Profiles of pro-opiomelanocortin and encoded peptides, and their processing enzymes in equine pituitary pars intermedia dysfunction
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Abstract:
Equine pituitary pars intermedia dysfunction (PPID) is characterized by hyperplasia of the pars intermedia (PI) melanotrophs of the pituitary gland (PG), and increased production of proopiomelanocortin (POMC). POMC is cleaved by prohormone convertase 1 (PC1) to produce adrenocorticotropic hormone (ACTH), and further processing of ACTH by PC2 to produce alpha-melanocyte stimulating hormone (α-MSH) and corticotropin-like intermediate peptide (CLIP). High plasma ACTH concentrations in horses with PPID might be related to reduced conversion of ACTH to α-MSH by PCs. The hypothesis of this study was that PC1 and PC2 expression in the pituitary gland are altered in PPID, resulting in an abnormal relative abundance of POMC derived proteins. The objectives of this study were to identify the partial sequences of equine POMC, PC1, and PC2 mRNAs; and to determine whether the expression of POMC, PC1, and PC2 mRNAs in whole pituitary extracts, and POMC-protein in the cavernous sinus blood of horses are altered in PPID. We confirmed (RT-PCR and sequencing) that the partial sequences obtained match the corresponding regions of predicted equine POMC, PC1 and PC2 sequences. The expression (quantification by RT-qPCR) of POMC, PC1 and PC2 mRNAs were found upregulated in the pituitary of horses with PPID. Plasma (measured using RIA/ELISA) ACTH and α-MSH were elevated in PPID horses. These results indicate distinct differences in gene and protein expression of POMC and its intermediates, and processing enzymes in PPID. It provides evidence to support the notion that local, pituitary-specific inadequacies in prohormone processing likely contribute to equine PPID.Keywords:
Proopiomelanocortin
Pars intermedia
Prohormone convertase
Corticotropic cell
Proopiomelanocortin
Corticotropin-releasing hormone
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Prohormone convertases (PCs) are thought to represent the major proteinases involved in the biosynthetic processing of peptide hormone precursors to bioactive peptide products. The maturation of PC2 requires the aid of a helper protein, 7B2, in order for the zymogen to become an active enzyme species. The 7B2 and PC2 nulls should thus be functionally equivalent with regard to deficits in precursor processing. In this article, we have examined this proposition through the study of proopiomelanocortin (POMC) biosynthesis and granule content in both null models. RIA data indicate that both PC2 and 7B2 nulls lack pituitary α-MSH; interestingly, 7B2 nulls are still able to generate β-endorphin from β-lipotropin, whereas PC2 nulls contain little if any β-endorphin. Labeling experiments demonstrate a build-up of POMC, high molecular weight intermediates, and intact ACTH, as well as the disappearance of α-MSH, in both null models. Electron microscopy of neurointermediate lobe melanotrophs reveals the presence of a significantly greater number of secretory granules in both 7B2 and PC2 nulls compared with wild-type controls. However, PC2 null melanotrophs contain twice as many granules as 7B2 null melanotrophs. Another difference between the two null models is a relatively enhanced accumulation of precursors in the PC2 null compared with the 7B2 null; these include not only PC2 substrates, but also presumed PC1 substrates. These data indicate that the two nulls are not phenotypically equivalent.
Proopiomelanocortin
Prohormone convertase
Null cell
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Proopiomelanocortin (POMC) is the precursor protein of different hormones and neuropeptides, and the POMC-derived peptides are produced through proteolytic cleavage. Prohormone convertase PC1 and PC2 are enzymes responsible for the cleavage of the POMC prohormone. The coexpression of POMC, PC1, and PC2 genes was previously described in the brain and the pituitary gland of Rana esculenta and Xenopus laevis, but no data are available for the gonad. The present work demonstrates a gonadal POMC convertase gene expression in Rana esculenta and Xenopus laevis.
Proopiomelanocortin
Prohormone convertase
Cleavage (geology)
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Equine pituitary pars intermedia dysfunction (PPID) is characterized by hyperplasia of the pars intermedia (PI) melanotrophs of the pituitary gland (PG), and increased production of proopiomelanocortin (POMC). POMC is cleaved by prohormone convertase 1 (PC1) to produce adrenocorticotropic hormone (ACTH), and further processing of ACTH by PC2 to produce alpha-melanocyte stimulating hormone (α-MSH) and corticotropin-like intermediate peptide (CLIP). High plasma ACTH concentrations in horses with PPID might be related to reduced conversion of ACTH to α-MSH by PCs. The hypothesis of this study was that PC1 and PC2 expression in the pituitary gland are altered in PPID, resulting in an abnormal relative abundance of POMC derived proteins. The objectives of this study were to identify the partial sequences of equine POMC, PC1, and PC2 mRNAs; and to determine whether the expression of POMC, PC1, and PC2 mRNAs in whole pituitary extracts, and POMC-protein in the cavernous sinus blood of horses are altered in PPID. We confirmed (RT-PCR and sequencing) that the partial sequences obtained match the corresponding regions of predicted equine POMC, PC1 and PC2 sequences. The expression (quantification by RT-qPCR) of POMC, PC1 and PC2 mRNAs were found upregulated in the pituitary of horses with PPID. Plasma (measured using RIA/ELISA) ACTH and α-MSH were elevated in PPID horses. These results indicate distinct differences in gene and protein expression of POMC and its intermediates, and processing enzymes in PPID. It provides evidence to support the notion that local, pituitary-specific inadequacies in prohormone processing likely contribute to equine PPID.
Proopiomelanocortin
Pars intermedia
Prohormone convertase
Corticotropic cell
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We have combined gene cloning with an assay for prohormone biosynthesis and processing in Xenopus oocytes to identify the genes that encode mammalian prohormone processing enzymes. The coinjection of RNA encoding murine prohormone convertase 1 (mPC1), a mammalian endoprotease, along with proopiomelanocortin RNA into an oocyte results in the appropriate cleavage after paired basic residues in the proopiomelanocortin polyprotein necessary to generate corticotropin. The ability of mPC1 to generate corticotropin, along with the observation that mPC1 is specifically expressed in endocrine and neuronal cells, suggests that the mPC1 gene encodes the endopeptidase responsible for the pathway of proopiomelanocortin cleavage observed in the anterior pituitary.
Prohormone convertase
Proopiomelanocortin
Cleavage (geology)
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Proopiomelanocortin
Corticotropic cell
Prohormone convertase
Cushing's disease
Pituitary Tumors
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Pars intermedia
Corticotropic cell
Proopiomelanocortin
Neuroendocrinology
Hypothalamic Hormones
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Recent evidence demonstrated that posttranslational processing of neuropeptides is critical in the pathogenesis of obesity. Leptin or other physiological changes affects the biosynthesis and processing of many peptides hormones as well as the regulation of the family of prohormone convertases responsible for the maturation of these hormones. Regulation of energy balance by leptin involves regulation of several proneuropeptides such as proTRH and proopiomelanocortin. These proneuropeptide precursors require for their maturation proteolytic cleavage by the prohormone convertases 1 and 2 (PC1/3 and PC2). Because biosynthesis of mature peptides in response to leptin requires prohormone processing, it is hypothesized that leptin might regulate hypothalamic PC1/3 and PC2 expression, ultimately leading to coordinated processing of prohormones into mature peptides. Leptin has been shown to increase PC1/3 and PC2 promoter activities, and starvation of rats, leading to low serum leptin levels, resulted in a decrease in PC1/3 and PC2 gene and protein expression in the paraventricular and arcuate nucleus of the hypothalamus. Changes in nutritional status also changes proopiomelanocortin processing in the nucleus of the solitary tract, but this is not reversed by leptin. The PCs are also physiologically regulated by states of hyperthyroidism, hyperglycemia, inflammation, and suckling, and a recently discovered nescient helix-loop-helix-2 transcription factor is the first one to show an ability to regulate the transcription of PC1/3 and PC2. Therefore, the coupled regulation of proneuropeptide/processing enzymes may be a common process, by which cells generate more effective processing of prohormones into mature peptides.
Proopiomelanocortin
Prohormone convertase
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Corticotropic cell
Proopiomelanocortin
Pars intermedia
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In the adult pituitary, anterior lobe corticotrophs and intermediate lobe melanotrophs differentially process proopiomelanocortin (POMC). Within the corticotrophs, POMC is processed mainly to corticotropin (ACTH) and beta-lipotropin, while alpha-melanotropin (alpha MSH) and beta-endorphin are the major end products in the melanotrophs. The observed transient presence of alpha MSH-like immunoreactivity during ontogeny suggested an age-dependent variation in POMC processing in the adenohypophysis. In this tissue, cell-specific POMC products are likely the result of differential expression of the two known prohormone convertases PC1 and PC2. In the present ontogeny study done in the mouse intermediate and anterior pituitary, we examined how the expression pattern of PC1 and PC2 mRNA transcripts correlates with that of ACTH and alpha MSH-like immunoreactivities. Our data demonstrated that both PC1 and PC2 transcripts can be detected in the presumptive adenohypophysis starting on embryonic day 15 (E15). In the intermediate lobe, PC1 and PC2 mRNAs appear on E18 and E16, respectively, and their levels increased during ontogeny, reaching maximal expression in the adult. Similarly, PC1 expression in the anterior pituitary increased from E15 to adulthood. However, PC2 mRNA expression peaked between postnatal days 1 (P1) and 14 (P14) and then decreased to adult levels. The distribution of PC1 and PC2 immunoreactivity is nicely correlated with the in situ hybridization data. In the anterior lobe, during the P1-P14 postnatal period, PC2 immunoreactivity was detected within cells synthesizing an alpha MSH-like peptide(s). This observation substantiates our earlier biochemical data suggesting that PC2 is the important convertase in the processing of POMC into alpha MSH. Furthermore, the demonstrated variation in the relative ratio of PC1/PC2 expression during ontogeny rationalizes the observed plasticity of POMC processing in the adenohypophysis. It is expected that beta-endorphin processing will follow that of alpha MSH.
Proopiomelanocortin
Corticotropic cell
Colocalization
Prohormone convertase
Pars intermedia
Alpha (finance)
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Citations (129)