Delineating the signals by which repetitive deformation stimulates intestinal epithelial migration across fibronectin

Abstract

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 PI3K is central to this pathway. We subjected Caco-2 and intestinal epithelial cell-6 cells to 10 cycles/min deformation on flexible fibronectin-coated membranes, assayed migration by wound closure, and signaling by immunoblots. Strain stimulated PI3K, AKT, glycogen synthase kinase (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 nonmuscle 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.

Fig. 1.

Role of PI3K, AKT, glycogen synthase kinase (GSK), and p38 in the motogenic effect of strain. A: strain stimulates phosphorylation of the p85 subunit of PI3K at 30 min in Caco-2 cells (n = 4, *P < 0.03). Phosphorylation of AKT (B), GSK (C), and p38 (D) occurs as early as 15 min of strain (n ≥ 6, *P < 0.02). Inhibiting these signals in Caco-2 cells with the PI3K inhibitor LY294002 (20 μM), AKT inhibitor IV (1 μM), GSK inhibitor SB415286 (20 μM), or p38 inhibitor SB203580 (20 μM) (E) blocked the motogenic effect of strain (solid bars) similarly to nonmuscle myosin II inhibition with blebbistatin (85.5 μM) compared with wound closure in static cells (open bars). Data summarize results of 6–36 holes per experiment from at least 3 independent experiments (*P < 0.05, **P < 0.001). F: isoform-specific siRNA reduced AKT1 or AKT2 78 and 65%, respectively (blots). Reducing either AKT1 or AKT2 reversed the motogenic effect of strain across fibronectin (graph, n ≥ 25, *P < 0.0001). t-, total; p-, phospho; NT1, nontargeting control SiRNA.

Fig. 2.

Signal interaction in response to strain. Downstream signals were studied in Caco-2 cells under static (open bars) and cyclic strain (solid bars) conditions during upstream signal inhibition. A: blocking PI3K blocks strain-induced extracellular signal-related kinase (ERK) activation. Blocking AKT does not (n = 10, *P < 0.05, **P < 0.01). B: similarly, PI3K inhibition prevents strain-induced p38 activation, whereas AKT inhibition does not (n ≥ 8, *P < 0.05, **P < 0.01). Furthermore, strain-induced GSK phosphorylation was blocked by PI3K inhibition (C, n = 9, *P < 0.01), AKT inhibition (D, n = 8, *P < 0.0001), and ERK inhibition (E, n = 10, *P < 0.0001). F: p38 inhibition did not block GSK phosphorylation (n = 6, *P < 0.001).

Fig. 3.

Intestinal epithelial cell (IEC)-6 migration. Inhibiting PI3K, AKT, ERK, and p38 prevents strain stimulation of IEC-6 migration across fibronectin (n = 16, *P < 0.00001). Open bars represent static conditions, and solid bars 24 h of strain.

Fig. 4.

Upstream control of AKT activation. PI3K inhibition (A) blocked AKT activation (n = 9, *P < 0.001), but neither Src inhibition (B, n = 6, *P < 0.04) nor Src reduction by siRNA (C) prevented strain-induced AKT activation (n = 10, *P < 0.05) in Caco-2 cells. However, reducing focal adhesion kinase (FAK) or Rac1 by siRNA prevented strain-induced phosphorylation of AKT (D, n = 5, *P < 0.05) and PI3K p85 subunit (E, n = 7, *P < 0.001). siRNA transfection achieved ∼60–80% knockdown as shown by immunoblot (F).

Fig. 5.

Specific FAK phosphorylation sites selectively control AKT activation by strain. Caco-2 cells were cotransfected with HA-tagged wild-type (wt) or point-mutated FAK constructs and HA-tagged AKT2 and subjected to static or cyclic strain conditions before immunoprecipitation of the HA antigen and immunoblot for phospho-AKT. Cotransfection with FAK Y397F or Y576/577F did not alter strain-induced AKT activation (A, n = 12, *P < 0.03), but FAK Y925F cotransfection blocked strain-induced AKT activation (B, n = 3, *P < 0.05, **P < 0.01). HA, hemagglutinin.

Fig. 6.

The role of myosin light chain (MLC) kinase in the motogenic effect of strain. A: p38 inhibition (SB203580) did not block MLC phosphorylation in response to strain (n = 4, *P < 0.03), but inhibiting PI3K (LY294002) or AKT (B), GSK (SB415286, C) or ERK (PD98059, D) blocked strain-associated MLC phosphorylation (n ≥ 4, *P < 0.002) in Caco-2 cells.

Fig. 7.

Effects of upstream kinases on MLC phosphorylation. Caco-2 MLC phosphorylation was not prevented by Src inhibition by PP2 (A, n = 9, *P < 0.002) or Src reduction by siRNA (B, n = 12, *P < 0.05), but reducing FAK (C) or Rac1 (D) by siRNA prevented strain-stimulated MLC phosphorylation (n ≥ 7, *P < 0.01, **P < 0.0001) in Caco-2 cells.