Human Caco-2 Intestinal Epithelial Motility Is Associated with Tyrosine Kinase and Cytoskeletal Focal Adhesion Kinase Signals

Liu, Y.-W.; Sanders, Matthew A.; Basson, Marc D.

doi:10.1006/jsre.1998.5369

Cited by 23 publications

(20 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Finally, homogeneous populations of static and motile cells were generated for biochemical studies, e.g., FAK protein expression and stability, by varying seeding density so that at 4 days after plating, one population of cells was static and confluent (13,000/cm 2 initial density in 35 ϫ 10-mm dishes) and the other consisted of small islands of radially migrating cells (750/ cm 2 in 100 ϫ 20-mm dishes). Previous studies (20,21,54,55) have demonstrated that this model yields highly reproducible results and that the expression of some conventional differentiation markers is equivalent between these two cell populations.…”

Section: Methodsmentioning

confidence: 85%

Focal adhesion kinase protein levels in gut epithelial motility

Basson

Sanders

Gomez³

et al. 2006

American Journal of Physiology-Gastrointestinal and Liver Physi

View full text Add to dashboard Cite

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 ...

show abstract

Section: Methodsmentioning

confidence: 85%

Focal adhesion kinase protein levels in gut epithelial motility

Basson

Sanders

Gomez³

et al. 2006

American Journal of Physiology-Gastrointestinal and Liver Physi

View full text Add to dashboard Cite

show abstract

“…The amount of FAK protein phosphorylation is increased during cell migration. 33 The autophosphorylation of FAK at the tyrosine residue 397 is a marker for the activation of FAK. Phosphorylation creates an SH2 binding site for different signaling and adapter proteins.…”

Section: Discussionmentioning

confidence: 99%

Decreased Fibrogenesis After Treatment with Pirfenidone in a Newly Developed Mouse Model of Intestinal Fibrosis

Meier

Lutz

Cosín-Roger

et al. 2016

Inflammatory Bowel Diseases

View full text Add to dashboard Cite

BACKGROUND Fibrosis as a common problem in patients with Crohn's disease is a result of an imbalance toward excessive tissue repair. At present, there is no specific treatment option. Pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis with both antifibrotic and anti-inflammatory effects. We subsequently investigated the impact of pirfenidone treatment on development of fibrosis in a new mouse model of intestinal fibrosis. METHODS Small bowel resections from donor mice were transplanted subcutaneously into the neck of recipients. Animals received either pirfenidone (100 mg/kg, three times daily, orally) or vehicle. RESULTS After administration of pirfenidone, a significantly decreased collagen layer thickness was revealed as compared to vehicle (9.7 ± 1.0 versus 13.5 ± 1.5 µm, respectively, **P < 0.001). Transforming growth factor-and matrix metalloproteinase-9 were significantly decreased after treatment with pirfenidone as confirmed by real-time PCR (0.42 ± 0.13 versus 1.00 ± 0.21 and 0.46 ± 0.24 versus 1.00 ± 0.62 mRNA expression level relative to GAPDH, respectively, *P < 0.05). Significantly decreased transforming growth factor-after administration of pirfenidone was confirmed by Western blotting. CONCLUSION In our mouse model, intestinal fibrosis can be reliably induced and is developed within 7 days. Pirfenidone partially prevented the development of fibrosis, making it a potential treatment option against Crohn's disease-associated fibrosis.

show abstract

“…Activation of focal adhesion kinase in response to integrin ligation results in phosphorylation of adapter proteins including paxillin, Shc, and src kinases, and, further downstream, methylethylketone-dependent mitogen-activated protein kinase pathways particularly involving extracellular signalregulated kinase-1 and extracellular signal-regulated kinase-2 (44). Interference of focal adhesion kinase activity by a dominant negative strategy or inhibition of extracellular signal-regulated kinase 1/2 via methylethylketone inhibition has been shown to retard epithelial migration (10,45) The actin-myosin-based cytoskeleton is the central effector of cell migration. Cytoskeletal motor activity is mediated by conventional type II myosin, which is composed of two heavy chains, two essential light chains, and two regulatory light chains.…”

Section: Intracellular Mechanisms Of Epithelial Repairmentioning

confidence: 99%