| Abstrakt: |
Repetitive strain stimulates intestinal epithelial migration across fibronectin via focal adhesion kinase (FAK), Src, and extracellular signal-related kinase (ERK) although how these signals act and interact remains unclear. We hypothesized that P13K is central to this pathway. We subjected Caco-2 and intestinal epithelial cell-6 cells to 10 cycles/mm deformation on flexible fibronectin-coated membranes, assayed migration by wound closure, and signaling by immunoblots. Strain stimulated P13K, AKT, glycogen synthase kinase (GSK), and p38 phosphorylation. Blocking each kinase prevented strain stimulation of migration. Blocking P13K prevented strain-stimulated ERK and p38 phosphorylation. Blocking AKT did not. Downstream, blocking P13K, AKT, or ERK inhibited strain-induced GSK-Ser9 phosphorylation. Upstream of AKT, reducing FAK or Rac1 by siRNA blocked strain-stimulated AKT phosphorylation, but inhibiting Src by PP2 or siRNA did not. Transfection with FAK point mutants at Tyr397, Tyr576/577, or Tyr925 demonstrated that only FAK925 phosphorylation is required for strain-stimulated AKT phosphorylation. Myosin light chain activation by strain required FAK, RacI, P13K, AKT, GSK, and ERK but not Src or p38. Finally, blebbistatin, a nonmuscle myosin IT inhibitor, blocked the motogenic effect of strain down-stream of myosin light chain. Thus strain stimulates intestinal epithehal migration across fibronectin by a complex pathway including Src, FAK, Rac1, P13K, AKT, GSK, ERK, p38, myosin light chain, and myosin TI. [ABSTRACT FROM AUTHOR] |
