Given that migration of PMNs across mucosal surfaces contributes to epithelial cell dysfunction in a host of mucosal diseases (2
), the data provided here not only identify a new mechanism underlying the vectored release of HXA3
but also reveal interference with HXA3
synthesis or apical secretion from epithelial cells as novel therapeutic strategies for the treatment of mucosal inflammatory disorders such as IBD. We used S. typhimurium
as a tool to induce an acute intestinal inflammatory response and found that during active states of intestinal inflammation apical expression of the efflux transporter MRP2 is profoundly up-regulated, functionally active, and capable of using HXA3
as a substrate. The molecular basis of this observation is consistent with the role of the S. typhimurium
type III secreted product SipA, as this effector protein has been found to be both necessary and sufficient for induction of PMN transepithelial migration across model intestinal epithelia (13
). In keeping with its role as a virulence factor, our data now reveal that SipA mediates PMN transepithelial migration by directly affecting the release of HXA3
via modulation of MRP2 expression. Thus, identification of the molecular mechanism by which SipA activates PMN transepithelial migration through MRP2 defines a novel pro-inflammatory signaling cascade essential for the pathogenesis of S. typhimurium
that likely represents a paradigm of mucosal pathogen-elicited events.
Insights into pathogen-elicited active inflammation of the intestine may provide important information relating to mechanisms of disorders underlying IBD, which appear to be unrelated to pathogen colonization (43
). Therefore, we hypothesize that while evolution of such responses most likely targeted pathogens such as S. typhimurium
, aberrant activation of such pathways may lead to induction of mucosal inflammation associated with some chronic diseases of the intestine and airway. Consistent with this notion, we found a profound up-regulation of MRP2 at the apical surface of the colonic epithelium in murine models of chronic intestinal inflammation as well as in intestinal biopsies from patients presenting with active Crohn's disease and ulcerative colitis. Under normal healthy conditions, the most abundant constitutive expression of human and rat MRP2/MRP2
mRNA is found in the renal proximal tubule brush-border membrane and the hepatocyte canalicular membrane (44
). Significantly lower levels of MRP2 have been found in the small intestine with exclusive localization at the apical brush border membrane of villi (44
). Further, MRP2 expression decreases in intensity from the villus tip to the crypts, where no expression has been observed (44
). At this location, MRP2 is thought to play an important role in drug disposition. We now describe an unanticipated function of MRP2 as it is uniformly up-regulated at the villus tips of the apical surface of epithelial cells during active states of inflammation and plays a pivotal role in the inflammatory response.
then establishes a gradient through the tight junction complex to provide a chemotactic gradient used by PMNs to target the lumen of mucosal tissues at sites of inflammation (2
). The observation that HXA3
can serve as a substrate for the apical efflux transporter MRP2 fits well with the appreciation that other eicosanoids such as the cysteinyl leukotriene, LTC4
, a metabolite of the 5-LOX pathway, is one of the highest affinity MRP2 substrates characterized to date (29
). Detailed studies, however, are required to fully characterize and provide direct evidence for MRP2 transport of hepoxilin.
There are four enzymes that possess 12-LOX activity in humans and the most abundant LOX expressed in epithelial cells is 12/15-LOX. In humans, unlike mice, 12HpETE is not the major product of 12/15-LOX (ALOX15, 4:1 ratio of 15-H(p)ETE:12-H(p)ETE) (45
). However, 12/15-LOX is highly expressed in epithelial cells, including intestinal epithelial cells (45
), and the synthesis of 12-HpETE through this pathway is likely significant. At present it is unclear which 12-LOX enzymes specifically contribute to HXA3
production in the human mucosa, and thus the potential contribution of ALOX12, ALOX12B or ALOXE3 cannot be excluded. Our studies, however, determined that pharmacological inhibition of 12/15-LOX activity (both in vitro
and in vivo
) was found to not only block HXA3
synthesis but also the enhanced apical expression of MRP2 typically observed as part of the inflammatory process.
Our observation that 12/15-LOX activity is coupled to MRP2 up-regulation and apical localization implies that either a metabolite of the 12/15-LOX pathway or an active form of the enzyme itself participates in MRP2-related cellular changes associated with inflammation. However, it is unclear at this point in our investigation at what level or through what mechanism this association is achieved. Nevertheless, prior studies have demonstrated cyclooxygenase-2 expression enhances the functional activity of P-gp, providing a precedent of a causal link between arachidonic acid metabolism and expression of an ABC transporter (47
). Furthermore, given that epithelial cells appear to regulate 12/15-LOX and MRP2 expression in a coupled fashion, several novel therapeutic strategies are potentially available to treat inflammatory conditions, such as IBD, based upon regulation of 12/15-LOX and MRP2. Underscoring such important clinical implications, we show that treatment with baicalein, a 12-LOX inhibitor, which inhibits HXA3
synthesis as well as the enhanced apical expression of MRP2, resolved PMN transmigration/intestinal inflammation in a murine model of IBD.
Although our studies provide the first description that MRP2 is involved in the mechanisms that promote active states of intestinal inflammation (i.e. PMN recruitment), other ABC transporters, namely P-gp (MDR1), have been found to play a role in the development of colitis (48
), albeit by a completely different mechanism than MRP2. Consistent with this observation, we have recently determined that prolonged apical colonization of intestinal epithelial cells by wild-type S. typhimurium
(~4 h) leads not only to a profound functional decrease in P-gp but that the presence of P-gp adversely influences the ability of S. typhimurium
to invade host cells (21
). These results demonstrate that MRP2 and P-gp can be differentially regulated within the same tissue during a disease process. This observation supports the finding that during the progression of chronic renal failure MRP2 is up-regulated in both the kidney and liver, whereas P-gp remains unaffected (49
In summary, we are beginning to understand how the PMN chemoattractant HXA3
is secreted in a vectoral fashion through a unique efflux transport system located at the apical surface of the intestinal epithelium that involves the ABC transporter MRP2. This study demonstrates a critical link between apically-expressed MRP2 and the HXA3
biosynthetic pathway. Surprisingly, inhibition of an enzyme critical for the synthesis of HXA3
, 12/15-LOX, was found to also suppress the apical expression of MRP2 induced by inflammatory signals. This unanticipated coupling of 12/15-LOX and MRP2 identified in an in vitro
acute inflammatory model initiated by a pathogen was recapitulated in an in vivo
mouse model of IBD and was consistent with observations made in biopsy samples obtained from IBD patients. Since HXA3
has also been shown to be involved in pathogen-induced inflammation of both intestinal (2
) and pulmonary (7
) epithelial cells, it is likely that our results describe a generalized mechanism governing HXA3
synthesis and its subsequent apical release that could provide novel therapeutic strategies to impede and/or limit PMN involvement in deleterious events associated with a number of acute and chronic inflammatory conditions.