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

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-alpha (TGF-alpha) 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-alpha or in mice given a specific TACE inhibitor. Pharmacologic inhibition of AngII prevented TGF-alpha 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.

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.

Heparin binding epidermal growth factor in renal ischaemia/reperfusion injury.

The epidermal growth factor (EGF) receptor and its ligands are crucially involved in the renal response to ischaemia. We studied the heparin binding-epidermal growth factor (HB-EGF), a major ligand for the EGF receptor, in experimental and human ischaemia/reperfusion injury (IRI). HB-EGF mRNA and protein expression was studied in rat kidneys and cultured human tubular (HK-2) cells that were subjected to IRI and in human donor kidneys during transplantation. The effect of EGF receptor inhibition was investigated in vivo and in vitro. Furthermore, urinary HB-EGF protein excretion was studied after renal transplantation. Finally, HB-EGF KO and WT mice were subjected to IRI to study the role of HB-EGF in renal injury. HB-EGF mRNA was significantly up-regulated in the early phase of IRI in rats, cells, and human donor biopsies. Treatment with PKI-166 reduces macrophage accumulation and interstitial alpha-SMA in the early phase of IRI in rats. In vitro, PKI-166 causes a marked reduction in HB-EGF-induced cellular proliferation. Urinary HB-EGF is increased after transplantation compared with control urines from healthy subjects. HB-EGF KO mice subjected to IRI revealed significantly less morphological damage after IRI, compared with WT mice. We conclude that IRI results in early induction of HB-EGF mRNA and protein in vivo and in vitro. Absence of HB-EGF and inhibition of the EGF receptor in the early phase of IRI has protective effects, suggesting a modulating role for HB-EGF.

Heparin-binding epidermal growth factor-like growth factor signaling in flow-induced arterial remodeling.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is activated by reduced endothelial shear stress and stimulates smooth muscle cell proliferation in vitro. Moreover, HB-EGF is augmented at sites of intimal hyperplasia and atherosclerosis, conditions favored by low/disturbed shear stress. We thus tested whether HB-EGF contributes to low flow-induced negative hypertrophic remodeling (FINR) of a mouse carotid artery. Blood flow was surgically decreased in the left and increased in the right common carotid arteries. After 21 days, the left carotid artery exhibited lumen narrowing, thickening of intima-media and adventitia, and increased circumference that were inhibited by approximately 50% in HB-EGF(+/-) and approximately 90% in HB-EGF(-/-) mice. FINR was also inhibited by the EGF receptor inhibitor AG1478. In contrast, eutrophic outward remodeling of the right carotid artery was unaffected in HB-EGF(+/-) and HB-EGF(-/-) mice, nor by AG1478. FINR-induced proliferation and leukocyte accumulation were reduced in HB-EGF(-/-). FINR was associated with increased reactive oxygen species, increased expression of pro-HB-EGF and tumor necrosis factor alpha-converting enzyme (pro-HB-EGF sheddase), increased phosphorylation of EGF receptor and extracellular signal-regulated kinase 1/2, and increased nuclear factor kappaB activity. Apocynin and deletion of p47(phox) inhibited FINR, whereas deletion of HB-EGF abolished nuclear factor kappaB activation in smooth muscle cells. These findings suggest that HB-EGF signaling is required for low flow-induced hypertrophic remodeling and may participate in vascular wall disease and remodeling.

Amphiregulin is not essential for ovalbumin-induced acute airway inflammation in mice.

BACKGROUND: The number of amphiregulin (AR)-positive mast cells in the bronchial mucosa and the levels of AR in sputum from asthmatic patients have been reported to be increased. In addition, AR can promote mucin gene expression in human epithelial cells, suggesting that AR contributes to the pathogenesis of allergic asthma. METHODS: To elucidate the role of AR in the pathogenesis of asthma, we immunized AR-deficient mice with ovalbumin (OVA) and then induced airway inflammation in them after OVA inhalation. The OVA-induced airway inflammation was assessed on the basis of the lung histology, number of leukocytes in the bronchoalveolar lavage (BAL) fluid, Th2 cytokine levels in the BAL fluid and OVA-specific IgG1 and IgE levels in the serum and compared between AR-sufficient and -deficient mice. RESULTS: The OVA-induced airway inflammation was comparable in the AR-sufficient and -deficient mice. CONCLUSIONS: Amphiregulin is not essential for induction of acute airway inflammation by OVA in mice.

Amphiregulin is not essential for induction of contact hypersensitivity.

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.

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

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-alpha-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-alpha, 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.

Heparin-binding epidermal growth factor-like growth factor, collateral vessel development, and angiogenesis in skeletal muscle ischemia.

OBJECTIVE: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent mitogen for smooth muscle cells and has been implicated in atherosclerosis, tissue regeneration after ischemia, vascular development, and tumor angiogenesis. We examined the hypothesis that HB-EGF participates in angiogenesis and collateral growth in ischemia. METHODS AND RESULTS: During 3 weeks after femoral artery ligation, no attenuation occurred in recovery of hindlimb perfusion or distal saphenous artery flow in HB-EGF-null (HB-EGF(-/-)) versus wild-type mice. Lumen diameters of remodeled collaterals in gracilis muscle were similar by morphometry (87+/-8 versus 94+/-6 microm) and angiography, although medial thickening was reduced. Gastrocnemius muscle underwent comparable angiogenesis (41% and 33% increase in capillary-to-muscle fiber ratio). Renal renin mRNA, arterial pressure, and heart rate during anesthesia or conscious unrestrained conditions were similar between groups. These latter findings validate comparisons of perfusion data and also suggest that differences in arterial pressure and/or renin-angiotensin activity are not masking an otherwise inhibitory effect of HB-EGF absence. Four days after ligation, EGF receptor phosphorylation increased in muscle by 104% in wild-type but by only 30% in HB-EGF(-/-) mice. This argues against compensation by other EGF receptor ligands. CONCLUSIONS: Our results suggest that HB-EGF is not required for arteriogenesis or angiogenesis in hindlimb ischemia.

Substrate specificity and inducibility of TACE (tumour necrosis factor alpha-converting enzyme) revisited: the Ala-Val preference, and induced intrinsic activity.

Tumour necrosis factor alpha (TNF alpha)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor alpha, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions. In addition to substrate specificity, the mechanism underlying the increased rate of shedding caused by agents that activate cells remains poorly understood. Recent work in this area, utilizing a peptide substrate as a probe for cellular TACE activity, indicates that the intrinsic activity of the enzyme is somehow increased.

Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads.

A method has been developed for rapid and sensitive identification of epitope-containing peptides, based on direct MALDI-MS/MS analysis of epitope-containing peptides affinity bound to affinity beads. This technique provides sequence information of the epitope that allows unambiguous identification of the epitope either by database searching or de novo sequencing. With MALDI-MS, affinity beads with bound peptides can be placed directly on the MALDI target and analyzed. Coupling a MALDI source to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer allows direct sequencing of the bound peptides. In contrast to ESI-MS/MS, elution of the affinity-bound peptides followed by additional concentration and purification steps is not required, thus reducing the potential for sample loss. Direct mass spectrometric sequencing of affinity-bound peptides eliminates the need for chemical or enzymatic sequencing. Other advantages of this direct MALDI-MS/MS analysis of epitope-containing peptides bound to the affinity beads include its sensitivity (femtomole levels) and speed. In addition, direct analysis of peptides on affinity beads does not adversely affect the high mass accuracy of a QqTOF, and database searching can be performed on the MS/MS spectra obtained. In proof-of-principle experiments, this method has been demonstrated on beads containing immobilized antibodies against phosphotyrosine, the c-myc epitope tag, as well as immobilized avidin. Furthermore, de novo sequencing of epitope-containing peptides is demonstrated. The first application of this method was with anti-FLAG-tag affinity beads, where direct MALDI MS/MS was used to determine an unexpected enzymatic cleavage site on a growth factor protein.

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.

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 ADAM17, 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.

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.

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-alpha 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.

Selective roles for tumor necrosis factor alpha-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.