has some basal (constitutive) activity. Naturally occurring and laboratory-generated mutations have been identified, which results in either increased or decreased basal activities. Impaired basal signaling has been suggested to be a cause of dysregulated energy homeostasis and early-onset obesity, although several constitutively active mutations have also been identified from obese patients. AgRP and several small-molecule antagonists have been shown to be inverse agonists in the Gs-cAMP pathway. However, in the extracellular signal-regulated kinase (ERK) 1/2 pathway, we showed that these inverse agonists are potent agonists, demonstrating convincingly that they are biased ligands. We also showed that some mutations that do not cause constitutive activation in the Gs-cAMP pathway cause constitutive activation in the ERK1/2 pathway, suggesting that they are biased receptors. The physiological and potential pathophysiological relevance of the biased constitutive signaling in MC4R and therapeutic potential remain to be investigated.

Mitofusin 2 in POMC Neurons Connects ER Stress with Leptin Resistance and Energy Imbalance

Mitofusin 2 (MFN2) plays critical roles in both mitochondrial fusion and the establishment of mitochondria-endoplasmic reticulum (ER) interactions. Hypothalamic ER stress has emerged as a causative factor for the development of leptin resistance, but the underlying mechanisms are largely unknown. Here, we show that mitochondria-ER contacts in anorexigenic pro-opiomelanocortin (POMC) neurons in the hypothalamus are decreased in diet-induced obesity. POMC-specific ablation of Mfn2 resulted inloss of mitochondria-ER contacts, defective POMC processing, ER stress-induced leptin resistance, hyperphagia, reduced energy expenditure, and obesity. Pharmacological relieve of hypothalamic ER stress reversed these metabolic alterations. Our data establish MFN2 in POMC neurons as an essential regulator of systemic energy balance by fine-tuning the mitochondrial-ER axis homeostasis and function. This previously unrecognized role for MFN2 argues for a crucial involvement in mediating ER stress-induced leptin resistance.

Loss of NSCL-2 in Gonadotropin Releasing Hormone Neurons Leads to Reduction of Pro-Opiomelanocortin Neurons in Specific Hypothalamic Nuclei and Causes Visceral Obesity

Regulation of sexual reproduction and energy homeostasis are closely interconnected, but only few efforts were made to explore the impact of gonadotropic neurons on metabolic processes. We have used Nscl-2 mutant mice suffering from adult onset of obesity and hypogonadotropic hypogonadism to study effects of gonadotropin releasing hormone (GnRH) neurons on neuronal circuits controlling energy balance. Inactivation of Nscl-2 in GnRH neurons but not in pro-opiomelanocortin (POMC) neurons reduced POMC neurons and increased visceral fat mass, suggesting a critical role of GnRH cells in the regulation of POMC neurons. In contrast, absence of POMC processing in the majority of Nscl-2-deficient POMC neurons had no effect on energy homeostasis. Finally, we investigated the cellular basis of the reduction of GnRH neurons in NSCL-2 mutants using a lineage tracing approach. We found that loss of Nscl-2 results in aberrant migration of GnRH neurons in Nscl-2 mutant mice causing a lineage switch of ectopically located GnRH neurons.

Low serum nesfatin-1 levels may be a contributing factor for monogenic obesity due to prohormone convertase 1 deficiency

Leptin, leptin receptor, proopiomelanocortin, prohormone convertase 1, melanocortin-4 receptor, melanocortin-3 receptor, brain-derived neurothrophic factor, neurotrophic tyrosine receptor kinase type 2, and single minded 1 genes, which are the genes that have been shown to cause monogenic obesity, are related to the leptin–melanocortin system. Congenital prohormone convertase 1 deficiency is a very rare syndrome characterized by early-onset obesity, small intestinal dysfunction, impaired glucose homeostasis, and disrupted prohormone processing. Obesity phenotype seen in this syndrome is known to be due to reduced melanocortin signaling in the hypothalamus which arises as a result of impaired processing of proopiomelanocortin. However, prohormone convertase 1 cleaves a number of other neuropeptides involved in feeding behavior and impaired cleavage of these neuropeptides, particularly NUCB2/nesfatin, may be a contributing factor for development of obesity in these cases. Until now, in prohormone convertase 1 deficient patients, low serum nesfatin-1 levels have not been mentioned to be a possible cause of obesity, we suggest that in these patients low levels of anorexigenic peptid nesfatin-1 might be another facilitating factor for development of obesity.

Intermediate lobe immunoreactivity in a patient with suspected lymphocytic hypophysitis

Lymphocytic hypophysitis is an organ-specific autoimmune disease characterised by destruction of pituitary hormone-secreting cells due to attack by self-reactive T lymphocytes. The spectrum of pituitary autoantibodies characterised by indirect immunofluorescence (IF) in these patients has not been substantially defined. The purpose of this study was to determine the spectrum of pituitary autoantibodies in 16 lymphocytic hypophysitis patients. Pituitary sections were prepared from guinea pigs and sera from 16 lymphocytic hypophysitis patients (13 biopsy proven and 3 suspected cases) and 13 healthy controls were evaluated for immunoreactivity to the pituitary tissue by immunofluorescence. A single patient was found to have high titre pituitary autoantibodies against guinea pig pituitary tissue. Immunoreactivity was directed against cells of the intermediate lobe. We present the case report of the patient who is a 24year old woman that presented with headaches, polyuria and polydipsia. A uniformly enlarged pituitary mass was visible on MRI and a diagnosis of suspected lymphocytic hypophysitis was made. Based on our IF study, we postulate this patient has an autoimmune process directed towards the major cell type in the intermediate lobe, the melanotroph. Pre-adsorption with peptides representing adrenocorticotropic hormone, α-melanocyte stimulating hormone or β-endorphin did not affect the IF signal suggesting our patient's pituitary autoantibodies may target some other product of Proopiomelanocortin (POMC) processing, such as corticotrophin-like intermediate peptide or γ-lipoprotein. Alternatively, the autoantibodies may target a peptide completely unrelated to POMC processing.

A Novel Medical Treatment of Cushing's Due to Ectopic ACTH in a Patient With Neurofibromatosis Type 1

A 64-year-old male presented with neurofibromatosis 1 and Cushing’s syndrome. Clinically he was over weight, depressed with extensive skin bruising and hypertension. His 24 hours urinary metanephrines, urinary 5HIAA, gut peptides and chromgranin levels were normal. His renal function and renal MRI scan was also normal. His cortisol failed to suppress on overnight dexamethsone suppression test. His low dose dexamethasone suppression with CRH stimulation showed failure of suppression of cortisol to < 50 nmol/L and ACTH was measurable at 10 ng/L on day 3. There was no response of ACTH or cortisol to CRH stimulation. His ACTH precursors were high at 126 pmol/L consistent with defective pro-opiomelanocortin (POMC) processing suggesting an ectopic source of ACTH production. The MRI scan of his pituitary and CT scan of the adrenal glands was normal. His octreotide scan was negative. The source of his ectopic ACTH was most likely a large retroperitoneal plexiform neurofibroma seen on CT abdomen that had undergone malignant peripheral nerve sheath tumour transformation on histology. He was a poor surgical risk for tumour debulking procedure. In view of the available literature and role of c-kit signalling in neurofibromatosis, he was treated with Imitinib. Four months after the treatment his Cushings had resolved on biochemical testing. After a year his plexiform neurofibroma has not increased in size. To our knowledge, this is the first case of NF1 associated with clinical and biochemical features of Cushing’s secondary to ectopic ACTH due to MPNST in a plexiform neurofibroma and its resolution on treatment with imatinib.

Hypothalamic regulation of energy balance: a key role for DICER miRNA processing in arcuate POMC neurons

Hypothalamic regulation of energy balance: a key role for DICER miRNA processing in arcuate POMC neurons

β-Endorphin Antagonizes the Effects of α-MSH on Food Intake and Body Weight

Proopiomelanocortin (POMC) is posttranslationally processed to several peptides including α-MSH, a primary regulator of energy balance that inhibits food intake and stimulates energy expenditure. However, another POMC-derived peptide, β-endorphin (β-EP), has been shown to stimulate food intake. In this study we examined the effects of intracerebroventricular (icv) β-EP on food intake and its ability to antagonize the negative effects of α-MSH on energy balance in male rats. A single icv injection of β-EP stimulated food intake over a 2- to 6-h period during both the light and dark cycles. This effect was, however, not sustained with chronic icv β-EP infusion. In the next study, a subthreshold dose of β-EP was injected together with Nle(4), d-Phe(7) (NDP)-MSH after a 16-h fast, and the negative effects of NDP-MSH on refeeding and body weight gain were partially reversed. Finally, peptide interactions were studied in a chronic icv infusion model. Weight gain and food intake were significantly suppressed in the NDP-MSH group during the entire study. A subthreshold dose of β-EP antagonized these suppressive effects on food intake and weight gain for the first 3 d. However on d 4-7, β-EP no longer blocked these effects. Of note, the stimulatory effect of β-EP on feeding and its ability to antagonize MSH were specific for β-EP(1-31) and were not observed with β-EP(1-27). This study highlights the importance of understanding how the balance between α-MSH and β-EP is maintained and the potential role of differential POMC processing in regulating energy balance.

Reversible impairment of the processing of proopiomelanocortin into ACTH in pituitary enlargement suspected of lymphocytic hypophysitis

We describe a 64-year-old woman with a cystic pituitary mass presenting with central diabetes insipidus. Brain magnetic resonance imaging (MRI) with enhancement showed enlargement of the pituitary gland with cystic portions and thickening of the pituitary stalk with homogeneous enhancement. Combined anterior pituitary stimulation test and insulin-induced hypoglycemic test confirmed the diagnosis of panhypopituitarism, including adrenocortical insufficiency due to pituitary and hypothalamic dysfunction by stalk compression. Interestingly, the response of serum cortisol to CRH was low and delayed, in contrast to the marked increase in plasma ACTH. Molecular analysis of her plasma ACTH by Sephadex G75 gel exclusion chromatography coupled with radioimmunoassay (RIA) indicated a peak for high molecular weight ACTH, i.e., proACTH, in addition to that for 1-39 ACTH. Three years later, enlargement of the pituitary gland with cystic portions and thickening of the pituitary stalk disappeared completely, followed by the decrease in plasma proACTH level. By the results of endocrinological study and the change of pituitary MRI findings, lymphocytic hypophysitis was suggested. Synthesis of immature ACTH is generally thought to be due to impaired processing of the precursor proopiomelanocortin (POMC) through activation of prohormone convertase (PC)-1 by CRH. It is possible that the immature ACTH in this case was produced by impaired processing of the precursor POMC due to decreased CRH, dysfunction of corticotrophs in the anterior pituitary by compression of the normal pituitary, or antibodies targeting hypothalamic and/or pituitary cells. This report suggested that impaired processing of POMC may unusually play a role in adrenocortical insufficiency exhibited in lymphocytic hypophysitis.

N-Glycosylation controls trafficking, zymogen activation and substrate processing of proprotein convertases PC1/3 and subtilisin kexin isozyme-1

The limited proteolysis of proteins by the proprotein convertases (PCs) is a common means of producing bioactive proteins or peptides. The PCs are associated with numerous human pathologies and their activity can be reduced through the use of specific inhibitors. Here, we demonstrate an alternative approach to inhibiting PCs by altering their N-glycosylation. Through site-directed mutagenesis, we show that the convertase PC1/3 contains two N-glycans, only one of which is critical for its prosegment cleavage. The exact structure of PC1/3 N-glycans does not significantly affect its zymogen activation within endocrine cells, but glycosylation of Asn(146) is critical. Processing of the PC1/3's substrate proopiomelanocortin (POMC) was used in a cell-based assay to screen a collection of 45 compounds structurally related to known glycosidase inhibitors. Two 5-thiomannose-containing disaccharide derivatives were discovered to block PC1/3 and POMC processing into the analgesic peptide β-endorphin. These compounds also reduced the zymogen activation of the convertase subtilisin kexin isozyme-1 (SKI-1), blocked the processing of its substrate the sterol regulatory element-binding protein SREBP-2 and altered its glycosylation. Thus, modification of PC glycosylation may also be a means of blocking their activity, an effect which, in the case of SKI-1, may be of possible therapeutic use since SREBP-2 regulates sterol levels including cholesterol biosynthesis and its metabolism.

Genetic modifications of mouse proopiomelanocortin peptide processing

Pro-opiomelanocortin (POMC) is a prohormone which undergoes extensive tissue and cell specific post-translational processing producing a number of active peptides with diverse biological roles ranging from control of adrenal function to pigmentation to the regulation of feeding. One approach to unraveling the complexities of the POMC system is to engineer mouse mutants which lack specific POMC peptides. We describe here the design, generation, validation, and preliminary analysis of one such partial POMC mutant specifically lacking α-MSH. In contrast to POMC null mutant mice, mice lacking α-MSH in the presence of all other POMC peptides maintain adrenal structures and produce corticosterone comparable to wildtype littermates; however, they still have decreased levels of aldosterone, as found in POMC null mutant mice. Our findings demonstrate that α-MSH is not needed for maintenance of adrenal structure or for corticosterone production, but is needed for aldosterone production. These data demonstrate that mouse strains generated with precise genetic modifications of POMC peptide processing can answer questions about POMC peptide function. Further analysis of this and additional strains of mice with modified POMC peptide processing patterns will open up a novel avenue for studying the roles of individual POMC peptides.

Feather follicles express two classes of pro-opiomelanocortin (POMC) mRNA using alternative promoters in chickens

Feather coloration in chickens mainly depends on melanin produced by melanocytes located in the feather follicles. The melanocortin 1 receptor (MC1R) on follicular melanocytes regulates melanin synthesis; however, the source of the melanocortins that interact with the receptors remains unclear. In this study, we examine the potential expression of melanocortins and characterize the mRNAs for the precursor pro-opiomelanocortin (POMC) in chicken feather follicles. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of mRNAs for POMC, prohormone convertase 1 (PC1) and PC2, and western blotting detected adrenocorticotropic hormone (ACTH)-related products of POMC processing in feather follicles, suggesting that melanocortins are produced locally in the tissues of chickens. A combination of 5′RACE (rapid amplification of cDNA 5′ end), 3′RACE and RT-PCR analyzes identified two classes of POMC mRNA, class a and class b, which encode the same full-length POMC protein but have different non-coding leader exons. Class a mRNAs were expressed specifically in feather follicles, whereas class b mRNAs were expressed in the pituitary, hypothalamus, and various peripheral tissues that we examined. Within the feather follicles, the class a mRNAs were distributed in epidermal layers from middle to distal locations, whereas the class b mRNAs were mainly expressed in pulp at proximal locations. Our findings suggest that feather pigmentation is regulated by locally produced melanocortins, and indicate that the melanocortins encoded by the different classes of POMC mRNAs may play different intra-follicular roles in chickens. This is the first report that demonstrates alternative promoter usage generating different full-length POMC mRNAs in vertebrates.

V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells

The vacuolar (H(+))-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH-dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing.

α-Melanocyte–Stimulating Hormone Inhibits Tumor Necrosis Factor α–StimulatedMUC5ACExpression in Human Nasal Epithelial Cells

Mucin hypersecretion is an important clinical feature of several respiratory diseases, including asthma, cystic fibrosis, nasal allergy, rhinitis, and sinusitis. It has been shown that α-melanocyte-stimulating hormone (α-MSH), a proopiomelanocortin (POMC)-derived peptide, has immunomodulatory activities by inhibiting NF-κB activation induced by proinflammatory cytokines such as TNF-α. Because MUC5AC expression is known to be up-regulated by TNF-α via NF-κB activation, we evaluated the inhibitory effect of α-MSH on MUC5AC gene expression induced by TNF-α in normal human nasal epithelial (NHNE) cells. Melanocortin-1-receptor (MC-1R) was detected by RT-PCR, Western blotting, and immunofluorescent labeling in NHNE cells. α-MSH suppressed NF-κB/p65 phosphorylation induced by TNF-α as well as IkB-α degradation in a dose-dependent manner, as assessed by Western blotting. In addition, α-MSH inhibited TNF-α-induced nuclear translocation of NF-κB and NF-κB luciferase activity. Real-time quantitative PCR data showed that α-MSH inhibited TNF-α-induced expression of MUC5AC, and this effect of α-MSH was neutralized by knockdown of MC-1R using MC-1R shRNA lentivirus. Analyses using RT-PCR and Western blotting showed the expression of POMC and two key enzymes in the POMC processing, proprotein convertases (PC)1 and PC2, and 7B2, which is required for enzymatic activity of PC2, in normal human nasal mucosa. We conclude that α-MSH down-regulates MUC5AC expression by inhibiting TNF-α-induced NF-κB activity through MC-1R stimulation in NHNE cells and that normal human nasal mucosa possesses the POMC processing machinery. Therefore, α-MSH may be a promising candidate to decrease mucin overproduction initiated by NF-κB activation.

Signaling from the Secretory Granule to the Nucleus: Uhmk1 and PAM

Neurons and endocrine cells package peptides in secretory granules (large dense-core vesicles) for storage and stimulated release. Studies of peptidylglycine alpha-amidating monooxygenase (PAM), an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain (CD) of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by U2AF homology motif kinase 1 (Uhmk1) and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble CD fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously overexpressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wild-type mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels whereas expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent.

Local pulmonary opioid network in patients with lung cancer: a putative modulator of respiratory function

Recently, there has been growing interest in the opioid regulation of physiological respiratory function. However, evidence for a local opioid network that includes endogenous opioid peptides and their receptors is scarce. Tissue samples from patients with lung cancer were examined by immunohistochemistry to identify the components of the opioid network: β-endorphin (END); its precursor, proopiomelanocortin (POMC); the key processing enzymes prohormone convertase 1 and 2; carboxypeptidase E; and END’s corresponding opioid receptor, the u-opioid receptor (MOR). Additionally, we tested pulmonary function parameters in a patient with advanced lung cancer after inhalation of nebulized morphine. Confocal immunofluorescence microscopy revealed that the opioid precursor POMC colocalizes with its active peptide END, key processing enzymes and MOR in alveolar macrophages, sub-mucosal glands, cancerous cells, and pulmonary neuroendocrine cells within the bronchial epithelium. In addition, MOR was identified on sensory nerve endings within the bronchial epithelium. Furthermore, nebulized morphine improved pulmonary function parameters in advanced lung cancer. These findings provide evidence of a local opioid network in functionally important anatomical structures of the respiratory system; this network consists of all the machinery required for POMC processing into active peptides, such as END, and contains the receptors for END. Our findings indicate a need for further clinical trials to elucidate the modulatory function of peripheral endogenous opioids in the human lung.