Shedding Of Epidermal Growth Factor Receptor Ligands Research Articles

Unraveling the differential impact of PAHs and dioxin-like compounds on AKR1C3 reveals the EGFR extracellular domain as a critical determinant of the AHR response.

Polycyclic aromatic hydrocarbons (PAHs), dioxin-like compounds (DLCs) and structurally-related environmental pollutants may contribute to the pathogenesis of various diseases and disorders, primarily by activating the aryl hydrocarbon receptor (AHR) and modulating downstream cellular responses. Accordingly, AHR is considered an attractive molecular target for preventive and therapeutic measures. However, toxicological risk assessment of AHR-modulating compounds as well as drug development is complicated by the fact that different ligands elicit remarkably different AHR responses. By elucidating the differential effects of PAHs and DLCs on aldo–keto reductase 1C3 expression and associated prostaglandin D2 metabolism, we here provide evidence that the epidermal growth factor receptor (EGFR) substantially shapes AHR ligand-induced responses in human epithelial cells, i.e. primary and immortalized keratinocytes and breast cancer cells. Exposure to benzo[a]pyrene (B[a]P) and dioxin-like polychlorinated biphenyl (PCB) 126 resulted in a rapid c-Src-mediated phosphorylation of EGFR. Moreover, both AHR agonists stimulated protein kinase C activity and enhanced the ectodomain shedding of cell surface-bound EGFR ligands. However, only upon B[a]P treatment, this process resulted in an auto-/paracrine activation of EGFR and a subsequent induction of aldo–keto reductase 1C3 and 11-ketoreduction of prostaglandin D2. Receptor binding and internalization assays, docking analyses and mutational amino acid exchange confirmed that DLCs, but not B[a]P, bind to the EGFR extracellular domain, thereby blocking EGFR activation by growth factors. Finally, nanopore long-read RNA-seq revealed hundreds of genes, whose expression is regulated by B[a]P, but not by PCB126, and sensitive towards pharmacological EGFR inhibition. Our data provide novel mechanistic insights into the ligand response of AHR signaling and identify EGFR as an effector of environmental chemicals.

Epidermal Growth Factor Receptor Signaling Enhances the Proinflammatory Effects of Staphylococcus aureus Gamma-Toxin on the Mucosa.

Staphylococcus aureus (S. aureus) produces many different exotoxins including the gamma-toxins, HlgAB and HlgCB. Gamma-toxins form pores in both leukocyte and erythrocyte membranes, resulting in cell lysis. The genes encoding gamma-toxins are present in most strains of S. aureus, and are commonly expressed in clinical isolates recovered from menstrual Toxic Shock Syndrome (mTSS) patients. This study set out to investigate the cytotoxic and proinflammatory effects of gamma-toxins on vaginal epithelial surfaces. We found that both HlgAB and HlgCB were cytotoxic to cultured human vaginal epithelial cells (HVECs) and induced cytokine production at sub-cytotoxic doses. Cytokine production induced by gamma-toxin treatment of HVECs was found to involve epidermal growth factor receptor (EGFR) signaling and mediated by shedding of EGFR ligands from the cell surface. The gamma-toxin subunits displayed differential binding to HVECs (HlgA 93%, HlgB 97% and HlgC 28%) with both components (HlgAB or HlgCB) required for maximum detectable binding and significant stimulation of cytokine production. In studies using full thickness ex vivo porcine vaginal mucosa, HlgAB or HlgCB stimulated a dose-dependent cytokine response, which was reduced significantly by inhibition of EGFR signaling. The effects of gamma-toxins on porcine vaginal tissue and cultured HVECs were validated using ex vivo human ectocervical tissue. Collectively, these studies have identified the EGFR-signaling pathway as a key component in gamma-toxin-induced proinflammatory changes at epithelial surfaces and highlight a potential therapeutic target to diminish toxigenic effects of S. aureus infections.

Local Epidermal Growth Factor Receptor Signaling Mediates the Systemic Pathogenic Effects of Staphylococcus aureus Toxic Shock Syndrome.

Secreted factors of Staphylococcus aureus can activate host signaling from the epidermal growth factor receptor (EGFR). The superantigen toxic shock syndrome toxin-1 (TSST-1) contributes to mucosal cytokine production through a disintegrin and metalloproteinase (ADAM)-mediated shedding of EGFR ligands and subsequent EGFR activation. The secreted hemolysin, α-toxin, can also induce EGFR signaling and directly interacts with ADAM10, a sheddase of EGFR ligands. The current work explores the role of EGFR signaling in menstrual toxic shock syndrome (mTSS), a disease mediated by TSST-1. The data presented show that TSST-1 and α-toxin induce ADAM- and EGFR-dependent cytokine production from human vaginal epithelial cells. TSST-1 and α-toxin also induce cytokine production from an ex vivo porcine vaginal mucosa (PVM) model. EGFR signaling is responsible for the majority of IL-8 production from PVM in response to secreted toxins and live S. aureus. Finally, data are presented demonstrating that inhibition of EGFR signaling with the EGFR-specific tyrosine kinase inhibitor AG1478 significantly increases survival in a rabbit model of mTSS. These data indicate that EGFR signaling is critical for progression of an S. aureus exotoxin-mediated disease and may represent an attractive host target for therapeutics.

The Inflammasome and the Epidermal Growth Factor Receptor (EGFR) Are Involved in the Staphylococcus aureus-Mediated Induction of IL-1alpha and IL-1beta in Human Keratinocytes.

Staphylococcus (S.) aureus is an important pathogen causing various infections including those of the skin. Keratinocytes are able to sense invading S. aureus and to initiate a fast defense reaction by the rapid release of innate defense mediators such as antimicrobial peptides and cytokines. There is increasing evidence that the cytokines IL-1alpha and IL-1beta, which both signal through the IL-1 receptor, play an important role in cutaneous defense against S. aureus. The aim of this study was to gain more insight into the underlying mechanisms leading to the S. aureus-induced IL-1alpha and IL-1beta expression in keratinocytes. Infection of human primary keratinocytes with S. aureus led to the induction of gene expression and protein secretion of IL-1alpha and IL-1beta. Full S. aureus-induced IL-1 protein release required the inflammasome components caspase-1 and ASC (apoptosis-associated speck-like protein containing a CARD) whereas gene induction of IL-1alpha and IL-beta by S. aureus was not dependent on caspase-1 and ASC. Since patients receiving anti-cancer therapy by inhibition of the epidermal growth factor receptor (EGFR) often suffer from skin infections caused by S. aureus we additionally evaluated whether the EGFR pathway may be involved in the IL-1alpha and IL-1beta induction by S. aureus. Inactivation of the EGFR with a blocking antibody decreased the S. aureus-mediated IL-1alpha and IL-1beta induction in primary keratinocytes. Moreover, the use of siRNA experiments revealed that ADAM17 (A Disintegrin and A Metalloprotease 17), a metalloproteinase known to mediate the shedding and release of EGFR ligands, was required for full induction of IL-1alpha and IL-1beta in keratinocytes infected with S. aureus. A failure of keratinocytes to adequately upregulate IL-1alpha and IL-1beta may promote S. aureus skin infections.

Hypercapnia attenuates ventilator-induced lung injury via a disintegrin and metalloprotease-17.

Hypercapnic acidosis, common in mechanically ventilated patients, has been reported to exert both beneficial and harmful effects in models of lung injury. Understanding its effects at the molecular level may provide insight into mechanisms of injury and protection. The aim of this study was to establish the effects of hypercapnic acidosis on mitogen‐activated protein kinase (MAPK) activation, and determine the relevant signalling pathways. p44/42 MAPK activation in a murine model of ventilator‐induced lung injury (VILI) correlated with injury and was reduced in hypercapnia. When cultured rat alveolar epithelial cells were subjected to cyclic stretch, activation of p44/42 MAPK was dependent on epidermal growth factor receptor (EGFR) activity and on shedding of EGFR ligands; exposure to 12% CO2 without additional buffering blocked ligand shedding, as well as EGFR and p44/42 MAPK activation. The EGFR ligands are known substrates of the matrix metalloprotease ADAM17, suggesting stretch activates and hypercapnic acidosis blocks stretch‐mediated activation of ADAM17. This was corroborated in the isolated perfused mouse lung, where elevated CO2 also inhibited stretch‐activated shedding of the ADAM17 substrate TNFR1 from airway epithelial cells. Finally, in vivo confirmation was obtained in a two‐hit murine model of VILI where pharmacological inhibition of ADAM17 reduced both injury and p44/42 MAPK activation. Thus, ADAM17 is an important proximal mediator of VILI; its inhibition is one mechanism of hypercapnic protection and may be a target for clinical therapy.

Caveolin-1 is required for TGF-β-induced transactivation of the EGF receptor pathway in hepatocytes through the activation of the metalloprotease TACE/ADAM17.

Transforming growth factor-beta (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-β receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-β-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1−/− hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-β, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-β-induced survival signals in Cav1−/− hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-β was impaired in Cav1−/− hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1−/− hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1+/+ cells, which was not the case in Cav1−/− cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-β treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-β-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-β pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-β in liver cancer cells.

Autocrine ligands of the epithelial growth factor receptor mediate inflammatory responses to diesel exhaust particles

BackgroundDiesel exhaust is associated with cardiovascular and respiratory mortality and morbidity. Acute exposure leads to increased IL-8 expression and airway neutrophilia, however the mechanism of this response is unknown. Objectives: As cigarette smoke-induced IL-8 expression by epithelial cells involves transactivation of the epidermal growth factor receptor (EGFR), we studied the effects of diesel exhaust particles (DEP) on IL-8 release and the role of the EGFR.MethodsPrimary bronchial epithelial cells (PBEC) were exposed to DEPs or carbon black. IL-8 and EGFR ligand expression (transforming growth factor alpha (TGFα), heparin-binding EGF-like growth factor, and amphiregulin (AR)) were assessed by quantitative RT-PCR and ELISA.ResultsDEP, but not carbon black, caused a dose-dependent increase in mitogen-activated protein kinase (MAPK) activation and IL-8 expression, however above 50 μg/ml there was an increase in cytotoxicity. At 50 μg/ml, DEPs stimulated transcription and release of IL-8 and EGFR ligands. IL-8 release was blocked by EGFR neutralizing antibodies, an EGFR-selective tyrosine kinase inhibitor and by the metalloprotease inhibitor, GM6001, which blocks EGFR ligand shedding. Neutralizing antibodies to AR, TGFα and heparin-binding (HB)-EGF reduced DEP-induced IL-8 by >50%. Conclusion Expression of IL-8 in response to DEPs is dependent on EGFR activation and that autocrine production of EGFR ligands makes a substantial contribution to this response. Capsule Summary: This study identifies a mechanism whereby diesel particles stimulates IL-8 release from bronchial epithelial cells. This mechanism may help to explain the recruitment of neutrophils into the airways of people exposed to particulate air pollution.

T Cell–Induced Airway Smooth Muscle Cell Proliferation via the Epidermal Growth Factor Receptor

Allergic asthma is a heterogeneous disease with no curative therapies. T cells infiltrate the airway smooth muscle (ASM) layer and may be implicated in airway remodeling and the increase of ASM mass, a cardinal feature of asthma. The mechanism by which CD4(+) T cells drive airway remodeling remains unknown. This study sought to determine the T cell-mediated mechanism of ASM cell proliferation. We hypothesized that CD4(+) T cells adhere to ASM cells via CD44, and induce ASM cell proliferation through the activation of the epidermal growth factor receptor (EGFR). A coculture model showed that the contact of antigen-stimulated CD4(+) T cells with ASM cells induced high levels of EGFR ligand expression in CD4(+) T cells and the activation of matrix metalloproteinase (MMP)-9, required for the shedding of EGFR ligands. The inhibition of EGFR and MMP-9 prevented the increase of ASM cell proliferation after coculture. The hyaluronan receptor CD44 is the dominant mediator of the tight adherence of T cells to ASM and is colocalized with MMP-9 on the cell surface. Moreover, the neutralization of CD44 prevents ASM cell hyperplasia. These data provide a novel mechanism by which antigen-stimulated CD4(+) T cells induce the remodeling indicative of a direct trophic role for CD4(+) T cells.

Autocrine effect of EGFR ligands on the pro-inflammatory response induced by PM2.5 exposure in human bronchial epithelial cells

Human exposure to PM(2.5) (particulate matter with an aerodynamic diameter below 2.5 μm) is known to be responsible for airway inflammation and may also induce airway remodelling. In respiratory epithelial cells exposed to PM(2.5), releases of pro-inflammatory cytokines such as granulocyte macrophage-colony stimulating factor (GM-CSF) and growth factor ligands of the epidermal growth factor receptor (EGFR) are increased. The present study aimed at determining the involvement of EGFR ligands by autocrine effects in PM(2.5)-induced GM-CSF release. PM(2.5) exposure triggers GM-CSF release by human bronchial epithelial (HBE) cells. This release is dependent on EGFR activation by ligand binding as it is inhibited by AG1478, an inhibitor of EGFR tyrosine kinase activity as well as by a neutralizing anti-EGFR antibody. The use of conditioned medium from cells previously exposed to PM(2.5) demonstrates that PM(2.5)-exposed cells release soluble EGFR ligands able to induce GM-CSF release by an autocrine manner. It was further demonstrated by inhibiting tumour-necrosis factor-alpha converting enzyme (TACE) that is involved in some EGFR ligand shedding. TAPI-2 and GM-6001, two TACE inhibitors, prevented the PM(2.5)-induced GM-CSF release as well as the silencing of TACE by siRNA. We provide evidence that the pro-inflammatory response induced by PM(2.5) exposure on HBE cells, results from an autocrine effect of EGFR ligands released by TACE activity. This autocrine loop by eliciting and sustaining inflammation could contribute to exacerbation of airway remodelling in respiratory-compromised individuals.

Cell surface annexins regulate ADAM-mediated ectodomain shedding of proamphiregulin

A disintegrin and metalloproteinase (ADAM) is a family of enzymes involved in ectodomain shedding of various membrane proteins. However, the molecular mechanism underlying substrate recognition by ADAMs remains unknown. In this study, we successfully captured and analyzed cell surface transient assemblies between the transmembrane amphiregulin precursor (proAREG) and ADAM17 during an early shedding phase, which enabled the identification of cell surface annexins as components of their shedding complex. Annexin family members annexin A2 (ANXA2), A8, and A9 interacted with proAREG and ADAM17 on the cell surface. Shedding of proAREG was increased when ANXA2 was knocked down but decreased with ANXA8 and A9 knockdown, because of enhanced and impaired association with ADAM17, respectively. Knockdown of ANXA2 and A8 in primary keratinocytes altered wound-induced cell migration and ultraviolet B-induced phosphorylation of epidermal growth factor receptor (EGFR), suggesting that annexins play an essential role in the ADAM-mediated ectodomain shedding of EGFR ligands. On the basis of these data, we propose that annexins on the cell surface function as "shedding platform" proteins to determine the substrate selectivity of ADAM17, with possible therapeutic potential in ADAM-related diseases.

Neisseria gonorrhoeae-induced transactivation of EGFR enhances gonococcal invasion

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhoea, adheres to and invades into genital epithelial cells. Here, we investigate host components that are used by the bacteria for their entry into epithelial cells. We found that gonococcal microcolony formation on the surface of HEC-1-B cells disrupted the polarized, basolateral distribution of both epidermal growth factor receptor (EGFR) and ErbB2, a related family member, and induced their accumulation under the microcolonies at the apical membrane. Gonococcal infection increased EGFR and ErbB2 phosphorylation. The EGFR kinase inhibitor, AG1478, reduced gonococcal invasion by 80%, but had no effect on adherence or the recruitment of EGFR and ErbB2 to the microcolonies. Gonococcal inoculation upregulated the mRNA levels of several ligands of EGFR. Prevention of EGFR ligand shedding by blocking matrix metalloproteinase activation reduced gonococcal invasion without altering their adherence, while the addition of the EGFR ligand, HB-EGF, was able to restore invasion to 66% of control levels. These data indicate that N. gonorrhoeae modulates the activity and cellular distribution of host EGFR, facilitating their invasion. EGFR activation does not appear to be due to direct gonococcal binding to EGFR, but instead by its transactivation by gonococcal induced increases in EGFR ligands.

Activation of epidermal growth factor receptor signaling by the prostaglandin E2 receptor EP4 pathway during gastric tumorigenesis

Cyclooxygenase-2 (COX-2) plays an important role in tumorigenesis through prostaglandin E(2) (PGE(2)) biosynthesis. It has been shown by in vitro studies that PGE(2) signaling transactivates epidermal growth factor receptor (EGFR) through an intracellular mechanism. However, the mechanisms underlying PGE(2)-induced EGFR activation in in vivo tumors are still not fully understood. We previously constructed transgenic mice that develop gastric tumors caused by oncogenic activation and PGE(2) pathway induction. Importantly, expression of EGFR ligands, epiregulin, amphiregulin, heparin-binding EGF-like growth factor, and betacellulin, as well as a disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, and ADAM17 were significantly increased in the mouse gastric tumors in a PGE(2) pathway-dependent manner. These ADAMs can activate EGFR by ectodomain shedding of EGFR ligands. Notably, the extensive induction of EGFR ligands and ADAMs was suppressed by inhibition of the PGE(2) receptor EP4. Moreover, EP4 signaling induced expression of amphiregulin and epiregulin in activated macrophages, whereas EP4 pathway was required for basal expression of epiregulin in gastric epithelial cells. In contrast, ADAMs were not induced directly by PGE(2) in these cells, suggesting indirect mechanism possibly through PGE(2)-associated inflammatory responses. These results suggest that PGE(2) signaling through EP4 activates EGFR in gastric tumors through global induction of EGFR ligands and ADAMs in several cell types either by direct or indirect mechanism. Importantly, gastric tumorigenesis of the transgenic mice was significantly suppressed by combination treatment with EGFR and COX-2 inhibitors. Therefore, it is possible that inhibition of both COX-2/PGE(2) and EGFR pathways represents an effective strategy for preventing gastric cancer.

Caveolin-1 negatively regulates a metalloprotease-dependent epidermal growth factor receptor transactivation by angiotensin II

A metalloprotease, ADAM17, mediates the generation of mature ligands for the epidermal growth factor receptor (EGFR). This is the key signaling step by which angiotensin II (AngII) induces EGFR transactivation leading to hypertrophy and migration of vascular smooth muscle cells (VSMCs). However, the regulatory mechanism of ADAM17 activity remains largely unclear. Here we hypothesized that caveolin-1 (Cav1), the major structural protein of a caveolae, a membrane microdomain, is involved in the regulation of ADAM17. In cultured VSMCs, infection of adenovirus encoding Cav1 markedly inhibited AngII-induced EGFR ligand shedding, EGFR transactivation, ERK activation, hypertrophy and migration, but not intracellular Ca2+ elevation. Methyl-β-cyclodextrin and filipin, reagents that disrupt raft structure, both stimulated an EGFR ligand shedding and EGFR transactivation in VSMCs. In addition, non-detergent sucrose gradient membrane fractionations revealed that ADAM17 cofractionated with Cav1 in lipid rafts. These results suggest that lipid rafts and perhaps caveolae provide a negative regulatory environment for EGFR transactivation linked to vascular remodeling induced by AngII. These novel findings may provide important information to target cardiovascular diseases under the enhanced renin angiotensin system.

Epidermal growth factor receptor transactivation is implicated in IL-6-induced proliferation and ERK1/2 activation in non-transformed prostate epithelial cells

Epidermal growth factor receptor (EGF-R) is a receptor tyrosine kinase that can be activated by molecules other than its cognate ligands. This form of crosstalk called transactivation is frequently observed in both physiological and pathological cellular responses, yet it involves various mechanisms. Using the RWPE-1 cell line as a model of non-transformed prostate epithelial progenitor cells, we observed that interleukin-6 (IL-6) is able to promote cell proliferation and ERK1/2 activation provided that EGF-R kinase activity is not impaired. Treatment with GM6001, a general matrix metalloprotease inhibitor, indicated that IL-6 activates EGF-R through cleavage and release of membrane-anchored EGF-R ligands. Several inhibitors were used to test implication of “a disintegrin and metalloprotease” ADAM10 and ADAM17. GW280264X that targets both ADAM10 and ADAM17 blocked IL-6-induced proliferation and ERK1/2 phosphorylation with same potency as GM6001. However, ADAM10 inhibitor GI254023X and ADAM17 inhibitor TAPI-2 were less efficient in inhibiting response of RWPE-1 cells to IL-6, indicating possible cooperation of ADAM17 with ADAM10 or other metalloproteases. Accordingly, our findings suggest that IL-6 stimulates shedding of EGF-R ligands and transactivation of EGF-R in normal prostate epithelial cells, which may be an important mechanism to promote cell proliferation in inflammatory prostate.

Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice

The protease a disintegrin and metalloprotease (ADAM) 17 cleaves tumor necrosis factor (TNF), L-selectin, and epidermal growth factor receptor (EGF-R) ligands from the plasma membrane. ADAM17 is expressed in most tissues and is up-regulated during inflammation and cancer. ADAM17-deficient mice are not viable. Conditional ADAM17 knockout models demonstrated proinflammatory activities of ADAM17 in septic shock via shedding of TNF. We used a novel gene targeting strategy to generate mice with dramatically reduced ADAM17 levels in all tissues. The resulting mice called ADAM17ex/ex were viable, showed compromised shedding of ADAM17 substrates from the cell surface, and developed eye, heart, and skin defects as a consequence of impaired EGF-R signaling caused by failure of shedding of EGF-R ligands. Unexpectedly, although the intestine of unchallenged homozygous ADAM17ex/ex mice was normal, ADAM17ex/ex mice showed substantially increased susceptibility to inflammation in dextran sulfate sodium colitis. This was a result of impaired shedding of EGF-R ligands resulting in failure to phosphorylate STAT3 via the EGF-R and, consequently, in defective regeneration of epithelial cells and breakdown of the intestinal barrier. Besides regulating the systemic availability of the proinflammatory cytokine TNF, our results demonstrate that ADAM17 is needed for vital regenerative activities during the immune response. Thus, our mouse model will help investigate ADAM17 as a potential drug target.

Structure of the EGF receptor transactivation circuit integrates multiple signals with cell context.

Transactivation of the epidermal growth factor receptor (EGFR) is thought to be a process by which a variety of cellular inputs can be integrated into a single signaling pathway through either stimulated proteolysis (shedding) of membrane-anchored EGFR ligands or by modification of the activity of the EGFR. As a first step towards building a predictive model of the EGFR transactivation circuit, we quantitatively defined how signals from multiple agonists were integrated both upstream and downstream of the EGFR to regulate extracellular signal regulated kinase (ERK) activity in human mammary epithelial cells. By using a "non-binding" reporter of ligand shedding, we found that transactivation triggers a positive feedback loop from ERK back to the EGFR such that ligand shedding drives EGFR-stimulated ERK that in turn drives further ligand shedding. Importantly, activated Ras and ERK levels were nearly linear functions of ligand shedding and the effect of multiple, sub-saturating inputs was additive. Simulations showed that ERK-mediated feedback through ligand shedding resulted in a stable steady-state level of activated ERK, but also showed that the extracellular environment can modulate the level of feedback. Our results suggest that the transactivation circuit acts as a context-dependent integrator and amplifier of multiple extracellular signals and that signal integration can effectively occur at multiple points in the EGFR pathway.

Thromboxane A 2 receptor-mediated epidermal growth factor receptor transactivation: Involvement of PKC-δ and PKC-ɛ in the shedding of epidermal growth factor receptor ligands

We examined thromboxane A 2 receptor (TP)-mediated transactivation of epidermal growth factor receptor (EGFR) through the shedding of EGFR ligands. A TP agonist U46619 caused the phosphorylation of EGFR in 1321N1 human astrocytoma cells, which was inhibited by an EGFR selective inhibitor AG1478 and by a disintegrin and metalloproteinase (ADAM) inhibitor TAPI-2, indicating TP stimulation caused the EGFR transactivation through the EGFR ligand shedding. Since 1321N1 cells expressed heparin-binding EGF (HB-EGF) mRNA, the mechanism of TP-mediated EGFR transactivation was examined in HEK293 cells expressing alkaline phosphatase-conjugated HB-EGF and TP. U46619 caused the shedding of HB-EGF in a time- and concentration-dependent manner. The TP-mediated shedding was inhibited by a furin inhibitor CMK, TAP-2, dominant-negative Gα q, a G q/11 inhibitor YM254890, and also by a non-selective PKC inhibitor GF109203X and PKC down-regulation, but not by a conventional PKC inhibitor Gö6976. Furthermore, siRNAs of PKC-δ and PKC-ɛ inhibited U46619-induced HB-EGF shedding. Although BAPTA/AM had no effect on U46619-induced shedding of HB-EGF, EGTA inhibited it. These results suggest that TP-mediated EGFR transactivation is partially caused by shedding of HB-EGF, which involves furin and ADAM via novel types of PKCs (PKC-δ and PKC-ɛ) through Gα q/11 proteins in an extracellular Ca 2+-dependent manner.

Intestinal inflammation is coordinated by the metalloprotease ADAM17

Epidermal growth factor receptor (EGF-R) is a receptor tyrosine kinase that can be activated by molecules other than its cognate ligands. This form of crosstalk called transactivation is frequently observed in both physiological and pathological cellular responses, yet it involves various mechanisms. Using the RWPE-1 cell line as a model of non-transformed prostate epithelial progenitor cells, we observed that interleukin-6 (IL-6) is able to promote cell proliferation and ERK1/2 activation provided that EGF-R kinase activity is not impaired. Treatment with GM6001, a general matrix metalloprotease inhibitor, indicated that IL-6 activates EGF-R through cleavage and release of membrane-anchored EGF-R ligands. Several inhibitors were used to test implication of “a disintegrin and metalloprotease” ADAM10 and ADAM17. GW280264X that targets both ADAM10 and ADAM17 blocked IL-6-induced proliferation and ERK1/2 phosphorylation with same potency as GM6001. However, ADAM10 inhibitor GI254023X and ADAM17 inhibitor TAPI-2 were less efficient in inhibiting response of RWPE-1 cells to IL-6, indicating possible cooperation of ADAM17 with ADAM10 or other metalloproteases. Accordingly, our findings suggest that IL-6 stimulates shedding of EGF-R ligands and transactivation of EGF-R in normal prostate epithelial cells, which may be an important mechanism to promote cell proliferation in inflammatory prostate.

ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli

Activation of the NADPH oxidase homolog dual oxidase 1 (DUOX1) within the airway epithelium represents a key mechanism of innate airway host defense, through enhanced production of H2O2, which mediates cellular signaling pathways that regulate the production of various inflammatory mediators. Production of the CXC chemokine interleukin (IL)-8/CXCL8 forms a common epithelial response to many diverse stimuli, including bacterial and viral triggers, environmental oxidants, and other biological mediators, suggesting the potential involvement of a common signaling pathway that may involve DUOX1-dependent H2O2 production. Following previous reports showing that DUOX1 is activated by extracellular ATP and purinergic receptor stimulation, this study demonstrates that airway epithelial IL-8 production in response to several bacterial stimuli involves ATP release and DUOX1 activation. ATP-mediated DUOX1 activation resulted in the activation of ERK1/2 and NF-kappaB pathways, which was associated with epidermal growth factor receptor (EGFR) ligand shedding by ADAM17 (a disintegrin and metalloproteinase-17). Although ATP-mediated ADAM17 activation and IL-8 release were not prevented by extracellular H2O2 scavenging by catalase, these responses were attenuated by intracellular scavengers of H2O2 or related oxidants, suggesting an intracellular redox signaling mechanism. Both ADAM17 activation and IL-8 release were suppressed by inhibitors of EGFR/ERK1/2 signaling, which can regulate ADAM17 activity by serine/threonine phosphorylation. Collectively, our results indicate that ATP-mediated DUOX1 activation represents a common response mechanism to several environmental stimuli, involving H2O2-dependent EGFR/ERK activation, ADAM17 activation, and EGFR ligand shedding, leading to amplified epithelial EGFR activation and IL-8 production.

Up‐regulated expression of ADAM17 in gastrointestinal stromal tumors: coexpression with EGFR and EGFR ligands

Metalloproteinase activities of a disintegrin and metalloproteinases (ADAMs), matrix metalloproteinases (MMPs), and membrane type (MT-)MMPs are involved in many aspects of tumor biology. ADAMs are transmembrane proteins that cleave membrane-anchored proteins to release soluble factors, and thereby mediate important biological phenomena in tumors. The aim of this study was to analyze histopathology, expression and roles of metalloproteinases, especially ADAMs, in gastric gastrointestinal stromal tumor (GIST). Histopathology and immunohistochemical expression of ADAMs were examined in 89 gastric GISTs. In 11 GISTs, ADAM expression was examined at mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting, respectively. RT-PCR analysis showed frequent expression of ADAM9 (91%), ADAM10 (64%), ADAM17 (82%), MMP-2 (82%), and MT1-MMP (73%). However, ADAM17 and MMP-2 were the only metalloproteinases that were up-regulated in GISTs at the protein level compared with non-neoplastic gastric tissues. ADAM17 was immunohistochemically expressed in 93% of GIST versus 16% of normal gastric tissues. Furthermore, CD117-positive interstitial cells of Cajal in normal gastric tissues were all negative for ADAM17 with double immunostaining. Expressions of epidermal growth factor receptor (EGFR) and several EGFR ligands such as amphiregulin, heparin-binding epidermal growth factor (HB-EGF), betacellulin, and epiregulin were also demonstrated in GIST by RT-PCR. Protein expression of EGFR, phosphorylated EGFR, amphiregulin, and HB-EGF, both of which can be shed by ADAM17, was confirmed in tumors coexpressing ADAM17 by immunoblotting. Moreover, proteolytically cleaved soluble forms of amphiregulin were identified in tumor extracts. Considered together, the results suggest that ADAM17 may contribute to the progression and growth of GIST through shedding of EGFR ligands and consequent EGFR stimulation. ADAM17, as a major sheddase in GIST, could be potentially a suitable target in anticancer treatment of imatinib-resistant GISTs.