Abstract
The small neuroendocrine protein 7B2 is required for the production of active prohormone convertase 2 (PC2), an enzyme involved in the synthesis of peptide hormones, such as glucagon and proopiomelanocortin-derived α-melanocyte-stimulating hormone. However, whether 7B2 can dynamically modulate peptide production through regulation of PC2 activity remains unclear. Infection of the pancreatic alpha cell line α-TC6 with 7B2-encoding adenovirus efficiently increased production of glucagon, whereas siRNA-mediated knockdown of 7B2 significantly decreased stored glucagon. Furthermore, rescue of 7B2 expression in primary pituitary cultures prepared from 7B2 null mice restored melanocyte-stimulating hormone production, substantiating the role of 7B2 as a regulatory factor in peptide biosynthesis. In anterior pituitary and pancreatic beta cell lines, however, overexpression of 7B2 affected neither production nor secretion of peptides despite increased release of active PC2. In direct contrast, 7B2 overexpression decreased the secretion and increased the activity of PC2 within α-TC6 cells; the increased intracellular concentration of active PC2 within these cells may therefore account for the enhanced production of glucagon. In line with these findings, we found elevated circulating glucagon levels in 7B2-overexpressing cast/cast mice in vivo. Surprisingly, when proopiomelanocortin and proglucagon were co-expressed in either pituitary or pancreatic alpha cell lines, proglucagon processing was preferentially decreased when 7B2 was knocked down. Taken together, these results suggest that proglucagon cleavage has a greater dependence on PC2 activity than other precursors and moreover that 7B2-dependent routing of PC2 to secretory granules is cell line-specific. The manipulation of 7B2 could therefore represent an effective way to selectively regulate synthesis of certain PC2-dependent peptides.
Highlights
The small neuroendocrine protein 7B2 is required for the production of active prohormone convertase 2 (PC2), an enzyme involved in the synthesis of peptide hormones, such as glucagon and proopiomelanocortin-derived ␣-melanocyte-stimulating hormone
When proopiomelanocortin and proglucagon were co-expressed in either pituitary or pancreatic alpha cell lines, proglucagon processing was preferentially decreased when 7B2 was knocked down. These results suggest that proglucagon cleavage has a greater dependence on PC2 activity than other precursors and that 7B2-dependent routing of PC2 to secretory granules is cell line-specific
More recent work has shown that 7B2 acts to block the aggregation of proPC2 into unactivatable forms (15). 7B2 may subserve additional functions, because it is present in cells lacking PC2 (32) and circulates in blood (14, 21)
Summary
Preparation of Recombinant Adenovirus—Recombinant adenoviruses encoding either 27-kDa 7B2 or -galactosidase (as a control) were initially made by M. For ␣-MSH assays, 5 l of cell extracts, 50 l (for basal secretion) or 25 l (stimulated secretion) of medium samples from pituitary primary cell cultures were subjected to assay in duplicate using the ␣-MSH RIA kit (Phoenix Pharmaceuticals, Burlingame, CA) For ACTH assays, 5 l of a 1:20 dilution of cell extracts, 5 l (for basal secretion) or 2 l (stimulated secretion) of medium samples from pituitary primary cell cultures were subjected to assay in duplicate using the two-site Nichols human ACTH 1–39 assay kit (Nichols Institute, San Juan Capistrano, CA). 7B2 Adenoviral Infection Increases ACTH Processing to ␣-MSH in Primary Pituitary Cultures Prepared from 7B2 Null Mice—Primary cell cultures derived from pituitaries of 7B2 null mice were infected with either -galactosidase-encoding control virus or 27-kDa 7B2-encoding virus, respectively, labeled with radioactive methionine/cysteine; cell extracts were immunoprecipitated using antisera against PC2 and 7B2. Application of 7B2 siRNA to AtT-20/PC2 cultures failed to reduce cellular ␣-MSH levels (Fig. 4B)
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