Access Type
Open Access Dissertation
Date of Award
January 2012
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Anatomy and Cell Biology
First Advisor
Marc Basson
Abstract
The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. In vitro, cyclic strain promotes intestinal epithelial proliferation and induces an absorptive phenotype characterized by increased dipeptidyl dipeptidase (DPPIV) expression. Schlafen 3 is a novel gene recently associated with cellular differentiation. We sought to evaluate whether Schlafen 3 mediates the effects of strain on the differentiation of IEC-6 intestinal epithelial cells in the absence or presence of cyclic strain. Strain increased Schlafen 3 mRNA and protein. In cells transfected with a control non-targeting siRNA, strain increased DPPIV specific activity. However, Schlafen
3 reduction by siRNA decreased basal DPPIV and prevented any stimulation of DPPIV activity by strain. Schlafen 3 reduction also prevented DPPIV induction by sodium butyrate (1mM) or TGF-â (0.1ng/ml), two unrelated differentiating stimuli. However, Shlafen-3 reduction by siRNA did not prevent the mitogenic effect of strain or that of EGF. Blocking Src and PI-3-kinase prevented strain induction of Schlafen 3, but Schlafen 3 induction required activation of p38 but not ERK. These results suggest that cyclic strain induces an absorptive phenotype characterized by increased DPPIV activity via Src-, p38-, and PI-3-kinasedependent induction of Schlafen 3 in rat intestinal epithelial IEC-6 cells on collagen, while Schlafen 3 may also be a key factor in the induction of intestinal epithelial differentiation by other stimuli such as sodium butyrate or TGF-â. The induction of Schlafen 3 or its human homologues may modulate intestinal epithelial differentiation and preserve the gut mucosa during normal gut function.
Repetitive strain promotes intestinal epithelial migration across fibronectin in vitro, but signaling mediators for this are poorly understood. We hypothesized that integrin linked kinase (ILK) mediates strain-stimulated migration in intestinal epithelial cells cultured on fibronectin. ILK kinase activity increased rapidly five minutes after strain induction in both Caco-2 and IEC-6 cells. Wound closure in response to strain was reduced in ILK siRNA transfected Caco-2 cell monolayers compared to control siRNA transfected Caco-2 cells. Pharmacologic blockade of PI3K or Src or reducing Src by siRNA prevented strain activation of ILK. ILK coimmunoprecipitated with FAK, and this association was decreased by mutation of FAK Tyr925 but not FAK Tyr397. Strain induction of FAK Tyr925 phosphorylation but not FAK Tyr397 or FAK Tyr576 phosphorylation was blocked in ILK siRNA transfected cells. ILK-Src association was stimulated by strain and was blocked by the Src inhibitor PP2. Finally, ILK reduction by siRNA inhibited strain-induced phosphorylation of myosin light chain and Akt. These results suggest a strain-dependent signaling pathway in which ILK association with FAK and Src mediates the subsequent downstream strain-induced motogenic response, and suggest that ILK induction by repetitive deformation may contribute to recovery from mucosal injury and restoration of the mucosal barrier in patients with prolonged ileus. ILK may therefore be an important target for intervention to maintain the mucosa in such patients.
Recommended Citation
Yuan, Lisi, "Molecular mechanisms of strain-stimulated intestinal epithelial cell differentiation and migration" (2012). Wayne State University Dissertations. 489.
https://digitalcommons.wayne.edu/oa_dissertations/489