This review focuses on the role of ADAM-17 in disease. Since its debut as the tumor necrosis factor converting enzyme or TACE, ADAM-17 has been reported to be an indispensible regulator of almost every cellular event from proliferation to migration. The central role of ADAM-17 in cell regulation is rooted in its diverse array of substrates: cytokines, growth factors, and their receptors as well as adhesion molecules are activated or inactivated by their cleavage with ADAM-17. It is therefore not surprising that ADAM-17 is implicated in numerous human diseases including cancer, heart disease, diabetes, rheumatoid arthritis, kidney fibrosis, Alzheimer’s disease, and is a promising target for future treatments. The specific role of ADAM-17 in the pathophysiology of these diseases is very complex and depends on the cellular context. To exploit the therapeutic potential of ADAM-17, it is important to understand how its activity is regulated and how specific organs and cells can be targeted to inactivate or activate the enzyme.

Background & Aims Infection with H. pylori represses expression of the gastric H, K-ATPase α subunit (HKα), which could contribute to transient hypochlorhydria. CagL, a pilus protein component of the H. pylori type IV secretion system, binds to the integrin α5β1 to mediate translocation of virulence factors into the host cell and initiate signaling. α5β1 binds ADAM17, a metalloenzyme that catalyzes ectodomain shedding of receptor tyrosine kinase ligands. We investigated whether H. pylori-induced repression of HKα is mediated by CagL activation of ADAM17 and release of heparin-binding epidermal growth factor (HB-EGF). Methods HKα promoter and ADAM17 activity were measured in AGS gastric epithelial cells transfected with HKα promoter-reporter constructs or ADAM17-specific small interfering (si)RNAs and infected with H. pylori. HB-EGF secretion was measured by ELISA analysis and ADAM17 interaction with integrins were investigated by co-immunoprecipitation analyses. Results Infection of AGS cells with wild-type H. pylori or an H. pylori cagL-deficient isogenic mutant that also contained a wild-type version of cagL (P12ΔcagL/cagL) repressed HKα promoter-Luc reporter activity and stimulated ADAM17 activity. Both responses were inhibited by point mutations in the NF-κB binding site of HKα or by infection with P12ΔcagL. siRNA-mediated silencing of ADAM17 in AGS cells inhibited the repression of wild-type HKα promoter and reduced ADAM17 activity and HB-EGF production, compared to controls. Coimmunoprecipitation studies of AGS lysates showed that wild-type H. pylori disrupted ADAM17–α5β1 complexes. Conclusions During acute H. pylori infection, CagL dissociates ADAM17 from the integrin α5β1 and activates ADAM17-dependent, NF-κB–mediated repression of HKα. This might contribute to transient hypochlorhydria in patients with H. pylori infection.

Idiopathic pulmonary fibrosis (IPF) is a progressive clinical syndrome of fatal outcome. The lack of information about the signaling pathways that sustain fibrosis and the myofibroblast phenotype has prevented the development of targeted therapies for IPF. Our previous study showed that isolated fibrogenic lung fibroblasts have high endogenous levels of the hyaluronan receptor, CD44V6 (CD44 variant containing exon 6), which enhances the TGFβ1 autocrine signaling and induces fibroblasts to transdifferentiate into myofibroblasts. NADPH oxidase 4 (NOX4) enzyme, which catalyzes the reduction of O to hydrogen peroxide (HO), has been implicated in the cardiac and lung myofibroblast phenotype. However, whether CD44V6 regulates NOX4 to mediate tissue repair and fibrogenesis is not well-defined. The present study assessed the mechanism of how TGF-β-1-induced CD44V6 regulates the NOX4/reactive oxygen species (ROS) signaling that mediates the myofibroblast differentiation. Specifically, we found that NOX4/ROS regulates hyaluronan synthesis and the transcription of via an effect upon activity. Further, CD44V6 is part of a positive-feedback loop with TGFβ1/TGFβRI signaling that acts to increase NOX4/ROS production, which is required for myofibroblast differentiation, myofibroblast differentiation, myofibroblast extracellular matrix production, myofibroblast invasion, and myofibroblast contractility. Both NOX4 and CD44v6 are up-regulated in the lungs of mice subjected to experimental lung injury and in cases of human IPF. Genetic ( shRNA) or a small molecule inhibitor (CD44v6 peptide) targeting of CD44v6 abrogates fibrogenesis in murine models of lung injury. These studies support a function for CD44V6 in lung fibrosis and offer proof of concept for therapeutic targeting of CD44V6 in lung fibrosis disorders.

Background Parkinson’s disease (PD) is a progressive, neurodegenerative disorder with no disease-modifying therapies, and symptomatic treatments are often limited by debilitating side effects. In PD, locus coeruleus noradrenergic (LC-NE) neurons degenerate prior to substantia nigra dopaminergic (SN-DA) neurons. Vagus nerve stimulation (VNS) activates LC neurons, and decreases pro-inflammatory markers, allowing improvement of LC targets, making it a potential PD therapeutic. Objective To assess therapeutic potential of VNS in a PD model. Methods To mimic the progression of PD degeneration, rats received a systemic injection of noradrenergic neurotoxin DSP-4, followed one week later by bilateral intrastriatal injection of dopaminergic neurotoxin 6-hydroxydopamine. At this time, a subset of rats also had vagus cuffs implanted. After eleven days, rats received a precise VNS regimen twice a day for ten days, and locomotion was measured during each afternoon session. Immediately following final stimulation, rats were euthanized, and left dorsal striatum, bilateral SN and LC were sectioned for immunohistochemical detection of monoaminergic neurons (tyrosine hydroxylase, TH), α-synuclein, astrocytes (GFAP) and microglia (Iba-1). Results VNS significantly increased locomotion of lesioned rats. VNS also resulted in increased expression of TH in striatum, SN, and LC; decreased SN α-synuclein expression; and decreased expression of glial markers in the SN and LC of lesioned rats. Additionally, saline-treated rats after VNS, had higher LC TH and lower SN Iba-1. Conclusions Our findings of increased locomotion, beneficial effects on LC-NE and SN-DA neurons, decreased α-synuclein density in SN TH-positive neurons, and neuroinflammation suggest VNS has potential as a novel PD therapeutic.

ObjectiveRegulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.Methods and ResultsWe report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\TRPV4 channel mediated increase in intracellular calcium.ConclusionWe conclude that in the absence of cilia, an EGF activated TRPP2\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.

The intracellular processing of epidermal growth factor receptor (EGFR) induced by epidermal growth factor (EGF) and transforming growth factor-α (TGF-α has been studied meticulously, with the former resulting in EGFR degradation and the latter in EGFR recycling to the plasma membrane. However, little is known about how other EGF family growth factors affect the trafficking of the EGFR. Additionally, although both EGF and TGF-α have been shown to effectively induce initial c-Cbl (ubiquitin ligase)-mediated ubiquitination of the EGFR, limited information is available regarding the role of c-Cbl in the trafficking and signaling of recycling EGFR. Thus, in the present study we investigated the roles of c-Cbl in endogenous EGFR trafficking and signaling after stimulation with amphiregulin (AR). We demonstrated that a physiological concentration of AR induced recycling of endogenous EGFR to the plasma membrane, which correlated closely with transient association of EGFR with c-Cbl and transient EGFR ubiquitination. Most importantly, we used c-Cbl small interfering RNA (siRNA) duplexes and a c-Cbl dominant negative mutant to show that c-Cbl is critical for the efficient transition of EGFR from early endosomes to a recycling pathway, and that c-Cbl regulates the duration of extracellular-signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) phosphorylation. These data support novel functions of c-Cbl in mediating recycling of EGF receptors to the plasma membrane, as well as in mediating the duration of activation (transient vs. sustained) of ERK1/2 MAPK phosphorylation.

Opening of voltage dependent anion channels (VDAC) by the erastin-like compound X1 and the multikinase inhibitor sorafenib promotes oxidative stress and mitochondrial dysfunction in hepatocarcinoma cells. Here, we hypothesized that X1 and sorafenib induce mitochondrial dysfunction by increasing ROS formation and activating c-Jun N-terminal kinases (JNKs), leading to translocation of activated JNK to mitochondria. Both X1 and sorafenib increased production of reactive oxygen species (ROS) and activated JNK. X1 and sorafenib caused a drop in mitochondrial membrane potential (ΔΨ), a readout of mitochondrial metabolism, after 60 min. Mitochondrial depolarization after X1 and sorafenib occurred in parallel with JNK activation, increased superoxide (O 2•− ) production, decreased basal and oligomycin sensitive respiration, and decreased maximal respiratory capacity. Increased production of O 2 •− after X1 or sorafenib was abrogated by JNK inhibition and antioxidants. S3QEL specific inhibitor of site IIIQ o, at Complex III prevented depolarization induced by X1. JNK inhibition by JNK inhibitors VIII and SP600125 also prevented mitochondrial depolarization. After X1, activated JNK translocated to mitochondria as assessed by proximity ligation assays.

Background and aims: Matrix metalloproteinases (MMPs) are endopeptidases with roles in extracellular matrix remodelling, cell proliferation, and inflammatory processes. We showed previously that Helicobacter pylori infection of human gastric adenocarcinoma (AGS) cells increased epithelial secretion of epithelial MMP-1 and MMP-3 and bacterial secretion of MMP-3-like activity. In the present study, we sought to characterise the role of interleukin (IL)-1β in H pylori induced secretion of epithelial MMPs. Methods and results: AGS cells were treated with H pylori and/or IL-1β. Comparable IL-8 secretory responses (∼1700 ng/ml) measured by ELISA were induced by 2.0 ng/ml IL-1β and by H pylori at a multiplicity of infection (MOI) of 50. The same IL-1β and H pylori concentrations induced comparable increases in AGS cell caseinolytic activity at 60 kDa. MMP-3 monoclonal antibody immunoblots of AGS cell conditioned media detected immunoreactive bands at 71 kDa and 56 kDa. H pylori (MOI=50-100) induced dose dependent increases in both bands whereas IL-1β (0.2-2 ng/ml) induced dose dependent increases only in the 71 kDa band, which was identified as a MMP-3/TIMP-3 (tissue inhibitor of metalloproteinases 3) heterodimer. AGS/H pylori conditioned media expressed 24 times more MMP-3 activity than AGS/IL-1β conditioned media. There was a strong interaction between IL-1β and H pylori on MMP-3 secretion. Conclusions: We conclude that IL-1β induces gastric epithelial cell MMP-3 secretion, contributing to epithelial tissue destruction during H pylori infection. However, other bacterial/host factors are needed to mediate the full gastric epithelial cell MMP-3 secretory response induced by H pylori infection.