Losac and Lopap Recombinant Proteins from Lonomia obliqua Bristles Positively Modulate the Myoblast Proliferation Process

Abstract

The pursuit of better therapies for disorders creating deficiencies in skeletal muscle regeneration is in progress, and several biotoxins are used in skeletal muscle research. Since recombinant proteins derived from Lonomia obliqua bristles, recombinant Lonomia obliqua Stuart-factor activator (rLosac) and recombinant Lonomia obliqua prothrombin activator protease (rLopap) act as cytoprotective agents and promote cell survival, we hypothesize that both rLosac and rLopap favour the skeletal muscle regeneration process. In the present work, we investigate the ability of these recombinant proteins rLosac and rLopap to modulate the production of key mediators of the myogenic process. The expression of myogenic regulatory factors (MRFs), cell proliferation, the production of prostaglandin E2 (PGE₂) and the protein expression of cyclooxygenases COX-1 and COX-2 were evaluated in C2C12 mouse myoblasts pre-treated with rLosac and rLopap. We found an increased proliferation of myoblasts, stimulated by both recombinant proteins. Moreover, these proteins modulated PGE₂ release and MRFs activities. We also found an increased expression of the EP4 receptor in the proliferative phase of C2C12 cells, suggesting the involvement of this receptor in the effects of PGE₂ in these cells. Moreover, the recombinant proteins inhibited the release of IL-6 and PGE₂, which is induced by an inflammatory stimulus by IL-1β. This work reveals rLopap and rLosac as promising proteins to modulate processes involving tissue regeneration as occurs during skeletal muscle injury.

Keywords: differentiation; inflammation; myoblast; proliferation; prostaglandin E2.

FIGURE 1

Workflows for studies performed under (A) proliferation, (B) differentiation, and (C) inflammatory/proliferation conditions. FBS: fetal bovine serum; HS: horse serum; ITS: insulin-transferrin-sodium.

FIGURE 2

Recombinant proteins increased cell proliferation. C2C12 cells were treated with recombinant proteins in DMEM-1% ITS for 24 or 48 h, and then cell proliferation was assessed by incorporation of BrdU. Bar charts represent effects of control, rLosac and rLopap on BrdU incorporation at (A) 24 h and (B) 48 h. Data are shown as mean ± SEM (*p < 0.05 vs. control from one-way ANOVA and Dunnett’s post-test; n = 3 with technical duplicates).

FIGURE 3

rLosac and rLopap modified the expression of myogenic regulatory factors (MRFs) at the proliferative stage. C2C12 cells were treated with recombinant proteins in DMEM-1% ITS for 24 h at the proliferative stage, and then MRF expression was assessed by high content screening (HCS). (A) Representative images of MRFs expression obtained by HCS (scale bar: 50 μm). (B) The average intensity in the nucleus (black) and cytoplasm (yellow) of every factor at basal (control) and treated conditions. Data are shown as mean ± SEM (*p < 0.05 vs. control from Two-way ANOVA and Dunette’s post test; n = 3 with technical duplicates).

FIGURE 4

rLosac and rLopap modified the expression of MRFs at the commitment stage. Once the C2C12 cells reached the commitment stage were treated with recombinant proteins in DMEM-2% HS for 24 h, and then MFRs expression was assessed by HCS. (A) Representative images of MRFs expression obtained by HCS (scale bar: 50 μm). (B) The average intensity in the nucleus (black) and cytoplasm (yellow) of every factor at basal (control) and treated conditions. Data are shown as mean ± SEM (*p < 0.05 and **p < 0.01 vs. control from Two-way ANOVA and Dunnett’s post test; n = 3 with technical duplicates).

FIGURE 5

Recombinant Losac and Lopap affect the cyclooxygenase (COX) pathway. C2C12 cells were treated with rLosac or rLopap in DMEM-1% ITS for 24 h at the proliferative stage, and then Elisa and Western blotting respectively assessed prostaglandin E2 (PGE₂) release, and COX pathway protein expression. Bar charts represent effects of control, and recombinant proteins on (A) COX-1 and (B) COX-2 protein expression, (C) production of PGE₂ and (D) expression of EP4 receptor. Data are shown as mean ± SEM (*p < 0.05 and **p < 0.01 vs. control from one-way ANOVA and Dunnett’s post-test; n = 3).

FIGURE 6

Recombinant Losac and Lopap have potential anti-inflammatory activity. C2C12 cells were treated with rLosac or rLopap in DMEM-1% ITS for 24 h for 24 h at the proliferative stage, and then PGE₂ release, COXs proteins expression and interleukin 6 (IL-6) production were assessed by Elisa, Western blotting and flow cytometry respectively. Bar charts represent effects of control and recombinant proteins on the IL-1β-induced (A) COX-1 and (B) COX-2 protein expression, (C) PGE₂ production and (D) IL-6 production. Data are shown as mean ± SEM (*p < 0.05 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. IL-1β from one-way ANOVA with Dunnett’s post-test and unpaired t-test respectively; n = 3).