In the present study, we show that suppression of ezrin adversely influences the ability of S. Typhimurium to induce PMN transmigration because the apical surface expression of MRP2 also becomes compromised. As MRP2 facilitates the apical release of the PMN chemoattractant, HXA3, suppression of this efflux mechanism directly correlates with a decrease in S. Typhimurium-induced PMN migration. Thus, our study tested the hypothesis that during infection S. Typhimurium triggers the activation of ezrin as a means to regulate the localization of MRP2 to the apical surface, a function critical for release of HXA3, and hence the induction of PMN transepithelial migration. In support of this hypothesis we reveal three key findings.
First, we determined a link between the activation of ezrin and the ability of S. Typhimurium (in a SipA dependent manner) to not only induce PMN migration, but also to translocate MRP2 to the apical membrane surface. Our observations are consistent with recent reports documenting the involvement of ERM proteins in the apical localization of MRP2. As an example, the predominant ERM protein expressed in hepatocytes and bile canalicular is radixin, and this protein was shown to be necessary for the apical localization of MRP2 (Kikuchi et al., 2002
). Mutations in the human MRP2 gene results in conjugated hyperbilirubinemia, defective excretion of organic anions, and leads to a disease known as Dubin-Johnson syndrome (Wakusawa et al., 2003
). Remarkably, radixin deficiency in mice results in a similar phenotype with loss of MRP2 from the apical canalicular membranes, emphasizing the critical role played by radixin in directing the apical localization of MRP2 in the bile canalicular membrane (Kikuchi et al., 2002
). ERM proteins have also been reported to play a critical role in the modulation of membrane expression of P-glycoprotein (another member of the ABC family) in cells of human lymphoid origin (Luciani et al., 2002
). However, whether ERM regulation of apical membrane protein expression of members of the ABC family is a general phenomenon remains to be determined.
Another study has described the requirement of radixin in maintaining the structure, and function of the apical canalicular membrane in rat hepatocytes (Wang et al., 2006
). In keeping with this observation, in the developing mouse intestine, ezrin was shown to be essential for the establishment and/or maintenance of epithelial cell polarity, as well as villus morphogenesis (Saotome et al., 2004
). The same study also determined that ezrin regulates the localization and/or function of some apical membrane proteins required for normal intestinal functions, including MRP2. These studies are consistent with our findings where we demonstrate the ERM protein, ezrin, plays a critical role in regulating the apical surface localization of MRP2 during S.
Second, we have determined that infection of cells with wild-type S.
Typhimurium but not the SipA mutant strain resulted in increased ezrin:MRP2 interactions. These results suggest that ezrin is functioning as a scaffold where MRP2 binds and is transported to the apical cell surface, and offer an explanation of how ezrin may induce the apical transport of MRP2 during S.
Typhimurium infection. In rat intestinal epithelial cells and Caco-2 cells ezrin:MRP2 interactions have also been shown to be associated with apical surface localization of MRP2 (Yang et al., 2007
; Nakano et al., 2009
), supporting our observations described above. However, whether ezrin and MRP2 interact directly or indirectly through another adaptor protein remains to be determined.
Speculatively, a potential candidate protein that may function as a scaffold, mediating ezrin-MRP2 interaction in intestinal epithelial cells is NHERF-1 (Na+/H+ exchanger regulatory factor 1). NHERF-1 binds to both MRP2 and ezrin through its PDZ domain located on its C- and N-termini (Reczek et al., 1997
; Weinman et al., 1995
). Moreover, the PDZ domain of NHERF-1 has been reported to bind to many transport and membrane proteins including the CFTR, the β2
-adrenegic receptor, and the Na+/H+ exchanger-3 (NHE3) where it facilitates their cross linking to F-actin cytoskeleton (Bretscher et al., 2000
; Cao et al., 1999
; Reczek et al., 1997
). In the liver, NHERF-1 was also shown to directly bind to MRP2, an interaction that promoted the apical localization of MRP2 in hepatocytes (Li et al., 2010
). As NHERF-1 is also expressed in intestinal epithelial cells, its potential involvement in apical localization of MRP2 or ezrin-MRP2 interaction during S.
Typhimurium infection is an obvious extension of our current study.
Our third key finding revealed that S. Typhimurium infection resulted in the activation of ezrin via phosphorylation at Thr-567, and this occurred in a SipA dependent manner. As PKC is known to play a significant role in the signaling cascades leading to ezrin activation, we first addressed whether PKC was involved in activating ezrin during S. Typhimurium infection, and if so, which isoform was involved. We found that inhibitors specific to PKC-α decreased the apical localization of MRP2, attenuated ezrin-MRP2 interactions, and significantly reduced the phosphorylation of ezrin at Thr-567, suggesting that PKC-α activates ezrin during S. Typhimurium infection.
Several protein kinases including the p38 MAPK, protein kinase B (Akt2), phosphatidyl inositides and members of the PKC family have been implicated in ezrin activation/phosphorylation in normal physiology and upon stimulation (Zhao et al., 2004
; Shiue et al., 2005
; Rasmussen et al., 2008
; Nakano et al.
, 2007). While PKC iota (PKC-ι) has been shown to be involved in ezrin activation during normal intestinal epithelial differentiation, in response to initiation of Na+
-glucose co-transport (Wald et al
., 2007), there is a rapid increase in the apical membrane association of NHE3 and in its cytoskeletal association with ezrin, which parallels ezrin Thr-567 phosphorylation (Zhao et al., 2004
). In this study, the p38 MAP kinase was identified as the kinase involved in ezrin Thr-567 phosphorylation.
Another study reported a correlation between the apical localization of MRP2 in rat intestines with ezrin activation upon conventional PKC activation (Nakano et al., 2009
). However, we have previously described a critical role played by PKC-α in PMN transmigration whereby inhibition of PKC-α resulted in a decrease in PMN transepithelial migration during S.
Typhimurium infection (Silva et al., 2004
) consistent with the role of PKC-α in ezrin activation, apical translocation of MRP2, and HXA3
secretion. Thus, based on these results, we propose that S.
Typhimurium infection, through a SipA dependent mechanism, induces the activation of PKC-α (Silva et al., 2004
), which in turn phosphorylates ezrin at Thr-567. Phosphorylated ezrin then modulates apical surface localization of MRP2 in intestinal epithelial cells (). Taken together these results reiterate the central role played by PKC-α in Salmonella
induced enteritis suggesting PKC-α can be used as an important therapeutic target during S.
Working model by which S. Typhimurium induces the apical expression of MRP2
In conclusion, we have shown that S. Typhimurium infection, in a process mediated by SipA; induces ezrin activation via a PKC-α dependent mechanism and that ezrin activation is coupled to apical localization of MRP2. We also provide evidence that there is a direct correlation between apical membrane localization of MRP2 in intestinal epithelial cells and the ability to induce S. Typhimurium-induced PMN transmigration. These results indicate that during S. Typhimurium infection ezrin plays a critical role in the pathogenesis of S. Typhimurium induced enteritis. Whether other inducers of intestinal inflammation (i.e. autoimmune or idiopathic colitis such as Crohn's disease and ulcerative colitis) share a similar mechanism of inflammation remains to be determined.