Allergen-induced interleukin-18 promotes experimental eosinophilic oesophagitis in mice.

Elevated levels of interleukin (IL)-18 have been reported in a number of allergic diseases. We recently reported that IL-18 in the blood and IL-18Rα mRNA in the oesophagus are induced during human eosinophilic oesophagitis (EoE). Additionally, we earlier showed that invariant natural killer T (iNKT) cells are critical to EoE pathogenesis; however, the mechanism of iNKT cell activation in EoE is not well understood. Therefore, the current study focused on the hypothesis that allergen-induced IL-18 may have an important role in iNKT cell-mediated EoE pathogenesis. We first validated the human EoE findings of IL-18 in experimental EoE by examining blood levels of IL-18 and oesophageal IL-18Rα mRNA levels in aeroallergen- and food allergen-induced experimental mouse models of EoE. We demonstrate that blood IL-18 protein and oesophageal IL-18Rα mRNA are induced in the mouse model of EoE and that IL-18Rα is expressed by iNKT cells in the oesophagus. Intranasal delivery of rIL-18 induced both mast cells and eosinophilic inflammation in the oesophagus in a time- and dose-dependent manner. To establish the significance of IL-18 in EoE pathogenesis, we examined DOX-inducible rtTA-CC10-IL-18 bitransgenic mice that induce IL-18 protein expression in the oesophagus. Our analysis indicated that induction of IL-18 in these mice resulted in the development of many of the characteristics of EoE, including oesophageal intraepithelial eosinophilia, increased mast cells, oesophageal remodelling and fibrosis. The current study provides evidence that IL-18 may induce iNKT cell activation to release the eosinophil-activating cytokine IL-5, as IL-5-deficient mice and iNKT cell-deficient (CD1d null) mice do not induce EoE in response to intranasal IL-18 challenge. Taken together, these findings provide evidence that allergen-induced IL-18 has a significant role in promoting IL-5- and iNKT-dependent EoE pathogenesis.

Invariant natural killer T-cell neutralization is a possible novel therapy for human eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder that needs a potential therapeutic strategy. We earlier showed that iNKT cell-deficient mice are protected from allergen-induced EoE. Therefore, we now tested the hypothesis that iNKT cells are induced in the human EoE and is a novel possible target for the treatment of human EoE. Accordingly, we examine number of iNKT cells and eosinophils and expression of iNKT-associated cell surface receptors and chemokines by performing immunofluorescence, qPCR and ELISA in the esophageal biopsies and blood samples of normal subjects (comparison control) and EoE patients. Herein, we show that iNKT cell number, their receptor subcomponents Vα24 and Vß11 expression, and associated chemokine CXCL16 levels (or expression) are induced significantly in EoE patients compared with normal individuals. In addition, we show that CXCL16 levels (or expression) correlate with the mRNA levels of Vα24 receptor but not well with esophageal eosinophilia in human EoE. Of note, we show that in vivo activation of iNKT cells is sufficient to induce EoE in mice. Furthermore, we show that anti-mCD1d- and anti-hVα24Jα18-neutralizing antibody treatment protects allergen-induced experimental EoE. Taken together, we have shown first time that iNKT cells have a critical pathogenic role in human and experimental EoE. iNKT cell neutralization by humanized anti-CD1d and anti-Vα24Jα18 antibodies might be a novel and potential therapy for human EoE.

Allergen-induced resistin-like molecule-α promotes esophageal epithelial cell hyperplasia in eosinophilic esophagitis.

Resistin-like molecule (Relm)-α is a secreted, cysteine-rich protein belonging to a newly defined family of proteins, including resistin, Relm-ß, and Relm-γ. Although resistin was initially defined based on its insulin-resistance activity, the family members are highly induced in various inflammatory states. Earlier studies implicated Relm-α in insulin resistance, asthmatic responses, and intestinal inflammation; however, its function still remains an enigma. We now report that Relm-α is strongly induced in the esophagus in an allergen-challenged murine model of eosinophilic esophagitis (EoE). Furthermore, to understand the in vivo role of Relm-α, we generated Relm-α gene-inducible bitransgenic mice by using lung-specific CC-10 promoter (CC10-rtTA-Relm-α). We found Relm-α protein is significantly induced in the esophagus of CC10-rtTA-Relm-α bitransgenic mice exposed to doxycycline food. The most prominent effect observed by the induction of Relm-α is epithelial cell hyperplasia, basal layer thickness, accumulation of activated CD4(+) and CD4(-) T cell subsets, and eosinophilic inflammation in the esophagus. The in vitro experiments further confirm that Relm-α promotes primary epithelial cell proliferation but has no chemotactic activity for eosinophils. Taken together, our studies report for the first time that Relm-α induction in the esophagus has a major role in promoting epithelial cell hyperplasia and basal layer thickness, and the accumulation of activated CD4(+) and CD4(-) T cell subsets may be responsible for partial esophageal eosinophilia in the mouse models of EoE. Notably, the epithelial cell hyperplasia and basal layer thickness are the characteristic features commonly observed in human EoE.

Pathogenesis of allergen-induced eosinophilic esophagitis is independent of interleukin (IL)-13.

Several studies have shown that interleukin (IL)-13 is induced in the esophageal biopsies of eosinophilic esophagitis (EoE) patients and promotes esophageal eosinophilia in mice, following an IL-13 challenge. However, the role of IL-13 has not been clearly investigated in allergen-induced EoE. Accordingly, we tested the hypothesis that IL-13 is required in allergen-induced EoE. Mice deficient in IL-13, STAT (signal transducer and activator of transcription)6 and both IL-4/IL-13 genes with their respective controls were challenged with Aspergillus extract, and IL-5 gene deficient with their control were challenged with recombinant IL-13, intranasally. The lung and esophageal eosinophils, mast cells and collagen accumulation were examined. Herein, we report that intranasal delivery of IL-13 promotes IL-5-dependent esophageal eosinophilia. However, allergen-induced EoE is not impaired in the IL-13 gene-deficient mice. In addition, wild-type and IL-13 gene-deficient mice demonstrated a comparable level of mast cells and collagen accumulation in the esophagus, following allergen-induced experimental EoE. Similarly, we found that esophageal eosinophilia in IL-4/IL-13 double gene-deficient and STAT6 gene-deficient mice were also not reduced following allergen-induced experimental EoE. In contrast, lung eosinophilia was significantly reduced in mice deficient in IL-13, both IL-4/IL-13 and STAT6 genes following allergen challenge. In conclusion, our data establish that allergen-induced EoE pathogenesis is independent of IL-13, whereas IL-13 is required for allergen-induced lung eosinophilia.

Alpha(E)-Catenin induces SRF-dependent transcriptional activity through its C-terminal region and is partly RhoA/ROCK-dependent.

The ubiquitous alpha(E)-catenin is an essential actin cytoskeletal linker. The transcription factor, serum response factor (SRF), induces transcription via binding to the serum response element (SRE) in gene promoters, and in many cases responds to actin dynamics. Here, we report that alpha(E)-catenin expression in HEK293 cells activates the SRE.L transcriptional reporter, a reporter containing the isolated SRF-binding site, and a stably integrated SRE.L reporter in fibroblasts. alpha-Catenin-induced reporter activity appears only partly dependent on RhoA GTPase and Rho kinase function. alpha-Catenin expression has no effect on RhoA activation or localization, and alpha-catenin-induced SRE.L reporter activation is insensitive to the actin-modulating agent latrunculin B. Ectopic alpha-catenin expression was not sufficient to induce actin filament assembly as measured by stress fiber formation. SRE.L reporter is activated by the C-terminal approximately 300 residue region of alpha(E)-catenin. These results suggest induction of SRF-mediated transcription by alpha(E)-catenin either downstream of RhoA or via a parallel pathway.

Inhibition of serotonin-induced mitogenesis, migration, and ERK MAPK nuclear translocation in vascular smooth muscle cells by atorvastatin.

The HMG-CoA reductase inhibitors, statins, have pleiotropic effects which may include interference with the isoprenylation of Ras and Rho small GTPases. Statins have beneficial effects in animal models of pulmonary hypertension, although their mechanisms of action remain to be determined. Serotonin [5-hydroxytryptamine (5-HT)] is implicated in the process of pulmonary artery smooth muscle (PASM) remodeling as part of the pathophysiology of pulmonary hypertension. We examined the effect of atorvastatin on 5-HT-induced PASM cell responses. Atorvastatin dose dependently inhibits 5-HT-induced mitogenesis and migration of cultured bovine PASM cells. Inhibition by atorvastatin was reversed by mevalonate and geranylgeranylpyrophosphate (GGPP) supplement, suggesting that the statin targets a geranylgeranylated protein such as Rho. Concordantly, atorvastatin inhibits 5-HT-induced cellular RhoA activation, membrane localization, and Rho kinase-mediated phosphorylation of myosin phosphatase-1 subunit. Atorvastatin reduced activated RhoA-induced serum response factor-mediated reporter activity in HEK293 cells, indicating that atorvastatin inhibits Rho signaling, and this was reversed by GGPP. While 5-HT-induced ERK MAP and Akt kinase activation were unaffected by atorvastatin, 5-HT-induced ERK nuclear translocation was attenuated in a GGPP-dependent fashion. These studies suggest that atorvastatin inhibits 5-HT-induced PASM cell mitogenesis and migration through targeting isoprenylation which may, in part, attenuate the Rho pathway, a mechanism that may apply to statin effects on in vivo models of pulmonary hypertension.

Rab11a and myosin Vb are required for bile canalicular formation in WIF-B9 cells.

Hepatocytes polarize by forming functionally distinct sinusoidal (basolateral) and canalicular (apical) plasma membrane domains. Two distinct routes are used for delivery of membrane proteins to the canaliculus. Proteins having glycosylphosphatidylinositol anchors or single transmembrane domains are targeted to the sinusoidal plasma membrane from where they transcytose to the canalicular domain. In contrast, apical ATP-binding-cassette (ABC) transporters, which are required for energy-dependent biliary secretion of bile acids (ABCB11), phospholipids (ABCB4), and nonbile acid organic anions (ABCC2), lack initial residence in the basolateral plasma membrane and traffic directly from Golgi membranes to the canalicular membrane. While investigating mechanisms of apical targeting in WIF-B9 cells, a polarized hepatic epithelial cell line, we observed that rab11a is required for canalicular formation. Knockdown of rab11a or overexpression of the rab11a-GDP locked form prevented canalicular formation as did overexpression of the myosin Vb motorless tail domain. In WIF-B9 cells, which lack bile canaliculi, apical ABC transporters colocalized with transcytotic membrane proteins in rab11a-containing endosomes and, unlike the transcytotic markers, did not distribute to the plasma membrane. We propose that polarization of hepatocytes (i.e., canalicular biogenesis) requires recruitment of rab11a and myosin Vb to intracellular membranes that contain apical ABC transporters and transcytotic markers, permitting their targeting to the plasma membrane. In this model, polarization is initiated upon delivery of rab11a-myosin Vb-containing membranes to the surface, which causes plasma membrane at the site of delivery to differentiate into apical domain (bile canaliculus).

Galphaz inhibits serum response factor-dependent transcription by inhibiting Rho signaling.

Galpha12/13 or Galphaq signals induce activation of Rho GTPase, leading to serum response factor (SRF)-mediated gene transcription and actin cytoskeletal organization; however, less is known regarding how Rho pathway signals are down-regulated. Here we report that Galphaz signals inhibit serum response factor (SRF)-dependent transcription. Galphaz expression inhibits Galpha12/13-, Galphaq-, and Rho guanine nucleotide exchange factor (GEF)-induced serum response element (SRE) reporter activation in human embryonic kidney 293T and PC-12 cells. Expression of Galphaz mutants with defective fatty acylation has no inhibitory effect. Expression of Galphaz, but not Galphai, attenuates serum-induced SRE reporter activation, suggesting that Galphaz can down-regulate endogenous signals leading to SRF. Whereas Galphaz also blocks SRE reporter induction by the activated mutant RhoAL63, it does not affect Galpha12- or Rho GEF-induced RhoA activation or RhoAL63-GTP binding in vivo. Moreover, Galphaz does not inhibit SRE reporter induction by an activated form of Rho kinase. Because Galphaz inhibits RhoAL63/A188-induced reporter activation, phosphorylation of RhoA on serine 188 does not seem to be involved; furthermore, RhoA subcellular localization was not affected. Use of pharmacologic inhibitors implies that Galphaz-induced reduction of SRE reporter activation occurs via a mechanism other than adenylate cyclase modulation. These findings suggest that Galphaz signals may attenuate Rho-induced stimulation of SRF-mediated transcription.

Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8.

Caspases play a central role in apoptosis, a well-studied pathway of programmed cell death. Other programs of death potentially involving necrosis and autophagy may exist, but their relation to apoptosis and mechanisms of regulation remains unclear. We define a new molecular pathway in which activation of the receptor-interacting protein (a serine-threonine kinase) and Jun amino-terminal kinase induced cell death with the morphology of autophagy. Autophagic death required the genes ATG7 and beclin 1 and was induced by caspase-8 inhibition. Clinical therapies involving caspase inhibitors may arrest apoptosis but also have the unanticipated effect of promoting autophagic cell death.

Role of Lbc RhoGEF in Galpha12/13-induced signals to Rho GTPase.

Heterotrimeric Galpha12/13 signals induce cellular responses such as serum response element (SRE)-mediated gene transcription via Rho GTPase. Guanine nucleotide exchange factors (GEFs) are strong candidates for linking Galpha signals to Rho. For example, p115 RhoGEF transduces Galpha13 signals to Rho and inhibits Galpha12/13 signals via the RhoGEF LH domain which links to Galpha subunits. Here, we have evaluated the signaling capacity of Lbc RhoGEF in the context of Galpha12/13 signals. In vitro GEF assays indicate that baculoviral-expressed proto-Lbc has minimal exchange activity, implying that a stimulus is required for Lbc activity in vivo. Expression of a catalytically inactive proto-Lbc mutant in HEK293T cells attenuates Galpha12- and thrombin-induced activation of an SRE transcriptional reporter, and the levels of inhibition observed is similar to that obtained with an analogous p115 RhoGEF mutant. proto-Lbc mutant expression also led to decreased levels of Galpha12-induced RhoA activation in vivo. Complex formation between Galpha12 and Lbc forms was detected. Analysis of the Lbc peptide sequence reveals a previously undetected region which may link to Galpha subunit signals. These findings support a role for Lbc in Galpha12-induced signaling pathways to Rho.

Activated Galphaq family members induce Rho GTPase activation and Rho-dependent actin filament assembly.

Rho GTPase is required for actin filament assembly and serum response element (SRE)-dependent gene transcription. Certain G protein-coupled receptors (GPCRs) induce Rho-dependent responses, but the intermediary signaling steps are poorly understood. The heterotrimeric Galpha12 family can induce Rho-dependent responses. In contrast, there are conflicting reports on the role of the Galphaq family in Rho signaling. We report that expression of activated Galphaq members, or activation of endogenous Galphaq via GPCR stimulation, induces SRE reporter activation via Rho, and increased GTP-Rho levels. Moreover, microinjection of activated Galphaq in fibroblasts induces actin stress fiber formation via Rho. Galphaq functionally cooperates with Lbc Rho guanine nucleotide exchange factor. Overall, these findings indicate that Galphaq family signals are sufficient to induce Rho-dependent cellular responses.

Association of Lbc Rho guanine nucleotide exchange factor with alpha-catenin-related protein, alpha-catulin/CTNNAL1, supports serum response factor activation.

The Rho GTPase signaling pathway is required for actin cytoskeletal organization and serum response factor-dependent gene transcription. Lbc is a Rho-specific guanine nucleotide exchange factor that contains a modulatory C-terminal region. To elucidate Lbc regulatory mechanism(s), a yeast two-hybrid screen for proteins that interact with the Lbc C-terminal region was carried out, resulting in multiple isolation of cDNAs encoding the same 734-amino acid Lbc interacting protein. The Lbc interacting protein has homology with the alpha-catenin cell adhesion component and is identical to the alpha-catenin-like alpha-catulin protein of unknown function. The human alpha-catulin gene (CTNNAL1) maps to 9q31-32. Here we identify the predicted endogenous alpha-catulin product, document alpha-catulin and Lbc co-expression in multiple human cell lines, and show alpha-catulin and Lbc subcellular co-fractionation and intracellular localization. The required regions for Lbc and alpha-catulin interaction were mapped, and complex formation between Lbc and alpha-catulin in mammalian cells was detected. Functionally, alpha-catulin co-expression leads to increased Lbc-induced serum response factor activation in vivo as measured by a transcriptional reporter assay. Furthermore, alpha-catulin co-expression enhances Lbc-induced GTP-Rho formation in vivo. These results support the concept that the recently identified alpha-catulin protein may modulate Rho pathway signaling in vivo by providing a scaffold for the Lbc Rho guanine nucleotide exchange factor.