Endogenous Semaphorin-7A Impedes Human Lung Fibroblast Differentiation

Abstract

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).Herein, we show that non-fibrotic HLF expressed high levels of cell surface semaphorin-7A with little dependence on the percentage of serum or recombinant TGF-ß1. Semaphorin-7A siRNA strongly decreased semaphorin-7A mRNA expression and reduced cell surface semaphorin-7A. Reduction of semaphorin-7A induced increased proliferation and migration of non-fibrotic HLF. Also, independent of the presence of TGF-ß1, the decline of semaphorin-7A by siRNA was associated with increased α-smooth muscle actin production and gene expression of periostin, fibronectin, laminin, and serum response factor (SRF), indicating differentiation into a myofibroblast. Conversely, overexpression of semaphorin-7A in the NIH3T3 fibroblast cell line reduced the production of pro-fibrotic markers. The inverse association between semaphorin-7A and pro-fibrotic fibroblast markers was further analyzed using HLF from idiopathic pulmonary fibrosis (IPF) (n = 6) and non-fibrotic (n = 7) lungs. Using these 13 fibroblast lines, we observed that semaphorin-7A and periostin expression were inversely correlated. In conclusion, our study indicates that endogenous semaphorin-7A in HLF plays a role in maintaining fibroblast homeostasis by preventing up-regulation of pro-fibrotic genes. Therefore, endogenous and exogenous semaphorin-7A may have opposite effects on the fibroblast phenotype.

RNA-sequencing analysis of lung primary fibroblast response to eosinophil-degranulation products predicts downstream effects on inflammation, tissue remodeling and lipid metabolism

“…Human lung fibroblasts (HLF) were isolated as described previously [ 30 , 31 ] using de-identified tissue samples from thoracic surgical resection specimens. These samples were collected and characterized in collaboration with the biobanking services run by the Carbone Cancer Center Translational Science BioCore at the University of Wisconsin-Madison, under Institutional Review Board approval.…”

IPF pathogenesis is dependent upon TGFβ induction of IGF‐1

“…Human fetal lung fibroblast cell lines (MRC5 and IMR-90) were maintained in Eagle's Minimum Essential Medium (EMEM; ATCC, Manassas, VA, USA) containing 20% FBS and 1% P/S. Deidentified patient primary cells including normal human lung fibroblasts (NHLF) and human IPF fibroblasts characterized in two previously published studies 33,34 and obtained from Dr Nathan Sandbo from the University of Wisconsin-Madison Department of Medicine (TSB BioBank IRB #2011-0521) and Dr Carol Feghali-Bostwick from the Medical University of South Carolina (University of Pittsburgh IRB #970946) were cultured in DMEM medium containing 10% FBS and 1% P/S. Unless stated otherwise, murine and human fibroblast cells were seeded at 2 × 10 6 cells/100 mm plate, serum starved for 18-24 hours in DMEM supplemented with 0.1% FBS and 1% P/S (murine fibroblasts) or EMEM supplemented with no FBS and 1% P/S (human fibroblasts) prior to 2-hour pretreatment with inhibitor and/or followed by growth factor stimulation for the indicated time.…”

Studies of postpartum mammary gland involution reveal novel pro-metastatic mechanisms

“…SEMA7A can affect ECM remodeling through its ability to recruit fibroblasts and immune cells to fibrotic sites, and it has been further implicated in fibrosis models in the liver, kidney, and in glial scar formation [103–108] . Contradictory to this role, when endogenously expressed on fibroblasts, SEMA7A can maintain fibroblast homeostasis and reduce pro-fibrotic markers [109] , indicating context dependent roles for SEMA7A-mediated signaling. In cancer, fibrillar collagen coordinates upregulation of COX-2 on tumor cells, further promoting tumor cell invasion and metastasis [7] , and we have published that COX-2 drives SEMA7A expression [49] .…”