Hormones, Neurotransmitters, Growth Factors, Receptors, and Signaling (cid:2) -PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding

(cid:2) -PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding. Am J Physiol Gastrointest Liver Physiol 314: G399–G407, 2018. Early gut mucosal restitution is a process by which intestinal epithelial cells (IECs) migrate over the wounded area, and its defective regulation occurs commonly in various critical pathological conditions. This rapid reepithelialization is mediated by different activating small GTP-binding proteins, but the exact mechanism underlying this process remains largely unknown. Recently, it has been reported that interaction between p21-activated kinase-interacting exchange factor ( (cid:2) -PIX) and G protein-coupled receptor kinase-interacting protein 1 (GIT1) activates small GTPases and plays an important role in the regulation of cell motility. Here, we show that induced association of (cid:2) -PIX with GIT1 is essential for the stimulation of IEC migration after wounding by activating Rac1. Levels of (cid:2) -PIX and GIT1 proteins and their association in differentiated IECs (line of IEC-Cdx2L1) were much higher than those observed in undifferentiated IECs (line of IEC-6), which was associated with an increase in IEC migration after wounding. Decreased levels of endogenous (cid:2) -PIX by its gene-silencing destabilized (cid:2) -PIX/GIT1 complexes, repressed Rac1 activity and inhibited cell migration over the wounded area. In contrast, ectopic overexpression of (cid:2) -PIX increased the levels of (cid:2) -PIX/GIT1 complexes, stimulated Rac1 activity, and enhanced intestinal epithelial restitution. Increased levels of cellular polyamines also stimulated (cid:2) -PIX/ GIT1 association, increased Rac1 activity, and promoted the epithelial restitution. Moreover, polyamine depletion decreased cellular abundances of (cid:2) -PIX/GIT1 complex and repressed IEC migration after wounding, which was rescued by ectopic overexpression of (cid:2) -PIX or GIT1. These results indicate that (cid:2) -PIX/GIT1/Rac1 association is necessary for stimulation of IEC migration after wounding and that this signaling pathway is tightly regulated by cellular polyamines. NEW & NOTEWORTHY Our current study demonstrates that induced association of (cid:2) -PIX with GIT1 is essential for the stimulation of intestinal epithelial restitution by activating Rac1, and this signaling pathway is tightly regulated by cellular polyamines. reaction buffer containing biotin-alkyne reagent and CuSO 4 for 20 min, and the biotin-alkyne-azide-modiﬁed protein complex was pulled down using paramagnetic streptavidin-conjugated Dynabeads. The pull-down material was resolved by 10% SDS-PAGE and analyzed by Western immunoblotting analysis using antibody against (cid:2) -PIX, GIT1, or GAPDH. Measurement of cell migration. Migration assays were carried out as described in our earlier publications (6, 32, 40, 41, 53). Cells were plated at 6.25 (cid:6) 10 4 /cm 2 in DMEM containing FBS on 60-mm dishes thinly coated with Matrigel according to the manufacturer’s instruc- tions (BD Biosciences, Bedford, MA) and were incubated as described for stock cultures. Cells were fed on day 2 , and cell migration was assayed on day 4 . To initiate migration, the cell layer was scratched with a single-edge razor blade cut to ~27 mm in length. The scratch was made over the diameter of the dish and extended over an area 7–10 mm wide. The migrating cells in six contiguous 0.1-mm squares were counted at (cid:6) 100 magniﬁcation beginning at the scratch line and extending as far out as the cells had migrated. All experi- ments were carried out in triplicates, and the results were reported as number of migrating cells per millimeter of scratch. one three n 3). and repeated three signiﬁcance between ANOVA. of signiﬁcance Duncan’s multiple range test and of P (cid:9) 0.05 signiﬁcant.