Fig. 2.

Involvement of p42/44 MAPK in URB597- and AA-5HT-induced migration of MSCs. A, B: Time-course of p42/44 MAPK phosphorylation by URB597 (A) and AA-5HT (B) (Western blot analyses). Cells were treated with vehicle, 10 μM URB597 (A) or 10 μM AA-5HT (B), for the indicated times. C, D: Concentration-dependent activation of p42/44 MAPK by URB597 (C) and AA-5HT (D) (Western blot analyses). Cells were treated for 2 h (C, URB597) or 1 h (D, AA-5HT) with vehicle or the indicated concentrations of the test substances. To indicate activation of p42/44 MAPK, values obtained from densitometric band analyses of the phosphorylated form were normalized to those of nonphosphorylated p42/44 MAPK. β-actin was used as loading control. E, F: Analysis of FAAH inhibitor-induced promigratory effects in the presence of a p42/44 MAPK signaling pathway blocker and a p38 MAPK blocker. Cells were pretreated with the upstream inhibitor of p42/44 activation, PD98059 (PD, 10 μM), or the p38 MAPK inhibitor SB203580 (SB, 10 μM) for 1 h and subsequently incubated with vehicle, URB597 (E, 10 μM) or AA-5HT (F, 10 μM) for another 6 h before quantification of migration by Boyden chamber assays. Data represent mean ± SEM compared with vehicle control (100%) of n = 15 experiments with cells from five donors (A, 15 min and 360 min), n = 9 experiments with cells from three donors (A, 30 min), n = 21 experiments with cells from seven donors (A, 60 min and 120 min), n = 6 experiments with cells from two donors (B–D), n = 16 experiments with cells from four donors (E, F). *P < 0.05 versus vehicle, Student’s t-test (A, B) or one-way ANOVA plus post hoc Dunnett (C, D) or Bonferroni test (E, F). ^#P < 0.05 versus respective FAAH inhibitor in the absence of PD98059; one-way ANOVA plus post hoc Bonferroni test (E, F).