Shedding Of Heparin-binding Epidermal Growth Factor Research Articles

Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain.

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

Epithelial cell ADAM17 activation by Helicobacter pylori: role of ADAM17 C-terminus and Threonine-735 phosphorylation

Helicobacter pylori transactivates the epidermal growth factor receptor (EGFR) on gastric epithelial cells via a signalling cascade involving a disintegrin and metalloprotease 17 (ADAM17) cleavage of membrane bound heparin binding-epidermal growth factor (HB-EGF). The effects of H. pylori on ADAM17 C-terminus in epithelial cells have been examined. Total cellular ADAM17 and surface expression of ADAM17 were significantly increased by H. pylori in AGS gastric epithelial cells. These changes were associated with ADAM17 C-terminal phosphorylation at T375 and S791. AGS cells lacking the ADAM17 C-terminal domain induced significantly attenuated cleavage of HB-EGF and were also unable to upregulate HB-EGF and EGFR transcripts to the same extent as cells expressing full length ADAM17. In mitotic unstimulated AGS and ADAM17 over-expressing AGS cells, ADAM17 was highly T735 phosphorylated indicating ADAM17 T735 phosphorylation is modified during the cell cycle. In conclusion, H. pylori induced ADAM17 C-terminal T735 and/or S791 phosphorylation in gastric epithelial cells are likely to be an important trigger inducing ADAM17 activation and shedding of HB-EGF leading to EGFR transactivation. ADAM17 over-expression in gastric cancer represents a potential target for therapeutic intervention.

A Disintegrin and Metalloenzyme (ADAM) 17 Activation Is Regulated by α5β1 Integrin in Kidney Mesangial Cells

BackgroundThe disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5β1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation.Methodology/Principal FindingsWe showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5β1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5β1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5β1 integrin interaction. In cell-free assays, we showed that purified α5β1 integrin and β1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of β1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of β1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing β1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT.Conclusions/SignificanceOur data show for the first time that β1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5β1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases.

LIV-1 Promotes Prostate Cancer Epithelial-to-Mesenchymal Transition and Metastasis through HB-EGF Shedding and EGFR-Mediated ERK Signaling

LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there was no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In the present communication, we found increased LIV-1 expression in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives human prostate cancer EMT in an androgen-refractory prostate cancer cells (ARCaP) prostate cancer bone metastasis model. LIV-1, when overexpressed in ARCaPE (derivative cells of ARCaP with epithelial phenotype) cells, promoted EMT irreversibly. LIV-1 overexpressed ARCaPE cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaPE cells that elicited constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promoted EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases.

Cyclosporin A and tacrolimus induce renal Erk1/2 pathway via ROS-induced and metalloproteinase-dependent EGF-receptor signaling

We previously demonstrated that the widely used immunosuppressive drugs cyclosporin A (CsA) and tacrolimus (FK506), independent of immunophilin binding, can activate profibrogenic transforming growth factor β (TGFβ)/Smad signaling cascades in rat renal mesangial cells (MC). Here we report that both peptidyl-prolyl cis/trans isomerase (PPIase) inhibitors activate the extracellular-signaling regulated kinase (ERK) a member of the mitogen activated protein kinase (MAPK) and induce a rapid and transient increase in ERK phosphorylation. The MEK inhibitor U0126, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), a cell-permeant superoxide dismutase (SOD) and stigmatellin, an inhibitor of mitochondrial cytochrome bc1 complex strongly attenuated the increase in ERK1/2 phosphorylation triggered by PPIase inhibitors. Moreover, neutralizing antibodies against heparin binding-epidermal growth factor (HB-EGF), and inhibition of the EGF receptor by either small interfering (si)RNA or AG1478, demonstrate that ERK activation by both PPIase inhibitors is mediated via HB-EGF-induced EGF receptor (EGFR) tyrosine kinase activation. The strong inhibitory effects achieved by GM6001 and TAPI-2 furthermore implicate the involvement of a desintegrin and metalloproteinase 17 (ADAM17). Concomitantly, the PPIase inhibitor-induced ADAM17 secretase activity was significantly reduced by SOD and stigmatellin thus suggesting that mitochondrial ROS play a primary role in PPIase inhibitor-induced and ADAM17-mediated HB-EGF shedding. Functionally, both immunosuppressants caused a strong increase in MC proliferation which was similarly impeded when cells were treated in the presence of NAC, TAPI-2 or AG1478, respectively. Our data suggest that CsA and FK506, via ROS-dependent and ADAM17-catalyzed HB-EGF shedding induce the mitogenic ERK1/2 signaling cascade in renal MC.

ADAM17 up-regulation in renal transplant dysfunction and non-transplant-related renal fibrosis

Interstitial fibrosis and tubular atrophy (IF/TA) is an important cause of renal function loss and ischaemia-reperfusion (I/R) injury is considered to play an important role in its pathophysiology. The aim of the present study was to investigate the role of a disintegrin and metalloproteinase 17 (ADAM17) in human renal allograft disease and in experimental I/R injury of the kidney. We studied the expression of ADAM17 messenger RNA (mRNA) in IF/TA and control kidneys by reverse transcription-polymerase chain reaction and in situ hybridization. Moreover, we assessed ADAM17-mediated heparin-binding epidermal growth factor (HB-EGF) shedding in immortalized human cells. Finally, we studied the effect of pharmacological ADAM17 inhibition in a model of renal I/R injury in rats. ADAM17 mRNA was up-regulated in IF/TA when compared to control kidneys. In normal kidneys, ADAM17 mRNA was weakly expressed in proximal tubules, peritubular capillaries, glomerular endothelium and parietal epithelium. In IF/TA, tubular, capillary and glomerular ADAM17 expression was strongly enhanced with de novo expression in the mesangium. In interstitial fibrotic lesions, we observed co-localization of ADAM17 with HB-EGF protein. In vitro, inhibition of ADAM17 with TNF484 resulted in a dose-dependent reduction of HB-EGF shedding in phorbol 12-myrisate 13-acetate-stimulated cells and non-stimulated cells. In vivo, ADAM17 inhibition significantly reduced the number of glomerular and interstitial macrophages at Day 4 of reperfusion. In conclusion, HB-EGF co-expresses with ADAM17 in renal interstitial fibrosis, suggesting a potential interaction in IF/TA. Targeting ADAM17 to reduce epidermal growth factor receptor phosphorylation could be a promising way of intervention in human renal disease.

Migration of growth factor-stimulated epithelial and endothelial cells depends on EGFR transactivation by ADAM17.

The fibroblast growth factor receptor 2-IIIb (FGFR2b) and the vascular endothelial growth factor receptor 2 (VEGFR2) are tyrosine kinases that can promote cell migration and proliferation and have important roles in embryonic development and cancer. Here we show that FGF7/FGFR2b-dependent activation of epidermal growth factor receptor (EGFR)/ERK1/2 signalling and cell migration in epithelial cells require stimulation of the membrane-anchored metalloproteinase ADAM17 and release of heparin-binding epidermal growth factor (HB-EGF). Moreover, VEGF-A/VEGFR2-induced migration of human umbilical vein endothelial cells also depends on EGFR/ERK1/2 signalling and shedding of the ADAM17 substrate HB-EGF. The pathway used by the FGF7/FGFR2b signalling axis to stimulate shedding of substrates of ADAM17, including ligands of the EGFR, involves Src, p38 mitogen-activated protein-kinase and PI3K, but does not require the cytoplasmic domain of ADAM17. Based on these findings, ADAM17 emerges as a central component in a triple membrane-spanning pathway between FGFR2b or VEGFR2 and EGFR/ERK1/2 that is required for cell migration in keratinocytes and presumably also in endothelial cells.

Lysophosphatidic acid stimulates epidermal growth factor-family ectodomain shedding and paracrine signaling from human lung fibroblasts

Lysophospatidic acid (LPA) is a bioactive lipid mediator implicated in tissue repair and wound healing. It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types involved in wound healing. We hypothesized that human pulmonary fibroblasts stimulated by LPA would exhibit ectodomain shedding of epidermal growth factor receptor (EGFR) ligands that signal to lung epithelial cells. To test this hypothesis, we used alkaline phosphatase-tagged EGFR ligand plasmids transfected into lung fibroblasts, and enzyme-linked immunosorbent assays to detect shedding of native ligands. LPA induced shedding of alkaline phosphatase-tagged heparin-binding epidermal growth factor (HB-EGF), amphiregulin, and transforming growth factor-a; non-transfected fibroblasts shed amphiregulin and HBEGF under baseline conditions, and increased shedding of HB-EGF in response to LPA. Treatment of fibroblasts with LPA resulted in elevated phosphorylation of extracellular signal-regulated kinase 1/2, enhanced expression of mRNA for c-fos, HB-EGF and amphiregulin, and enhanced proliferation at 96 hours. However, none of these fibroblast responses to LPA required ectodomain shedding or EGFR activity. To test the ability of LPA to stimulate paracrine signaling from fibroblasts, we transferred conditioned medium from LPA-stimulated cells, and found enhanced EGFR and extracellular signal-regulated kinase 1/2 phosphorylation in reporter A549 cells in excess of what could be accounted for by transferred LPA alone. These data show that LPA mediates EGF-family ectodomain shedding, resulting in enhanced paracrine signaling from lung fibroblasts to epithelial cells.

Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, gastrin and matrix metalloproteinase-7

Background and aimsHelicobacter pylori (H pylori) infection is a major risk factor in the development of distal gastric adenocarcinoma. Development of the invasive phenotype is associated with the phenomenon of...

A cyclic peptide derived from α-fetoprotein inhibits the proliferative effects of the epidermal growth factor and estradiol in MCF7 cells

A cyclic peptide derived from the active domain of alpha-fetoprotein (AFP) significantly inhibited the proliferation of MCF7 cells stimulated with the epidermal growth factor (EGF) or estradiol (E2). The action of these three agents on cell growth was independent of the presence of calf serum in the culture medium. Our results demonstrated that the cyclic peptide interfered markedly with the regulation of MAPK by activated c-erbB2. The cyclic peptide showed no effect on the E2-stimulated release of matrix metalloproteinases 2 and 9 nor on the shedding of heparin-binding EGF into the culture medium. We propose that the AFP-derived cyclic peptide represents a valuable novel antiproliferative agent for treating breast cancer.

Phosphorylation, desensitization and internalization of human α 1B-adrenoceptors induced by insulin-like growth factor-I

The effect of insulin-like growth factor-I (IGF-I) on human α 1B-adrenoceptor function, phosphorylation state and cellular location was studied. Rat-1 fibroblasts were transfected with a plasmid construction containing enhanced green fluorescent protein joined to the carboxyl terminus of the human α 1B-adrenoceptor. Receptors were identified by radioligand binding and photoaffinity labeling, and were immunoprecipitated with an antiserum generated against the enhanced green fluorescent protein. The receptor was functional, as evidenced by noradrenaline action on intracellular calcium and inositol phosphate production. IGF-I had no significant effect by itself on these parameters but markedly reduced the effects of noradrenaline. IGF-I induced α 1B-adrenoceptor phosphorylation, which was markedly reduced by the following agents: pertussis toxin, a metalloproteinase inhibitor, diphtheria toxin mutant CRM 197, an epidermal growth factor (EGF) receptor intrinsic kinase activity inhibitor, and by phosphoinositide 3-kinase and protein kinase C inhibitors. IGF-I action appears to involve activation of a pertussis toxin-sensitive G protein, shedding of heparin-binding EGF and autocrine activation of EGF receptors. G protein subunits and phosphotyrosine residues stimulate phosphoinositide 3-kinase activity leading to activation of protein kinase C, which in turn phosphorylates α 1B-adrenoceptors. Confocal fluorescent microscopy showed that α 1B-adrenoceptors fussed to the green fluorescent protein were located in plasma membrane and intracellular vesicles in the basal state. IGF-I induced receptor redistribution favoring the intracellular location; this effect was blocked by hypertonic sucrose and concanavalin A. Our data show that IGF-I induces α 1B-adrenoceptor desensitization associated to receptor phosphorylation and internalization.

G Protein Coupling and Second Messenger Generation Are Indispensable for Metalloprotease-dependent, Heparin-binding Epidermal Growth Factor Shedding through Angiotensin II Type-1 Receptor

A G protein-coupled receptor agonist, angiotensin II (AngII), induces epidermal growth factor (EGF) receptor (EGFR) transactivation possibly through metalloprotease-dependent, heparin-binding EGF (HB-EGF) shedding. Here, we have investigated signal transduction of this process by using COS7 cells expressing an AngII receptor, AT1. In these cells AngII-induced EGFR transactivation was completely inhibited by pretreatment with a selective HB-EGF inhibitor, or with a metalloprotease inhibitor. We also developed a COS7 cell line permanently expressing a HB-EGF construct tagged with alkaline phosphatase, which enabled us to measure HB-EGF shedding quantitatively. In the COS7 cell line AngII stimulated release of HB-EGF. This effect was mimicked by treatment either with a phospholipase C activator, a Ca2+ ionophore, a metalloprotease activator, or H2O2. Conversely, pretreatment with an intracellular Ca2+ antagonist or an antioxidant blocked AngII-induced HB-EGF shedding. Moreover, infection of an adenovirus encoding an inhibitor of G(q) markedly reduced EGFR transactivation and HB-EGF shedding through AT1. In this regard, AngII-stimulated HB-EGF shedding was abolished in an AT1 mutant that lacks G(q) protein coupling. However, in cells expressing AT1 mutants that retain G(q) protein coupling, AngII is still able to induce HB-EGF shedding. Finally, the AngII-induced EGFR transactivation was attenuated in COS7 cells overexpressing a catalytically inactive mutant of ADAM17. From these data we conclude that AngII stimulates a metalloprotease ADAM17-dependent HB-EGF shedding through AT1/G(q)/phospholipase C-mediated elevation of intracellular Ca2+ and reactive oxygen species production, representing a key mechanism indispensable for EGFR transactivation.

Agonist-induced activation of matrix metalloproteinase-7 promotes vasoconstriction through the epidermal growth factor-receptor pathway.

Matrix metalloproteinase (MMP)-dependent shedding of heparin-binding epidermal growth factor (HB-EGF) and subsequent activation of the EGF receptor (EGFR) in the cardiovasculature is emerging as a unique mechanism signaling growth effects of diverse G protein-coupled receptors (GPCRs). Among these GPCRs are adrenoceptors and angiotensin receptors that contribute to the pathogenesis of hypertension through their vasoconstrictive and growth effects. Focusing on alpha(1b)-adrenoceptors, we suggest here that MMP-dependent activation of the EGFR promotes vasoconstriction as well as growth. We identified MMP-7 as a major HB-EGF sheddase in rat mesenteric arteries and alpha(1b)-adrenoceptors, angiotensin receptors, and hypertension-stimulated MMP-7 activity. Adrenoceptors stimulated EGFR autophosphorylation in arteries, and this transactivation was opposed by the MMP-7 inhibitor GM6001 as well as MMP-7-specific antibodies. In isolated microperfused arteries, blockade of EGFR transactivation with inhibitors of the EGFR (AG1478 and PD153035), HB-EGF (CRM197 and neutralizing antibodies), or MMPs (doxycycline) inhibited adrenergic vasoconstriction. In spontaneously hypertensive rats but not in normotensive rats, the inhibition of MMPs with doxycycline (19.2 mg/d from week 7 until week 12) reduced systolic blood pressure and attenuated HB-EGF shedding in the mesenteric arteries. These findings suggest a previously unknown mechanism of vasoregulation whereby agonists of certain GPCRs (such as adrenoceptors and angiotensin receptors) activate MMPs (such as MMP-7) that shed EGFR ligands (such as HB-EGF), which then activate the EGFR, thereby promoting vasoconstriction as well as growth. Because this mechanism is triggered by agonists typically overexpressed in hypertension, its blockade may have therapeutic potential for simultaneously inhibiting pathological vasoconstriction and growth in hypertensive disorders.

Role of the epidermal growth factor receptor in signaling strain-dependent activation of the brain natriuretic peptide gene.

The epidermal growth factor receptor (EGFR) and ectoshedding of heparin-binding epidermal growth factor (HBEGF), an EGFR ligand, have been linked to the development of cardiac myocyte hypertrophy. However, the precise role that the liganded EGFR plays in the transcriptional activation of the gene program that accompanies hypertrophy remains undefined. Utilizing the human (h) BNP gene as a model of hypertrophy-dependent gene activation, we show that activation of the EGFR plays an important role in mediating mechanical strain-dependent stimulation of the hBNP promoter. Strain promotes endothelin (ET) generation through NAD(P)H oxidase-dependent production of reactive oxygen species. ET in turn induces metalloproteinase-mediated cleavage of pro-HBEGF and ectoshedding of HBEGF, which activates the EGFR and stimulates hBNP promoter activity. HBEGF also stimulates other phenotypic markers of hypertrophy including protein synthesis and sarcomeric assembly. The antioxidant N-acetylcysteine or the NAD(P)H oxidase inhibitor, apocynin, inhibited strain-dependent activation of the ET-1 promoter, HBEGF shedding, and hBNP promoter activation. The metalloproteinase inhibitor, GM-6001, prevented the induction of HBEGF ectoshedding and the hBNP promoter response to strain, suggesting a critical role for the metalloproteinase-dependent cleavage event in signaling the strain response. These findings suggest that metalloproteinase activity as an essential step in this pathway may prove to be a relevant therapeutic target in the management of cardiac hypertrophy.

Lysophosphatidic acid-regulated mitogenic ERK signaling in androgen-insensitive prostate cancer PC-3 cells.

Advanced and recurrent prostate tumors contain elevated levels of activated extracellular signal-regulated kinases 1 and 2 (ERK) in comparison to early-stage or benign specimens, and inhibition of ERK activation attenuates growth factor-dependent proliferation of prostate cells, suggesting a potential regulatory role for ERK in prostate tumorigenesis. Factors responsible for ERK activation in prostate cells are not well defined. Here, we show positive cooperative interaction between the G protein-coupled lysophosphatidic acid (LPA) and tyrosine kinase epidermal growth factor (EGF) receptors in androgen-insensitive prostate cancer PC-3 cells. Pre-treatment of the PC-3 cells with LPA decreases the dose of EGF required to elicit maximal activation of EGFR. Furthermore, treatment with LPA alone induces the rapid (maximal signal within 2 min) tyrosine phosphorylation of EGFR, and subsequent (maximal signal after 5 min) activation of ERK, suggesting that EGFR activation precedes ERK phosphorylation and may constitute a required component for signal relay from the LPA receptor to ERK. Accordingly, we show that inhibition of EGFR kinase activity attenuates the LPA-regulated ERK activation. In addition, we find that the LPA-regulated tyrosine phosphorylation of EGFR and activation of ERK are attenuated by batimastat, a generic inhibitor of matrix metalloproteinases (MMP). However, unlike the situation in fibroblasts, we find that the LPA-induced transactivation of EGFR in PC-3 cells is not mediated by shedding of heparin-binding EGF. Together, our data show that LPA and EGF cooperate to induce mitogenic signaling in prostate cancer cells in an MMP-regulated activation of the ERK pathway.

Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy.

G-protein-coupled receptor (GPCR) agonists are well-known inducers of cardiac hypertrophy. We found that the shedding of heparin-binding epidermal growth factor (HB-EGF) resulting from metalloproteinase activation and subsequent transactivation of the epidermal growth factor receptor occurred when cardiomyocytes were stimulated by GPCR agonists, leading to cardiac hypertrophy. A new inhibitor of HB-EGF shedding, KB-R7785, blocked this signaling. We cloned a disintegrin and metalloprotease 12 (ADAM12) as a specific enzyme to shed HB-EGF in the heart and found that dominant-negative expression of ADAM12 abrogated this signaling. KB-R7785 bound directly to ADAM12, suggesting that inhibition of ADAM12 blocked the shedding of HB-EGF. In mice with cardiac hypertrophy, KB-R7785 inhibited the shedding of HB-EGF and attenuated hypertrophic changes. These data suggest that shedding of HB-EGF by ADAM12 plays an important role in cardiac hypertrophy, and that inhibition of HB-EGF shedding could be a potent therapeutic strategy for cardiac hypertrophy.