Muscle Cell Proliferation During Muscle Healing Can Be Stimulated By Mast Cell

Abstract

Mature skeletal muscle can regenerate because myoblasts persist in adult muscle in the form of satellite and stem cells. In injured muscles, resolution of inflammation leads to the secretion of mitogenic and growth factors activating these cells which proliferate to repair muscle. Mast cells were shown to contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase, a protease secreted by activated mast cells. Tryptase binds to a protease-activated receptor (PAR-2), which induces the expression of cyclo-oxygenase-2 (COX-2) and the production of cyclopentenone prostaglandins, which interact with nuclear receptors and activate cell proliferation. PURPOSE: To evaluate if mast cell tryptase can stimulate skeletal muscle cell proliferation and determine if this effect is dependent on PAR-2 receptors. METHODS: L6 muscle cells were first cultured in medium containing 10% serum and then submitted to proliferation assays with various tryptase concentrations in 1% serum. Control group received medium only. Cell number was determined with CellTiter assay 48h post-seeding. To show that the effect of tryptase on proliferation was related to PAR-2 activation, L6 cells were incubated with the synthetic PAR-2 agonist SLIGRL and control wells with the reverse sequence peptide. Western blot analyses were used to study the impact of tryptase stimulation and cell detachment procedure on COX-2 expression in L6 cells. RESULTS: Cell proliferation was significantly increased by tryptase when compared to control (43.6 ± 2.8%, p<0.05) and in a dose-dependent manner. The synthetic agonist SLIGRL was less potent but also stimulated proliferation (13.3 ± 2.7%), confirming the implication of PAR-2 in this signaling cascade. Our results showed that COX-2 expression was increased several folds in these experiments but this effect was likely related to the cell detachment procedure per se rather than being induced by tryptase; no correlation was found with the increase in proliferation observed. CONCLUSION: Tryptase can accelerate proliferation of L6 muscle cells in vitro and this effect is dependent on the activation of PAR-2. The implication of COX-2 in this mechanism has yet to be confirmed and is presently under investigation. Supported by grants from NSERC and CIHR.