Focal adhesion kinase protein levels in gut epithelial motility.
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 >/=30% 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.
Basson
Marc D
MD
Chief, Surgical Service, John D. Dingell VA Medical Center, 4646 John R. St., Detroit, MI 48201-1932, USA. marc.basson@va.gov
Sanders
Matthew A
MA
Gomez
Ruben
R
Hatfield
James
J
Vanderheide
Richard
R
Thamilselvan
Vijayalakshmi
V
Zhang
Jianhu
J
Walsh
Mary F
MF
D000818:Animals; D018938:Caco-2 Cells; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D005769:Gastrointestinal Motility / Q000502:physiology*; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology* / Q000502:physiology*; D051381:Rats
D051416:Focal Adhesion Protein-Tyrosine Kinases