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Abstract
BackgroundMast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2). The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade.MethodsProliferation of L6 rat skeletal myoblasts stimulated with PAR-2 agonists (tryptase, trypsin and SLIGKV) was assessed. The specificity of the tryptase effect was evaluated with a specific inhibitor, APC-366. Western blot analyses were used to evaluate the expression and functionality of PAR-2 receptor and to assess the expression of COX-2. COX-2 activity was evaluated with a commercial activity assay kit and by measurement of PGF2α production. Proliferation assays were also performed in presence of different prostaglandins (PGs).ResultsTryptase increased L6 myoblast proliferation by 35% above control group and this effect was completely inhibited by APC-366. We confirmed the expression of PAR-2 receptor in vivo in skeletal muscle cells and in satellite cells and in vitro in L6 cells, where PAR-2 was found to be functional. Trypsin and SLIGKV increased L6 cells proliferation by 76% and 26% above control, respectively. COX-2 activity was increased following stimulation with PAR-2 agonist but its expression remained unchanged. Inhibition of COX-2 activity by NS-398 abolished the stimulation of cell proliferation induced by tryptase and trypsin. Finally, 15-deoxy-Δ-12,14-prostaglandin J2 (15Δ-PGJ2), a product of COX-2-derived prostaglandin D2, stimulated myoblast proliferation, but not PGE2 and PGF2α.ConclusionsTaken together, our data show that tryptase can stimulate myoblast proliferation and this effect is part of a signaling cascade dependent on PAR-2 activation and on the downstream activation of COX-2.
Highlights
Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2)
Proliferation assay For measurement of proliferation rate, cells were seeded onto 96-well plate at a density of 3,000 cells/well in 65 μL of a-Mimimum Essential Medium (a-MEM) containing 1% fetal bovine serum (FBS) and treated with or without tryptase (Sigma-Aldrich; St-Louis (MO), USA), trypsin (Sigma-Aldrich), PAR-2 synthetic activating peptide SLIGKV (Bachem Americas; Torrance (CA), USA), or prostaglandins PGF2a, PGE2 or 15Δ-PGJ2 (Cayman Chemical; Ann Arbor (MI), USA), at various doses for 48 h
Implication of PAR-2 and COX-2 in L6 myoblast proliferation Since it has been demonstrated in fibroblasts that COX-2 is part of the signaling cascade leading to the stimulation of proliferation following PAR-2 activation, we investigated its putative role in the modulation of L6 myoblast proliferation
Summary
Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2). The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. There is an orchestrated recruitment of inflammatory cells, which produce proand/or anti-inflammatory signals leading initially to the removal of the damaged tissue and subsequently to the resolution of inflammation and tissue repair [1,2,3,4,5]. Activated myoblasts migrate to the site of injury, proliferate, differentiate and fuse into myotubes to form new myofibers, eventually leading to restoration of form and function of the damaged muscle. Macrophages, mast cells, endothelial cells, fibroblasts and muscle fibers themselves can produce growth and transcription factors that influence myoblasts during muscle regeneration
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