Searching Novel Therapeutic Targets for Scleroderma: P2X7-Receptor Is Up-regulated and Promotes a Fibrogenic Phenotype in Systemic Sclerosis Fibroblasts.

Daniela Gentile,Franco Laghi-Pasini,Pier L Capecchi,Alessandra Gamberucci,Silvia Del-Ry,Alessandra Alì,Enrico Selvi,Maria A Morales,Veronica Della-Latta,Rosella Fulceri,Mariarita Natale,Paolo Taddeucci,Francesca Vanni,Pietro E Lazzerini,David J Abraham,Manuela Cabiati

doi:10.3389/fphar.2017.00638

Abstract

Objectives: Systemic sclerosis (SSc) is a connective tissue disorder presenting fibrosis of the skin and internal organs, for which no effective treatments are currently available. Increasing evidence indicates that the P2X7 receptor (P2X7R), a nucleotide-gated ionotropic channel primarily involved in the inflammatory response, may also have a key role in the development of tissue fibrosis in different body districts. This study was aimed at investigating P2X7R expression and function in promoting a fibrogenic phenotype in dermal fibroblasts from SSc patients, also analyzing putative underlying mechanistic pathways.Methods: Fibroblasts were isolated by skin biopsy from 9 SSc patients and 8 healthy controls. P2X7R expression, and function (cytosolic free Ca2+ fluxes, α-smooth muscle actin [α-SMA] expression, cell migration, and collagen release) were studied. Moreover, the role of cytokine (interleukin-1β, interleukin-6) and connective tissue growth factor (CTGF) production, and extracellular signal-regulated kinases (ERK) activation in mediating P2X7R-dependent pro-fibrotic effects in SSc fibroblasts was evaluated.Results: P2X7R expression and Ca2+ permeability induced by the selective P2X7R agonist 2′-3′-O-(4-benzoylbenzoyl)ATP (BzATP) were markedly higher in SSc than control fibroblasts. Moreover, increased αSMA expression, cell migration, CTGF, and collagen release were observed in lipopolysaccharides-primed SSc fibroblasts after BzATP stimulation. While P2X7-induced cytokine changes did not affect collagen production, it was completely abrogated by inhibition of the ERK pathway.Conclusion: In SSc fibroblasts, P2X7R is overexpressed and its stimulation induces Ca2+-signaling activation and a fibrogenic phenotype characterized by increased migration and collagen production. These data point to the P2X7R as a potential, novel therapeutic target for controlling exaggerated collagen deposition and tissue fibrosis in patients with SSc.

Highlights

Systemic sclerosis (SSc; scleroderma) is a connective tissue disease (CTD) presenting with three major aspects: small vessel disease, autoantibody production, and fibroblasts dysfunction leading to exaggerated deposition of collagen and extracellular matrix (ECM), tissue fibrosis and organ dysfunction (Allanore et al, 2015)
Both the percentage of P2X7-positive cells (46 ± 7 vs. 35 ± 6%, p = 0.023; Figure 1B) and, the total amount of the P2X7 receptor (P2X7R) expression was significantly higher in SSc than healthy fibroblasts, as demonstrated by the ∼3-times higher values of mean fluorescence intensity (MFI; 6.5 ± 2.6 vs. 2.2 ± 0.3, p = 0.0087; Figure 1C)
Such a phenomenon was not associated with an increased P2X7R gene expression by the cells, as real time-PCR analysis indicated that the levels of P2X7R mRNA were not significantly different in SSc patients when compared to healthy controls (2.6 ± 2.5 vs. 1.9 ± 1.1 fold-increase, p = 0.58; Figure 1D)

Summary

Introduction

Systemic sclerosis (SSc; scleroderma) is a connective tissue disease (CTD) presenting with three major aspects: small vessel disease, autoantibody production, and fibroblasts dysfunction leading to exaggerated deposition of collagen and extracellular matrix (ECM), tissue fibrosis and organ dysfunction (Allanore et al, 2015). The P2X7 receptor (P2X7R) is a nucleotide-gated ion channel involved in the inflammatory response triggered by the release of ATP from damaged cells It is largely expressed in monocytes where it enhances the synthesis and/or promotes the release of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines, interleukin-1β (IL-1β) and interleukin-18, by activating the K+-dependent NALP3 inflammasome/caspase pathway (Di Virgilio, 2007; Castrichini et al, 2014; Gicquel et al, 2015), and possibly interleukin-6 (IL-6), via the Ca2+dependent MAP-Kinase/early growth response-1 gene systems, egr-1 (Solini et al, 1999; Caporali et al, 2008; Friedle et al, 2011). Accumulating data demonstrated that the P2X7R is expressed in fibroblastic cells (Solini et al, 1999; Caporali et al, 2008; Friedle et al, 2011), promoting lung, kidney, pancreas, and cardiac fibrosis in animal models (Gentile et al, 2015)

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