S. boulardii Enhances Cell Restitution Figure 9. Sb activates FAK, paxillin and MAPK. (A) HCT-8/E11 cells were allowed to adhere on type I collagen after Sb pretreatment. The phosphorylation of tyrosine residues in FAK (Tyr-397 FAK), paxillin (Tyr-118 paxillin) and Thr-202/Tyr-204 in MAPK (Erk1 and Erk2) was determined after cell lysis at the indicated times of cell adhesion. Samples were analyzed by western blot analysis. Equal amounts of protein were analyzed and loading amounts were verified by probing the blot with anti-FAK, anti-paxillin or anti-Erk1/Erk2 Abs. The intensity of the bands was measured after scanning of the autoradiograms. Values presented on the blots correspond to percentage of band intensity compared to control. (B and C) HCT-8/ E11 cell monolayers were wounded and incubated with or without Sb supernatant. The monolayers were further incubated with or without PD 98059, a MAPK inhibitor (B) or with or without PF 573228, a FAK inhibitor (C), 1 hour before wounding and during cell migration. Wound closure was determined as described in Methods. Results are expressed as the percentage of wound repair. Data represent the mean+SD of 3 separate experiments. ** P,.01. doi:10.1371/journal.pone.0018427.g009 Sb has been shown to generate transduction pathways, including activation of Erk1 and Erk2 in intestinal mucosal cells [11]. In the present study, we demonstrated that Sb can modulate pathways leading to cell migration. A large number of molecules, including FAK, have been suggested as candidates for recruitment in adhesion structures upon integrin activation. Furthermore these molecules may serve as conduits for the transmission of the force that is necessary for cell migration and for bidirectional signaling between the cell interior and its environment [37]. FAK activation leads to the stimulation of other signaling proteins such as paxillin, thereby activating various signaling pathways crucial in the regulation of cell adhesion and migration [37]. In the present study, we demonstrated that Sb secretes factor(s) that alter the FAK/paxillin signaling pathways. In mice, relocalization of a2b1 integrin to the basal domain of the enterocyte following Sb treatment is associated to paxillin phosphorylation of the Y118 residue of the same domain. Also Sb increased the tyrosine phosphorylation levels of FAK, paxillin and Erk1/2. Furthermore, Sb-treated cells exhibited focal structures that appeared thicker when compared to control cells. These observations clearly indicate that cell treatment with Sb resulted in a strengthening of a2b1/collagen interactions as shown by enhanced outside-in signaling and focal adhesion reorganization. Sb has been proposed to act as a shuttle able to liberate at least 1500 as yet not fully characterized molecules during intestinal transit [38]. This large number of secreted peptidic and non- Figure 10. Sb modulates integrin signaling pathway both in vitro and in vivo. HCT-8/E11 cells were pretreated (B) or not (A) for 2 hours with Sb supernatant, and then allowed to adhere to type I collagen-coated surfaces (3 mg/ml) for 30 minutes. After fixation and permeabilization, cells were stained with an anti-paxillin Ab, the binding of which was revealed with an AlexaFluor 488-conjugated anti-mouse secondary Ab. Scale bar: 20 mm. HCT-8/E11 cell monolayers were wounded and incubated without (C) or with (D) Sb supernatant for 5 hours. Paxilin was revealed using a specific anti-paxillin Ab. Scale bar: 20 mm. Intestinal tissues from control (E) or Sb force-fed mice (F) were immunostained with an Ab against Tyr-118 paxillin. Stainings were analyzed using a Leica confocal microscope. Scale bar: 100 mm. doi:10.1371/journal.pone.0018427.g010 PLoS ONE | www.plosone.org 10 March 2011 | Volume 6 | Issue 3 | e18427