Basolateral EGF receptor sorting regulated by functionally distinct mechanisms in renal epithelial cells.

Proliferation of epithelial tissues is controlled by polarized distribution of signaling receptors including the EGF receptor (EGFR). In kidney, EGFRs are segregated from soluble ligands present in apical fluid of nephrons by selective targeting to basolateral membranes. We have shown previously that the epithelial-specific clathrin adaptor AP1B mediates basolateral EGFR sorting in established epithelia. Here we show that protein kinase C (PKC)-dependent phosphorylation of Thr654 regulates EGFR polarity as epithelial cells form new cell-cell junctional complexes. The AP1B-dependent pathway does not override a PKC-resistant T654A mutation, and conversely AP1B-defective EGFRs sort basolaterally by a PKC-dependent mechanism, in polarizing cells. Surprisingly, EGFR mutations that interfere with these different sorting pathways also produce very distinct phenotypes in three-dimensional organotypic cultures. Thus EGFRs execute different functions depending on the basolateral sorting route. Many renal disorders have defects in cell polarity and the notion that apically mislocalized EGFRs promote proliferation is still an attractive model to explain many aspects of polycystic kidney disease. Our data suggest EGFR also integrates various aspects of polarity by switching between different basolateral sorting programs in developing epithelial cells. Fundamental knowledge of basic mechanisms governing EGFR sorting therefore provides new insights into pathogenesis and advances drug discovery for these renal disorders.

Flagellin is required for salmonella-induced expression of heat shock protein Hsp25 in intestinal epithelium.

Flagellin is a bacterial protein responsible for activation of Toll-like receptor 5 (TLR5), which we hypothesize is involved in Salmonella's induction of cytoprotective heat shock proteins in intestinal epithelial cells. Flagellin induces the cytoprotective heat shock protein Hsp25 in different intestinal epithelial cell lines and in mouse intestine. Flagellin induces Hsp25 expression in a time-dependent manner in vitro. This effect is transcriptional, as confirmed by luciferase reporter assays and actinomycin D treatment. In addition, Hsp25 induction requires p38 MAPK activation and is only observed when flagellin is added to the basolateral side of polarized intestinal epithelial cells, consistent with the known location of TLR5. Flagellin-mediated Hsp25 induction is associated with increased protective effects against oxidant stress, an effect that is at least partially mediated by p38 MAPK. Use of small interfering RNA against Hsp25 demonstrates that flagellin-mediated protection against oxidant stress is to some degree mediated through Hsp25 induction. This suggests that, by protecting against oxidant injury, the induction of Hsp25 expression by flagellin may contribute to intestinal homeostasis. In a coculture cell model and in a mouse model of Salmonella enterica Serovar Typhimurium infection, not only does infection with wild-type and a flagellin-deletion mutant strain of Salmonella show that flagellin induces Hsp25 in vivo, but it also demonstrates that in the case of live Salmonella infection, flagellin serves as a major stimulus for the induction of Hsp25 expression. These data provide evidence that flagellin is required for Salmonella-mediated induction of Hsp25 expression in intestinal epithelium.

Flagellin-induced tolerance of the Toll-like receptor 5 signaling pathway in polarized intestinal epithelial cells.

Salmonella typhimurium is a gram-negative enteric pathogen that invades the mucosal epithelium and is associated with diarrheal illness in humans. Flagellin from S. typhimurium and other gram-negative bacteria has been shown to be the predominant proinflammatory mediator through activation of the basolateral Toll-like receptor 5 (TLR5). Recent evidence has shown that prior exposure can render immune cells tolerant to subsequent challenges by TLR ligands. Accordingly, we examined whether prior exposure to purified flagellin would render human intestinal epithelial cells insensitive to future contact. We found that flagellin-induced tolerance is common to polarized epithelial cells and prevents further activation of proinflammatory signaling cascades by both purified flagellin and Salmonella bacteria but does not affect TNF-alpha stimulation of the same pathways. Flagellin tolerance is a rapid process that does not require protein synthesis, and that occurs within 1 to 2 h of flagellin exposure. Prolonged flagellin exposure blocks activation of the NF-kappaB, MAPK, and phosphoinositol 3-kinase signaling pathways and results in the internalization of a fraction of the basolateral TLR5 without affecting the polarity or total expression of TLR5. After removal of flagellin, cells require more than 24 h to fully recover their ability to mount a normal proinflammatory response. We have found that activation of phosphoinositol 3-kinase and Akt by flagellin has a small damping effect in the early stages of flagellin signaling but is not responsible for tolerance. Our study indicates that inhibition of TLR5-associated IL-1 receptor-associated kinase-4 activity occurs during the development of flagellin tolerance and is likely to be the cause of tolerance.

Crosstalk between NF-kappaB and beta-catenin pathways in bacterial-colonized intestinal epithelial cells.

Salmonella-epithelial cell interactions are known to activate the proinflammatory NF-kappaB signaling pathway and have recently been found to also influence the beta-catenin signaling pathway, an important regulator of epithelial cell proliferation and differentiation. Here, using polarized epithelial cell models, we demonstrate that these same bacteria-mediated effects also direct the molecular crosstalk between the NF-kappaB and beta-catenin signaling pathways. Convergence of these two pathways is a result of the direct interaction between the NF-kappaB p50 subunit and beta-catenin. We show that PhoP(c), the avirulent derivative of a wild-type Salmonella strain, attenuates NF-kappaB activity by stabilizing the association of beta-catenin with NF-kappaB. In cell lines expressing constitutively active beta-catenin, IkappaBalpha protein was indirectly stabilized and NF-kappaB activity was repressed after wild-type Salmonella colonization. Accordingly, constitutively active beta-catenin was found to inhibit the secretion of IL-8. Thus our findings strongly suggest that the crosstalk between the beta-catenin and NF-kappaB signaling pathways is an important regulator of intestinal inflammation.

RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin.

Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJs) and adherens junctions (AJs) are regulated in part by their affiliation with the F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1, and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. With a tetracycline-repressible system to achieve regulated expression in Madin-Darby canine kidney (MDCK) epithelial cells, we used dominant-negative (DN) and constitutively active (CA) forms of RhoA, Rac1, and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2, and junctional adhesion molecule (JAM)-1 were dramatically redistributed in the presence of CA RhoA or CA Cdc42, whereas only claudins-1 and -2 were redistributed in response to CA Rac1. DN Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, whereas DN Cdc42 influenced only claudin-2 and DN RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN Rac1 induced a reduction in E-cadherin detergent solubility. All CA GTPases increased the detergent solubility of claudins-1 and -2, but CA RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active/resting GTPase levels rather than simply loss or gain of GTPase activity.

Bacterial activation of beta-catenin signaling in human epithelia.

The mucosal lining of the human intestine is constantly bathed in a milieu of commensal gut flora, the vast majority of these being nonpathogenic microorganisms. Here, we demonstrate that microbial-epithelial cell interactions not only affect proinflammatory pathways but also influence beta-catenin signaling, a key component in regulating epithelial cell proliferation. The nonpathogenic Salmonella strain PhoP(c) activates the beta-catenin signaling pathway of human epithelia via a blockade of beta-catenin degradation. Normal beta-catenin ubiquitination necessary for constitutive beta-catenin degradation is abolished, allowing the accumulation and translocation of beta-catenin to the nucleus. Transcriptional activation mediated by the beta-catenin/T cell factor complex increases c-myc expression and enhances cell proliferation. We also show that the Salmonella effector protein AvrA is involved in modulating this beta-catenin activation. These data suggest that nonvirulent bacterial-epithelial interactions can influence beta-catenin signaling and cell growth control in a manner previously unsuspected.

Cdc42 and Rac1 regulate late events in Salmonella typhimurium-induced interleukin-8 secretion from polarized epithelial cells.

Salmonella typhimurium colonization of the intestinal epithelium initiates biochemical cross-talk between pathogen and host that results in the secretion of chemokines, such as interleukin (IL)-8, that direct neutrophil migration to the site of infection. In nonpolarized cells, Rac1 and Cdc42 have been shown to regulate both bacterial invasion and signaling events leading to nuclear responses and IL-8 secretion. However, because the underlying actin cytoskeleton and the associated signaling machinery are distributed much differently in polarized epithelial cells, we used polarized Madin-Darby canine kidney monolayers to investigate the role of Rac1 and Cdc42 in S. typhimurium-induced pro-inflammatory responses in the more physiologically relevant polarized state. In Madin-Darby canine kidney monolayers expressing dominant-negative Rac1 or Cdc42, both Salmonella- and tumor necrosis factor alpha-induced activation of NFkappaB and mitogen-activated protein kinase signaling cascades proceeded normally, but IL-8 secretion was inhibited. We found that Rac1 and Cdc42 were not involved in early pro-inflammatory signaling events, as in nonpolarized cells, but rather regulated the basolateral exocytosis and secretion of IL-8. In contrast, dominant-negative Rac1 inhibited apical actin pedestal formation, indicating that pedestal formation and nuclear signaling for pro-inflammatory activation are not linked. These findings indicate that there are significant differences in the requirements of pathogen-induced host cell signaling pathways in polarized and nonpolarized cells.

The Salmonella typhimurium flagellar basal body protein FliE is required for flagellin production and to induce a proinflammatory response in epithelial cells.

During apical colonization by Salmonella typhimurium, intestinal epithelial cells orchestrate a proinflammatory response that involves secretion of chemoattractants, predominantly interleukin-8, which coordinate neutrophil trans-epithelial migration at the site of infection. This host-pathogen interaction requires several S. typhimurium genes. To identify novel genes that participate in this pathogen-induced proinflammatory response, we created S. typhimurium Tn-10 transposon mutants and identified a single mutant with Tn-10 insertional inactivation within the fliE flagellar locus that was able to adhere to and invade intestinal epithelial cells normally but was unable to induce interleukin-8 secretion in host cells. The fliE-deficient mutant failed to secrete flagellin and lacked any surface assembly of flagellae. Unlike wild-type S. typhimurium, the fliE-deficient mutant did not activate the IkappaBalpha/NF-kappaB signaling pathway or induce the coordinated trans-epithelial migration of isolated human neutrophils. Transcomplementation of the fliE-deficient mutant with a wild-type fliE-harboring plasmid restored all defects and produced a wild-type S. typhimurium phenotype. Furthermore, functional down-regulation of basolateral TLR5 completely inhibited the monolayers' ability to respond to both wild-type S. typhimurium and purified flagellin but had no affect on tumor necrosis factor alpha-induced responses. We therefore conclude that S. typhimurium fliE is essential for flagellin secretion, flagellar assembly, and S. typhimurium-induced proinflammatory responses through basolateral TLR5 and is consistent with the emerging model of S. typhimurium flagellin-induced inflammation.