Proprotein Convertase 1/3 (PC1/3) in the Rat Alveolar Macrophage Cell Line NR8383: Localization, Trafficking and Effects on Cytokine Secretion

Hugo Gagnon,Sarah Refaie,Michel Salzet,Roxane Desjardins,Robert Day,Sandra Gagnon

doi:10.1371/journal.pone.0061557

Hugo Gagnon, Sarah Refaie + Show 4 more

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https://doi.org/10.1371/journal.pone.0061557

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Abstract

The proprotein convertase 1/3 (PC1/3) is an important post-translational processing enzyme for the activation of precursor proteins within the regulated secretory pathway. Well characterized for its role in the neural and endocrine systems, we recently reported an unconventional role of PC1/3 as a modulator of the Toll-like receptor innate immune response. There are only a few reports that have studied PC1/3 expression in macrophages, and more investigation is needed to better characterize its function. These studies would greatly benefit from model cell lines. Our study aims to identify and characterize PC1/3 in a relevant model macrophage cell line and to determine the links between PC1/3 and innate immune cellular responses. We describe the rat alveolar cell line, NR8383, as expressing PC1/3 and the most common Toll-like receptors. In NR8383 cells, PC1/3 is localized at the Trans-Golgi network and traffics to lysosome related vesicles upon lipopolysaccharide stimulation. Moreover, we report the co-localization of PC1/3 and Toll-like receptor 4 upon lipopolysaccharide stimulation. Down regulation of PC1/3 by shRNA produce a similar phenotype in NR8383 to what we previously reported in isolated peritoneal macrophages. PC1/3 shRNA induced changes in the cellular organization and expression of the specific trafficking regulator RAB GTPase. As a consequence, NR8383 down-regulated for PC1/3, present an abnormal cytokine secretion profile. We conclude that the NR8383 cell line represents a good model to study PC1/3 in macrophages and we present PC1/3 as an important regulator of vesicle trafficking and secretion in macrophages.

Highlights

Post-translational modifications are important processes that contribute to the biological regulation of proteins
The human macrophage cell line THP-1 was shown to express proprotein convertase 1/3 (PC1/3), THP-1 cells need to be stimulated with phorbol 12myristate 13-acetate (PMA) to induce PC1/3 expression [10]
NR8383 cells were shown to be sensitive to TLR ligands, notably LPS [24] and polyI:C [41], making them a good cellular model in which to examine the role of PC1/3 in TLRbased activation of macrophages

Summary

Introduction

Post-translational modifications are important processes that contribute to the biological regulation of proteins. One such modification is the endoproteolysis of precursor proteins, which can lead to activation, inactivation or functional changes [1]. This cleavage process can be extensive or limited to a few bonds by specific convertases and is followed by amino-terminal, internal and carboxy-terminal modification into smaller biologically active polypeptides [2,3]. In a recent study [12], we characterized an innate immune related phenotype for the PC1/3 knockout (KO) mice challenged with lipopolysaccharides (LPS), which triggers a cascade of events following the stimulation of Toll-like receptor 4 (TLR4) [13,14,15]. We concluded that PC1/3 regulates the cytokine response and the innate immune response

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