Amphiregulin exosomes increase cancer cell invasion

Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands including EGF, TGF-α (TGFα), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin, and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells 4-fold over TGFα or HB-EGF exosomes and 5-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGFα or HB-EGF. An average of 24?AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between nontransformed and transformed cells is unknown. Exosomes from DLD-1?colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, nontransformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes might contribute to diverse cancer phenomena such as field effect and priming of the metastatic niche.     

A Potent Anti-HB-EGF Monoclonal Antibody Inhibits Cancer Cell Proliferation and Multiple Angiogenic Activities of HB-EGF

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathological functions. Modulation of HB-EGF activity might have a therapeutic potential in the oncology area. We explored the therapeutic possibilities by characterizing the in vitro biological activity of anti-HB-EGF monoclonal antibody Y-142. EGF receptor (EGFR) ligand and species specificities of Y-142 were tested. Neutralizing activities of Y-142 against HB-EGF were evaluated in EGFR and ERBB4 signaling. Biological activities of Y-142 were assessed in cancer cell proliferation and angiogenesis assays and compared with the anti-EGFR antibody cetuximab, the HB-EGF inhibitor CRM197, and the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab. The binding epitope was determined with alanine scanning. Y-142 recognized HB-EGF as well as the EGFR ligand amphiregulin, and bound specifically to human HB-EGF, but not to rodent HB-EGF. In addition, Y-142 neutralized HB-EGF-induced phosphorylation of EGFR and ERBB4, and blocked their downstream ERK1/2 and AKT signaling. We also found that Y-142 inhibited HB-EGF-induced cancer cell proliferation, endothelial cell proliferation, tube formation, and VEGF production more effectively than cetuximab and CRM197 and that Y-142 was superior to bevacizumab in the inhibition of HB-EGF-induced tube formation. Six amino acids in the EGF-like domain were identified as the Y-142 binding epitope. Among the six amino acids, the combination of F115 and Y123 determined the amphiregulin cross-reactivity and that F115 accounted for the species selectivity. Furthermore, it was suggested that the potent neutralizing activity of Y-142 was derived from its recognition of R142 and Y123 and its high affinity to HB-EGF. Y-142 has a potent HB-EGF neutralizing activity that modulates multiple biological activities of HB-EGF including cancer cell proliferation and angiogenic activities. Y-142 may have a potential to be developed into a therapeutic agent for the treatment of HB-EGF-dependent cancers.     

Epidermal growth factor receptor pathway analysis identifies amphiregulin as a key factor for cisplatin resistance of human breast cancer cells

The use of platinum complexes for the therapy of breast cancer is an emerging new treatment modality. To gain insight into the mechanisms underlying cisplatin resistance in breast cancer, we used estrogen receptor-positive MCF-7 cells as a model system. We generated cisplatin-resistant MCF-7 cells and determined the functional status of epidermal growth factor receptor (EGFR), MAPK, and AKT signaling pathways by phosphoreceptor tyrosine kinase and phospho-MAPK arrays. The cisplatin-resistant MCF-7 cells are characterized by increased EGFR phosphorylation, high levels of AKT1 kinase activity, and ERK1 phosphorylation. In contrast, the JNK and p38 MAPK modules of the MAPK signaling pathway were inactive. These conditions were associated with inactivation of the p53 pathway and increased BCL-2 expression. We investigated the expression of genes encoding the ligands for the ERBB signaling cascade and found a selective up-regulation of amphiregulin expression, which occurred at later stages of cisplatin resistance development. Amphiregulin is a specific ligand of the EGFR (ERBB1) and a potent mitogen for epithelial cells. After exposure to cisplatin, the resistant MCF-7 cells secreted amphiregulin protein over extended periods of time, and knockdown of amphiregulin expression by specific short interfering RNA resulted in a nearly complete reversion of the resistant phenotype. To demonstrate the generality and importance of our findings, we examined amphiregulin expression and cisplatin resistance in a variety of human breast cancer cell lines and found a highly significant correlation. In contrast, amphiregulin levels did not significantly correlate with cisplatin resistance in a panel of lung cancer cell lines. We have thus identified a novel function of amphiregulin for cisplatin resistance in human breast cancer cells.     

Amphiregulin and hepatocyte-derived extracellular matrix regulate proliferation and autocrine growth factor expression in colon cancer cell lines of varying liver-colonizing capability

We studied the effect of two members of the epidermal growth factor (EGF) family--amphiregulin and heparin-binding EGF-like growth factor (HB-EGF)-on cell proliferation, growth factor and growth factor receptor expression, and cell differentiation in two human colon cell lines of varying liver-colonizing potential. The effect of amphiregulin and HB-EGF was assessed both in cells grown on plastic, as well as on cells grown on hepatocyte-derived extracellular matrix (ECM). We found that both colon cell lines were sensitive to HB-EGF stimulation of cell proliferation. Amphiregulin inhibited cell proliferation in KM12 cells and stimulated the strongly metastatic cell line KM12SM to a slight extent. When the cells were cultured on hepatocyte-derived ECM, amphiregulin inhibited the weakly metastatic KM12 and stimulated the growth of KM12SM. HB-EGF synergistically acted with hepatocyte-derived ECM to enhance cell proliferation in both colon cell lines. Expression of ligands of the EGF family, such as transforming growth factor-alpha (TGF-alpha) and amphiregulin, was decreased in both cell lines when cultured on ECM. Hepatocyte-derived ECM decreased expression of cripto in KM12 and increased it in KM12SM cells. Neither cripto nor TGF-alpha mRNA levels was affected by growing the cells in the presence of amphiregulin. However, amphiregulin increased expression of its own mRNA in the weakly metastatic KM12 and decreased it in the strongly metastatic KM12SM when the cells were cultured on plastic. Amphiregulin and HB-EGF stimulated expression of erb-B2 in both cell lines cultured on plastic. Surprisingly, when the cells were grown on hepatocyte-derived ECM, amphiregulin inhibited erb-B2 expression in both cell lines. We observed no effect of amphiregulin on cell differentiation as assessed by alkaline phosphatase expression. Our studies demonstrate one mechanism that could play a role in site-specific metastasis. We found an inhibitory response to an autocrine growth factor in the context of hepatocyte-derived ECM in a weakly metastatic cell and a stimulatory effect of the same growth factor when strongly metastatic cells were cultured on the same ECM.     

Ventilation-Induced Increases in EGFR Ligand mRNA Are Not Altered by Intra-Amniotic LPS or Ureaplasma in Preterm Lambs

Chorioamnionitis and mechanical ventilation are associated with bronchopulmonary dysplasia (BPD) in preterm infants. Mechanical ventilation at birth activates both inflammatory and acute phase responses. These responses can be partially modulated by previous exposure to intra-amniotic (IA) LPS or Ureaplasma parvum (UP). Epidermal growth factor receptor (EGFR) ligands participate in lung development, and angiotensin converting enzyme (ACE) 1 and ACE2 contribute to lung inflammation. We asked whether brief mechanical ventilation at birth altered EGFR and ACE pathways and if antenatal exposure to IA LPS or UP could modulate these effects. Ewes were exposed to IA injections of UP, LPS or saline multiple days prior to preterm delivery at 85% gestation. Lambs were either immediately euthanized or mechanically ventilated for 2 to 3 hr. IA UP and LPS cause modest changes in the EGFR ligands amphiregulin (AREG), epiregulin (EREG), heparin binding epidermal growth factor (HB-EGF), and betacellulin (BTC) mRNA expression. Mechanical ventilation greatly increased mRNA expression of AREG, EREG, and HB-EGF, with no additional increases resulting from IA LPS or UP. With ventilation AREG and EREG mRNA localized to cells in terminal airspace. EGFR mRNA also increased with mechanical ventilation. IA UP and LPS decreased ACE1 mRNA and increased ACE2 mRNA, resulting in a 4 fold change in the ACE1/ACE2 ratio. Mechanical ventilation with large tidal volumes increased both ACE1 and ACE2 expression. The alterations seen in ACE with IA exposures and EGFR pathways with mechanical ventilation may contribute to the development of BPD in preterm infants.     

Induction of heparin-binding EGF-like growth factor and activation of EGF receptor in imatinib mesylate-treated squamous carcinoma cells

Imatinib mesylate is a tyrosine kinase inhibitor of the ABL, platelet-derived growth factor receptor (PDGFR), and c-kit kinases. Inhibition of BCR-ABL and c-kit accounts for its clinical activity in leukemia and sarcoma, respectively. In this report, we describe other cellular targets for imatinib. Treatment of head and neck squamous carcinoma cells with clinically relevant concentrations of imatinib-induced changes in cell morphology and growth similar to changes associated with epidermal growth factor receptor (EGFR) activation. Imatinib-induced changes were blocked with the EGFR antagonist cetuximab, which suggested direct involvement of EGFR in this process. Western blot analysis of cells incubated with imatinib demonstrated activation of EGFR and downstream signaling that was reduced by inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1) and EGFR, but not Her2/ErbB2. An in vitro kinase assay showed that imatinib did not directly affect EGFR kinase activity, suggesting involvement of EGFR-activating molecules. Inhibitors and neutralizing antibodies against heparin-binding epidermal growth factor-like growth factor (HB-EGF), and to a lesser extent transforming growth factor-alpha, reduced imatinib-mediated mitogen activated protein kinase (MAPK) activation. Imatinib stimulated the rapid release of soluble HB-EGF and the subsequent induction of membrane-bound HB-EGF, which correlated with biphasic MAPK activation. Together, these results suggested that imatinib affects EGFR activation and signaling pathways through rapid release and increased expression of endogenous EGFR-activating ligands. Although, imatinib primarily inhibits tyrosine kinases, it also stimulates the activity of EGFR tyrosine kinase in head and neck squamous tumors. This finding demonstrates the need for careful use of this drug in cancer patients.     

5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-alpha-converting enzyme (TACE) activation and heparin-bound epidermal growth factor-like growth factor (HB-EGF) shedding in mesangial cells

In this study, we present multiple lines of evidence to support a critical role for heparin-bound EGF (epidermal growth factor)-like growth factor (HB-EGF) and tumor necrosis factor-alpha-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT(2A) receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells. ERK phosphorylation was attenuated by 1) neutralizing EGFR antibodies and the EGFR kinase inhibitor, AG1478, 2) neutralizing HB-EGF, but not amphiregulin, antibodies, heparin, or CM197, and 3) pharmacological inhibitors of matrix-degrading metalloproteinases or TACE small interfering RNA. Exogenously administered HB-EGF stimulated ERK phosphorylation. Additionally, TACE was co-immunoprecipitated with HB-EGF. Small interfering RNA against TACE also blocked 5-HT-induced increases in ERK phosphorylation, HB-EGF shedding, and DNA content. In aggregate, this work supports a pathway map that can be depicted as follows: 5-HT --> 5-HT(2A) receptor --> TACE --> HB-EGF shedding --> EGFR --> ERK --> increased DNA content. To our knowledge, this is the first time that TACE has been implicated in 5-HT-induced EGFR transactivation or in proliferation induced by a G protein-coupled receptor in native cells in culture.     

Juxtacrine activation of epidermal growth factor (EGF) receptor by membrane-anchored heparin-binding EGF-like growth factor protects epithelial cells from anoikis while maintaining an epithelial phenotype

Loss of cell-matrix adhesion is often associated with acute epithelial injury, suggesting that "anoikis" may be an important contributor to cell death. Resistance against anoikis is a key characteristic of transformed cells. When nontransformed epithelia are injured, activation of the epidermal growth factor (EGF) receptor (EGFR) by paracrine/autocrine release of soluble ligands can induce a prosurvival program, but there is generally evidence for concomitant dedifferentiation. The EGFR ligand, heparin-binding EGF-like growth factor (HB-EGF), is synthesized as a membrane-anchored precursor that can activate the EGFR via juxtacrine signaling or can be released and act as a soluble growth factor. In Madin-Darby canine kidney cells, expression of membrane-anchored HB-EGF increases cell-cell and cell-matrix adhesion. Therefore, these studies were designed to test the effects of juxtacrine HB-EGF signaling upon cell survival and epithelial integrity when cells are denied proper cell-matrix interactions. Cells expressing a noncleavable mutated form of membrane-anchored HB-EGF demonstrated increased survival from anoikis, formed larger cell aggregates, and maintained epithelial characteristics even following prolonged detachment from the substratum. Physical association between membrane-anchored HB-EGF and EGFR was observed. Signaling studies indicated synergistic effects of EGFR activation and phosphatidylinositol 3-kinase signaling to regulate apoptotic and survival pathways. In contrast, although administration of exogenous EGF partially suppressed anoikis in wild type cells, it also led to an increased expression of mesenchymal markers, suggesting dedifferentiation. Taken together, we propose a novel role for membrane-anchored HB-EGF in the cytoprotection of epithelial cells.     

Histamine stimulation of MMP-1(collagenase-1) secretion and gene expression in gastric epithelial cells: role of EGFR transactivation and the MAP kinase pathway

BACKGROUND AND AIMS: GPCR stimulation by various ligands including histamine has been shown to transactivate the epidermal growth factor receptor (EGFR). This study examines the functional interactions between the H2 receptor and the EGFR in the regulation of matrix metalloproteinase-1 (MMP-1) secretion and gene expressions in cultured gastric epithelial cells. METHODS: AGS cells were incubated for up to 24 h with either histamine or heparin binding-epidermal growth factor (HB-EGF) and MMP-1 release was determined by immunoassay. MMP-1 responses to histamine and HB-EGF were further tested by the use of H2 receptor antagonist, EGFR inhibitor and mitogen activator protein kinase (MAPK) inhibitor. The role of EGFR in MMP-1 release was further tested in cells transfected with specific EGFR siRNA. EGFR and ERK1/2 phosphorylation was determined by Western blot analysis. MMP-1 gene expression was determined by RNase protection assay (RPA). RESULTS: Histamine and HB-EGF caused a dose-dependent release of MMP-1 with maximal responses that were 2.7- and 4.5-fold greater, respectively, than control, P<0.001. Famotidine prevented histamine-mediated MMP-1 release and AG1478 and EGFR siRNA completely inhibited MMP-1 secretion stimulated by both histamine and HB-EGF. Both histamine and HB-EGF stimulation of MMP-1 release was associated with activation of ERK1/2. MAPK inhibition also prevented histamine-and HB-EGF-induced MMP-1 secretion. Results of MMP-1 gene expression, either stimulatory or inhibitory, paralleled responses to MMP-1 secretion. CONCLUSION: Histamine stimulation of the H2 receptor on AGS cells evoked MMP-1 secretion and gene up regulation that was dependent on transactivation of the EGFR and downstream activation of MAPK.     

IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage proHB-EGF in human colon carcinoma cells

Interleukin-8 (IL-8) has been reported to promote tumor cell growth in colon cancer cells after binding to its receptors, which are members of the G-protein coupled receptor (GPCR) family. Recent studies demonstrated that stimulation of GPCR can induce shedding of epidermal growth factor (EGF) ligands via activation of a disintegrin and metalloprotease (ADAM), with subsequent transactivation of the EGF receptor (EGFR). In this study, we investigated mechanisms of cell proliferation and migration stimulated by IL-8 in a human colon carcinoma cell line (Caco2). IL-8 increased DNA synthesis of Caco2 in a dose dependent manner and this was inhibited by ADAM, EGFR kinase, and MEK inhibitors. IL-8 transiently induced EGFR tyrosine phosphorylation after 5-90 min and this was completely inhibited by ADAM inhibitor. Neutralizing antibody against HB-EGF as a key ligand for EGFR also blocked transactivation of EGFR and cell proliferation by IL-8. Since IL-8-induced cell migration was further suppressed by the ADAM inhibitor and the HB-EGF neutralizing antibody, our data indicate that IL-8 induces cell proliferation and migration by an ADAM-dependent pathway, and that HB-EGF plays an important role as the major ligand for this pathway.     

Amphiregulin regulates the production of human chorionic gonadotropin in trophoblasts

AimsThe aim of this study was to investigate the significance of epidermal growth factor receptor (EGFR) ligands produced in syncytiotrophoblasts during normal pregnancy.Main methodsWe examined the expression of EGFR ligands in human pregnancy by real-time PCR, and analyzed the relationship between EGFR ligands and human chorionic gonadotropin (hCG) or human placental lactogen in amniotic fluid by ELISA. In addition, we also examined the EGFR ligands in syncytiotrophoblasts and the amount of hCG secretion in JAR, JEG3 and BeWo cells in the presence of each EGFR ligand.Key findingsIn order to identify possible candidates among the EGFR ligands, we examined the predominant expression of an EGFR ligand in the chorionic villi and amniotic fluid during normal pregnancy, and analyzed the relationship between EGFR ligands and hCG in trophoblastic model cells. Amphiregulin was primarily expressed throughout human pregnancy and stimulated the secretion of hCG, indicating that amphiregulin is a key molecule among EGFR ligands.SignificanceAmphiregulin may play a pivotal role in the development or maturation of placenta.     

Involvement of TNF-α Converting Enzyme in the Development of Psoriasis-Like Lesions in a Mouse Model

TNF-α plays a crucial role in psoriasis; therefore, TNF inhibition has become a gold standard for the treatment of psoriasis. TNF-α is processed from a membrane-bound form by TNF-α converting enzyme (TACE) to soluble form, which exerts a number of biological activities. EGF receptor (EGFR) ligands, including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin and transforming growth factor (TGF)-α are also TACE substrates and are psoriasis-associated growth factors. Vascular endothelial growth factor (VEGF), one of the downstream molecules of EGFR and TNF signaling, plays a key role in angiogenesis for developing psoriasis. In the present study, to assess the possible role of TACE in the pathogenesis of psoriasis, we investigated the involvement of TACE in TPA-induced psoriasis-like lesions in K5.Stat3C mice, which represent a mouse model of psoriasis. In this mouse model, TNF-α, amphiregulin, HB-EGF and TGF-α were significantly up-regulated in the skin lesions, similar to human psoriasis. Treatment of K5.Stat3C mice with TNF-α or EGFR inhibitors attenuated the skin lesions, suggesting the roles of TACE substrates in psoriasis. Furthermore, the skin lesions of K5.Stat3C mice showed down-regulation of tissue inhibitor of metalloproteinase-3, an endogenous inhibitor of TACE, and an increase in soluble TNF-α. A TACE inhibitor abrogated EGFR ligand-dependent keratinocyte proliferation and VEGF production in vitro, suggesting that TACE was involved in both epidermal hyperplasia and angiogenesis during psoriasis development. These results strongly suggest that TACE contributes to the development of psoriatic lesions through releasing two kinds of psoriasis mediators, TNF-α and EGFR ligands. Therefore, TACE could be a potential therapeutic target for the treatment of psoriasis.     

Decreased autocrine EGFR signaling in metastatic breast cancer cells inhibits tumor growth in bone and mammary fat pad

Breast cancer metastasis to bone triggers a vicious cycle of tumor growth linked to osteolysis. Breast cancer cells and osteoblasts express the epidermal growth factor receptor (EGFR) and produce ErbB family ligands, suggesting participation of these growth factors in autocrine and paracrine signaling within the bone microenvironment. EGFR ligand expression was profiled in the bone metastatic MDA-MB-231 cells (MDA-231), and agonist-induced signaling was examined in both breast cancer and osteoblast-like cells. Both paracrine and autocrine EGFR signaling were inhibited with a neutralizing amphiregulin antibody, PAR34, whereas shRNA to the EGFR was used to specifically block autocrine signaling in MDA-231 cells. The impact of these was evaluated with proliferation, migration and gene expression assays. Breast cancer metastasis to bone was modeled in female athymic nude mice with intratibial inoculation of MDA-231 cells, and cancer cell-bone marrow co-cultures. EGFR knockdown, but not PAR34 treatment, decreased osteoclasts formed in vitro (p<0.01), reduced osteolytic lesion tumor volume (p<0.01), increased survivorship in vivo (p<0.001), and resulted in decreased MDA-231 growth in the fat pad (p<0.01). Fat pad shEGFR-MDA-231 tumors produced in nude mice had increased necrotic areas and decreased CD31-positive vasculature. shEGFR-MDA-231 cells also produced decreased levels of the proangiogenic molecules macrophage colony stimulating factor-1 (MCSF-1) and matrix metalloproteinase 9 (MMP9), both of which were decreased by EGFR inhibitors in a panel of EGFR-positive breast cancer cells. Thus, inhibiting autocrine EGFR signaling in breast cancer cells may provide a means for reducing paracrine factor production that facilitates microenvironment support in the bone and mammary gland.     

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.     

Upregulation of endogenous heparin-binding EGF-like growth factor and its role as a survival factor in skeletal myotubes.

To investigate the role of heparin-binding EGF-like growth factor (HB-EGF) in skeletal muscle, we studied its function in skeletal myotubes in vitro using mouse C2C12 cells. Expression levels of membrane-anchored HB-EGF (proHB-EGF) protein were increased specifically during their differentiation among epidermal growth factor receptor (EGFR) ligands. Production levels of EGFR on the cell surface were constant. Tyrosine phosphorylation of EGFR, however, was constitutively increased during differentiation. Quenching of endogenous HB-EGF significantly rendered myotubes sensitive to apoptotic cell death induced by hypoxic stress, suggesting that proHB-EGF in the skeletal muscle is specifically upregulated to function as a survival factor.     

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.     

ADAM-mediated ectodomain shedding of HB-EGF in receptor cross-talk

All ligands of the epidermal growth factor receptor (EGFR) which has important roles in development and disease, are shed from the plasma membrane by metalloproteases. The ectodomain shedding of EGFR ligands has emerged as a critical component in the functional activation of EGFR in the interreceptor cross-talk. Identification of the sheddases for EGFR ligands using mouse embryonic cells lacking candidate sheddases (a disintegrin and metalloprotease; ADAM) has revealed that ADAM10, -12 and -17 are the sheddases of the EGFR ligands in response to various shedding stimulants such as GPCR agonists, growth factors, cytokines, osmotic stress, wounding and phorbol ester. Among the EGFR ligands, heparin-binding EGF-like growth factor (HB-EGF) is a representative ligand to understand the pathophysiological roles of the ectodomain shedding in wound healing, cardiac diseases, etc. Here we focus on the ectodomain shedding of HB-EGF by ADAMs, which is not only a key event of receptor cross-talk but also a novel intercellular signaling by the carboxy-terminal fragment (CTF signal).