Lysophosphatidic acid, but neither clenbuterol nor salbutamol, stimulates increases in ERK-1/2 phosphorylation which is not associated with an appreciable increase in proliferation

Extracellular signal-related kinases (ERK-1/2) are important signaling components for the regulation of cell proliferation. Both beta-adrenergic receptor agonists (beta-AA) and lysophosphatidic acid (LPA) have been shown to stimulate phosphorylation of ERK-1/2 in several cell lines. The beta-AA have been used to stimulate skeletal muscle growth in livestock, yet there are still questions about the signaling mechanisms involved. The effects of LPA on skeletal muscle have not been investigated. The objective of these studies was to evaluate the effects of beta-AA and LPA on ERK-1/2 phosphorylation and proliferation in muscle cell lines. In both studies, proliferation was assessed by [3H]thymidine incorporation and ERK-1/2 activation was determined by western blot analysis and a cell cycle analysis was conducted by flow cytometry. In study one, muscle-derived cells were treated with 2x10 -11 M clenbuterol. Clenbuterol treatment did not increase [ 3H]thymidine incorporation and had no effect on ERK-1/2 activation (P<0.05). Clenbuterol treatment also did not increase the percentage of cells in the S or G2/M phases of the cell cycle. In study two, the effect of LPA on proliferation and ERK-1/2 activation in C2C 12 myoblasts was assessed. LPA treatment induced increases in [ 3H]thymidine incorporation were similar to that observed with 10% fetal bovine serum (FBS; P>0.05) with the exception of the lowest concentration of LPA (12.5 muM; P<0.05). Lysophosphatidic acid stimulated a rapid increase in ERK-1/2 phosphorylation which was attenuated by 2h. Likewise, 10% FBS stimulated phosphorylation of ERK-1/2, but the increase in phosphorylation was sustained past 4h. Analysis of cell cycle showed LPA induced a transient increase in the percentage of cells in S phase at 12h, but this increase was abated by 24h. These data demonstrate that although LPA treatment does induce an increase in ERK-1/2 phosphorylation, neither beta-AA or LPA treatment resulted in an appreciable change in proliferation of muscle cell lines, suggesting that LPA treatment and ERK-1/2 activation are not sufficient to induce persistent cell growth in vitro.