Date Published: January 17, 2017
Publisher: Public Library of Science
Author(s): Stephane Esnault, Elizabeth E. Torr, Ksenija Bernau, Mats W. Johansson, Elizabeth A. Kelly, Nathan Sandbo, Nizar N. Jarjour, Collynn Woeller.
Semaphorin-7A is a glycosylphosphatidylinositol-anchored protein, initially characterized as an axon guidance protein. Semaphorin-7A also contributes to immune cell regulation and may be an essential pro-fibrotic factor when expressed by non-fibroblast cell types (exogenous). In mouse models, semaphorin-7A was shown to be important for TGF-ß1-induced pulmonary fibrosis characterized by myofibroblast accumulation and extracellular matrix deposition, but the cell-specific role of semaphorin-7A was not examined in fibroblasts. The purpose of this study is to determine semaphorin-7A expression by fibroblasts and to investigate the function of endogenously expressed semaphorin-7A in primary human lung fibroblasts (HLF).
Pulmonary fibrosis is a complication of many chronic respiratory disorders, and is best illustrated in idiopathic pulmonary fibrosis (IPF) [1,2]. Pulmonary fibrosis is characterized by the accumulation of extracellular matrix (ECM)-producing fibroblasts, termed myofibroblasts [3,4]. In addition to producing ECM proteins such as periostin, collagen, and fibronectin, myofibroblasts form robust actin stress fibers, express smooth muscle α-actin (α-SMA) and exhibit enhanced contractile activity similar to smooth muscle cells [5,6]. The change of phenotype from fibroblast into myofibroblast can be induced by transforming growth factor-ß (TGF-ß1), under the control of the SMAD proteins and serum response factor (SRF) [7,8]. Moreover, myofibroblasts display enhanced proliferation, migration, and extracellular matrix formation compared to fibroblasts [9–11].
We first examined the expression of semaphorin-7A on the cell surface of non-fibrotic HLF by flow cytometry. Fig 1A shows high staining for surface semaphorin-7A in HLF cultured in 1% FBS, 10% FBS or with TGF-ß1. In order to begin analyzing the function of endogenous semaphorin-7A on fibroblasts, we reduced the production of semaphorin-7A in fibroblasts using semaphorin-7A-siRNA. The efficacy of the semaphorin-7A-siRNA on its target is shown on S1 Fig. The specific level of gene expression reduction was compared to an irrelevant control-siRNA provided by the manufacturer and to the effect on the non-targeted semaphorin-7A receptor, plexin C1. The semaphorin-7A-siRNA reduced the level of semaphorin-7A mRNA by ~90% and had no effect on plexin-C1 mRNA. Importantly, semaphorin-7A-siRNA reduced HLF semaphorin-7A protein as determined by flow cytometry (Fig 1B) and the diffuse membrane staining seen by immunocytochemistry (Fig 1C). Immunocytochemistry also demonstrated localization of semaphorin-7A to the cell edge in some cells, including structures resembling filipodia and the lamellipodia (semaphorin-7A in green, S2 Fig).
Fibroblast proliferation, migration and differentiation constitute major hallmarks of tissue fibrosis. In this study, we identify endogenous semaphorin-7A as a constrainer of these pro-fibrotic characteristics, and an inhibitor of the production of the myofibroblast differentiation markers α-SMA, periostin and fibronectin. In contrast to other studies, semaphorin-7A-mediated effects were independent of exogenous TGF-ß.