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.     

Epidermal growth factor family in rhesus monkey uterus during the menstrual cycle and early pregnancy

This study examines immunohistochemically the presence of EGF, TGFalpha, HB-EGF, AR, and EGFR, members of the EGF family in the monkey uterus during the menstrual cycle and early pregnancy. EGF, TGFalpha, HB-EGF, AR, and EGFR were mainly localized in glandular and luminal epithelium. TGFalpha, HB-EGF, and AR staining were stronger in the glandular epithelium closer to the myometrium than in that closer to the luminal epithelium. The level of EGF, TGFalpha, HB-EGF, AR, and EGFR staining was low on days 1 and 6, and began to increase on day 9 of the menstrual cycle. A high level of EGF, and EGFR staining was maintained on days 16, 20, and 25 of the menstrual cycle. The highest levels of TGFalpha, AR, and HB-EGF staining were seen on days 16 and 20 of the menstrual cycle. In early pregnancy, a low level of EGF, TGFalpha, HB-EGF, AR, and EGFR staining appeared on days 1 and 2 of pregnancy, and then gradually increased from day 3 of pregnancy. The highest levels of EGF, TGFalpha, HB-EGF, and EGFR were detected on days 9, and 11 of pregnancy. Our data suggest that the EGF family may play a role in monkey implantation. Mol. Reprod. Dev. 55:164-174, 2000.     

Epiregulin is more potent than EGF or TGFalpha in promoting in vitro wound closure due to enhanced ERK/MAPK activation

Epiregulin (EPR) is a broad specificity EGF family member that activates ErbB1 and ErbB4 homodimers and all possible heterodimeric ErbB complexes. We have previously shown that topical EPR enhances the repair of murine excisional wounds. The purpose of this study was to determine whether EPR was more effective than EGF or TGFalpha in promoting in vitro wound closure and to compare the EPR induced signal transduction pathways with those activated by EGF and TGFalpha. Normal human epidermal keratinocytes or A431 cells were scratch wounded and treated for 24 h with varying doses of EPR, EGF or TGFalpha. Five-fold lower doses of EPR were significantly better than EGF or TGFalpha in stimulating in vitro wound closure. Mitomycin-c reduced EPR induced wound closure by 59%, versus a 9% and 25% decrease in EGF and TGFalpha induced closure. The ERK/MAPK inhibitor PD-98059 decreased EPR induced wound closure by 88%. By contrast, the PLC inhibitor U-73122, only reduced the EPR induced response by 21%. Immunoblot analysis revealed that 2 nM EPR stimulated a six-fold increase in p-ERK1/2, whereas 10 nM EGF or TGFalpha stimulated only a 3- and 2.5-fold increase in p-ERK1/2. When compared with EGF or TGFalpha, EPR is a more potent and more effective inducer of in vitro wound closure due to its ability to promote significantly greater ERK/MAPK activation.     

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.     

c-Jun regulates eyelid closure and skin tumor development through EGFR signaling

To investigate the function of c-Jun during skin development and skin tumor formation, we conditionally inactivated c-jun in the epidermis. Mice lacking c-jun in keratinocytes (c-jun(Deltaep)) develop normal skin but express reduced levels of EGFR in the eyelids, leading to open eyes at birth, as observed in EGFR null mice. Primary keratinocytes from c-jun(Deltaep) mice proliferate poorly, show increased differentiation, and form prominent cortical actin bundles, most likely because of decreased expression of EGFR and its ligand HB-EGF. In the absence of c-Jun, tumor-prone K5-SOS-F transgenic mice develop smaller papillomas, with reduced expression of EGFR in basal keratinocytes. Thus, using three experimental systems, we show that EGFR and HB-EGF are regulated by c-Jun, which controls eyelid development, keratinocyte proliferation, and skin tumor formation.     

TGFalpha and EGFR in ovine preimplantation embryos and effects on development

The present study aimed to assess location and relative amounts of transforming growth factor alpha (TGFalpha) and its receptor (EGFR) in ovine oocytes and preimplantation embryos by using immunohistochemical technique that was graded on a relative scale of 0-3, with 0 representing absence of staining, and 3 exhibiting prominent staining, and to evaluate the effects of TGFalpha/EGF on in vitro development of preimplantation embryos by adding different concentrations of EGF and TGFalpha to culture medium. The results showed that EGFR was abundant in cell plasma membranes in immature and mature oocytes, cumulus cells of immature cumulus-oocyte complexes (COC), fertilized oocytes and at different stages of embryo development. However, the relative amounts in inner cell mass (ICM) (1+) was less than that in trophectoderm (TE) cells (2+) at the blastocysts stage. The staining pattern for TGFalpha was a similar to EGFR. However, the staining for TGFalpha slightly increased in the fertilized oocytes (1-2+) as compared to immature and mature oocytes (1+). TGFalpha was mainly detected in the cytoplasm close to the membrane in both ICM and trophectoderm (TE) cells. The developmental rate of 8-cell stage embryos cultured with 5 ng/ml TGFalpha was increased as compared to other treatments (P<0.05). There was no significant difference in the rate of development of blastocysts cultured with 5 ng/ml TGFalpha, 20 ng/ml EGF, 20 ng/ml EGF+5 ng/ml TGFalpha or the control treatment (P>0.05). In addition, there was no significant difference in the number of cells in blastocyst stage as compared with different treatments (P>0.05). However, TGFalpha alone enhanced cell survival rated (P<0.01) and reduced apoptosis. We concluded that TGFalpha can improve development of ovine preimplantation embryos at the 8-cell and blastocyst stages in vitro.     

A Lactobacillus rhamnosus GG-derived Soluble Protein,p40, Stimulates Ligand Release from Intestinal Epithelial Cells to Transactivate Epidermal Growth Factor Receptor

p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis, and preserves barrier function by transactivation of the EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study is to determine the mechanisms by which p40 transactivates the EGFR in intestinal epithelial cells. Here we show that p40-conditioned medium activates EGFR in young adult mouse colon epithelial cells and human colonic epithelial cell line, T84 cells. p40 up-regulates a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) catalytic activity, and broad spectrum metalloproteinase inhibitors block EGFR transactivation by p40 in these two cell lines. In ADAM17-deficient mouse colonic epithelial (ADAM17^−/− MCE) cells, p40 transactivation of EGFR is blocked, but can be rescued by re-expression with WT ADAM17. Furthermore, p40 stimulates release of heparin binding (HB)-EGF, but not transforming growth factor (TGF)α or amphiregulin, in young adult mouse colon cells and ADAM17^−/− MCE cells overexpressing WT ADAM17. Knockdown of HB-EGF expression by siRNA suppresses p40 effects on transactivating EGFR and Akt, preventing apoptosis, and preserving tight junction function. The effects of p40 on HB-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGFR activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion. Thus, these data define a mechanism of a probiotic-derived soluble protein in modulating intestinal epithelial cell homeostasis through ADAM17-mediated HB-EGF release, leading to transactivation of EGFR.     

Effects of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), and 2,3,7,8-tetrachlorodibenzo-p-dioxin on fusion of embryonic palates in serum-free organ culture using wild-type, EGF knockout, and TGFalpha knockout mouse strains

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing cleft palate (CP). TCDD exposure disrupts expression of epidermal growth factor (EGF) receptor, EGF, and transforming growth factor-alpha (TGFalpha) in the palate and affects proliferation and differentiation of medial epithelial cells. EGF knockout embryos are less susceptible to the induction of CP by TCDD. This study used palate organ culture to examine the hypothesis that EGF enables a response to TCDD. METHODS: The midfacial tissues from wild-type (WT), EGF knockout, C57BL/6J, and TGFalpha knockout embryos were placed in organ culture on gestational day (GD) 12. Palatal explants were cultured for 4 days in serum-free Bigger's (BGJ) medium with 0.1% dimethyl sulfoxide (DMSO) or 1 x 10(-8) M TCDD with or without 2 ng of EGF/ml, 1 or 2 ng of TGFalpha/ml. Effects on palatal fusion were evaluated on day 4 of culture. EGF levels in explants and medium were determined using Luminex technology. RESULTS: In serum-free, control medium, palates from all of the strains fused. EGF knockout palates cultured with TCDD (no EGF) fused, but those cultured with TCDD + 2 ng of EGF/ml failed to fuse (p < 0.05 vs. control or TCDD without EGF). TGFalpha knockout palates failed to fuse when cultured with TCDD + 2 ng of TGFalpha/ml. EGF levels increased in tissue and accumulated in the medium after 24 hr of culture. CONCLUSIONS: This study demonstrated that providing EGF to the palates of EGF knockout mice restored the response to TCDD. These studies support the hypothesis that the mechanism for induction of CP by TCDD is mediated via the EGFR pathway.     

ErbB and HB-EGF signaling in heart development and function

The epidermal growth factor (EGF)-ErbB signaling network is composed of multiple ligands of the EGF family and four tyrosine kinase receptors of the ErbB family. In higher vertebrates, these four receptors bind a multitude of ligands. Ligand binding induces the formation of various homo- and heterodimers of ErbB, potentially providing for a high degree of signal diversity. ErbB receptors and their ligands are expressed in a variety of tissues throughout development. Recent advances in gene targeting strategies in mice have revealed that the EGF-ErbB signaling network has fundamental roles in development, proliferation, differentiation, and homeostasis in mammals. The heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR/ErbB1) and ErbB4. Recent studies using several mutant mice lacking HB-EGF expression have revealed that HB-EGF has a critical role in normal heart function and in normal cardiac valve formation in conjunction with ErbB receptors. HB-EGF signaling through ErbB2 is essential for the maintenance of homeostasis in the adult heart, whereas HB-EGF signaling through EGFR is required during cardiac valve development. In this review, we introduce and discuss the role of ErbB receptors in heart function and development, focusing on the physiological function of HB-EGF in these processes.     

5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-alpha-converting enzyme (TACE) activation and heparin-bound epidermal growth factor-like growth factor (HB-EGF) shedding in mesangial cells

In this study, we present multiple lines of evidence to support a critical role for heparin-bound EGF (epidermal growth factor)-like growth factor (HB-EGF) and tumor necrosis factor-alpha-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT(2A) receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells. ERK phosphorylation was attenuated by 1) neutralizing EGFR antibodies and the EGFR kinase inhibitor, AG1478, 2) neutralizing HB-EGF, but not amphiregulin, antibodies, heparin, or CM197, and 3) pharmacological inhibitors of matrix-degrading metalloproteinases or TACE small interfering RNA. Exogenously administered HB-EGF stimulated ERK phosphorylation. Additionally, TACE was co-immunoprecipitated with HB-EGF. Small interfering RNA against TACE also blocked 5-HT-induced increases in ERK phosphorylation, HB-EGF shedding, and DNA content. In aggregate, this work supports a pathway map that can be depicted as follows: 5-HT --> 5-HT(2A) receptor --> TACE --> HB-EGF shedding --> EGFR --> ERK --> increased DNA content. To our knowledge, this is the first time that TACE has been implicated in 5-HT-induced EGFR transactivation or in proliferation induced by a G protein-coupled receptor in native cells in culture.     

Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding

"Transactivation" of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrine/paracrine shedding of heparin-binding EGF (HB-EGF). HB-EGF shedding involves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, alpha(2A)-adrenergic receptors (ARs) stimulate ERK phosphorylation via two distinct pathways, a transactivation pathway that involves the release of HB-EGF and the EGFR and an alternate pathway that is independent of both HB-EGF and the EGFR. We have developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells. In this system, alpha(2A)AR expressing "donor" cells are co-cultured with "acceptor" cells lacking the alpha(2A)AR. Each population expresses a uniquely epitope-tagged ERK2 protein, allowing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the alpha(2)AR selective agonist UK14304 rapidly increases ERK2 phosphorylation in both the donor and the acceptor cells. The acceptor cell response is sensitive to inhibitors of both the EGFR and HB-EGF, indicating that it results from the release of HB-EGF from the alpha(2A)AR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after alpha(2A)AR stimulation requires Gbetagamma subunits upstream and dynamin-dependent endocytosis downstream of HB-EGF shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.     

Metalloproteinases and transforming growth factor-alpha mediate substance P-induced mitogen-activated protein kinase activation and proliferation in human colonocytes

Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of proinflammatory cytokines from colonic epithelial cells. SP also stimulates cell proliferation, a critical event in tissue healing during chronic colitis, via transactivation of the epidermal growth factor (EGF) receptor (EGFR) and activation of mitogen-activated protein kinase (MAPK). Here we examined the mechanism by which SP induces EGFR and MAPK activation. We used non-transformed human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) as well as untransfected U373 MG cells expressing high levels of endogenous NK-1R. Exposure of both cell lines to SP (10(-7) m) stimulated EGFR activation (1 min) followed by extracellular signal-regulated protein kinase (ERK1/2) activation (2-5 min). SP-induced ERK1/2 activation was blocked by pretreatment with the metalloproteinase inhibitor Batimastat/GM6001, the EGFR phosphorylation inhibitor AG1478, and the tumor necrosis factor-alpha-converting enzyme (TACE) inhibitor TAPI-1. Pretreatment with antibodies against potential EGFR ligands suggested that transforming growth factor-alpha (TGFalpha), but not the other EGFR ligands EGF, heparin-binding EGF, or amphiregulin, mediates SP-induced EGFR transactivation. SP stimulated TGFalpha release into the extracellular space that was measurable within 2 min, and this release was inhibited by metalloproteinase inhibitors and the TACE inhibitor TAPI-1. SP also induced MAPK-mediated cell proliferation that was inhibited by TACE, matrix metalloproteinase (MMP), EGFR, and MEK1 inhibitors. Thus, in human colonocytes, NK-1R-induced EGFR and MAPK activation and cell proliferation involve matrix metalloproteinases (most likely TACE) and the release of TGFalpha. These signaling mechanisms may be involved in the protective effects of NK-1R in chronic colitis.     

Transforming growth factor-alpha short-circuits downregulation of the epidermal growth factor receptor

Transforming growth factor-alpha (TGFalpha) is an epidermal growth factor receptor (EGFR) ligand which is distinguished from EGF by its acid-labile structure and potent transforming function. We recently reported that TGFalpha induces less efficient EGFR heterodimerization and downregulation than does EGF (Gulliford et al., 1997, Oncogene, 15:2219-2223). Here we use isoform-specific EGFR and ErbB2 antibodies to show that the duration of EGFR signalling induced by a single TGFalpha exposure is less than that induced by equimolar EGF. The protein trafficking inhibitor brefeldin A (BFA) reduces the duration of EGF signalling to an extent similar to that seen with TGFalpha alone; the effects of TGFalpha and BFA on EGFR degradation are opposite, however, with TGFalpha sparing EGFR from downregulation but BFA accelerating EGF-dependent receptor loss. This suggests that BFA blocks EGFR recycling and thus shortens EGF-dependent receptor signalling, whereas TGFalpha shortens receptor signalling and thus blocks EGFR downregulation. Consistent with this, repeated application of TGFalpha is accompanied by prolonged EGFR expression and signalling, whereas similar application of EGF causes receptor downregulation and signal termination. These findings indicate that constitutive secretion of pH-labile TGFalpha may perpetuate EGFR signalling by permitting early oligomer dissociation and dephosphorylation within acidic endosomes, thereby extinguishing a phosphotyrosine-based downregulation signal and creating an irreversible autocrine growth loop.     

Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R beta subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR.Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.     

Heparin binding epidermal growth factor-like growth factor is a transforming growth factor beta-regulated gene in intestinal epithelial cells.

Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an EGF-related peptide with prominent effects on cell growth and migration. We explored potentially unique characteristics of HB-EGF in the intestinal epithelial cell line RIE-1. HB-EGF stimulated [(3)H]thymidine incorporation to a level equivalent to transforming growth factor alpha (TGFalpha). HB-EGF also rapidly activated MAPK and induced cyclin D1 in mid-G1 with kinetics similar to TGFalpha. Unlike TGFalpha, HB-EGF mRNA was induced within 1 h by a variety of stimuli, including TGFbeta1. Maximal induction by TGFbeta (7-fold) occurred within 2 h of treatment. Actinomycin D decay curves showed that TGFbeta1 had no effect on HB-EGF mRNA half-life (T(1/2) 20 min). Induction of HB-EGF by TGFbeta1 was not affected by pretreatment with the MEK inhibitor PD-98059 while inhibition of protein kinase C either partially (calphostin C) or completely (staurosporin) blocked induction. Our results suggest that major differences exist in the regulation of the closely related EGF family members TGFalpha and HB-EGF. TGFbeta and HB-EGF, structurally unrelated peptides with potent effects on wound healing, may function coordinately to mediate responses to wounding or cell injury in the intestinal epithelium.     

Role of metalloproteinase-dependent EGF receptor activation in alpha-adrenoceptor-stimulated MAP kinase phosphorylation in GT1-7 neurons

Adrenoceptors (ARs) are involved in the regulation of gonadotropin-releasing hormone (GnRH) release from native and immortalized hypothalamic (GT1-7) neurons. However, the AR-mediated signaling mechanisms and their functional significance in these cells are not known. Stimulation of GT1-7 cells with the alpha1-AR agonist, phenylephrine (Phe), causes phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinases that is mediated by protein kinase C (PKC)-dependent transactivation of the epidermal growth factor receptor (EGF-R). Phe stimulation causes shedding of the soluble ligand, heparin-binding EGF (HB-EGF), as a consequence of matrix metalloproteinase (MMP) activation. Phe-induced phosphorylation of the EGF-R, and subsequently of Shc and ERK1/2, was attenuated by inhibition of MMP or HB-EGF with the selective inhibitor, CRM197, or by a neutralizing antibody. In contrast, phosphorylation of the EGF-R, Shc and ERK1/2 by EGF and HB-EGF was independent of PKC and MMP activity. Moreover, inhibition of Src attenuated ERK1/2 responses by Phe, but not by HB-EGF and EGF, indicating that Src acts upstream of the EGF-R. Consistent with a potential role of reactive oxygen species (ROS), Phe-induced phosphorylation of EGF-R was attenuated by the antioxidant, N-acetylcysteine. These data suggest that activation of the alpha1-AR causes phosphorylation of ERK1/2 through activation of PKC, ROS and Src, and shedding of HB-EGF, which binds to and activates the EGF-R.