Abstract
Intestinal epithelial cells are subject to repetitive deformation during peristalsis and villous motility, whereas the mucosa atrophies during sepsis or ileus when such stimuli are abnormal. Such repetitive deformation stimulates intestinal epithelial proliferation via focal adhesion kinase (FAK) and extracellular signal-regulated kinases (ERK). However, the upstream mediators of these effects are unknown. We investigated whether Src and Rac1 mediate deformation-induced FAK and ERK phosphorylation and proliferation in human Caco-2 and rat IEC-6 intestinal epithelial cells. Cells cultured on collagen-I were subjected to an average 10% cyclic strain at 10 cycles/min. Cyclic strain activated Rac1 and induced Rac1 translocation to cell membranes. Mechanical strain also induced rapid sustained phosphorylation of c-Src at Tyr(418), Rac1 at Ser(71), FAK at Tyr(397) and Tyr(576), and ERK1/2 at Thr(202)/Tyr(204). The mitogenic effect of cyclic strain was blocked by inhibition of Src (PP2 or short interfering RNA) or Rac1 (NSC23766). Src or Rac1 inhibition also prevented strain-induced FAK phosphorylation at Tyr(576) and ERK phosphorylation but not FAK phosphorylation at Tyr(397). Reducing FAK using short interfering RNA blocked strain-induced mitogenicity and attenuated ERK phosphorylation but not Src or Rac1 phosphorylation. Src inhibition blocked strain-induced Rac1 phosphorylation, but Rac inhibition did not alter Src phosphorylation. Transfection of a two-tyrosine phosphorylation-deficient FAK mutant Y576F/Y577F prevented activation of cotransfected myc-ERK2 by cyclic strain. Repetitive deformation induced by peristalsis or villus motility may support the gut mucosa by a pathway involving Src, Rac1, FAK, and ERK. This pathway may present important targets for interventions to prevent mucosal atrophy during prolonged ileus or fasting.
Highlights
The intestinal mucosa is subjected to a wide variety of physical forces during normal function and in pathophysiologic states [1]
We have reported previously that rhythmic mechanical strain-induced deformation activates focal adhesion kinase (FAK)2 and extracellular signalregulated kinases (ERK1/2) in human Caco-2 intestinal epithelial cells [15] and tyrosine kinase signaling in rat small and large bowel mucosa in vivo [17]
Caco-2 cells cotransfected with FAK and extracellular signal-regulated kinases (ERK) were lysed in immunoprecipitation buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 mM Na3VO4, 50 mM NaF, 10 mM sodium pyrophosphate, 2 g/ml aprotinin, and 2 g/ml leupeptin), and protein was measured by BCA as above. 20 l of protein G-Sepharose was preblocked with 1% heat-inactivated bovine serum albumin in phosphate-buffered saline (PBS) for 1 h at room temperature for each immunoprecipitation reaction
Summary
Src and Rac Mediate Mitogenic Effects of Strain [31, 34]. Rac involvement in regulating the FAK and ERK activation that mediates the mitogenic effects of repetitive mechanical strain in intestinal epithelium is not well understood. We sought to determine what upstream mediators might be responsible for such FAK and ERK activation. We sought to determine whether strain activation of Src and the small G-protein Rac mediates strain-induced FAK and ERK activation and cell proliferation in human (Caco-2) and rat (IEC-6) intestinal epithelial cells. We characterized time-dependent sitespecific FAK phosphorylation and Rac and Src activation in response to repetitive deformation in Caco-2 intestinal epithelial cells in order to begin tracing a mechanotransduced pathway that links these signals into a mitogenic cascade
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