Rac1 promotes intestinal epithelial restitution by increasing Ca²⁺ influx through interaction with phospholipase C-γ1 after wounding

Abstract

Intestinal mucosal restitution occurs as a consequence of epithelial cell migration and reseals superficial wounds after injury. This rapid reepithelialization is mediated in part by a phospholipase C-gamma1 (PLC-gamma1)-induced Ca(2+) signaling, but the exact mechanism underlying such signaling and its regulation remains elusive. The small GTP-binding protein Rac1 functions as a pivotal regulator of several signaling networks and plays an important role in regulating cell motility. The current study tests the hypothesis that Rac1 modulates intestinal epithelial cell migration after wounding by altering PLC-gamma1-induced Ca(2+) signaling. Inhibition of Rac1 activity by treatment with its inhibitor NSC-23766 or Rac1 silencing with small interfering RNA decreased store depletion-induced Ca(2+) influx and suppressed cell migration during restitution, whereas ectopic overexpression of Rac1 increased Ca(2+) influx and promoted cell migration. Rac1 physically interacted with PLC-gamma1 and formed Rac1/PLC-gamma1 complex in intestinal epithelial cells. PLC-gamma1 silencing in cells overexpressing Rac1 prevented stimulation of store depletion-induced Ca(2+) influx and cell migration after wounding. Polyamine depletion inhibited expression of both Rac1 and PLC-gamma1, decreased Rac1/PLC-gamma1 complex levels, reduced Ca(2+) influx, and repressed cell migration. Overexpression of Rac1 alone failed to rescue Ca(2+) influx after store depletion and cell migration in polyamine-deficient cells, because it did not alter PLC-gamma1 levels. These results indicate that Rac1 promotes intestinal epithelial cell migration after wounding by increasing Ca(2+) influx as a result of its interaction with PLC-gamma1.

Rac1/osmosensing scaffold for MEKK3 contributes via phospholipase C-γ1 to activation of the osmoprotective transcription factor NFAT5

“…Not finding that MAPK pathways are involved in the activation of TonEBP/ OREBP by Rac1, we turned our attention to PLC-γ1 based on reports that PLC-γ1 contributes to activation of TonEBP/ OREBP (3) and that Rac1 can activate PLC-γ1 (16,17). We compared the effect of Rac1 in PLC-γ1-null MEFs to its effect in the same cells in which PLC-γ1 is reconstituted (18).…”

“…Having found that regulation of TonEBP/OREBP by Rac1 is not mediated by MKK3 and p38, we tested whether mediation is via PLC-γ1, which contributes to regulation of TonEBP/OREBP (3), interacts directly with Rac1 (16,25), and can be a downstream target of Rac1 (16,17). In addition, Rac1 often acts in combination with PLC-γ1 to regulate cellular signaling events (16,25,26). In the present study, we find that caRac1 and dominant-negative Rac1 do not affect TonEBP/ OREBP transcriptional activity in PLC-γ1-null MEFs unless PLC-γ1 is reconstituted (Fig.…”

Signaling Pathways in the Activation of Mast Cells Cocultured with Astrocytes and Colocalization of Both Cells in Experimental Allergic Encephalomyelitis

“…3A, 4A). Rac1 activation increases Ca 2+ influx in epithelial cells (36), and mast cells activate PKCs, MAPKs, and NF-kB/AP-1 signal pathways (28). PKC regulates NF-kB-dependent transcription (59).…”

“…The 8-oxo-dG is a Rac1/2 and cdc42 inhibitor (33-35) Rac1 increases Ca 2+ influx in epithelial cells (36). The 8-oxo-dG pretreatment inhibited Rho activation (Fig.…”

Polyamines regulate E-cadherin transcription through c-Myc modulating intestinal epithelial barrier function

“…Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO 2, and passages 15-20 were used in experiments. IEC-6 cells at passages 15-20 exhibit a stable phenotype (9,42).…”