Heparin-binding EGF-like Growth Factor Signaling Research Articles

The astrocyte-produced growth factor HB-EGF limits autoimmune CNS pathology

Central nervous system (CNS)-resident cells such as microglia, oligodendrocytes and astrocytes are gaining increasing attention in respect to their contribution to CNS pathologies including multiple sclerosis (MS). Several studies have demonstrated the involvement of pro-inflammatory glial subsets in the pathogenesis and propagation of inflammatory events in MS and its animal models. However, it has only recently become clear that the underlying heterogeneity of astrocytes and microglia can not only drive inflammation, but also lead to its resolution through direct and indirect mechanisms. Failure of these tissue-protective mechanisms may potentiate disease and increase the risk of conversion to progressive stages of MS, for which currently available therapies are limited. Using proteomic analyses of cerebrospinal fluid specimens from patients with MS in combination with experimental studies, we here identify Heparin-binding EGF-like growth factor (HB-EGF) as a central mediator of tissue-protective and anti-inflammatory effects important for the recovery from acute inflammatory lesions in CNS autoimmunity. Hypoxic conditions drive the rapid upregulation of HB-EGF by astrocytes during early CNS inflammation, while pro-inflammatory conditions suppress trophic HB-EGF signaling through epigenetic modifications. Finally, we demonstrate both anti-inflammatory and tissue-protective effects of HB-EGF in a broad variety of cell types in vitro and use intranasal administration of HB-EGF in acute and post-acute stages of autoimmune neuroinflammation to attenuate disease in a preclinical mouse model of MS. Altogether, we identify astrocyte-derived HB-EGF and its epigenetic regulation as a modulator of autoimmune CNS inflammation and potential therapeutic target in MS.

MicroRNA-194-5p/Heparin-binding EGF-like growth factor signaling mediates dexamethasone-induced activation of pseudorabies virus in rat pheochromocytoma cells

Pseudorabies virus (PRV) is a neurotropic virus, which infects a wide range of mammals. The activity of PRV is gradually suppressed in hosts that have tolerated the primary infection. Increased glucocorticoid levels resulting from stressful stimuli overcome repression of PRV activity. However, the host cell mechanism involved in the activation processes under stressful conditions remains unclear. In this study, infection of rat PC-12 pheochromocytoma cells with neuronal properties using PRV at a multiplicity of infection (MOI) = 1 for 24 h made the activity of PRV be the relatively repressed state, and then incubation with 0.5 μM of the corticosteroid dexamethasone (DEX) for 4 h overcomes the relative repression of PRV activity. RNA-seq deep sequencing and bioinformatics analyses revealed different microRNA and mRNA profiles of PC-12 cells with/without PRV and/or DEX treatment. qRT-PCR and western blot analyses confirmed the negative regulatory relationship of miRNA-194–5p and its target heparin-binding EGF-like growth factor (Hbegf); a dual-luciferase reporter assay revealed that Hbegf is directly targeted by miRNA-194–5p. Further, miRNA-194–5p mock transfection contributed to PRV activation, Hbegf was downregulated in DEX-treated PRV infection cells, and Hbegf overexpression contributed to returning activated PRV to the repression state. Moreover, miRNA-194–5p overexpression resulted in reduced levels of HBEGF, c-JUN, and p-EGFR, whereas Hbegf overexpression suppressed the reduction caused by miRNA-194–5p overexpression. Overall, this study is the first to report that changes in the miR-194–5p-HBEGF/EGFR pathway in neurons are involved in DEX-induced activation of PRV, laying a foundation for the clinical prevention of stress-induced PRV activation.

HB-EGF Signaling Is Required for Glucose-Induced Pancreatic β-Cell Proliferation in Rats.

The molecular mechanisms of β-cell compensation to metabolic stress are poorly understood. We previously observed that nutrient-induced β-cell proliferation in rats is dependent on epidermal growth factor receptor (EGFR) signaling. The aim of this study was to determine the role of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) in the β-cell proliferative response to glucose, a β-cell mitogen and key regulator of β-cell mass in response to increased insulin demand. We show that exposure of isolated rat and human islets to HB-EGF stimulates β-cell proliferation. In rat islets, inhibition of EGFR or HB-EGF blocks the proliferative response not only to HB-EGF but also to glucose. Furthermore, knockdown of HB-EGF in rat islets blocks β-cell proliferation in response to glucose ex vivo and in vivo in transplanted glucose-infused rats. Mechanistically, we demonstrate that HB-EGF mRNA levels are increased in β-cells in response to glucose in a carbohydrate-response element–binding protein (ChREBP)–dependent manner. In addition, chromatin immunoprecipitation studies identified ChREBP binding sites in proximity to the HB-EGF gene. Finally, inhibition of Src family kinases, known to be involved in HB-EGF processing, abrogated glucose-induced β-cell proliferation. Our findings identify a novel glucose/HB-EGF/EGFR axis implicated in β-cell compensation to increased metabolic demand.

Role of Heparin-Binding Epidermal Growth Factor-Like Growth Factor in Oxidative Stress-Associated Metabolic Diseases.

Heparin-binding EGF-like growth factor (HB-EGF) is an EGF family member that interacts with epidermal growth factor receptor (EGFR) and ERBB4. Since HB-EGF was first identified as a novel growth factor secreted from a human macrophage cell line, numerous pathological and physiological functions related to cell proliferation, migration, and inflammation have been reported. Notably, the expression of HB-EGF is sensitively upregulated by oxidative stress in the endothelial cells and functions for auto- and paracrine-EGFR signaling. Overnutrition and obesity cause elevation of HB-EGF expression and EGFR signaling in the hepatic and vascular systems. Modulations of HB-EGF signaling showed a series of protections against phenotypes related to metabolic syndrome and advanced metabolic diseases, suggesting HB-EGF as a potential target against metabolic diseases.

Peptides derived from a short stretch of diphtheria toxin bind to heparin-binding epidermal growth factor-like growth factor

Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) is the receptor for diphtheria toxin (DT). Mutated or truncated, non-toxic DT has been used earlier for HB-EGF-targeted drug delivery and to modulate HB-EGF signaling. In the present work, we have synthesized a peptide corresponding to a 26 amino acid long stretch of the receptor-binding domain of DT. This region of DT makes multiple contacts with HB-EGF and has residues critical for binding to HB-EGF. We show that this peptide and two of its mutants bind to HB-EGF. We have also created recombinant proteins by fusing Maltose-binding Protein (MBP) with these peptides. These recombinant MBP-tagged peptides bind to HB-EGF with affinities in the range of 10−7 to 10−8 M. We have observed that these MBP-tagged peptides can modulate molecular signaling of HB-EGF. Therefore, this 26 amino acid long stretch of DT can be considered as an independent functional segment for binding to HB-EGF. Peptides corresponding to this region may be used for HB-EGF targeted cellular delivery of molecular cargo or to modulate HB-EGF signaling.

Rare mutations of ADAM17 from TOFs induce hypertrophy in human embryonic stem cell-derived cardiomyocytes via HB-EGF signaling.

Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart defects (CHDs). The right ventricular hypertrophy is associated with the survival rate of patients with repaired TOF. However, very little is known concerning its genetic etiology. Based on mouse model studies, a disintergrin and metalloprotease 10/17 (ADAM10 and ADAM17) are the key enzymes for the NOTCH and ErbB pathways, which are critical pathways for heart development. Mutations in these two genes have not been previously reported in human TOF patients. In this study, we sequenced ADAM10 and ADAM17 in a Han Chinese CHD cohort comprised of 80 TOF patients, 286 other CHD patients, and 480 matched healthy controls. Three missense variants of ADAM17 were only identified in 80 TOF patients, two of which (Y42D and L659P) are novel and not found in the Exome Aggregation Consortium (ExAC) database. Point mutation knock-in (KI) and ADAM17 knock-out (KO) human embryonic stem cells (hESCs) were generated by CRISPR/Cas9 and programmed to differentiate into cardiomyocytes (CMs). Y42D or L659P KI cells or complete KO cells all developed hypertrophy with disorganized sarcomeres. RNA-seq results showed that phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt), which is downstream of epidermal growth factor receptor (EGFR) signaling, was affected in both ADAM17 KO and KI hESC-CMs. In vitro experiments showed that these two mutations are loss-of-function mutations in shedding heparin-binding EGF-like growth factor (HB-EGF) but not NOTCH signaling. Our results revealed that CM hypertrophy in TOF could be the result of mutations in ADAM17 which affects HB-EGF/ErbB signaling.

Heparin-binding EGF-like growth factor (HB-EGF) antisense oligonucleotide protected against hyperlipidemia-associated atherosclerosis

Background and aimsHeparin-binding EGF-like growth factor (HB-EGF) is a representative EGF family member that interacts with EGFR under diverse stress environment. Previously, we reported that the HB-EGF-targeting using antisense oligonucleotide (ASO) effectively suppressed an aortic aneurysm in the vessel wall and circulatory lipid levels. In this study, we further examined the effects of the HB-EGF ASO administration on the development of hyperlipidemia-associated atherosclerosis using an atherogenic mouse model. Methods and resultsThe male and female LDLR deficient mice under Western diet containing 21% fat and 0.2% cholesterol content were cotreated with control and HB-EGF ASOs for 12 weeks. We observed that the HB-EGF ASO administration effectively downregulated circulatory VLDL- and LDL-associated lipid levels in circulation; concordantly, the HB-EGF targeting effectively suppressed the development of atherosclerosis in the aorta. An EGFR blocker BIBX1382 administration suppressed the hepatic TG secretion rate, suggesting a positive role of the HB-EGF signaling for the hepatic VLDL production. We newly observed that there was a significant improvement of the insulin sensitivity by the HB-EGF ASO administration in a mouse model under the Western diet as demonstrated by the improvement of the glucose and insulin tolerances. ConclusionThe HB-EGF ASO administration effectively downregulated circulatory lipid levels by suppressing hepatic VLDL production rate, which leads to effective protection against atherosclerosis in the vascular wall.

The actions of sildenafil on trophoblast survival is dependent on HBEGF signaling

Sildenafil (Viagra) has proved useful for alleviating fetal growth restriction, but we have found that it has direct effects on trophoblast invasion and survival during oxidative stress. However, the mechanism is not completely understood. Heparin-binding EGF-like growth factor (HBEGF) has similar effects on human trophoblast cells. Therefore, we examined whether there is a link between sildenafil and HBEGF signaling.

Abstract 40: HB-EGF‑mediated CITED4 signaling in lung cancer

Abstract Abnormal activation of the EGF receptor (EGFR) signaling is a hallmark of lung cancer. However, the role of heparin-binding EGF-like growth factor (HB-EGF), one of EGFR ligands, in lung tumorigenesis has been unappreciated. In this study, we showed that HB-EGF was highly expressed in a subgroup of lung cancer cells, the proliferation of which was dependent on HB-EGF signaling. Members of CITED family proteins respond to various cytokine signalings, regulating cell proliferation; among them, we identified CITED4 as a downstream effecter of HB-EGF. We revealed that CITED4 was upregulated by HB-EGF through a STAT3 dependent pathway. Moreover, CITED4-silencing attenuated HB-EGF-mediated lung cancer cell proliferation. ChIP analysis and promoter reporter assay showed that STAT3 bound CITED4 promoter, enhancing CITED4 transcription. Co-immunoprecipitation analysis identified MYC as a CITED4-interacting protein. Furthermore, we found that CITED4 expression promoted MYC-mediated transcription of cyclin D1 promoter, leading to increased G1/S cell cycle transition. Thus, our findings support that HB-EGF-CITED4 signaling cascade plays a critical role in aberrant proliferation in lung cancer with potential as a biomarker for lung cancer progression. Citation Format: Cheng-Han Hsieh, Yu-Ting Chou, Cheng-Wen Wu. HB-EGF‑mediated CITED4 signaling in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 40. doi:10.1158/1538-7445.AM2015-40

Clarifying the Role of MAP Kinases in the Molecular Pathway That Regulates HBEGF Mobilization for Trophoblast Survival at Low O2 during the First Trimester of Pregnancy

Human trophoblast cells survive at low concentrations of O2 during the first trimester of pregnancy and avoid apoptosis associated with placenta-mediated disorders. Heparin-binding EGF-like growth factor (HBEGF) is upregulated post-transcriptionally in trophoblast cells exposed to low O2, and its downstream signaling prevents apoptosis. HBEGF signaling is associated with MAP kinase (MAPK) activation, which regulates HBEGF biosynthesis downstream of ERBB receptors in a positive feedback loop. MAPKs (ERK, p38 and JNK) could be regulated during hypoxia, inducing transcription of metalloproteases, including MMP2, which is required to shed the extracellular domain of pro-HBEGF and initiate HBEGF signaling.

HCV core protein promotes heparin binding EGF‐like growth factor expression and activates Akt

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction and is closely associated with the development of human hepatocellular carcinoma (HCC). Among HCV components, core protein is implicated in cell growth regulation, and we previously demonstrated that HCV core protein interacted with 14-3-3 protein and activated the kinase Raf-1 and mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK) pathway. In the present study, we investigated the expression levels and function of downstream molecules in the MAPK/ERK signaling pathway in cells expressing HCV core protein. Heparin-binding EGF-like growth factor (HB-EGF) mRNA, in HepG2 cells stably expressing HCV core protein, was detected by RT-PCR. The soluble HB-EGF in culture media was measured by heparin agarose chromatography/Western blot analysis. Immunodetection of Akt and IKK and IB, in HeLa cells and HepG2 cells expressing HCV core protein, were performed with neutralizing antibody for HB-EGF, phospatidylinositol-3-kinase [PI(3)K] inhibitor and dominant-negative mutant of Ras (DN-Ras). HB-EGF expression was significantly elevated in cells expressing HCV core protein. HCV core protein activated Akt through the Ras/PI(3)K pathway by autocrine secretion of HB-EGF. Also, HCV core protein activated IKK through Ras/PI(3)K/Akt pathway by autocrine secretion of HB-EGF. As the Ras/PI(3)K/Akt pathway is critical in anti-apoptotic HB-EGF signaling, we examined the possible role of this pathway in cells expressing HCV core protein. In addition, we investigated the relationship between IB kinases (IKK) and Akt in cells expressing HCV core protein, since IKKs are known to be activated by HCV core protein and by Akt in the presence of potent mitogen. We showed that HCV core protein promoted autocrine secretion of HB-EGF and activated Akt through the Ras/PI(3)K pathway. This model indicates a new approach to mechanism of proliferation and anti-apoptosis in HCC. HCV core protein is a potent activator of mitogenic and anti-apoptotic signaling involved in hepatocarcinogenesis.

Maternal heparin-binding-EGF deficiency limits pregnancy success in mice

An intimate discourse between the blastocyst and uterus is essential for successful implantation. However, the molecular basis of this interaction is not clearly understood. Exploiting genomic Hbegf mutant mice, we show here that maternal deficiency of heparin-binding EGF-like growth factor (HB-EGF) defers on-time implantation, leading to compromised pregnancy outcome. We also demonstrate that amphiregulin, but not epiregulin, partially compensates for the loss of HB-EGF during implantation. In search of the mechanism of this compensation, we found that reduced preimplantation estrogen secretion from ovarian HB-EGF deficiency is a cause of sustained expression of uterine amphiregulin before the initiation of implantation. To explore the significance specifically of uterine HB-EGF in implantation, we examined this event in mice with conditional deletion of uterine HB-EGF and found that this specific loss of HB-EGF in the uterus still defers on-time implantation without altering preimplantation ovarian estrogen secretion. The observation of normal induction of uterine amphiregulin surrounding the blastocyst at the time of attachment in these conditional mutant mice suggests a compensatory role of amphiregulin for uterine loss of HB-EGF, preventing complete failure of pregnancy. Our study provides genetic evidence that HB-EGF is critical for normal implantation. This finding has high clinical relevance, because HB-EGF signaling is known to be important for human implantation.

Juxtacrine Activation of Epidermal Growth Factor (EGF) Receptor by Membrane-anchored Heparin-binding EGF-like Growth Factor Protects Epithelial Cells from Anoikis While Maintaining an Epithelial Phenotype

Loss of cell-matrix adhesion is often associated with acute epithelial injury, suggesting that "anoikis" may be an important contributor to cell death. Resistance against anoikis is a key characteristic of transformed cells. When nontransformed epithelia are injured, activation of the epidermal growth factor (EGF) receptor (EGFR) by paracrine/autocrine release of soluble ligands can induce a prosurvival program, but there is generally evidence for concomitant dedifferentiation. The EGFR ligand, heparin-binding EGF-like growth factor (HB-EGF), is synthesized as a membrane-anchored precursor that can activate the EGFR via juxtacrine signaling or can be released and act as a soluble growth factor. In Madin-Darby canine kidney cells, expression of membrane-anchored HB-EGF increases cell-cell and cell-matrix adhesion. Therefore, these studies were designed to test the effects of juxtacrine HB-EGF signaling upon cell survival and epithelial integrity when cells are denied proper cell-matrix interactions. Cells expressing a noncleavable mutated form of membrane-anchored HB-EGF demonstrated increased survival from anoikis, formed larger cell aggregates, and maintained epithelial characteristics even following prolonged detachment from the substratum. Physical association between membrane-anchored HB-EGF and EGFR was observed. Signaling studies indicated synergistic effects of EGFR activation and phosphatidylinositol 3-kinase signaling to regulate apoptotic and survival pathways. In contrast, although administration of exogenous EGF partially suppressed anoikis in wild type cells, it also led to an increased expression of mesenchymal markers, suggesting dedifferentiation. Taken together, we propose a novel role for membrane-anchored HB-EGF in the cytoprotection of epithelial cells.

Heparin-binding EGF-like growth factor mediates oxyhemoglobin-induced suppression of voltage-dependent potassium channels in rabbit cerebral artery myocytes

Oxyhemoglobin (OxyHb) can suppress voltage-dependent K(+) channel (K(V)) currents through protein tyrosine kinase activation, which may contribute to cerebral vasospasm following subarachnoid hemorrhage. Here we have tested the hypothesis that shedding of heparin-binding EGF-like growth factor (HB-EGF) and the resulting activation of the tyrosine kinase EGF receptor (EGFR) underlie OxyHb-induced K(V) channel suppression in the cerebral vasculature. With the use of the conventional whole cell patch-clamp technique, two EGFR ligands, EGF and HB-EGF, were found to mimic OxyHb-induced K(V) suppression in rabbit cerebral artery myocytes. K(V) current suppression by OxyHb or EGF ligands was eliminated by a specific EGFR inhibitor, AG-1478, but was unaffected by PKC inhibition. Compounds (heparin and CRM-197) that specifically interfere with HB-EGF signaling eliminated OxyHb-induced K(V) suppression, suggesting that HB-EGF is the EGFR ligand involved in this pathway. HB-EGF exists as a precursor protein that, when cleaved by matrix metalloproteases (MMPs), causes EGFR activation. MMP activation was detected in OxyHb-treated arteries by gelatin zymography. Furthermore, the MMP inhibitor (GM-6001) abolished OxyHb-induced K(V) current suppression. We also observed K(V) current suppression due to EGFR activation in human cerebral artery myocytes. In conclusion, these data demonstrate that OxyHb induces MMP activation, causing HB-EGF shedding and enhanced EGFR activity, ultimately leading to K(V) channel suppression. We propose that EGFR-mediated K(V) suppression contributes to vascular pathologies, such as cerebral vasospasm, and may play a more widespread role in the regulation of regional blood flow and peripheral resistance.

Lysophosphatidic Acid Promoting Corneal Epithelial Wound Healing by Transactivation of Epidermal Growth Factor Receptor

To identify the underlying mechanisms by which lipid mediator lysophosphatidic acid (LPA) acts as a growth factor in stimulating extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3K) during corneal epithelial wound healing. Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence or absence of an epidermal growth factor receptor (EGFR) inhibitor (tyrphostin AG1478), a matrix metalloproteinase inhibitor (GM6001), or a heparin-binding EGF-like growth factor (HB-EGF) antagonist (CRM197) with or without LPA. EGFR activation was analyzed by immunoprecipitation using EGFR antibodies and Western blotting with phosphotyrosine antibodies. Phosphorylation of ERK and AKT (a major substrate of PI3K) was analyzed by Western blotting with antibodies specific to the phosphorylated proteins. Wound- and LPA-induced shedding of HB-EGF was assessed by measuring the release of alkaline phosphatase (AP) in a stable THCE cell line that expressed HB-EGF with AP inserted in the heparin-binding site. In organ and cell culture models, LPA enhanced corneal epithelial wound healing. LPA-stimulated and spontaneous wound closure was attenuated by AG1478, GM6001, or CRM197. Consistent with the effects on epithelial migration, these inhibitors, as well as the Src kinase inhibitor (PP2), retarded LPA-induced activation of EGFR and its downstream effectors ERK and AKT in THCE cells. Unlike exogenously added HB-EGF, LPA stimulated moderate EGFR phosphorylation; the level of phosphorylated EGFR was similar to that induced by wounding. However, LPA appeared to prolong wound-induced EGFR signaling. The release of HB-EGF assessed by AP activity increased significantly in response to wounding, LPA, or both, and the release of HB-EGF-AP induced by LPA was inhibited by PP2 and GM6001. LPA accelerates corneal epithelial wound healing through its ability to induce autocrine HB-EGF signaling. Transactivation of EGFR by LPA represents a convergent signaling pathway accessible to stimuli such as growth factors and ligands of G-protein-coupled receptors in response to pathophysiological challenge in human corneal epithelial cells.

Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor.

Heparin-binding EGF-like growth factor (HBEGF), which is expressed in the placenta during normal pregnancy, is down regulated in pre-eclampsia, a human pregnancy disorder associated with poor trophoblast differentiation and survival. This growth factor protects against apoptosis during stress, suggesting a role in trophoblast survival in the relatively low O(2) ( approximately 2%) environment of the first trimester conceptus. Using a well-characterized human first trimester cytotrophoblast cell line, we found that a 4-hour exposure to 2% O(2) upregulates HBEGF synthesis and secretion independently of an increase in its mRNA. Five other expressed members of the EGF family are largely unaffected. At 2% O(2), signaling via HER1 or HER4, known HBEGF receptors, is required for both HBEGF upregulation and protection against apoptosis. This positive-feedback loop is dependent on metalloproteinase-mediated cleavage and shedding of the HBEGF ectodomain. The restoration of trophoblast survival by the addition of soluble HBEGF in cultures exposed to low O(2) and metalloproteinase inhibitor suggests that the effects of HBEGF are mediated by autocrine/paracrine, rather than juxtacrine, signaling. Our results provide evidence that a post-transcriptional mechanism induced in trophoblasts by low O(2) rapidly amplifies HBEGF signaling to inhibit apoptosis. These findings have a high clinical significance, as the downregulation of HBEGF in pre-eclampsia is likely to be a contributing factor leading to the demise of trophoblasts.

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.

Lysophosphatidic acid regulates murine blastocyst development by transactivation of receptors for heparin-binding EGF-like growth factor

Transient elevation of intracellular calcium (Cai2+) by various means accelerates murine preimplantation development and trophoblast differentiation. Several G-protein-coupled receptors (GPCRs), including the lysophosphatidic acid (LPA) receptor (LPAR), induce Cai2+ transients and transactivate the EGF receptor (ErbB1) through mobilization of EGF family members, including heparin-binding EGF-like growth factor (HB-EGF). Because HB-EGF accelerates blastocyst differentiation in vitro, we examined whether crosstalk between LPA and HB-EGF regulates peri-implantation development. During mouse blastocyst differentiation, embryos expressed LPAR1 mRNA constitutively, LPAR2 only in late stage blastocysts and no LPAR3. Consistent with a mechanism based on Cai2+ signaling, LPA rapidly accelerated the rate of trophoblast outgrowth, an index of blastocyst differentiation, and chelation of Cai2+ with BAPTA-AM blocked LPA stimulation. Interfering with HB-EGF signaling through ErbB1 or ErbB4 also attenuated LPA stimulation. We established that mouse blastocysts indeed express HB-EGF and that LPA induces the transient accumulation of HB-EGF on the embryo surface, which was blocked by treatment with either BAPTA-AM or the protein trafficking inhibitor, brefeldin A. We conclude that LPA accelerates blastocyst differentiation through its ability to induce Cai2+ transients and HB-EGF autocrine signaling. Transactivation of ErbB1 or ErbB4 by HB-EGF could represent a convergent signaling pathway accessed in the trophoblast by stimuli that mobilize Cai2+.

Lysophosphatidic acid regulates murine blastocyst development by transactivation of receptors for heparin-binding EGF-like growth factor

Transient elevation of intracellular calcium (Ca i 2+) by various means accelerates murine preimplantation development and trophoblast differentiation. Several G-protein-coupled receptors (GPCRs), including the lysophosphatidic acid (LPA) receptor (LPAR), induce Ca i 2+ transients and transactivate the EGF receptor (ErbB1) through mobilization of EGF family members, including heparin-binding EGF-like growth factor (HB-EGF). Because HB-EGF accelerates blastocyst differentiation in vitro, we examined whether crosstalk between LPA and HB-EGF regulates peri-implantation development. During mouse blastocyst differentiation, embryos expressed LPAR 1 mRNA constitutively, LPAR 2 only in late stage blastocysts and no LPAR 3. Consistent with a mechanism based on Ca i 2+ signaling, LPA rapidly accelerated the rate of trophoblast outgrowth, an index of blastocyst differentiation, and chelation of Ca i 2+ with BAPTA-AM blocked LPA stimulation. Interfering with HB-EGF signaling through ErbB1 or ErbB4 also attenuated LPA stimulation. We established that mouse blastocysts indeed express HB-EGF and that LPA induces the transient accumulation of HB-EGF on the embryo surface, which was blocked by treatment with either BAPTA-AM or the protein trafficking inhibitor, brefeldin A. We conclude that LPA accelerates blastocyst differentiation through its ability to induce Ca i 2+ transients and HB-EGF autocrine signaling. Transactivation of ErbB1 or ErbB4 by HB-EGF could represent a convergent signaling pathway accessed in the trophoblast by stimuli that mobilize Ca i 2+.

Dual source and target of heparin-binding EGF-like growth factor during the onset of implantation in the hamster.

Heparin binding EGF-like growth factor (HB-EGF), encoded by the Hegfl gene, is considered as an important mediator of embryo-uterine interactions during implantation in mice. However, it is unknown whether HB-EGF is important for implantation in species with different steroid hormonal requirements. In mice and rats, maternal ovarian estrogen and progesterone (P(4)) are essential to implantation. In contrast, blastocyst implantation can occur in hamsters in the presence of P(4) alone. To ascertain whether HB-EGF plays any role in implantation in hamsters, we examined the expression, regulation and signaling of HB-EGF in the hamster embryo and uterus during the periimplantation period. We demonstrate that both the blastocyst and uterus express HB-EGF during implantation. Hegfl is expressed solely in the uterine luminal epithelium surrounding the blastocyst prior to and during the initiation of implantation. Hypophysectomized P(4)-treated pregnant hamsters also showed a similar pattern of implantation-specific Hegfl expression. These results suggest that uterine Hegfl expression at the implantation site is driven by either signals emanating from the blastocyst or maternal P(4), but not by maternal estrogen. However, in ovariectomized hamsters, uterine induction of Hegfl requires the presence of estrogen and activation of its nuclear receptor (ER), but not P(4). This observation suggests an intriguing possibility that an estrogenic or unidentified signal from the blastocyst is the trigger for uterine HB-EGF expression. An auto-induction of Hegfl in the uterus by blastocyst-derived HB-EGF is also a possibility. We further observed that HB-EGF induces autophosphorylation of ErbB1 and ErbB4 in the uterus and blastocyst. Taken together, we propose that HB-EGF production and signaling by the blastocyst and uterus orchestrate the 'two-way' molecular signaling to initiate the process of implantation in hamsters.