Heparin-binding EGF-like growth factor: a juxtacrine growth factor

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family growth factors, is synthesized as a membrane-anchored form (proHB-EGF). Proteolytic cleavage of proHB-EGF at the extracellular domain yields the soluble form of HB-EGF (sHB-EGF). ProHB-EGF is not only the precursor molecule for sHB-EGF but also a biologically active molecule itself. Recent studies indicate that proHB-EGF has unique properties distinct from the soluble form. ProHB-EGF forms a complex with membrane proteins including a tetramembrane spanning protein: CD9, an adhesion molecule integrin: 3β1, and heparan sulfate proteoglycans. The complex is localized at the cell–cell contact site, suggesting that proHB-EGF may function in cell-to-cell signaling by a juxtacrine mechanism. In an in vitro model system, proHB-EGF showed growth inhibitory activity, while sHB-EGF was growth stimulatory. Ectodomain shedding, conversion of the membrane-anchored form into the soluble form, is regulated by multiple signaling pathways. All these characteristics imply that proHB-EGF and sHB-EGF are used in different ways. In vivo functions of sHB-EGF and proHB-EGF have been largely undefined, but recent studies implicate them in a variety of physiological processes including blastocyst implantation and wound healing.     

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.     

BAG-1 is a novel cytoplasmic binding partner of the membrane form of heparin-binding EGF-like growth factor: a unique role for proHB-EGF in cell survival regulation

Several cell functions related to growth and survival regulation have been attributed specifically to the membrane form of heparin-binding EGF-like growth factor (proHB-EGF), rather than to the diffusible, processed HB-EGF isoform. These findings suggest the existence of a functional binding partner specifically for the membrane form of the growth factor. In this study we have identified the prosurvival cochaperone, BAG-1, as a protein that interacts with the cytoplasmic tail domain of proHB-EGF. Interaction between BAG-1 and the 24-amino acid proHB-EGF cytoplasmic tail was initially identified in a yeast two-hybrid screen and was confirmed in mammalian cells. The proHB-EGF tail bound BAG-1 in an hsp70-independent manner and within a 97-amino acid segment that includes the ubiquitin homology domain in BAG-1 but does not include the hsp70 binding site. Effects of BAG-1 and proHB-EGF co-expression were demonstrated in cell adhesion and cell survival assays and in quantitative assays of regulated secretion of soluble HB-EGF. Because the BAG-1 binding site is not present on the mature, diffusible form of the growth factor, these findings suggest a new mechanism by which proHB-EGF, in isolation from the diffusible form, can mediate cell signaling events. In addition, because effects of BAG-1 on regulated secretion of soluble HB-EGF were also identified, this interaction has the potential to alter the signaling capabilities of both the membrane-anchored and the diffusible forms of the growth factor.     

Identification of serum factor inducing ectodomain shedding of proHB-EGF and sStudies of noncleavable mutants of proHB-EGF

The ectodomain of the transmembrane form of HB-EGF (proHB-EGF) is cleaved at the cell surface by proteases, yielding a soluble growth factor. A number of stimuli, including TPA, accelerate this cleavage. However, proHB-EGF is shed constitutively under normal culture conditions without any particular stimuli. We demonstrate here that constitutive cleavage resulted largely from factor(s) contained in supplemented FCS in a culture medium. Analysis of serum factors, including digestion with enzymes, separation by thin layer chromatography, and shedding assay with purified phospholipids, revealed that lysophosphatidic acid (LPA) is a major factor in FCS for stimulation of proHB-EGF shedding. We also studied here ectodomain shedding of two kinds of mutant form of proHB-EGF which have a single amino acid substitution around the putative cleavage sites. These mutant forms showed resistance to stimuli of both TPA and LPA, suggesting that proHB-EGF is cleaved at the similar site by stimulation with TPA and LPA.     

Roles of Dppa2 in the regulation of the present status and future of pluripotent stem cells

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.     

Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain-truncated form (HBdeltatm) of the molecule. HB(uc/uc) mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBdeltatm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.     

Identification of the Cancer Cell Proliferation and Survival Functions of proHB-EGF by Using an Anti-HB-EGF Antibody

Purpose

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The membrane-bound proHB-EGF is known to be a precursor of the soluble form of HB-EGF (sHB-EGF), which promotes cell proliferation and survival. While the functions of sHB-EGF have been extensively studied, it is not yet fully understood if proHB-EGF is also involved in cellular signaling events. In this study, we utilized the anti-HB-EGF monoclonal antibodies Y-142 and Y-073, which have differential specificities toward proHB-EGF, in order to elucidate proHB-EGF functions in cancer cells.

Experimental Design

The biological activities of proHB-EGF were assessed in cell proliferation, caspase activation, and juxtacrine activity assays by using a 3D spheroid culture of NUGC-3 cells.

Results

Y-142 and Y-073 exhibited similar binding and neutralizing activities for sHB-EGF. However, only Y-142 bound to proHB-EGF. We could detect the function of endogenously expressed proHB-EGF in a 3D spheroid culture. Blocking proHB-EGF with Y-142 reduced spheroid formation, suppressed cell proliferation, and increased caspase activation in the 3D spheroid culture of NUGC-3 cells.

Conclusions

Our results show that proHB-EGF acts as a cell proliferation and cell survival factor in cancer cells. The results suggest that proHB-EGF may play an important role in tumor progression.     

肝素结合表皮生长因子及其在恶性血液细胞中的表达与作用

肝素结合表皮生长因子（HB—EGF）是EGF家族的成员之一,可以在多种组织和细胞中表达,参与多种病理生理过程。不管是它的前体蛋白（proHB—EGF）还是剪切后的形成的sHB—EGF和HB—EGF—C都对细胞的生长和迁移具有重要的影响。但由于HB—EGF72．其受体在血液系统肿瘤细胞中表达水平并不高,所以相关报道并不多见。随着相关研究的深入,HB—EGF在骨髓瘤和白血病细胞中的表达得到证实,而且对上述两种细胞具有诱导增殖的潜力,但具体机制大多仍未澄清。相信伴随着对这些机制的认识,会为骨髓瘤和白血病的治疗提供新的思路。     

Heparin-binding EGF-like growth factor in the human prostate: Synthesis predominantly by interstitial and vascular smooth muscle cells and action as a carcinoma cell mitogen

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an activating ligand for the EGF receptor (HER1/ErbB1) and the high-affinity receptor for diphtheria toxin (DT) in its transmembrane form (proHB-EGF). HB-EGF was immunolocalized within human benign and malignant prostatic tissues, using monospecific antibodies directed against the mature protein and against the cytoplasmic domain of proHB-EGF. Prostate carcinoma cells, normal glandular epithelial cells, undifferentiated fibroblasts, and inflammatory cells were not decorated by the anti-HB-EGF antibodies; however, interstitial and vascular smooth muscle cells were highly reactive, indicating that the smooth muscle compartments are the major sites of synthesis and localization of HB-EGF within the prostate. In marked contrast to prostatic epithelium, proHB-EGF was immunolocalized to seminal vesicle epithelium, indicating differential regulation of HB-EGF synthesis within various epithelia of the reproductive tract. HB-EGF was not overexpressed in this series of cancer tissues, in comparison to the benign tissues. In experiments with LNCaP human prostate carcinoma cells, HB-EGF was similar in potency to epidermal growth factor (EGF) in stimulating cell growth. Exogenous HB-EGF and EGF each activated HER1 and HER3 receptor tyrosine kinases and induced tyrosine phosphorylation of cellular proteins to a similar extent. LNCaP cells expressed detectable but low levels of HB-EGF mRNA; however, proHB-EGF was detected at the cell surface indirectly by demonstration of specific sensitivity to DT. HB-EGF is the first HER1 ligand to be identified predominantly as a smooth muscle cell product in the human prostate. Further, the observation that HB-EGF is similar to EGF in mitogenic potency for human prostate carcinoma cells suggests that it may be one of the hypothesized stromal mediators of prostate cancer growth. J. Cell. Biochem. 68:328-338, 1998. © 1998 Wiley-Liss, Inc.     

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

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

Importance of the major extracellular domain of CD9 and the epidermal growth factor (EGF)-like domain of heparin-binding EGF-like growth factor for up-regulation of binding and activity

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors. The membrane-anchored form of HB-EGF (proHB-EGF) is mitogenically active to neighboring cells as well as being a precursor of the soluble form. In addition to its mitogenic activity, proHB-EGF has the property of binding to diphtheria toxin (DT), serving as the specific receptor for DT. Tetramembrane-spanning protein CD9, a member of the TM4 superfamily, is physically associated with proHB-EGF at the cell surface and up-regulates both mitogenic and DT binding activities of proHB-EGF. To understand this up-regulation mechanism, we studied essential regions of both CD9 and proHB-EGF for up-regulation. Immunoprecipitation experiments revealed that not only CD9 but also other TM4 proteins including CD63, CD81, and CD82 associate with proHB-EGF on the cell surface. However, these TM4 proteins did not up-regulate DT binding activity of proHB-EGF. Transfection of a series of chimeric constructs comprising CD9 and CD81 showed that the major extracellular domain of CD9 is essential for up-regulation. Assays of DT binding activity and juxtacrine mitogenic activity of the deletion mutants of proHB-EGF and chimeric molecules, derived from proHB-EGF and TGF-alpha, showed that the essential domain of proHB-EGF for up-regulation is the EGF-like domain. These results indicate that the interaction of the extracellular domains of both molecules is important for up-regulation.     

Metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor and its pathophysiological roles

Heparin-binding EGF-like growth factor (HB-EGF) exists as a membrane-anchored form (proHB-EGF) and as its soluble cleaved product (sHB-EGF). The conversion (ectodomain shedding) of proHB-EGF to sHB-EGF is tightly regulated by specific metalloproteinases. Ectodomain shedding plays a central role in GPCR-mediated EGFR transactivation. Antagonizing metalloproteinases can inhibit EGFR transactivation and might be of therapeutic value, for example in cardiac hypertrophy, skin remodeling and tumor growth.     

Possible role of coexpression of CD9 with membrane-anchored heparin-binding EGF-like growth factor and amphiregulin in cultured human keratinocyte growth

CD9 is a protein with 4 transmembrane domains, and functions as a cell surface antigen. We have previously reported that CD9 functions as an up-regulator of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) activity, which is a potent mitogen as well as a soluble HB-EGF. Anti-CD9 antibodies can neutralize the juxtacrine activity of proHB-EGF when both CD9 and proHB-EGF are coexpressed. We demonstrated here: (1) the CD9 gene was transcribed and translated in the cultured human keratinocytes; (2) anti-CD9 antibody inhibited the approximately 50% growth of human keratinocytes in culture; (3) CD9 was coprecipitated with proHB-EGF and membrane-anchored amphiregulin (proAR), and (4) the transient coexpression of CD9 with proHB-EGF or proAR in mouse L cells up-regulated their juxtacrine growth factor activities. These results suggest that CD9 would make a heterodimer and/or trimer complex with proHB-EGF and proAR, and might cooperate with proHB-EGF and proAR for human keratinocyte growth in a juxtacrine manner. J. Cell. Physiol. 171:291–298, 1997. © 1997 Wiley-Liss, Inc.     

Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) and diphtheria toxin receptor-associated protein (DRAP27)/CD9 form a complex with integrin alpha 3 beta 1 at cell-cell contact sites

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors, which interact with EGF receptor to exert mitogenic activity. The membrane-anchored form of HB- EGF, proHB-EGF, is biologically active, providing mitogenic stimulation to neighboring cells in a juxtacrine mode. ProHB-EGF forms a complex with diphtheria toxin receptor-associated protein (DRAP27)/CD9, a tetra membrane-spanning protein that upregulates the juxtacrine mitogenic activity of proHB-EGF. We explored whether other proteins associate with DRAP27/CD9 and proHB-EGF. Immunoprecipitation with anti-DRAP27/CD9 resulted in preferential coprecipitation of integrin alpha 3 beta 1 from Vero cell, A431 cell and MG63 cell lysates. Anti-integrin alpha 3 or anti-integrin beta 1 coprecipitated DRAP27/CD9 from the same cell lysates. Chemical cross-linking confirmed the physical association of DRAP27/CD9 and integrin alpha 3 beta 1. Using Vero-H cells, which overexpress HB-EGF, we also demonstrated the association of proHB-EGF with DRAP27/CD9 and integrin alpha 3 beta 1. Moreover, colocalization of proHB-EGF, DRAP27/CD9, and integrin alpha 3 beta 1 at cell-cell contact sites was observed by double-immunofluorescence staining. At cell-cell contact sites, DRAP27/CD9 was highly coincident with alpha- catenin and vinculin, suggesting that DRAP27/CD9, proHB-EGF, and integrin alpha 3 beta 1 are colocalized with adherence junction- locating proteins. These results indicate that direct interaction of growth factors and cell adhesion molecules may control cell proliferation during the cell-cell adhesion process.     

Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C

The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca²⁺ionophore, ionomycin, suggesting the involvement of extracellular Ca²⁺ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca²⁺ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca²⁺ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC. J. Cell. Biochem. 69:143–153, 1998. © 1998 Wiley-Liss, Inc.