Soluble Heparin-binding Epidermal Growth Factor-like Growth Factor Research Articles

Antiproliferative and apoptotic effects of anti-human HB-EGF neutralizing polyclonal antibodies in vitro.

To obtain HB-EGF neutralizing polyclonal antibodies and test their anti-proliferative properties in vitro. Lab rabbits and mice were used for immunization with recombinant HB-EGF. The effect of generated polyclonal antibodies on viability and apoptosis of human epidermoid carcinoma derived A431cell line was assessed using MTT and Annexin V-propidium iodide assays. Rabbit polyclonal anti-HB-EGF serum could block binding of soluble HB-EGF to epidermal growth factor receptor/human epidermal growth factor receptor. Also, anti-HB-EGF antibodies could bind to surface of A431cells which express abnormally high levels of membrane bound proHB-EGF and its receptor. It has been shown that immune serum with polyclonal antibodies against HB-EGF was able to block the mitogenic activation of the cells with HB-EGF and cause apoptotic cell death. Inhibition of HB-EGF activity with neutralizing polyclonal antibodies can effectively inhibit mitogenic activation and cause apoptosis of cancer cells with significant epidermal growth factor receptor overexpression.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

Abstract 1182: Heparin-binding epidermal growth factor-like growth factor is a pro-differentiating factor in neuroblastoma

Abstract Neuroblastoma is the most common cancer in infancy. Current therapies are only modestly effective; patients with high-risk disease have less than a 50% chance of survival. High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low Schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble proteins, including heparan sulfate proteoglycans (HSPGs), which act to promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent pro-differentiating factor in neuroblastoma. Using microarray analysis, HBEGF mRNA expression is decreased in neuroblastoma compared to benign disease, correlating to an increase in mortality. HBEGF protein is expressed only in stromal compartments of tumor specimens, with tissue from high-stage disease having decreased stroma and HBEGF. Soluble HBEGF increased neuroblast differentiation and decreased proliferation, while HBEGF knockdown decreased neuroblast differentiation. In patient samples, HBEGF expression correlates with SOX10, a neural crest differentiation marker, and the cell cycle inhibitor, CDKN1A. Alternatively, HBEGF negatively correlates with ASCL1, a primitive neuroectodermal marker, and the cell cycle promoter CCND1. HSPGs, including TβRIII, GPC1, GPC3, and SDC3 further promote the differentiating effects of HBEGF treatment by forming a complex with the epidermal growth factor receptor (EGFR) via glycosaminoglycan modifications, leading to activation of the Erk1/2 pathway and upregulation of the inhibitor of DNA binding transcription factor, whose expression correlates with HBEGF in patient samples. Inhibition of EGFR with erlotinib, lapatinib, and gefitinib diminish HBEGF-induced differentiation. These data identify a novel member of the pro-differentiating secretome and support the use of heparan sulfate mimetics in neuroblastoma, while cautioning against the use of EGFR inhibitors for neuroblastoma treatment. Citation Format: Angela L. Gaviglio, Gerard C. Blobe. Heparin-binding epidermal growth factor-like growth factor is a pro-differentiating factor in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1182.

A heparin-modified thermoresponsive surface with heparin-binding epidermal growth factor-like growth factor for maintaining hepatic functions invitro and harvesting hepatocyte sheets.

A heparin-modified thermoresponsive surface bound with heparin-binding epidermal growth factor-like growth factor (HB-EGF) was designed to allow creation of transferrable and functional hepatocyte sheets. A heparin-modified thermoresponsive surface was prepared by covalently tethering heparin onto poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide)-grafted tissue culture polystyrene surfaces (Heparin-IC). HB-EGFs were able to stably bind to heparin-IC via affinity interaction. The survival of primary rat hepatocytes was maintained through HB-EGF-bound heparin-IC (HB-EGF/heparin-IC). Moreover, cultured rat primary hepatocytes on HB-EGF/heparin-IC exhibited higher albumin-secretion than hepatocytes cultured on PIPAAm-grafted and collagen-coated surfaces with soluble HB-EGF in the culture medium, regardless of whether soluble EGF was added. These results suggested that HB-EGF/heparin-IC is able to effectively maintain hepatic function via continuous signaling of HB-EGF. After a 4-day cultivation, the cultured hepatocytes on HB-EGF/heparin-IC detached as a cell sheet with fibronectin and HB-EGF only after the temperature was lowered to 20 °C. In addition, higher expression of hepatocyte-specific genes (albumin, hepatocyte nuclear factor 4 alpha, coagulation factor VII, and coagulation factor IX) in hepatocyte sheets was detected on HB-EGF/heparin-IC than on a PIPAAm surface with soluble HB-EGF, indicating that HB-EGF/heparin-IC suppressed the dedifferentiation of cultured hepatocytes. Hence, heparin-modified thermoresponsive surfaces bound with HB-EGF facilitate the fabrication of transferrable hepatocyte sheets with intact hepatic functions and have the potential to provide an in vitro culture system using functional hepatocyte sheet tissues, which may serve as an effective hepatocyte-based tissue engineering platform for liver disease treatments.

Involvement of reactive oxygen species in stimuli-induced shedding of heparin-binding epidermal growth factor-like growth factor.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a critical growth factor for a number of physiological and pathological processes, such as wound healing, atherosclerosis and cancer proliferation. HB-EGF is synthesized as a membrane form (proHB-EGF), and is shedded at the cell surface to yield soluble HB-EGF, resulting in making it active. In this study, the involvement of reactive oxygen species (ROS) in stimuli-induced shedding of HB-EGF was investigated using monkey kidney Vero cells overexpressing HB-EGF (Vero-H cells). 12-O-tetradecanoylphorbol-13-acetate (TPA), lysophosphatidic acid (LPA) as a ligand for seventransmembrane G protein coupled receptors (GPCR) and sorbitol as stress induced shedding of HB-EGF mediated protein kinase C (PKC)-δ, mitogen-activated protein kinase (MAPK) and p38MAPK, respectively. These stimuli-induced sheddings of HB-EGF were inhibited by N-acetyl-L-cysteine (NAC), suggesting the involvement of ROS. As specific inhibitors of these protein kinases inhibited the shedding of HB-EGF, these signaling pathways seem to be independent, respectively. In contrast, γ-ray irradiation did not induce shedding although it did increase intracellular ROS levels. Taken together, these results suggest that the synergistic generation of ROS and the activation of protein kinase are required to promote stimuli-induced shedding of HB-EGF.

Ectodomain shedding of HB-EGF: A potential target for cancer therapy

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored protein, known as proHB-EGF. ProHB-EGF is cleaved by metalloproteases through a process referred to as 'ectodomain shedding', resulting in the formation of soluble HB-EGF. Both proHB-EGF and soluble HB-EGF are biologically active; the former acts on neighbouring cells through juxtacrine signalling, whereas the latter can move to distant locations. Elevated HB-EGF expression has been observed in ovarian and some other cancers. CRM197, a diphtheria toxin (DT) mutant, binds directly to the epidermal growth factor (EGF)-like domain and represses the mitogenic activity of HB-EGF. Recently, monoclonal antibodies (mAbs) specific for human HB-EGF were generated by immunizing HB-EGF-deficient mice with human HB-EGF (Hamaoka et al. (2010) J. Biochem. 148, 55-69). Most of the mAbs can bind to the EGF-like domain of HB-EGF, but fail to inhibit the mitogenic activity of soluble HB-EGF. However, some mAbs prevented the ectodomain shedding of proHB-EGF and inhibited the proliferation of EGF receptor-expressing cells stimulated by proHB-EGF-expressing cells. Hamaoka et al. showed that CRM197 prevents the ectodomain shedding of proHB-EGF. Thus, these mAbs function as specific inhibitors for the ectodomain shedding of HB-EGF and may be useful for treating cancers exhibiting elevated levels of HB-EGF.

Transmembrane and Soluble Isoforms of Heparin-Binding Epidermal Growth Factor–Like Growth Factor Regulate Distinct Processes in the Pancreas

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is produced as a type-I, single-pass transmembrane protein that can be cleaved to release a diffusible peptide. HB-EGF, often overexpressed in damaged or diseased epithelium, is normally expressed in pancreatic islets, but its function is not understood. To understand the function of each isoform of HB-EGF, we made transgenes expressing either a constitutively transmembrane or a constitutively secreted protein. The transmembrane isoform was not an inert precursor protein, but a functional molecule, downregulating the glucose-sensing apparatus of pancreatic islets. Conversely, the secreted form of HB-EGF improved islet function, but had severe fibrotic and neoplastic effects on surrounding tissues. Each isoform had a more severe phenotype than that of full-length HB-EGF, even though the full-length protein was efficiently cleaved, thus producing both isoforms, suggesting that a level of regulation was lost by separating the isoforms. This work demonstrates that islet function depends on the ratio of cleaved to uncleaved HB-EGF and that the transmembrane intermediate, while deleterious to islet function, is necessary to restrict action of soluble HB-EGF away from surrounding tissue.

Heparin‐binding epidermal growth factor‐like growth factor functionally antagonizes interstitial cystitis antiproliferative factor via mitogen‐activated protein kinase pathway activation

To delineate the mechanism underlying the potential functional relationship between interstitial cystitis antiproliferative factor (APF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF), as APF has previously been shown to decrease the proliferation rate of normal bladder epithelial cells and the amount of HB-EGF produced by these cells. APF-responsive T24 transitional carcinoma bladder cells were treated with high-pressure liquid chromatography-purified native APF with or without HB-EGF to determine the involvement of signalling pathways and proliferation by Western blot analysis, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (Erk)/MAPK assays, and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Cyclic stretch induced the secretion of HB-EGF from T24 cells overexpressing the HB-EGF precursor, resulting in enhanced proliferation. T24 cells treated with APF had increased p38MAPK activity and suppressed cell growth, events that were both reversed by treatment with a p38MAPK-selective inhibitor. Activation of Erk/MAPK by HB-EGF was inhibited by APF, and APF did not stimulate p38MAPK in the presence of soluble HB-EGF or when cells overexpressed constitutively secreted HB-EGF. Lastly, APF inhibitory effects on cell growth were attenuated by HB-EGF. These results indicate that HB-EGF and APF are functionally antagonistic and signal through parallel MAPK signalling pathways in bladder cells.

Activity-dependent shedding of heparin-binding EGF-like growth factor in brain neurons

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is initially produced as a membrane-anchored precursor (pro-HB-EGF) and subsequently liberated from the cell membrane through ectodomain shedding. Here, we characterized the molecular regulation of pro-HB-EGF shedding in the central nervous system. Cultured neocortical or hippocampal neurons were transfected with the alkaline-phosphatase-tagged pro-HB-EGF gene and stimulated with various neurotransmitters. Both kainate and N-methlyl- d-aspartate, but not agonists for metabotropic glutamate receptors, promoted pro-HB-EGF shedding and HB-EGF release, which were attenuated by an exocytosis blocker and metalloproteinase inhibitors. In the brain of transgenic mice over-expressing human pro-HB-EGF, kainate-induced seizure activity decreased content of pro-HB-EGF-like immunoreactivity and conversely increased levels of soluble HB-EGF. There was concomitant phosphorylation of EGF receptors (ErbB1) following seizures, suggesting that seizure activities liberated HB-EGF and activated neighboring ErbB1 receptors. Therefore, we propose that glutamatergic neurotransmission in the central nervous system plays a crucial role in regulating ectodomain shedding of pro-HB-EGF.

Lysophosphatidic Acid, a Disintegrin and Metalloprotease-17 and Heparin-Binding Epidermal Growth Factor–Like Growth Factor in Ovarian Cancer: The First Word, Not the Last

Cancer of the ovary is the leading cause of death for women with gynecologic malignancies. Over 90% of ovarian cancers derive from malignant transformation of the ovarian surface epithelium (OSE), from which cells disseminate into the peritoneal cavity, invade locally or spread via lymphatics,

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.

Mechanism for activation of heparin-binding EGF-like growth factor induced by stimuli

Heparin-binding epidermal growth factor-like growth factor(HB-EGF), which belongs to the EGF family, is a critical growth factor for a number of physiological and pathological processes, such as wound healing and cardiac hypertrophy. HB-EGF is synthesized as a membrane-anchored form(pro-HB-EGF), and pro-HB-EGF is cleaved at the cell surface to yield soluble HB-EGF by a mechanism called "ectodomain shedding". Soluble HB-EGF has mitogenic activity. Ectodomain shedding of proHB-EGF is induced by stimuli, including 12-O-tetradecanoylphorbol-13-acetate(TPA), a ligand for seven-transmembrane G protein-coupled receptors(GPCR), stress and inflammatory cytokine. Lysophosphatidic acid(LPA), a ligand for GPCR, stimulates the shedding of proHB-EGF, which constitutes a GPCR-mediated transactivation of the EGF receptor. Ras-Raf-MEK signal and the small GTPase Rac are essential for the LPA-induced shedding. On the other hand, protein kinase C and ADAM 9 protease are essential for the TPA-induced shedding. Furthermore, p38 MAPK is essential for the stress- and IL-1 beta-induced shedding. Finally there is a mechanism for activation of HB-EGF regulated by the environment in the living body.

The Stress- and Inflammatory Cytokine-induced Ectodomain Shedding of Heparin-binding Epidermal Growth Factor-like Growth Factor Is Mediated by p38 MAPK, Distinct from the 12-O-Tetradecanoylphorbol-13-acetate- and Lysophosphatidic Acid-induced Signaling Cascades

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a critical growth factor for a number of physiological and pathological processes. HB-EGF is synthesized as a membrane-anchored form (pro-HB-EGF), and pro-HB-EGF is cleaved at the cell surface to yield soluble HB-EGF by a mechanism called "ectodomain shedding." We show here that the ectodomain shedding of pro-HB-EGF in Vero cells is induced by various stress-inducing stimuli, including UV light, osmotic pressure, hyperoxidation, and translation inhibitors. The pro-inflammatory cytokine interleukin-1beta also stimulated the ectodomain shedding of pro-HB-EGF. An inhibitor of p38 MAPK (SB203580) or the expression of a dominant-negative (dn) form of p38 MAPK inhibited the stress-induced ectodomain shedding of pro-HB-EGF, whereas an inhibitor of JNK (SP600125) or the expression of dnJNK1 did not. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and lysophosphatidic acid (LPA) are also potent inducers of pro-HB-EGF shedding in Vero cells. Stress-induced pro-HB-EGF shedding was not inhibited by the inhibitors of TPA- or LPA-induced pro-HB-EGF shedding or by dn forms of molecules involved in the TPA- or LPA-induced pro-HB-EGF shedding pathway. Reciprocally, SB203580 or dnp38 MAPK did not inhibit TPA- or LPA-induced pro-HB-EGF shedding. These results indicate that stress-induced pro-HB-EGF shedding is mediated by p38 MAPK and that the signaling pathway induced by stress is distinct from the TPA- or LPA-induced pro-HB-EGF shedding pathway.

Liver regeneration in heparin-binding EGF-like growth factor transgenic mice after partial hepatectomy

Background & Aims: Heparin-binding epidermal growth factor–like growth factor (HB-EGF), a member of the EGF family, is synthesized in the form of a membrane-anchored precursor (proHB-EGF), which subsequently is processed proteolytically to mature HB-EGF. This study describes the effects of HB-EGF on liver regeneration after 70% partial hepatectomy in proHB-EGF transgenic mice with liver-specific expression. Methods & Results: No significant differences in liver/body weight ratios and in bromodeoxyuridine (BrdU)-labeling index (the ratios of BrdU-positive hepatocyte nuclei) were found between adult transgenic and wild-type mice. However, in regenerating liver after partial hepatectomy, transgenic mice had higher liver/body weight ratios than wild-type mice and at 120 hours reached a level equal to that before partial hepatectomy. The BrdU-labeling index was about 5 times higher in the livers of transgenic mice compared with the wild type (51.5% vs. 10.2%, respectively; P < 0.01) at 48 hours after partial hepatectomy. Activation of microtubule-associated protein kinase after partial hepatectomy was higher and earlier in the transgenic mice as compared with the wild-type mice. Soluble HB-EGF was increased in the liver (at 8 min) after partial hepatectomy, indicating that the shedding of proHB-EGF occurred after partial hepatectomy. Conclusions: The transgenic expression of HB-EGF accelerates the proliferation of hepatocytes after partial hepatectomy, suggesting that HB-EGF functions as a hepatotrophic factor in vivo.GASTROENTEROLOGY 2003;124:701-707

Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching

Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching. The developing metanephros is characterized by growth and differentiation of the ureteric bud and the surrounding mesenchymal tissue. These processes can be influenced by several growth factors, including epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). We examined whether another member of the EGF family of growth factors, heparin-binding epidermal growth factor (HB-EGF), might act as a morphogen in renal epithelial tubulogenesis. Expression of HB-EGF mRNA and immunoreactive protein were examined in fetal, neonatal and adult rat kidneys. For in vitro studies of tubulogenesis, a rat renal epithelial cell line (NRK52E) stably transfected with proHB-EGF (NRKproHB-EGF) was treated with TPA for 30 minutes, washed with 2 mol/L NaCl to remove soluble HB-EGF trapped by cell surface heparan sulfate proteoglycan and replated onto plastic dishes in the absence of fetal calf serum. In further experiments, NRKproHB-EGF were suspended in a type I collagen gel in serum-free media. Northern blot analysis indicated that HB-EGF was strongly expressed in embryonic rat kidney (embryonic days 18-20) and was still increased in the neonatal kidney (day 10), compared to the low basal levels in adult kidney. Immunohistochemical analysis confirmed that immunoreactive HB-EGF expression in the fetal rat kidney was localized predominantly to the ureteric bud. When NRKproHB-EGF were plated onto plastic substrata, they became progressively flattened and enlarged and exhibited filopoidia. By 10 hours after plating, NRKproHB-EGF began to migrate and subsequently developed cell-cell contact and fully established tubular-like structures. Immunoelectron microscopy revealed that the initial recovery of cellular proHB-EGF was localized predominantly to areas of cell-cell attachment. No tubule-like structures were observed in similarly treated NRK52E cells transfected with the vector alone. In collagen gels, NRKproHB-EGF developed short tubule-like structures in the absence of TPA treatment, but with simultaneous TPA treatment, longer and more arborized structures developed. MMP-1 mRNA and immunoreactive protein increased in the TPA-treated cells, suggesting that protein kinase C-mediated collagenase activity was important for the observed tubulogenesis. However, inhibition of EGF receptor tyrosine kinase with AG 1478 significantly blunted the TPA-induced tubulogenesis by NRKproHB-EGF grown in collagen gels. These results indicate that membrane-bound HB-EGF can mediate both epithelial cell branching and cell motility. Localization of proHB-EGF to the site of cell-cell contact and development of tubule-like structures in collagen gels suggests that proHB-EGF may be an important morphogen for renal epithelial cells.

Heparin-binding EGF-like growth factor mediates the biological effects of P450 arachidonate epoxygenase metabolites in epithelial cells.

In addition to its important functions in detoxification of foreign chemicals and biosynthesis of steroid hormones, the cytochrome P450 enzyme system metabolizes arachidonate to 14,15-epoxyeicosatrienoic acid (14,15-EET). This study demonstrates that a P450 arachidonate epoxygenase metabolite can activate cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and delineates an essential role for HB-EGF in the mitogenic effects of this lipid mediator. Blockade of HB-EGF processing or EGF receptor (EGFR) inhibited 14,15-EET-stimulated early mitogenic signals and DNA synthesis. 14,15-EET failed to induce mitogenesis in cell lines expressing minimal HB-EGF, whereas 14,15-EET induced soluble HB-EGF release into the conditioned media of cell lines that both express high levels of HB-EGF and display mitogenic response to this lipid mediator. Moreover, transfection of a bacterial 14,15-epoxygenase established intracellular endogenous 14,15-EET biosynthesis in cultured cell systems, which allowed direct confirmation of involvement of EGFR transactivation in the endogenous 14,15-EET-mediated mitogenic signaling pathway. This mechanism involves EET-dependent activation of metalloproteinases and release of the potent mitogenic EGFR ligand, HB-EGF.

Induction of collecting duct morphogenesis in vitro by heparin-binding epidermal growth factor-like growth factor.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor family of growth factors, is synthesized as a membrane-an-chored precursor (proHB-EGF) that is capable of stimulating adjacent cells in a juxtacrine manner. ProHB-EGF is cleaved in a protein kinase C-dependent process, to yield the soluble form. It was observed that HB-EGF acts as a morphogen for the collecting duct system in developing kidneys. HB-EGF protein was expressed in the ureteric bud of embryonic kidneys. Cultured mouse ureteric bud cells (UBC) produced HB-EGF via protein kinase C activation. After stimulation with phorbol ester (12-O-tetradecanoylphorbol-13-acetate) or recombinant soluble HB-EGF, UBC cultured in three-dimensional collagen gels formed short tubules with varied abundant branches. When proHB-EGF-transfected UBC were stimulated with 12-O-tetradecanoylphorbol-13-acetate and cultured in collagen gels, they exhibited linear growth, forming long tubular structures with few branches at the time of appearance of proHB-EGF on the cell surface. The structures exhibited a strong resemblance to the early branching ureteric bud of embryonic kidneys. When UBC were cultured in the presence of transforming growth factor-beta and soluble HB-EGF, they formed long tubules and few branches, similar to the structures observed in proHB-EGF-transfected UBC. These cells exhibited apical-basolateral polarization and expression of the water channel aquaporin-2. These findings indicate that soluble HB-EGF and proHB-EGF induce branching tubulogenesis in UBC in different ways. Juxtacrine activation by proHB-EGF or the synergic action of soluble HB-EGF with transforming growth factor-beta is important for well balanced morphogenesis of the collecting duct system.

Granulocyte-Macrophage Colony-Stimulating Factor Induces Expression of Heparin-Binding Epidermal Growth Factor–Like Growth Factor/Diphtheria Toxin Receptor and Sensitivity to Diphtheria Toxin in Human Neutrophils

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a widely expressed EGF superfamily member that induces mitogenic and/or chemotactic activities toward different cell types through binding to EGF receptors 1 or 4. Membrane-bound HB-EGF exerts growth activity and adhesion capabilities and possesses the unique property of being the receptor for diphtheria toxin (DT). Using molecular and functional techniques, we show that human polymorphonuclear granulocytes (PMN), which did not express HB-EGF in resting conditions, expressed it at mRNA and protein level, following incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Other classic agonists for PMN (including lipopolysaccharide, phagocytable particles, tumor necrosis factor-alpha, or G-CSF) failed to induce HB-EGF. The effects of GM-CSF on HB-EGF mRNA levels were concentration-dependent, reached a plateau after 1 to 2 hours of stimulation, and did not require protein synthesis. After GM-CSF treatment, membrane-bound HB-EGF was detected by flow cytometry. At the same time, PMN acquired sensitivity to the apoptosis-promoting effect of DT, which, moreover, specifically suppressed the GM-CSF-induced priming of formyl-methionyl-leucyl-phenylalanine-stimulated superoxide anion release. Finally, soluble HB-EGF was detected in the PMN culture medium by a specific enzyme-linked immunosorbent assay. Thus, we provide evidence that HB-EGF is specifically inducible by GM-CSF in PMN and represents a novel peptide to be included in the repertoire of PMN-derived cytokines.

Matrix Metalloproteinase-3 Releases Active Heparin-binding EGF-like Growth Factor by Cleavage at a Specific Juxtamembrane Site

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored precursor that is cleaved to release the soluble mature growth factor. The two forms are active as juxtacrine and paracrine/autocrine growth factors, respectively. The enzymes that process the HB-EGF transmembrane form are unknown. Accordingly, an in vitro assay was established using a fusion protein in which alkaline phosphatase (AP) replaced the transmembrane and cytoplasmic domains of HB-EGF (HB-EGF JM-AP). The fusion protein was anchored to agarose beads coated with anti-AP antibodies. Several matrix metalloproteinases (MMPs) were tested for the ability to release soluble HB-EGF in the in vitro system. MMP-3 released soluble 12-kDa immunoreactive and mitogenic HB-EGF within 30 min. On the other hand neither MMP-2 nor MMP-9 had any cleavage activities. A non-cleavable mutant was prepared by replacing the juxtamembrane (JM) region of HB-EGF with the JM region of CD4. The mutant HB-EGF, which in its full-length form was as active a juxtacrine growth factor as was the wild type HB-EGF in vivo, was not cleaved by MMP-3 in the in vitro assay. The C-terminal portion of the cleaved HB-EGF JM-AP that remained attached to the anti-AP beads was N-terminally sequenced and the MMP-3 cleavage site was determined to be Glu151-Asn152, a site within the JM domain. MMP-3 treatment also released soluble HB-EGF in vivo from MC2 cells expressing transmembrane HB-EGF precursor, at a level of about 2-fold above control. It was concluded that MMP-3 cleaves HB-EGF at a specific site in the JM domain and that this enzyme might regulate the conversion of HB-EGF from being a juxtacrine to a paracrine/autocrine growth factor.

Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity.

Vero cell heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF precursor (proHB-EGF). Localization and processing of proHB-EGF, both constitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, was examined in Vero cells overexpressing recombinant HB-EGF (Vero H cells). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the interface of cell to cell contact. Cell surface biotinylation and immunoprecipitation detected a 20- to 30-kDa heterogeneous proHB-EGF species. Vero H cell surface proHB-EGF turned over constitutively with a half-life of 1.5 h. Some of the 20- to 30-kDa cell surface-associated proHB-EGF was processed and a 14-kDa species of bioactive HB-EGF was released slowly, but most of the proHB-EGF was internalized, displaying a diffuse immunofluorescent staining pattern and accumulation of proHB-EGF in endosomes. Addition of TPA induced a rapid processing of proHB-EGF at a Pro148-Val149 site with a half-life of 7min. The TPA effect was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is maximal at 30 min after addition of TPA and that proHB-EGF is resynthesized and the initial cell surface levels are regained within 12-24 h. Loss of cell surface proHB-EGF was concomitant with appearance of 14- and 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-associated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in coculture. Processing of proHB-EGF resulted in loss of juxtacrine activity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivity by converting it from a cell-surface juxtacrine growth factor to a processed, released soluble paracrine growth factor.