Obliteration of α‐melanocyte‐stimulating hormone derived from POMC in pituitary and brains of PC2‐deficient mice

Abstract

Alpha-melanocyte-stimulating hormone (a-MSH) is a neuropeptide expressed in pituitary and brain that is known to regulate energy balance, appetite control, and neuroimmune functions. The biosynthesis of a-MSH requires proteolytic processing of the proopiomelanocortin (POMC) precursor. Therefore, this study investigated the in vivo role of the prohormone convertase 2 (PC2) processing enzyme for production of a-MSH in PC2-deficient mice. Specific detection of a-MSH utilized radioimmunoassay (RIA) that does not crossreact with the POMC precursor, and which does not crossreact with other adrenocorticotropin hormone (ACTH) and b-endorphin peptide products derived from POMC. a-MSH in PC2-deficient mice was essentially obliterated in pituitary, hypothalamus, cortex, and other brain regions (collectively), compared to wild-type controls. These results demonstrate the critical requirement of PC2 for the production of a-MSH. The absence of a-MSH was accompanied by accumulation of ACTH, ACTH-containing imtermediates, and POMC precursor. ACTH was increased in pituitary and hypothalamus of PC2-deficient mice, evaluated by RIA and reversed-phase high pressure liquid chromatography (RP-HPLC). Accumulation of ACTH demonstrates its role as a PC2 substrate that can be converted for a-MSH production. Further analyses of POMC-derived intermediates in pituitary, conducted by denaturing western blot conditions, showed accumulation of ACTH-containing intermediates in pituitaries of PC2-deficient mice, which implicate participation of such intermediates as PC2 substrates. Moreover, accumulation of POMC was observed in PC2-deficient mice by western blots with anti-ACTH and anti-b-endorphin. In addition, increased b-endorphin 1)31 was observed in pituitary and hypothalamus of PC2-deficient mice, suggesting b-endorphin 1)31 as a substrate for PC2 in these tissues. Overall, these studies demonstrated that the PC2 processing enzyme is critical for the in vivo production of a-MSH in pituitary and brain. Keywords: alpha-melanocyte-stimulating hormone, brain, pituitary, prohormone convertase, proopiomelanocortin. The neuropeptide a-MSH (melanocyte-stimulating hormone) is derived from its POMC (proopiomelanocortin) precursor (Roberts et al. 1979;Chang et al. 1980) by proteolytic processing within neuroendocrine secretory vesicles of pituitary and brain (Steiner et al. 1992;Hook et al. 1994;Cawley et al. 1998;Seidah et al. 1999). Specific proteolytic processing of POMC at multibasic residues generates distinct neuropeptide products that consist of a-MSH, ACTH (adrenocorticotropin hormone), and b-endorphin. These POMC products are abundant in pituitary and brain where they are secreted for the regulation of pituitary and brain functions that include energy balance, appetite control, and neuroimmune functions (Ichiyama et al. 2000;Williams et al. 2001;Pritchard et al. 2002;Zimanyi and Pelleymounter 2003 These authors contributed equally to this study.Abbreviations used: ACTH, adrenocorticotropin hormone; a-MSH, amelanocyte-stimulating horm...

Identification of positional candidate genes for body weight and adiposity in subcongenic mice

“…PC2-null mice weigh 5-18% less than their wild-type counterparts with no gross anatomic disturbances (16). They lack pituitary and brain ␣-MSH and demonstrate high levels of ACTH in the pituitary and hypothalamus (24,32) due to the inability to process ␣-MSH from ACTH.…”

Proteases for Processing Proneuropeptides into Peptide Neurotransmitters and Hormones

“…Among many neuropeptides examined (56-65), α-MSH (derived from POMC) was nearly absent in PC2 deficient mice (56). Results of neuropeptide studies in PC2 null mice revealed three characteristic types of findings.…”

“…Extensive comparisons of multiple neuropeptides in different tissues of PC2 null mice demonstrated the selective effects of PC2 (56)(57)(58)(59)(60)(61)(62)(63)(64)(65). Among many neuropeptides examined (56-65), α-MSH (derived from POMC) was nearly absent in PC2 deficient mice (56).…”

60 YEARS OF POMC: From the prohormone theory to pro-opiomelanocortin and to proprotein convertases (PCSK1 to PCSK9)

“…PC1/3-deficient mice exhibited severe impairment of ACTH production and compensatory accumulation of POMC mRNA in the pituitary (Zhu et al 2002). Using refined immunological techniques with specific antibodies (Miller et al 2003) showed that the pituitary and hypothalamus of PC2-deficient mice lacked α-MSH, accumulating ACTH, ACTH-containing intermediates, and POMC as well as β-endorphin(1-31). The impaired processing was largely confirmed by mass spectrometry-based peptidomics (Wardman & Fricker 2011).…”