The ADAM17–amphiregulin–EGFR Axis in Mammary Development and Cancer

In order to fulfill its function of producing and delivering sufficient milk to newborn mammalian offspring, the mammary gland first has to form an extensive ductal network. As in all phases of mammary development, hormonal cues elicit local intra- and inter-cellular signaling cascades that regulate ductal growth and differentiation. Among other things, ductal development requires the epidermal growth factor receptor (EGFR), its ligand amphiregulin (AREG), and the transmembrane metalloproteinase AD-AM17, which can cleave and release AREG from the cell surface so that it may interact with its receptor. Tissue recombination and transplantation studies demonstrate that EGFR phosphorylation and ductal development proceed only when ADAM17 and AREG are expressed on mammary epithelial cells and EGFR is present on stromal cells, and that local administration of soluble AREG can rescue the development of ADAM17-deficient transplants. Thus proper mammary morphogenesis requires the ADAM17-mediated release of AREG from ductal epithelial cells, the subsequent activation of EGFR on stromal cells, and EGFR-dependent stromal responses that in return elicit a new set of epithelial responses, all culminating in the formation of a fully functional ductal tree. This, however, raises new issues concerning what may act upstream, downstream or in parallel with the ADAM17–AREG–EGFR axis, how it may become hijacked or corrupted during the onset and evolution of cancer, and how such ill effects may be confronted

Selective Roles for Tumor Necrosis Factor α-converting Enzyme/ADAM17 in the Shedding of the Epidermal Growth Factor Receptor Ligand Family: THE JUXTAMEMBRANE STALK DETERMINES CLEAVAGE EFFICIENCY

Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors. Previously, we established that tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) is a physiologic transforming growth factor-alpha (TGF-alpha) sheddase, and we also demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACE-deficient EC-2 fibroblasts. Here we extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse pro-HB-EGF and pro-AR ectodomains in vitro. For pro-HB-EGF, this site matched the C terminus of the purified human growth factor, and we speculate that the AR cleavage site is also physiologically relevant. In contrast, ADAM9 and -10, both implicated in HB-EGF shedding, failed to cleave the ectodomain or cleaved at a nonphysiologic site, respectively. Cotransfection of TACE in EC-2 cells enhanced phorbol myristate acetate-induced but not constitutive shedding of epiregulin and had no effect on betacellulin (BTC) processing. Additionally, soluble TACE did not cleave the juxtamembrane stalks of either pro-BTC or pro-epiregulin ectodomains in vitro. Substitution of the shorter pro-BTC juxtamembrane stalk or truncation of the pro-TGF-alpha stalk to match the pro-BTC length reduced TGF-alpha shedding from transfected cells to background levels, whereas substitution of the pro-BTC P2-P2' sequence reduced TGF-alpha shedding less dramatically. Conversely, substitution of the pro-TGF-alpha stalk or lengthening of the pro-BTC stalk, especially when combined with substitution of the pro-TGF-alpha P2-P2' sequence, markedly increased BTC shedding. These results indicate that efficient TACE cleavage is determined by a combination of stalk length and scissile bond sequence

TNF-α–Converting Enzyme/A Disintegrin and Metalloprotease−17 Mediates Mechanotransduction in Murine Tracheal Epithelial Cells

Bronchoconstriction applies compressive stress to airway epithelial cells. We show that the application of compressive stress to cultured murine tracheal epithelial cells elicits the increased phosphorylation of extracellular signal–regulated kinase (ERK) and Akt through an epidermal growth factor receptor (EGFR)–dependent process, consistent with previous observations of the bronchoconstriction-induced activation of EGFR in both human and murine airways. Mechanotransduction requires metalloprotease activity, indicating a pivotal role for proteolytic EGF-family ligand shedding. However, cells derived from mice with targeted deletions of the EGFR ligands Tgfα and Hb-egf showed only modest decreases in responses, even when combined with neutralizing antibodies to the EGFR ligands epiregulin and amphiregulin, suggesting redundant or compensatory roles for individual EGF family members in mechanotransduction. In contrast, cells harvested from mice with a conditional deletion of the gene encoding the TNF-α–converting enzyme (TACE/ADAM17), a sheddase for multiple EGF-family proligands, displayed a near-complete attenuation of ERK and Akt phosphorylation responses and compressive stress–induced gene regulation. Our data provide strong evidence that TACE plays a critical central role in the transduction of compressive stress

Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling

Heparin-binding epidermal growth factor (HB-EGF) and betacellulin (BTC) are activating ligands for EGF receptor (EGFR/ErbB1) and ErbB4. To identify their physiological functions, we disrupted mouse HB-EGF and BTC alleles by homologous recombination. Most HB-EGF(–/–) mice died before weaning, and survivors had enlarged, dysfunctional hearts and reduced lifespans. Although BTC(–/–) mice were viable and fertile and displayed no overt defects, the lifespan of double null HB-EGF(–/–)/BTC(–/–) mice was further reduced, apparently due to accelerated heart failure. HB-EGF(–/–) newborns had enlarged and malformed semilunar and atrioventricular heart valves, and hypoplastic, poorly differentiated lungs. Defective cardiac valvulogenesis was the result of abnormal mesenchymal cell proliferation during remodeling, and was associated with dramatic increases in activated Smad1/5/8. Consistent with the phenotype, HB-EGF transcripts were localized to endocardial cells lining the margins of wild-type valves. Similarly defective valvulogenesis was observed in newborn mice lacking EGFR and tumor necrosis factor-α converting enzyme (TACE). These results suggest that cardiac valvulogenesis is dependent on EGFR activation by TACE-derived soluble HB-EGF, and that EGFR signaling is required to regulate bone morphogenetic protein signaling in this context

Angiotensin II and EGF receptor cross-talk in chronic kidney diseases: a new therapeutic approach. Nat Med 11

Mechanisms of progression of chronic renal diseases, a major healthcare burden, are poorly understood. Angiotensin II (AngII), the major renin-angiotensin system effector, is known to be involved in renal deterioration, but the molecular pathways are still unknown. Here, we show that mice overexpressing a dominant negative isoform of epidermal growth factor receptor (EGFR) were protected from renal lesions during chronic AngII infusion. Transforming growth factor-α (TGF-α) and its sheddase, TACE (also known as ADAM17), were induced by AngII treatment, TACE was redistributed to apical membranes and EGFR was phosphorylated. AngII-induced lesions were substantially reduced in mice lacking TGF-α or in mice given a specific TACE inhibitor. Pharmacologic inhibition of AngII prevented TGF-α and TACE accumulation as well as renal lesions after nephron reduction. These findings indicate a crucial role for AngII-dependent EGFR transactivation in renal deterioration and identify in TACE inhibitors a new therapeutic strategy for preventing progression of chronic renal diseases. Regardless of etiology, most human kidney diseases are characterized by an initial injury, followed by progression of renal lesions to complete parenchymal destruction and end-stage renal failure 1 . Clinical and experimental studies have shown that angiotensin II (AngII), the major renin-angiotensin system effector, has an important role in the biological process leading to renal deterioration. Indeed, pharmacological inhibition of the renin-angiotension system attenuates development of renal lesions in several experimental models of renal injury 2 and retards progressive loss of renal function in individuals with chronic kidney disease (CKD) 3 . Conversely, individuals with genetic variants associated with higher renin-angiotensin system activity are at increased risk for progression of chronic renal failure 4 . It has been suggested that AngII causes renal injury through renal hemodynamic effects and stimulation of kidney growth and matrix deposition 5 , but the molecular pathways underlying these phenomena remain largely unidentified. AngII acts on at least two structurally and pharmacologically distinct G-protein-coupled receptors (GPCRs), AT 1 and AT 2 (ref. 6). Renal cells predominantly express AT 1 receptors, which mediate the majority of known AngII actions 7 . AT 1 receptors activate Gq-phospholipase C to generate inositol triphosphate and diacylglycerol, thereby increasing intracellular calcium and stimulating protein kinase C 8 . Additionally, activation of AT 1 receptors promotes tyrosine phosphorylation and stimulates mitogen-activated protein kinases and proliferation 9,10 . How AT 1 receptors, which lack intrinsic tyrosine kinase activity, induce these events is unclear, but recent evidence suggests that 'transactivation' of the epidermal growth factor receptor (EGFR) is involved 11 , and may require several intermediary signaling molecules including Ca 2+ , protein kinase C and cytosolic tyrosine kinases 9 . Recently, it has been shown that metalloprotease-dependent release of EGFR ligands from cells is also involved in GCPR-induced EGFR transactivation 12 . Whether and by which molecular mechanisms AngII transactivates EGFR in renal cells during kidney diseases is unknown. EGFR binds members of a family of growth factors, comprised of EGF, transforming growth factor-α (TGF-α), heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin and epigen 13 . All family members are synthesized as membrane-anchored precursors that can be processed by specific metalloproteases to release soluble bioactive factors from the cell surface 13 . In the kidney, both EGFR and its ligands are abundantly expressed along the nephron, suggesting a paracrine-autocrine syste

Amphiregulin is Not Essential for Induction of Contact Hypersensitivity

ABSTRACT: Background: Amphiregulin (AR) is expressed in Th2 cells, rather than Th1 cells, and plays an important role in Th2 cell/cytokine-mediated host defense against nematodes. We also found earlier that AR mRNA expression was strongly upregulated in inflamed tissue during Th2 cell/cytokine-mediated fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS), suggesting a contribution of AR to the induction of those responses. Methods: To elucidate the role of AR in the induction of FITC- or dinitrofluorobenzene (DNFB)-induced CHS, AR-deficient mice were sensitized and/or challenged with FITC or DNFB epicutaneously. The levels of FITC- mediated skin dendritic cell (DC) migration and FITC-specific lymph node cell proliferation and cytokine production were assessed by flow cytometry, [3H]-thymidine incorporation and ELISA, respectively, after FITC sensitization. The degree of ear swelling, the activities of myeloperoxidase (MPO) and eosinophil peroxidase (EPO) in inflammatory sites and the levels of FITC-specific immunoglobulin (Ig) in sera were determined by histological analysis, colorimetric assay and ELISA, respectively, after FITC challenge. Results: DC migration and FITC-specific lymph node cell proliferation and cytokine production were normal in the AR-deficient mice. Ear swelling, tissue MPO and EPO activities and FITC-specific serum Ig levels were also similar in AR-deficient and -sufficient mice. Conclusions: Amphiregulin is not essential for the induction of FITC- or DNFB-induced CHS responses in mice. KEY WORDS: amphiregulin, contact dermatitis, EGF, mast cells, Th2 cell/cytokin

Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis

Rapidly progressive glomerulonephritis (RPGN) is a life-threatening clinical syndrome and a morphological manifestation of severe glomerular injury that is marked by a proliferative histological pattern ('crescents') with accumulation of T cells and macrophages and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the epidermal growth factor receptor (EGFR, also known as ErbB1) in mice with RPGN. In HB-EGF-deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 d after the induction of experimental RPGN. This suggests that targeting the HB-EGF-EGFR pathway could also be beneficial in treatment of human RPGN

Timp3 deficiency in insulin receptor–haploinsufficient mice promotes diabetes and vascular inflammation via increased TNF-α

Activation of inflammatory pathways may contribute to the beginning and the progression of both atherosclerosis and type 2 diabetes. Here we report a novel interaction between insulin action and control of inflammation, resulting in glucose intolerance and vascular inflammation and amenable to therapeutic modulation. In insulin receptor heterozygous (Insr(+/–)) mice, we identified the deficiency of tissue inhibitor of metalloproteinase 3 (Timp3, an inhibitor of both TNF-α–converting enzyme [TACE] and MMPs) as a common bond between glucose intolerance and vascular inflammation. Among Insr(+/–) mice, those that develop diabetes have reduced Timp3 and increased TACE activity. Unchecked TACE activity causes an increase in levels of soluble TNF-α, which subsequently promotes diabetes and vascular inflammation. Double heterozygous Insr(+/–)Timp3(+/–) mice develop mild hyperglycemia and hyperinsulinemia at 3 months and overt glucose intolerance and hyperinsulinemia at 6 months. A therapeutic role for Timp3/TACE modulation is supported by the observation that pharmacological inhibition of TACE led to marked reduction of hyperglycemia and vascular inflammation in Insr(+/–) diabetic mice, as well as by the observation of increased insulin sensitivity in Tace(+/–) mice compared with WT mice. Our results suggest that an interplay between reduced insulin action and unchecked TACE activity promotes diabetes and vascular inflammation