Dual roles of syndecan-4 in regulating chicken fibrosis in vitro

Abstract

Introduction: Wooden Breast (WB) is a myopathy affecting the skeletal breast muscle (Pectoralis major) in broiler chickens and is characterized by muscle fiber damage and varying degrees of fibrosis, ECM remodeling and inflammation. We have previously shown that the expression of syndecan-4 (SDC4), a transmembrane proteoglycan, was increased in WB poultry skeletal muscle tissue. Furthermore, the ectodomain shedding of SDC4 by matrix metalloproteinases (MMPs) was increased. While SDC4 has been previously implicated as a key driver for regulating myofibroblast activity in mechanically induced fibrosis in cardiac tissue, its specific role and shedding activity in chicken fibroblasts in relation to WB myopathy remain poorly understood. Methods: In vitro overexpression system was used to mimic the previously detected increased SDC4 levels in WB and to further investigate fibrotic markers and syndecans at the gene and protein levels. Furthermore, we used blocking peptides derived from the SDC4 ectodomain and investigated their effect on SDC4 shedding and fibrotic markers. Additionally, TGF-β1 treatment, the main trigger of myofibroblasts, fibrosis, and cytokines, was used to investigate the connection between SDC4 shedding and fibrosis. Results and Discussion: Overexpression of SDC4 in chicken fibroblasts reduced the gene and protein expression of fibrotic markers collagen I, collagen III and MMP-2 but simultaneously increased TGFB1 and IL1B. SDC4 overexpression also modulated intracellular proteins connected to fibrosis-relevant signaling pathways, with increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt. Moreover, we could observe a decreased production of ribosomal protein S6 and β-catenin. SDC4 overexpression induced shedding of a 15 kDa SDC4 fragment, while a 20 kDa fragment was produced at similar level regardless of overexpression. Modulation of ectodomain shedding using blocking peptides targeting various ectodomain regions (amino acids 76-106 and 100-130) significantly reduced only the production of the 20 kDa endogenous fragment. When we stimulated the cells with TGF-β1, an increase in gene expression of fibrotic marker and the 20 kDa SDC4 fragment was observed. Taken together, our results suggesting a potential dual function where the full-length SDC4, produced by overexpression, may act as an anti-fibrotic regulator, while the TGF-β1 induced shed ectodomain fragments could promote pro-fibrotic and inflammatory processes.