Submitted on November 11, 2008
Revised on January 13, 2009
Accepted on January 20, 2009
Delineating the signals by which repetitive deformation stimulates intestinal epithelial migration across fibronectin
Christopher P Gayer1, Lakshmi S Chaturvedi2, Shouye Wang3, Brittany Alston1, and Marc D Basson4*
1 Wayne State University
2 Wany State University school of Medicine
3 John D. Dingell VA Medical Center/Wayne State University
4 Michigan State University
* To whom correspondence should be addressed. E-mail: marc.basson{at}hc.msu.edu.
Repetitive strain stimulates intestinal epithelial migration across fibronectin via FAK, Src, and ERK, although how these signals act and interact remains unclear. We hypothesized that PI3K is central to this pathway. We subjected Caco-2 and IEC-6 cells to 10 cycles/minute deformation on flexible fibronectin-coated membranes, assayed migration by wound closure, and signaling by immunoblots. Strain stimulated PI3K, AKT, GSK, and p38 phosphorylation. Blocking each kinase prevented strain stimulation of migration. Blocking PI3K prevented strain-stimulated ERK and p38 phosphorylation. Blocking AKT did not. Downstream, blocking PI3K, 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, Rac1, PI3K, AKT, GSK, and ERK but not Src or p38. Finally, blebbistatin, a non-muscle myosin II inhibitor, blocked the motogenic effect of strain downstream of myosin light chain. Thus, strain stimulates intestinal epithelial migration across fibronectin by a complex pathway including Src, FAK, Rac1, PI3K, AKT, GSK, ERK, p38, myosin light chain, and myosin II.
