Focal adhesion kinase protein levels in gut epithelial motility

Abstract

Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK 397 ) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by Ն30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK 397 phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.healing; migration; restitution; ulcer ALTHOUGH MOST PREVALENT in the stomach and duodenum, ulcers are found throughout the gastrointestinal tract. Regardless of site, mucosal ulcer healing is a complex process that involves restitution by migration of epithelial cells from the wound margins and eventual filling of the defect by proliferating epithelial and connective tissue cells (31, 43). Various factors have been shown to stimulate epithelial restitution and proliferation, but the intracellular signal proteins that control epithelial cellular migration are less well understood. Among these signals, activation of focal adhesion kinase (FAK) has been linked to gastric wound healing in vivo (41,44). FAK is a tyrosine kinase that associates with the cytoplasmic tail of clustered integrins in focal adhesion complexes. On activation, autophosphorylation of FAK on tyrosine 397 is followed by further phosphorylation of FAK itself and other proteins (36,56). Although FAK was originally described as being rapidly activated on integrin-mediated cell-matrix adhesion, this kinase has now been reported to be activated during epithelial cell motility and phosphorylated at both serine and tyrosine residues by many factors including occupancy of trans-activating G protein-coupled receptors (GPCRs) (10,23,35,51). Modulating FAK activity ...

Transforming Growth Factor-β Stimulates Intestinal Epithelial Focal Adhesion Kinase Synthesis via Smad- and p38-Dependent Mechanisms

“…31 Briefly, RNA was isolated from control and TGF-␤-treated Caco-2 and IEC-6 cells with the Qiagen Total RNA isolation kit (Qiagen, Valencia, CA) with digestion of DNA with DNase I. Complete digestion was tested for by standard PCR with primers for the control 18S gene (5Ј-CGGC-TACCACATCCAAGGAA-3Ј and 5Ј-GCTCGAATTAC-CGCGGCT-3Ј; Integrated DNA Technologies, Coralville, IA).…”

“…5,22,31 We now sought to determine whether FAK protein at the edge of healing gut mucosa decreases similarly in vivo. We stained for FAK and activated FAK 397 in 30 benign human gastric and ischemic and malignant colonic ulcers.…”

“…22 Indeed, intestinal epithelial cell motility modulates FAK protein abundance at the mRNA level in both human Caco-2 and rat nontransformed IEC-6 intestinal epithelial cells. 31 Such changes in FAK protein levels dramatically alter the amount of phosphorylated (active) FAK available within motile cells independently of (and more substantially than) motility-driven changes in the proportion of activated FAK.…”

“…We found that FAK and FAK 397 immunoreactivity were indeed lower in epithelial cells at the leading, migrating edge of the ulcer, consistent with our previously reported in vitro observations in Caco-2 and IEC-6 cells. 5,22,31 However, the epithelial region immediately behind the ulcer margin exhibited dramatically enhanced FAK protein immunostaining that then tapered off distant from the ulcer. Phosphorylated FAK 397 immunoreactivity followed the same pattern, suggesting that such changes in FAK protein critically regulate the amount of activated FAK present in vivo.…”

A simpli ed cell culture model for research on intestinal in ammation

“…Cells were grown to confluence in 75-cm 2 flasks. After serumstarving for 12 h, flasks were used in the multiple scrape model as previously described to mimic the conditions of migration assay (5). After scratching, cells were incubated for 12 h with either LPS or the cytokine mixture.…”