Figure 2. Anti-apoptopic property of HSP27-inducible fraction. Time course of inactive full-length caspase-3 and -9 degradation in Caco2/BBE induced by 1 mM staurosporine treatment in the presence or absence of HSP27-inducible fraction. doi:10.1371/journal.pone.0023278.g002 antagonists of integrin, echistatin, Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP) and Gly-Arg-Gly-Asp-Thr-Ser (GRGDTS), which contain an Arg-Gly-Asp (RGD) sequence recognized by integrin ß [19,20], completely inhibited the protective effect for the intestinal barrier function by poly P (Figures 5C, D). On the other hand, Ser-Thr-Asp-Gly-Arg-Gly (STDGRG), the reverse sequence of GRGDTS had no inhibitory activity, and integrin antagonists themselves did not increase the [3H]-mannitol flux induced by an oxidant (Figures 5D). Furthermore, the human integrin-a3b1 molecule inhibited the protective action by poly P (Figure 5E), indicating the almost all free poly P in the lumen was captured by the integrin molecule. P38 MAPK phosphorylation by poly P was inhibited by RGD peptide (Figure 7C). This poly P-induced F- actin stability against oxidative stress was reversed with the addition of the integrin antagonist GRGDS (Figures 6A–F). To confirm the ability of poly P on the binding with integrin b, we prepared a 32P-labeled poly P. Two mg/ml of a recombinant human integrin a3b1 or aVb3 were incubated with 32P-labeled poly P for 1 hour at the room temperature. Integrins were then precipitated with anti-integrin b1 or b3 antibody. Collected protein G beads were washed three times with phosphate buffered saline (PBS), and then the radioactivity of immunoprecipitated 32P-labeled poly P was measured. The radioactivity of the sample precipitated with anti-integrin b1 antibody was significantly increased while that with anti-integrin b3 antibody was similar as the control. This demonstrates that poly P possess the ability to bind to integrin b1 (Figure 7D). Growth factors have been known to protect the gastrointestinal epithelium from a variety of insults. Epidermal growth factor (EGF) prevents acetaldehyde-induced disruption of tight junctions in the Caco-2 cell monolayer by PLCc-dependent protein kinase C activation [21]. Moreover, it has been reported that poly P activated the mitogenic action of growth factors such as fibroblast growth factor by enhancing their physical interactions [22]. However, in our study, neither EGF receptor inhibitor AG1478 (1 mM) nor platelet-derived growth factor receptor inhibitor AG1296 (1 mM) suppressed the protective action by poly P (Figure 5G). Collectively, integrin–p38 MAPK signaling pathway is mediat- ed in the actions of poly P, which is responsible for protecting the intestinal epithelia. Polyphosphate Maintains Intestinal Homeostasis Poly P improved intestinal injuries and survival rate of mice treated with a lethal dose of dextrin sodium sulfate (DSS) HSP27 has been shown to have an anti-inflammatory property [23]. After we confirmed TNF-a–induced phosphorylation of NF- kB and degradation of IkB was inhibited by HSP27-inducible fraction (Figure 8), the effect of poly P on a DSS-enteritis model, in which the barrier function of the intestinal epithelia was injured and led to increased exposure to luminal antigen [24], was analyzed. C57Bl/6 mice were orally administered 4% DSS and intrarectally administered 10 mg of poly P every day throughout the experimental period. Poly P-treated mice survived longer compared to PBS-treated control mice (Figure 9A). The cumulative survival rate of the poly P-treated mice was significantly higher than that of PBS-treated mice. In addition, C57Bl/6 mice were orally administered 3% DSS, and intrarectally administered 0 or 10 mg of poly P every day for seven days, after which the mice were euthanized. The mice that were intrarectally administered 10 mg of poly P did not show any significant colon shrinkage compared to normal mice, although the colon length in PBS-treated mice was significantly decreased by oral administra- tion of 3% DSS (Figure 9B). Hematoxylin-eosin (H&E) staining of the colonic sections showed that poly P-treated mice experienced improvements in intestinal injuries compared to PBS-treated mice (Figure 9C). The proinflammatory cytokines IL-1b and IL-6 have been reported to be upregulated in the acute DSS colitis model [25,26]. Treatment with 3% DSS for seven days caused a significant increase in the proinflammatory cytokines IL-1b and IL-6 in mice colons compared to those of mice who were administered distilled water. On the other hand, intrarectal administration of 10 mg of poly P prevented DSS-induced proinflammatory cytokine upregulation (Figure 9D). Discussion This study, for the first time, demonstrated that poly P, a common product of lactobacilli and other bacteria associated with intestinal microflora, is responsible for probiotic actions that protect the intestinal epithelia from oxidant stress and improve epithelial injury due to excess inflammation. It is noteworthy that this probiotic action is required to activate the integrin–p38 MAPK pathway, which is a novel system for mediating host– bacterial interactions. Inorganic poly P is a polymer composed of tens to hundreds of phosphate residues linked by phosphoanhy- dride bonds. Poly P is ubiquitous in nature and widely distributed among microorganisms [27]. PPK plays an important role in poly P metabolism in bacteria. This enzyme reversibly catalyzes the polymerization of the terminal phosphate of ATP into a poly P chain [28]. Our study suggests that poly P secreted from many probiotic and commensal bacteria mediates the maintenance of intestinal homeostasis because they carry PPK and the poly P- degrading exopolyphosphatase gene in their genome. This study demonstrated that the protective effect of poly P on intestinal epithelia was eliminated by inhibitors of integrins or p38 MAPK (Figure 5). In addition, p38 MAPK phosphorylation by poly P was also inhibited by the integrin inhibitor (Figure 7C). Furthermore, we confirmed that integrin b1 and b3 molecules were apically expressed in mouse small intestine (Figure 7B), as previously reported [29,30], and poly P specifically binds to integrin b1 (Figure 7D). These results suggest that poly P develops a robust intestinal barrier function through interaction with integrin b1, followed by the p38 pathway activation. Integrins are obligate heterodimers containing two distinct chains called a and b subunits [31], which mediate attachment PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23278