Distinct ADAM metalloproteinases regulate G protein-coupled receptor-induced cell proliferation and survival

Cross-talk between G protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signaling systems is widely established in a variety of normal and transformed cell types. Here, we demonstrate that the EGFR transactivation signal requires metalloproteinase cleavage of epidermal growth factor-like growth factor precursors in fibroblasts, ACHN kidney, and TccSup bladder carcinoma cells. Furthermore, we present evidence that blockade of the metalloproteinase-disintegrin tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17) by a dominant negative ADAM17 mutant prevents angiotensin II-stimulated pro-HB-EGF cleavage, EGFR activation, and cell proliferation in ACHN tumor cells. Moreover, we found that in TccSup cancer cells, the lysophosphatidic acid-induced transactivation signal is mediated by ADAM15, demonstrating that distinct combinations of growth factor precursors and ADAMs (a disintegrin and metalloproteinases) regulate GPCR-EGFR cross-talk pathways in cell lines derived from urogenital cancer. Our data show further that activation of ADAMs results in discrete cellular responses; whereas GPCR agonists promote activation of the Ras/MAPK pathway and cell proliferation via the EGFR in fibroblasts and ACHN cells, EGFR transactivation pathways regulate activation of the survival mediator Akt/protein kinase B and the susceptibility of fibroblasts and TccSup bladder carcinoma cells to proapoptotic signals such as serum deprivation, death receptor stimulation, and the chemotherapeutic drug doxorubicin. Thus, ADAM15 and -17 function as effectors of GPCR-mediated signaling and define critical characteristics of cancer cells.     

ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17

The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H₂O₂ participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y₂ receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y₂R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y₂R activation as a proximal step in EGFR transactivation and downstream signaling.     

Mammary ductal morphogenesis requires paracrine activation of stromal EGFR via ADAM17-dependent shedding of epithelial amphiregulin

Epithelial-mesenchymal crosstalk is essential for tissue morphogenesis, but incompletely understood. Postnatal mammary gland development requires epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AREG), which generally must be cleaved from its transmembrane form in order to function. As the transmembrane metalloproteinase ADAM17 can process AREG in culture and Adam17(-/-) mice tend to phenocopy Egfr(-/-) mice, we examined the role of each of these molecules in mammary development. Tissue recombination and transplantation studies revealed that EGFR phosphorylation and ductal development occur only when ADAM17 and AREG are expressed on mammary epithelial cells, whereas EGFR is required stromally, and that local AREG administration can rescue Adam17(-/-) transplants. Several EGFR agonists also stimulated Adam17(-/-) mammary organoid growth in culture, but only AREG was expressed abundantly in the developing ductal system in vivo. Thus, ADAM17 plays a crucial role in mammary morphogenesis by releasing AREG from mammary epithelial cells, thereby eliciting paracrine activation of stromal EGFR and reciprocal responses that regulate mammary epithelial development.     

A Lactobacillus rhamnosus GG-derived Soluble Protein,p40, Stimulates Ligand Release from Intestinal Epithelial Cells to Transactivate Epidermal Growth Factor Receptor

p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis, and preserves barrier function by transactivation of the EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study is to determine the mechanisms by which p40 transactivates the EGFR in intestinal epithelial cells. Here we show that p40-conditioned medium activates EGFR in young adult mouse colon epithelial cells and human colonic epithelial cell line, T84 cells. p40 up-regulates a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) catalytic activity, and broad spectrum metalloproteinase inhibitors block EGFR transactivation by p40 in these two cell lines. In ADAM17-deficient mouse colonic epithelial (ADAM17^−/− MCE) cells, p40 transactivation of EGFR is blocked, but can be rescued by re-expression with WT ADAM17. Furthermore, p40 stimulates release of heparin binding (HB)-EGF, but not transforming growth factor (TGF)α or amphiregulin, in young adult mouse colon cells and ADAM17^−/− MCE cells overexpressing WT ADAM17. Knockdown of HB-EGF expression by siRNA suppresses p40 effects on transactivating EGFR and Akt, preventing apoptosis, and preserving tight junction function. The effects of p40 on HB-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGFR activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion. Thus, these data define a mechanism of a probiotic-derived soluble protein in modulating intestinal epithelial cell homeostasis through ADAM17-mediated HB-EGF release, leading to transactivation of EGFR.     

Tobacco smoke-induced lung cell proliferation mediated by tumor necrosis factor alpha-converting enzyme and amphiregulin

Cells dividing at the time of carcinogen exposure are at particular risk for neoplasia. Tobacco smoke contains numerous carcinogens, and we find that smoke, in the absence of exogenous growth factors, is capable of stimulating cell proliferation. The smoke-triggered mechanism includes the generation of oxygen radicals, which in turn stimulate tumor necrosis factor alpha-converting enzyme (a disintegrin and metalloproteinase (ADAM) 17) to cleave transmembrane amphiregulin, a ligand for the epidermal growth factor receptor (EGFR). The binding of amphiregulin to EGFR then stimulates proliferation of lung epithelial cells. These results shed light on the pathogenesis of lung cancer, suggest novel drug targets for the reduction of cancer risk in smokers, and provide insight into how EGFR integrates responses to diverse noxious stimuli.     

Transcriptional mechanisms regulating alveolar epithelial cell-specific CCL5 secretion in pulmonary tuberculosis

CCL5 (or RANTES (regulated upon activation, normal T cell expressed and secreted)) recruits T lymphocytes and monocytes. The source and regulation of CCL5 in pulmonary tuberculosis are unclear. Infection of the human alveolar epithelial cell line (A549) by Mycobacterium tuberculosis caused no CCL5 secretion and little monocyte secretion. Conditioned medium from tuberculosis-infected human monocytes (CoMTB) stimulated significant CCL5 secretion from A549 cells and from primary alveolar, but not upper airway, epithelial cells. Differential responsiveness of small airway and normal human bronchial epithelial cells to CoMTB but not to conditioned medium from unstimulated human monocytes was specific to CCL5 and not to CXCL8. CoMTB induced CCL5 mRNA accumulation in A549 cells and induced nuclear translocation of nuclear factor kappaB (NFkappaB) subunits p50, p65, and c-rel at 1 h; nuclear binding of activator protein (AP)-1 (c-Fos, FosB, and c-Jun) at 4-8 h; and binding of NF-interleukin (IL)-6 at 24 h. CCL5 promoter-reporter analysis using deletion and site-specific mutagenesis constructs demonstrated a key role for AP-1, NF-IL-6, and NFkappaB in driving CoMTB-induced promoter activity. The IL-1 receptor antagonist inhibited A549 and small airway epithelial cell CCL5 secretion, gene expression, and promoter activity. CoMTB contained IL-1beta, and recombinant IL-1beta reproduced CoMTB effects. Monocyte alveolar, but not upper airway, epithelial cell networks in pulmonary tuberculosis cause AP-1-, NF-IL-6-, and NFkappaB-dependent CCL5 secretion. IL-1beta is the critical regulator of tuberculosis-stimulated CCL5 secretion in the lung.     

The role of the Th2 CC chemokine ligand CCL17 in pulmonary fibrosis

Increasing evidence suggests that the development of pulmonary fibrosis is a Th2-mediated process. We hypothesized that the CC chemokines that are associated with a Th2 profile (CCL17 and CCL22) have an important role in the development of pulmonary fibrosis. We measured CCL17 and CCL22 during the pathogenesis of bleomycin-induced pulmonary fibrosis. We found that both CCL17 and CCL22 were significantly elevated through day 20 as compared with control mice. Peak expression of CCL22 preceded the peak levels of CCL17, as measured by real-time quantitative PCR. CCR4 is the receptor for CCL17 and CCL22 therefore, to further characterize the role of CCL17 and CCL22, we measured CCR4 mRNA in lung tissue of bleomycin-treated mice by real-time quantitative PCR. CCR4 was significantly elevated in bleomycin-treated mice as compared with control mice. Immunolocalization demonstrated that CCR4 was expressed predominantly on macrophages. Neutralization of CCL17, but not CCL22, led to a reduction in pulmonary fibrosis. Immunolocalization of bleomycin-treated lung tissue and human idiopathic pulmonary fibrosis tissue specimens showed that epithelial cells expressed CCL17. These findings demonstrate a central role for Th2 chemokines and the macrophage in the pathogenesis of pulmonary fibrosis and are further support for the role of a Th2 phenotype in the pathogenesis of pulmonary fibrosis.     

Neuregulin-1-human epidermal receptor-2 signaling is a central regulator of pulmonary epithelial permeability and acute lung injury

The mechanisms behind the loss of epithelial barrier function leading to alveolar flooding in acute lung injury (ALI) are incompletely understood. We hypothesized that the tyrosine kinase receptor human epidermal growth factor receptor-2 (HER2) would be activated in an inflammatory setting and participate in ALI. Interleukin-1β (IL-1β) exposure resulted in HER2 activation in human epithelial cells and markedly increased conductance across a monolayer of airway epithelial cells. Upon HER2 blockade, conductance changes were significantly decreased. Mechanistic studies revealed that HER2 trans-activation by IL-1β required a disintegrin and metalloprotease 17 (ADAM17)-dependent shedding of the ligand neuregulin-1 (NRG-1). In murine models of ALI, NRG-1-HER2 signaling was activated, and ADAM17 blockade resulted in decreased NRG-1 shedding, HER2 activation, and lung injury in vivo. Finally, NRG-1 was detectable and elevated in pulmonary edema fluid from patients with ALI. These results suggest that the ADAM17-NRG-1-HER2 axis modulates the alveolar epithelial barrier and contributes to the pathophysiology of ALI.     

A disintegrin and metalloproteinase 17 (ADAM17) mediates epidermal growth factor receptor transactivation by angiotensin II on hepatic stellate cells

Aims

Epidermal growth factor receptor (EGFR) transactivation induced by angiotensin II (Ang II) participates in the progression of various diseases. A disintegrin and metalloproteinase 17 (ADAM17) is thought to promote renal fibrosis, cardiac hypertrophy with fibrosis and atherosclerosis by activation of the EGFR through secretion of EGFR ligands. The purpose of this study was to investigate whether Ang II-induced EGFR transactivation occurs on hepatic stellate cells (HSCs) and whether the reaction is mediated via ADAM17.

Main methods

Ang II-induced EGFR transactivation and cellular proliferation of the human HSC line LI90 were investigated using Western blotting and ATP assay, respectively. Ang II-induced secretion of mature amphiregulin into the cell culture medium was evaluated by enzyme-linked immunosorbent assay (ELISA).

Key findings

An inhibitor of ADAM17, TAPI-1, as well as antagonists of EGFR and angiotensin II type-1 receptor (AT1), attenuated Ang II-induced EGFR transactivation and proliferation of LI90 cells. Furthermore, silencing of ADAM17 inhibited Ang II-induced secretion of mature amphiregulin in addition to EGFR transactivation.

Significance

These results indicate that ADAM17 mediates Ang II-induced EGFR transactivation on HSCs, and that this process may participate in the progression of liver fibrosis.     

A Disintegrin and Metalloproteinase 17 (ADAM17) Mediates Inflammation-induced Shedding of Syndecan-1 and -4 by Lung Epithelial Cells

Syndecans are cell surface proteoglycans that bind and modulate various proinflammatory mediators and can be proteolytically shed from the cell surface. Within the lung, syndecan-1 and -4 are expressed as transmembrane proteins on epithelial cells and released in the bronchoalveolar fluid during inflammation. We here characterize the mechanism leading to the generation of soluble syndecan-1 and -4 in cultured epithelial cells and murine lung tissue. We show that the bladder carcinoma epithelial cell line ECV304, the lung epithelial cell line A459 and primary alveolar epithelial cells express and constitutively release syndecan-1 and -4. This release involves the activity of the disintegrin-like metalloproteinase ADAM17 as demonstrated by use of specific inhibitors and lentivirally transduced shRNA. Stimulation of epithelial cells with PMA, thrombin, or proinflammatory cytokines (TNFα/IFNγ) led to the down-regulation of surface-expressed syndecan-1 and -4, which was associated with a significant increase of soluble syndecans and cell-associated cleavage fragments. The enhanced syndecan release was not related to gene induction of syndecans or ADAM17, but rather due to increased ADAM17 activity. Soluble syndecan-1 and -4 were also released into the bronchoalveolar fluid of mice. Treatment with TNFα/IFNγ increased ADAM17 activity and syndecan release in murine lungs. Both constitutive and induced syndecan shedding was prevented by the ADAM17 inhibitor. ADAM17 may therefore be an important regulator of syndecan functions on inflamed lung epithelium.     

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.     

Lung cancer-derived galectin-1 enhances tumorigenic potentiation of tumor-associated dendritic cells by expressing heparin-binding EGF-like growth factor

The interaction between cancer cells and their microenvironment is a vicious cycle that enhances the survival and progression of cancer, resulting in metastasis. This study is the first to indicate that lung cancer-derived galectin-1 secretion is responsible for stimulating tumor-associated dendritic cells (TADCs) production of mature heparin-binding EGF-like growth factor (HB-EGF), which, in turn, increases cancer progression. Treatment of galectin-1, present in large amounts in lung cancer conditioned medium and lung cancer patient sera, mimicked the inductive effect of lung cancer conditioned medium on the expression and ectodomain shedding of HB-EGF by TNFα-converting enzyme/a disintegrin and metalloproteinase 9 (ADAM9) and ADAM17. Significant up-regulation of HB-EGF has been seen in tumor-infiltrating CD11c(+) dendritic cells in human lung cancer samples. Active cleavage of HB-EGF in TADCs by ADAM9 and ADAM17 is associated with increased protein kinase C δ and Lyn signaling. Enhancement of HB-EGF production in TADCs increased the proliferation, migration, and epithelial-to-mesenchymal transition abilities of lung cancer. In contrast, inhibiting HB-EGF by siRNA suppressed TADC-mediated cancer progression. Moreover, mice injected with galectin-1 knockdown Lewis lung carcinoma showed decreased expression and ectodomain shedding of HB-EGF and reduced incidence of cancer development, resulting in increased survival rates. We demonstrate here for the first time that human and mouse DCs are a source of HB-EGF, an EGFR ligand with tumorigenic properties. Antagonists of the effect of lung cancer-derived galectin-1 on DCs and anti-HB-EGF blocking antibodies could, therefore, have therapeutic potential as antitumor agents.     

Inhibitory monoclonal antibody targeting ADAM17 expressed on cancer cells

ADAM17 is upregulated in many cancers and in turn activates signaling pathways, including EGFR/ErbB, as well as those underlying resistance to targeted anti-EGFR therapies. Due to its central role in oncogenic pathways and drug resistance mechanisms, specific and efficacious monoclonal antibodies against ADAM17 could be useful for a broad patient population with solid tumors. Hence, we describe here an inhibitory anti-ADAM17 monoclonal antibody, named D8P1C1, that preferentially recognizes ADAM17 on cancer cells. D8P1C1 inhibits the catalytic activity of ADAM17 in a fluorescence-based peptide cleavage assay, as well as the proliferation of a range of cancer cell lines, including breast, ovarian, glioma, colon and the lung adenocarcinoma. In mouse models of triple-negative breast cancer and ovarian cancer, treatment with the mAb results in 78% and 45% tumor growth inhibition, respectively. Negative staining electron microscopy analysis of the ADAM17 ectodomain in complex with D8P1C1 reveals that the mAb binds the ADAM17 protease domain, consistent with its ability to inhibit the ADAM17 catalytic activity. Collectively, our results demonstrate the therapeutic potential of the D8P1C1 mAb to treat solid tumors.     

Polarization of ADAM17‐driven EGFR signalling in electric field‐guided collective migration of epidermal sheets

Endogenous electric field is considered to play an important role in promoting collective migration of epidermis to the wound centre. However, most studies are focused on the effect of bioelectric field on the movement and migration of single epithelial cell; the molecular mechanisms about collective migration of epidermal monolayers remain unclear. Here, we found that EFs dramatically promoted the collective migration of HaCaT cells towards the anode, activated the sheddase activity of ADAM17 and increased the phosphorylation level of EGFR. Moreover, EGFR phosphorylation and HB‐EGF shedding level were significantly decreased by the ADAM17 inhibitor TAPI‐2 or siADAM17 under EFs, which subsequently attenuated the directed migration of HaCaT sheets. Notably, the inhibition of EF‐regulated collective migration by siADAM17 was rescued by addition of recombinant HB‐EGF. Furthermore, we observed that F‐actin was dynamically polarized along the leading edge of the migrated sheets under EFs and that this polarization was regulated by ADAM17/HB‐EGF/EGFR signalling. In conclusion, our study indicated that ADAM17 contributed to the collective directional movement of the epidermal monolayer by driving HB‐EGF release and activating EGFR under EFs, and this pathway also mediated the polarization of F‐actin in migrating sheets, which is essential in directional migration.     

表皮生长因子受体与肺脏发育的关系

表皮生长因子受体(Epidermal growth factor receptor,EGFR)是一种跨膜蛋白受体,是ErbB家族成员之一,具有酪氨酸激酶活性。EGFR与相应的配体结合引起EGFR形成同源或异源二聚体启动胞内信号转导,激活下游多种信号转导途径,产生生物学效应,RAS/RAF/MEK/ERK通路与细胞增殖、分化和凋亡有关;PI3K/PDK1/AKT通路与细胞的迁移和粘附有关。EGFR能促进肺泡II型上皮细胞的成熟和肺表面活性物质的合成、分泌。EGFR对哺乳动物肺脏的作用呈现时空效应及剂量依赖效应,EGFR的下调表达则会引起肺脏发育不成熟;而EGFR过度表达促进肺肿瘤细胞的增殖、侵袭和转移。文章综述了EGFR及其调节信号通路的研究进展,以及EGFR与动物肺脏发育不成熟和肺癌之间的关系。     

CCL20/CCR6 feedback exaggerates epidermal growth factor receptor-dependent MUC5AC mucin production in human airway epithelial (NCI-H292) cells

Mucous hypersecretion is an important feature of obstructive airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Multiple stimuli induce mucin production via activation of an epidermal growth factor receptor (EGFR) cascade, but the mechanisms that exaggerate mucin production in obstructive airway diseases remain unknown. In this study, we show that binding of CCL20, a G protein-coupled receptor (GPCR) ligand that is upregulated in the airways of subjects with obstructive airway diseases, to its unique GPCR CCR6 induces MUC5AC mucin production in human airway epithelial (NCI-H292) cells via metalloprotease TNF-α-converting enzyme (TACE)-dependent EGFR activation. We also show that EGFR activation by its potent ligand TGF-α induces reactivation of EGFR via binding of endogenously produced CCL20 to its receptor CCR6 in NCI-H292 cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating mucin production in the NCI-H292 cells. In NCI-H292 cells, TGF-α stimulation induced two phases of EGFR phosphorylation (EGFR-P). The second EGFR-P was TACE-dependent and was responsible for most of the total mucin induced by TGF-α. Binding of endogenously produced CCL20 to CCR6 increased the second EGFR-P and subsequent mucin production induced by TGF-α. In NHBE cells, TGF-α-induced EGFR activation did not lead to significant CCL20 production or to EGFR rephosphorylation, and less mucin was produced. We conclude that NCI-H292 cells but not NHBE cells produce CCL20 in response to EGFR activation, which leads to a second phase of EGFR-P and subsequent exaggerated mucin production. These findings have potentially important therapeutic implications in obstructive airway diseases.