Activation of ETB receptors regulates the abundance of ET-1 mRNA in vascular endothelial cells.

BACKGROUND AND PURPOSE: The factors that influence the cellular levels of endothelin-1 (ET-1) include transcription, mRNA localization, stability and translation, post-translational maturation of preproET-1 and degradation of ET-1. We investigated the regulation of ET-1 mRNA abundance by extracellular ET-1 in porcine aortic endothelial cells (PAECs). EXPERIMENTAL APPROACH: Passsage one cultures of PAECs were incubated in starving medium in the presence or absence of ET-1 and antagonists or pharmacological inhibitors. PreproET-1 mRNA, endothelin-1 promoter activity, Erk and p38 MAPK activation were determined. KEY RESULTS: Exogenous ET-1 reduced cellular ET-1 mRNA content: a reduction of 10 000-fold was observed after 4 h. ET-1 simultaneously reduced the stability of ET-1 mRNA and increased the loading of RNA Polymerase II at the endothelin-1 promoter. In the absence of exogenous ET-1, the ETB-selective antagonist, BQ788, increased ET-1 mRNA. An ETA-selective antagonist had no effect. ET-1 mRNA returned to control levels within 24 h. Whereas activation of p38 MAPK induced by ET-1 peaked at 30 min and returned to control levels within 90 min, Erk1/2 remained active after 4 h of stimulation. Inhibition of p38 MAPK prevented the ET-1-induced decrease in ET-1 mRNA. In contrast, Erk1/2 inhibition increased ET-1 mRNA. Similarly, inhibition of receptor internalization increased ET-1 mRNA in the presence or absence of exogenous ET-1. CONCLUSIONS AND IMPLICATIONS: These results suggest that extracellular ET-1 regulates the abundance of ET-1 mRNA in PAECs, in an ETB receptor-dependent manner, by modulating both mRNA stability and transcription via mechanisms involving receptor endocytosis and both ERK and p38 MAPK pathways.

Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization.

Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin/kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT downward arrowSL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor.

The subtilisin/kexin family of precursor convertases. Emphasis on PC1, PC2/7B2, POMC and the novel enzyme SKI-1.

Proopiomelanocortin (POMC) is a precursor to various, bioactive peptides including ACTH, beta LPH, alpha MSH, and beta endorphin (beta END). Processing of POMC at dibasic residues is tissue-specific and is performed by either PC1 alone (resulting in ACTH and beta LPH, anterior pituitary corticotrophes) or by a combination of PC1 and PC2 (yielding alpha MSH and beta END, pituitary neurointermediate lobe and hypothalamus). The PC2-specific binding protein 7B2 is intimately involved in the zymogen activation of proPC2 into PC2. Structure-function studies of these enzymes demonstrated the presence of N- and C-terminal domains, as well as specific amino acids within the catalytic segment that influence the degree of activity of each enzyme and the interaction of PC2 with 7B2. The tissue distribution, plasticity of expression, and the multiple precursors that are differentially cleaved by PC1 and/or PC2, predict a wide array of combinatorial activities of these convertases within the endocrine and neuroendocrine system. The phenotypic consequences of the absence of genetic expression of either PC1 or PC2 are now explored using knockout mice and in human patients suffering from obesity and diabetes.

Residues unique to the pro-hormone convertase PC2 modulate its autoactivation, binding to 7B2 and enzymatic activity.

The prohormone convertase PC2 is one of the major subtilisin/kexin-like enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the members of the subtilisin/kexin-like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2-specific binding protein known as 7B2. We hypothesized that some of these unique properties of PC2 are dictated by the presence of PC2-specific amino acids, which in the six other known mammalian convertases are otherwise conserved but distinct. Accordingly, six sites were identified within the catalytic segment of PC2. Herein we report on the site-directed mutagenesis of Tyr194 and of the oxyanion hole Asp309 and the consequences of such mutations on the cellular expression and enzyme activity of PC2. The data show that the Y194D mutation markedly increases the ex vivo ability of PC2 to process proopiomelanocortin (POMC) into beta-endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these cells, expression of native PC2 does not result in the secretion of measurable in vitro activity against a pentapeptide fluorogenic substrate. In contrast, secreted Y194D-PC2 exhibited significant enzymatic activity, even in the absence of 7B2. Based on co-immunoprecipitations and Western blots, binding assays indicate that Tyr194 participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp309 is critical for the binding of proPC2 with pro7B2.