Dual intracellular signaling by proteolytic cleavage of membrane-anchored heparin-binding EGF-like growth factor

Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is synthesized as a membrane-anchored precursor (proHB-EGF) that is cleaved to release a soluble HB-EGF by specific metalloproteases. Proteolytic cleavage of proHB-EGF yields amino- and carboxy-terminal fragments (HB-EGF and HB-EGF-C). Recent studies indicate that the processing of proHB-EGF is strictly regulated and involved in a variety of biological processes and that not only HB-EGF but also HB-EGF-C functions as a signaling molecule. ProHB-EGF generates dual intracellular signaling molecules by its proteolytic cleavage.     

Cytoplasmic domain phosphorylation of heparin-binding EGF-like growth factor

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a transmembrane precursor protein that is anchored to the plasma membrane. The extracellular EGF-like domain acts as a mitogen and motogen upon ectodomain shedding, but the functional roles of the transmembrane and cytoplasmic domains are largely unknown. We demonstrate here that cytoplasmic domain of HB-EGF is phosphorylated by external stimuli, and that the phosphorylation site is involved in HB-EGF-dependent tumorigenesis. Treatment of Vero cells overexpressing human HB-EGF with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused ectodomain shedding of HB-EGF and generated two carboxyl (C)-terminal fragments with distinct electrophoretic mobilities. Mutation analysis showed that Ser207 in the cytoplasmic domain of HB-EGF is phosphorylated upon TPA stimulation, generating two C-terminal fragments with distinct phosphorylation states. Treatment of cells with lysophosphatidic acid, anisomycin, and calcium ionophore, all of which are known to induce ectodomain shedding, also caused phosphorylation of HB-EGF. Although ectodomain shedding and phosphorylation of HB-EGF occurred coordinately, Ala substitution of Ser207 had no effect on TPA-induced or constitutive ectodomain shedding. Injection of cells overexpressing HB-EGF into nude mice showed that Ala substitution of Ser207 reduced the tumorigenic activity of HB-EGF, even though the cell surface level and ectodomain shedding of HB-EGF were not affected by the mutation. Moreover, we found that the cytoplasmic domain of another EGFR ligand, transforming growth factor-alpha, is phosphorylated upon TPA stimulation. Thus, the present results suggest a novel role for the cytoplasmic domain of HB-EGF and other EGF family growth factors that is regulated by phosphorylation.     

Variation in the proregion structure of heparin-binding EGF-like growth factor precursors

In a previous study, we have isolated and characterized cDNA encoding a novel `short form' of heparin-binding EGF-like growth factor (SF HB-EGF) (Loukianov et al., 1997). In the present work, we have found that cDNA for SF HB-EGF and for full-length HB-EGF are each represented by two variants, which we refer to as L and P forms. The L form is the previously known form of HB-EGF cDNA and encodes a leucine in position 33. The P form described in this report, encodes a proline in codon 33. The L33P substitution is predicted to cause a significant alteration in the proregion structure of SF HB-EGF and HB-EGF.     

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-methyl-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.     

Regulated expression of heparin-binding EGF-like growth factor (HB-EGF) in the human endometrium: a potential paracrine role during implantation

Heparin-binding epidermal growth factor (HB-EGF) is a recently identified member of the EGF growth factor family found to be expressed in the uterus of both mouse and human at the time of implantation. In the present study, we investigated the expression patterns of HB-EGF in normal cycling endometrium and compared its expression with the fertility-associated endometrial epithelial biomarkers alpha(v)beta(3) integrin, leukemia inhibitory factor (LIF) and homeobox gene, HOXA-10. RNase protection assay (RPA) using RNA made from endometrium collected from different phases of the menstrual cycle demonstrated increased HB-EGF expression during the mid-secretory phase, a pattern similar to, but slightly preceding the expression of alpha(v)beta(3) integrin and HOXA-10. In vitro studies demonstrated stimulation of HB-EGF expression by estradiol-17beta (E(2)) and progesterone (P(4)) alone or in combination in stromal cells. Combined treatment with E(2) + P(4) was, however, required to stimulate epithelial HB-EGF expression. In vitro experiments demonstrated the ability of HB-EGF to stimulate epithelial expression of the key endometrial proteins including LIF, HOXA-10, and the beta(3) integrin subunit. Each has previously been demonstrated to be an important epithelial biomarker expressed during the implantation window. In addition, conditioned media from endometrial stromal cells treated with E(2) + P(4) + relaxin mimicked the stimulatory effect of HB-EGF on epithelial expression of the beta(3) integrin subunit. The stimulatory effect of the stromal-conditioned medium was blocked by antibodies that neutralize a known receptor for HB-EGF. These data suggest that uterine receptivity may be regulated in part by the stromal-derived HB-EGF.     

Short form of the heparin-binding EGF-like growth factor: Communication II

The structure of the green monkey Chlorocebus aethiops heparin-binding EGF-like growth factor (HB-EGF) gene was compared with that of the corresponding human gene. Exon 3a, characteristic of the short form of HB-EGF (SF-HB-EGF), was mapped between exons 3 and 4, approximately 700 bp away from the latter. In several human and simian cell lines, most of the SF-HB-EGF mRNA proved to lack exons 4 and 5, specific to the HB-EGF mRNA. In contrast to the HB-EGF mRNA, the SF-HB-EGF mRNA occurred predominantly in the P, rather than L, form, which codes for a protein with a different propeptide structure. Labeled SF-HB-EGF competed with HB-EGF and EGF for binding to the surface of A431 cells, suggesting its interaction with the specific EGF receptor. The results indicate that SF-HB-EGF plays a specific role in cell signaling.     

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.     

The short form of heparin-binding EGF-like growth factor. Part II

The structure of monkey (Chlorocebus aethiops) heparin-binding EGF-like growth factor (HB-EGF) gene has been investigated in this work in comparison with the known structure of human gene. It was shown that HB-EGF short form (SF-HB-EGF) specific exon 3a is mapped between exons 3 and 4 at distance 700 b.p. from exon 4. In a number of human and simian cell lines the main part of SF-HB-EGF mRNA does not contain HB-EGF mRNA specific exons 4 and 5. In comparison with HB-EGF mRNA in SF-HB-EGF mRNA P-form, but not L-form of is predominant, and this mRNA encodes a polypeptide with changed propeptide structure. Labeled SF-HB-EGF competes with HB-EGF and EGF for binding sites at A431 cell surface, which may be due to interaction with specific receptor. All the data suggest a specific role of SF-HB-EGF in cellular signalization.     

Stretch activates heparin-binding EGF-like growth factor expression in bladder smooth muscle cells

Cultured rat bladder smooth muscle cells (SMC) were grown oncollagen-coated silicone membranes and subjected to continuous cyclesof stretch-relaxation. Semiquantitative RT-PCR analysis revealed atime-dependent increase in heparin-binding epidermal growth factor(EGF)-like growth factor (HB-EGF) mRNA levels after stretch, withmaximal levels appearing after 4 h. Immunostaining for proHB-EGFrevealed higher levels of HB-EGF protein in the stretched than in thenonstretched SMC. The ANG II receptor type 1 antagonist losartanmarkedly suppressed stretch-activated HB-EGF expression. ANG II levelswere 3.3-fold higher in the stretch- than in thenon-stretch-conditioned media. Stretch stimulation of bladder SMC thathad been transiently transfected with an HB-EGF promoter-luciferaseexpression construct resulted in an 11-fold increase in reporteractivity. Mechanical stretch induced a 4.7-fold increase in tritiatedthymidine incorporation rate, and this was reduced by 25% in thepresence of losartan. We conclude that mechanical stretch activatesHB-EGF gene expression in bladder SMC and that this is mediated in partby autocrine ANG II secretion.

     

Expression of mRNA for a short form of heparin-binding EGF-like growth factor

In this paper we report the cloning and characterization of cDNA encoding a novel, short form of heparin-binding EGF-like growth factor (SF HB-EGF), and show expression of specific mRNA in various tissues and cell types. Our data suggest that SF HB-EGF mRNA is a product of alternative splicing. Like normal HB-EGF, SF HB-EGF contains the signal peptide, the propeptide, the heparin-binding domain and the first two conservative disulfide loops of the EGF unit. Instead of the third disulfide loop, the spacer, the transmembrane and the cytoplasmic domains, SF HB-EGF has a nine amino acid tail.     

An oxidative stress mechanism mediates chelerythrine-induced heparin-binding EGF-like growth factor ectodomain shedding

Regulated shedding of cell surface proteins is a mechanism for rapid activation of autocrine and paracrine signaling. Here we report that chelerythrine, a protein kinase C (PKC) inhibitor that possesses a variety of biological functions, is a potent inducer of heparin-binding epidermal growth factor-like growth factor (HB-EGF) shedding from the cell surface. Chelerythrine induced a time- and dose-dependent shedding of an HB-EGF-alkaline phosphatase (HB-EGF-AP) fusion protein expressed in MC2 rat prostate epithelial cells. The soluble form of HB-EGF-AP bound to heparin and exhibited potent biological activity as measured by DNA synthesis assay. Chelerythrine-induced HB-EGF shedding was metalloproteinase-(MMP-) mediated because specific MMP antagonists inhibited shedding by > or =60%. Chelerythrine stimulated production of reactive oxygen species, and antioxidants prevented chelerythrine-induced HB-EGF shedding, suggesting that the production of intracellular peroxides is necessary for this event. Consistent with this possibility, antioxidant- and MMP-inhibitable shedding was also demonstrated when hydrogen peroxide was used as an inducer. Although JNK/SAPK and p38 MAPK pathways were activated by chelerythine, these signaling mechanisms were not required to mediate the shedding event. However, JNK signaling was involved in chelerythrine-stimulated apoptosis. Our results suggest that HB-EGF shedding induced by chelerythrine is mediated predominantly via the production of reactive oxygen species.     

A short form of the heparin-binding EGF-like growth factor with a changed EGF domain

In all secreted proteins related to the epidermal growth factor (EGF), EGF domains that occur in a mature factor are each encoded by two exons, and those that do not, by one exon. During splicing, additional exon 3a can be inserted between exons 3 and 4, which code for the EGF domain of the mature heparin-binding EGF-like growth factor (HB-EGF). The resulting mRNA codes for the short form of HB-EGF (SF HB-EGF), which retains the signal peptide, the propeptide, and the heparin-binding domain. However, its EGF domain lacks the C-terminal subdomain essential for the interaction with the EGF receptor (EGFR). Structural analysis suggested that SF HB-EGF is a secreted polypeptide that has high affinity for heparin, but weakly, if at all, interacts with EGFR. Data obtained in three different systems indicated that SF HB-EGF possesses a mitogenic activity but utilizes a signal transduction pathway other than that of HB-EGF.     

Soluble form of heparin-binding EGF-like growth factor contributes to retinoic acid-induced epidermal hyperplasia

Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF-family, is thought to be important for keratinocyte functions. HB-EGF is first synthesized as a membrane-anchored form, and its soluble form is released by ectodomain shedding. Here we investigate the role of HB-EGF in epidermal hyperplasia induced by all-trans retinoic acid (tRA) treatment. HB-EGF is normally expressed in epidermis of normal adult mice at very low levels, but topical tRA treatment results in epidermal hyperplasia, concomitant with the strong induction of HB-EGF expression in the suprabasal layer. tRA-induced epidermal hyperplasia was reduced both in the keratinocyte-specific HB-EGF null mice (K5-HB(del/del)) and knock-in mice expressing the uncleavable mutant form of HB-EGF (HB(uc/uc)), as compared with wild-type HB-EGF knock-in mice (HB(lox/lox)). Among ErbB tyrosine kinase receptors, EGF receptor (EGFR) and ErbB2 were selectively activated by tRA treatment in skin from wild-type mice, while the activation of these ErbB receptors was significantly reduced in the skin of HB-EGF null mice. These results indicate that expression of HB-EGF and generation of its soluble form, followed by activation of EGFR and ErbB2, are pivotal processes in tRA-induced epidermal hyperplasia.     

Regulation of heparin-binding EGF-like growth factor expression in Ha-ras transformed human mammary epithelial cells

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA and protein expression is induced by EGF in MCF-10A nontransformed and Ha-ras transfected human mammary epithelial cells. The anti-EGF receptor (EGFR) blocking monoclonal antibody (MAb) 225 and the EGFR tyrosine kinase inhibitor PD153035 were able to inhibit the induction of HB-EGF mRNA levels in MCF-10A cells. However, the Ha-ras transformed MCF-10A cells were more refractory to inhibition by these agents and only a combination of the 225 MAb and PD153035 was able to significantly abrogate HB-EGF induction by EGF. The anti-erbB2 MAb L26 which interferes with heterodimer formation was able to block HB-EGF induction in response to EGF in MCF-10A cells and in the Ha-ras transformed cells only when used in combination with either the 225 MAb or PD153035. The MEK inhibitor PD90859 completely blocked EGF induction of HB-EGF mRNA levels in the nontransformed and Ha-ras transformed MCF-10A cells, which indicates that MAPK is involved in the signaling pathway of HB-EGF induction by EGF. An increase in the levels of HB-EGF may, therefore, be an important contributor to oncogenic transformation that is caused by Ha-ras overexpression in mammary epithelial cells. J. Cell. Physiol. 186:233-242, 2001. Published 2001 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.     

Carboxyl-terminal truncation of leucine76 converts heparin-binding EGF-like growth factor from a heparin-enhancible to a heparin-suppressible growth factor

Previous studies have indicated that heparin differentially regulates heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR) mitogenic activity. To further explore this phenomenon, these mitogens were compared under identical cell culture conditions in two different assays. The results of our present investigation demonstrated that AR-mediated mitogenic activity in the murine AKR-2B fibroblast-like cell line was inhibited by heparin, while HB-EGF activity was enhanced. However, the absolute effect of heparin appeared to be cell type specific since HB-EGF mitogenic activity was not dramatically affected by coincubation with heparin when tested on human dermal fibroblasts. Several studies have indicated that mutation of a conserved leucine in the carboxyl-terminal region of both EGF and transforming growth factor-α results in decreased affinity for EGF receptors. Since this leucine is present in the analogous position of HB-EGF, but absent in AR, we examined the effect of deleting this residue by carboxyl-terminal truncation of HB-EGF. Analysis of recombinant forms of HB-EGF demonstrated that HB-EGF can be converted to a heparin-inhibited growth factor if the putative mature form of the protein is truncated by two residues (leucine₇₆ and proline₇₇) at the carboxyl terminus. Further analysis demonstrated that only leucine₇₆ appears to be required for heparin-dependent enhancement of HB-EGF-mediated mitogenic activity, indicating that this amino acid may play a pivotal role in controlling the response of HB-EGF to heparin or related glycosaminoglycan sulfates. Our results also suggest that expression of different HB-EGF forms in vivo could result in the production of HB-EGFs with divergent responses to sulfated glycosaminoglycans and proteoglycans. © 1995 Wiley-Liss, Inc.     

The chemical synthesis and binding affinity to the EGF receptor of the EGF-like domain of heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family of growth factors, was isolated from the conditioned medium of macrophage-like cells. To investigate the effect of N- and C-terminal residues of the EGF-like domain of HB-EGF in the binding affinity to the EGF receptor on A431 cell. We synthesized HB-EGF(44-86) corresponding to the EGF-like domain of HB-EGF and its N- or C-terminal truncated peptides. Thermolytic digestion demonstrated three disulfide bond pairings of the EGF-like domain in HB-EGF is consistent with that of human-EGF and human-TGF-alpha. HB-EGF(44-86) showed high binding affinity to EGF-receptor, like human-EGF. The truncation of the C-terminal Leu86 residue from HB-EGF(44-86), HB-EGF(45-86) or HB-EGF(46-86) caused a drastic reduction in the binding affinity to the EGF receptor. These results suggest that the EGF-like domain of HB-EGF plays an important role in the binding to the EGF receptor, and its C-terminal Leu86 residue is necessary for binding with the EGF-receptor. In addition, the deletion of the two N-terminal residues (Asp44-Pro45) from HB-EGF(44-86) caused a 10-fold decrease in relative binding affinity to the EGF receptor. This indicates that the two N-terminal residues of the EGF-like domain of HB-EGF are necessary for its optimal binding affinity to the EGF receptor.     

The membrane protein CD9/DRAP 27 potentiates the juxtacrine growth factor activity of the membrane-anchored heparin-binding EGF-like growth factor

The membrane-anchored heparin-binding EGF-like growth factor precursor (proHB-EGF)/diphtheria toxin receptor (DTR) belongs to a class of transmembrane growth factors and physically associates with CD9/DRAP27 which is also a transmembrane protein. To evaluate the biological activities of proHB-EGF/DTR as a juxtacrine growth factor and the biological significance of its association with CD9/DRAP27, the mitogenic activity of proHB-EGF/DTR was analyzed using stable transfectants of mouse L cells expressing both human proHB-EGF/DTR and monkey CD9/DRAP27, or either one alone. Juxtacrine activity was assayed by measuring the ability of cells in co-culture to stimulate DNA synthesis in an EGF receptor ligand dependent cell line, EP170.7. LH-2 cells expressing human proHB-EGF/DTR stimulated EP170.7 cell growth moderately. However, LCH-1 cells, a stable co-transfectant expressing both human proHB-EGF/DTR and monkey CD9/DRAP27 cDNAs, dramatically unregulated the juxtacrine growth factor activity of proHB-EGF/DTR approximately 25 times over that of LH-2 cells even though both cell types expressed similar levels of proHB-EGF/DTR on the cell surface. Anti-CD9/DRAP27 antibodies which were not able to neutralize the mitogenic activity of soluble HB-EGF suppressed LCH-1 cell juxtacrine growth activity to the same extent as did anti-HB-EGF neutralizing antibodies and CRM 197, specific inhibitors of human HG-EGF. These findings suggest that optimal expression of the juxtacrine growth activity of proHB-EGF/DTR requires co-expression of CD9/DRAP27. These studies also indicate that growth factor potentiation effects which have been observed previously for soluble growth factors also occurs at the level of cell surface associated growth factors.     

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.