Data from: Sustained intestinal epithelial monolayer wound closure after transient application of a FAK-activating small molecule

M64HCl, which has drug-like properties, is a water-soluble Focal Adhesion
Kinase (FAK) activator that promotes murine mucosal healing after ischemic
or NSAID-induced injury. Since M64HCl has a short plasma half-life in vivo
(less than two hours), it has been administered as a continuous infusion
with osmotic minipumps in previous animal studies. However, the effects of
more transient exposure to M64HCl on monolayer wound closure remained
unclear. Herein, we compared the effects of shorter M64HCl treatment in
vitro to continuous treatment for 24 hours on monolayer wound closure. We
then investigated how long FAK activation and downstream ERK1/2 activation
persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl
concentrations immediately after washing measured by mass spectrometry
confirmed that M64HCl had been completely removed from the medium while
intracellular concentrations had been reduced by 95%. Three-hour and
four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration
over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not
increase cell number. Exposing cells twice with 2-hr exposures of M64HCl
during a 24-hour period had a similar effect. Both FAK inhibitor PF-573228
(10 µM) and ERK kinase (MEK) inhibitor PD98059 (20 µM) reduced basal wound
closure in the absence of M64HCl, and each completely prevented any
stimulation of wound closure by M64HCl. Rho kinase inhibitor Y-27632 (20
µM) stimulated Caco-2 monolayer wound closure but no further increase was
seen with M64HCl in the presence of Y-27632. M64HCl (100 nM) treatment for
3 hours stimulated Rho kinase activity.  M64HCl decreased F-actin in
Caco-2 cells. Furthermore, a two-hour treatment with M64HCl (100 nM)
stimulated sustained FAK activation and ERK1/2 activation for up to 16 and
hours 24 hours, respectively. These results suggest that transient M64HCl
treatment promotes prolonged intestinal epithelial monolayer wound closure
by stimulating sustained activation of the FAK/ERK1/2 pathway. Such
molecules may be useful to promote gastrointestinal mucosal repair even
with a relatively short half-life.