Heparin-binding EGF-like Growth Factor Gene Research Articles

Effect of pituitary adenylate cyclase-activating polypeptide supplementation, applied during or after vitrification on mouse embryo.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread occurrence and diverse functions. It occurs in high levels in the gonads suggesting a potential central role in reproduction. The aim of our study was to assess the effect of PACAP treatment during embryo vitrification on the developmental rate and the expression of the heparin-binding EGF-like growth factor gene (Hbegf). Mouse embryos, obtained from superovulated females were allocated into the four treatment groups. In EM1 and EM2, the embryos were prepared for vitrification in an Equilibration Solution that was supplemented with 1 or 2 μM PACAP1-38, respectively. The embyos in groups CM1 and CM2 were not treated prior to vitrification but were cultured in a medium supplemented with 1 or 2 μM PACAP1-38 after thawing. The Vitrified Control group consisted of embryos vitrified and thawed then cultured without PACAP1-38 treatment. A non-vitrified, non-treated Fresh Control group was also used. After 24 h of culture, the developmental rate of the embryos, as well as the relative expression level of the Hbegf gene, as determined by qPCR, were compared among groups. Higher developmental rate and Hbegf gene expression level were found in the embryos treated with a higher concentration of PACAP. These results indicate that PACAP treatment has a beneficial effect on the survival and development of vitrified/thawed mouse embryos.

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.

Observation of TNF-α, IL-10 and HB-EGF gene expression by peripheral blood CD14+ mononuclear cells: a case of guttate psoriatic patient

Introduction: Guttate is a type of psoriasis in which patients are sensitive to Streptococcus pneumoniae throughout innate immune responses. During the inflammation, tumour necrosis factor alpha (TNF-α), a well-known pro-inflammatory cytokine, is expressed; meanwhile interleukin 10 (IL-10) and heparin-binding EGF-like growth factor (HB-EGF), which are capable of inhibiting transcription of the TNF-α gene, are also prominent. Furthermore, HB-EGF only impacts fibroblasts and keratinocytes which promote psoriatic lesions. In this study, we looked for differences of TNF-α, IL-10 and HB-EGF expression between a psoriatic patient and a non-psoriatic relative. Methods: To achieve our target, peripheral blood mononuclear cells (PBMCs) expressing LPS receptors or CD14 (CD14+ cells) derived from a guttate patient, and the donor’s father (without psoriatic symptoms), were activated for 7 days by a lysate of Streptococcus pneumoniae for 24 hours before being harvested. Results: Results showed detectable mRNAs of TNF-α, IL-10 and HB-EGF from isolated CD14+ cells of guttate patient were more intensive expression than the non-psoriatic one at 24 hours after engaging the bacterial components. In addition, transcription of HB-EGF gene from the guttate patient was maintained over 168 hours, while its mRNA level from the non-psoriatic volunteer was only expressed within 24 hours. Conclusion: Finally, in initial results of inflammatory effects between strains, the Streptococcal lysate was seen to have stronger immune responses than the Staphylococcal lysate on the immune cells of the guttate psoriasis.

Heparin-binding EGF-like growth factor promotes neuronal nitric oxide synthase expression and protects the enteric nervous system after necrotizing enterocolitis

BackgroundNeonatal necrotizing enterocolitis (NEC) is associated with alterations of the enteric nervous system (ENS), with loss of neuronal nitric oxide synthase (nNOS)-expressing neurons in the intestine. The aim of this study was to investigate the roles of heparin-binding EGF-like growth factor (HB-EGF) in neural stem cell (NSC) differentiation, nNOS expression, and effects on ENS integrity during experimental NEC.MethodsThe effects of HB-EGF on NSC differentiation and nNOS production were determined using cultured enteric NSCs. Myenteric neuronal subpopulations were examined in HB-EGF knockout mice. Rat pups were exposed to experimental NEC, and the effects of HB-EGF treatment on nNOS production and intestinal neuronal apoptosis were determined.ResultsHB-EGF promotes NSC differentiation, with increased nNOS production in differentiated neurons and glial cells. Moreover, loss of nNOS-expressing neurons in the myenteric plexus and impaired neurite outgrowth were associated with absence of the HB-EGF gene. In addition, administration of HB-EGF preserves nNOS expression in the myenteric plexus and reduces enteric neuronal apoptosis during experimental NEC.ConclusionHB-EGF promotes the differentiation of enteric NSCs into neurons in a nitric oxide (NO)-dependent manner, and protects the ENS from NEC-induced injury, providing new insights into potential therapeutic strategies for the treatment of NEC in the future.

Abstract 625: Targeting of Heparin Binding EGF-like Growth Factor (HBEGF) Suppresses Hyperlipidemia and Atherosclerosis in LDL Receptor Deficient Mice

Objective: Elevation of apoB-containing lipoproteins is a well-established risk factor for the development of atherosclerosis. Previous reports showed that expression of heparin-binding EGF-like growth factor (HBEGF), a ligand of epidermal growth factor receptor (EGFR), is associated with atherosclerosis development. In this study, we examined in vivo effects of HBEGF targeting on hyperlipidemia-induced atherosclerosis by suppressing HBEGF expression using antisense oligonucleotide (ASO). Methods and Results: Female and male LDLR deficient mice were fed a high fat diet (HFD; 21% fat, 0.2% cholesterol) throughout the study. After 8 weeks of HFD feeding, mice were injected intraperitoneally with either control or HBEGF ASOs weekly for 12 weeks. At termination, we measured circulating lipid concentrations and atherosclerotic lesion size in the aorta. Compared to control ASO group, HBEGF ASO group had a significant reduction of circulating total cholesterol, triglyceride, and apoB-containing lipoprotein concentrations but no change of high-density lipoprotein (HDL) concentration. Importantly, HBEGF ASO injection significantly suppressed atherosclerosis in the aortic arch, thoracic, and abdominal aorta. HBEGF ASO suppressed sterol synthetic gene expression in the liver but elevated lipid contents in the liver. HBEGF gene silencing in a liver cell system induced downregulation of sterol regulatory element binding protein (SREBP) target genes including LDLR and Insig1. Conclusion: Targeting HBEGF using ASOs is an efficient approach to suppress dyslipidemia and hyperlipidemia-induced atherosclerosis. The differential gene expression analysis suggests that HBEGF ASO administration suppresses SREBP-regulated gene expression in the liver leading to downregulation of circulating cholesterol and TG concentrations.

Genome-wide association study reveals regions associated with gestation length in two pig populations.

Reproduction traits, such as gestation length (GLE), play an important role in dam line breeding in pigs. The objective of our study was to identify single nucleotide polymorphisms (SNPs) that are associated with GLE in two pig populations. Genotypes and deregressed breeding values were available for 2081 Dutch Landrace-based (DL) and 2301 Large White-based (LW) pigs. We identified two QTL regions for GLE, one in each population. For DL, three associated SNPs were detected in one QTL region spanning 0.52Mbp on Sus scrofa chromosome (SSC) 2. For LW, four associated SNPs were detected in one region of 0.14Mbp on SSC5. The region on SSC2 contains the heparin-binding EGF-like growth factor (HBEGF) gene, which promotes embryo implantation and has been described to be involved in embryo survival throughout gestation. The associated SNP can be used for marker-assisted selection in the studied populations, and further studies of the HBEGF gene are warranted to investigate its role in GLE.

HB‐EGF synthesis and release induced by cholesterol depletion of human epidermal keratinocytes is controlled by extracellular atp and involves both p38 and ERK1/2 signaling pathways

The heparin-binding EGF-like growth factor (HB-EGF) is an autocrine/paracrine keratinocyte growth factor, which binds to the epidermal growth factor (EGF) receptor family and plays a critical role during the re-epithelialization of cutaneous wound by stimulating the keratinocytes proliferation and migration. In this study, cellular stressing condition in autocrine cultures of human keratinocytes was induced by cholesterol depletion using methyl-beta-cyclodextrin (MβCD). MβCD treatment induces the expression and the release of HB-EGF. By analysis of the culture media, large amounts of cellular ATP were measured particularly after 1 h of MβCD treatment. To investigate whether ATP contributes to the expression of HB-EGF, the nonhydrolyzable ATP analogue, ATP-γ-S, was used to mimic the extracellular ATP released. We report that keratinocytes stimulated with ATP-γ-S induce HB-EGF expression and activate EGFR and ERK1/2. Using an antagonist of P2 purinergic receptors, we demonstrate that HB-EGF synthesis induced by lipid rafts disruption is dependent on ATP interaction with P2 purinergic receptors. Moreover, our data suggest that both MAPKs p38 and ERK1/2 are involved together or independently in the regulation of HB-EGF gene expression. These findings provide new insight into the signaling pathway by which HB-EGF is expressed after lipid rafts disruption. In summary, after lipid raft disruption, keratinocytes release large amount of extracellular ATP. ATP induces HB-EGF synthesis and release by interacting with the P2 purinergic receptor and through p38 and ERK1/2 signaling in response to a challenging environment. A release of ATP acts as an early stress response in keratinocytes.

EGFR and IL-1 Signaling Synergistically Promote Keratinocyte Antimicrobial Defenses in a Differentiation-Dependent Manner

Ligands of the EGF family regulate autocrine keratinocyte proliferation, and IL-1 family cytokines orchestrate epithelial defense responses. Although members of both families are overexpressed in wound healing and psoriasis, their roles in regulating the innate immune functions of keratinocytes remain incompletely explored. Using sensitive assays, we found significant increases of heparin-binding EGF-like growth factor, transforming growth factor-α, and amphiregulin mRNA and protein in lesional psoriasis compared with uninvolved or control skin. In normal human keratinocyte (NHK) monolayers, EGFR ligands were ineffective in inducing DEFB4, S100A7, and CCL20 mRNAs and human β-defensin (hBD)-2 peptide. Combined with IL-1α, however, EGFR ligands provoked 250 × more DEFB4 and CCL20 and a 9-fold rise in S100A7 mRNA relative to the EGFR ligand alone. This synergy was also reflected in secreted hBD-2 protein, both from NHK and reconstituted human epidermis. Keratinocyte differentiation was critical for these responses, as postconfluent NHK yielded mRNA and protein levels an order of magnitude greater than subconfluent cells. Differentiation also influenced signal transduction, with subconfluent cells using NF-κB and postconfluent cells using EGFR, MEK1/2, and p38. We propose that EGFR ligands are important modifiers of IL-1 activity, synergizing with IL-1 to stimulate epidermal production of hBD-2, S100A7, and CCL20, three of the most upregulated transcripts in psoriatic plaques.

Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

Primary objective. Heparin-binding EGF-like growth factor (HB-EGF) protects the intestine from damage in animals. Future clinical trials of HB-EGF may involve administration of repeated doses of HB-EGF. Since HB-EGF activates EGF receptors which have been implicated in tumor development, we examined the effects of HB-EGF overexpression in the intestine.Research design. We generated transgenic (TG) mice in which the human HB-EGF gene is driven by the villin promoter to overexpress HB-EGF along the crypt-villous axis from the duodenum to the colon.Results. HB-EGF TG mice have increased enterocyte proliferation balanced by increased enterocyte apoptosis. Despite prolonged overexpression of HB-EGF, no evidence of intestinal hyperplasia or tumor formation occurs. Although HB-EGF TG mice have no significant phenotypic alterations under basal conditions, they have increased resistance to intestinal injury.Conclusions. Prolonged intestinal HB-EGF overexpression results in no significant phenotypic alterations under basal conditions, but confers protection against intestinal injury.

In vivo hepatic HB-EGF gene transduction inhibits Fas-induced liver injury and induces liver regeneration in mice: A comparative study to HGF

It is unknown whether heparin-binding EGF-like growth factor (HB-EGF) can be a therapeutic agent, although previous studies suggested that HB-EGF might be a hepatotrophic factor. This study explores the potential of hepatic HB-EGF gene therapy in comparison with HGF. Mice received an intraperitoneal injection of the agonistic anti-Fas antibody 72 h after an intravenous injection of either adenoviral vector (1x10(11) particles) expressing human HB-EGF (Ad.HB-EGF), human HGF (Ad.HGF) or no gene (Ad.dE1.3), and were sacrificed 24 or 36 h later to assess liver injury and regeneration. Exogenous HB-EGF was predominantly localized on the membrane, suggesting the initial synthesis of proHB-EGF in hepatocytes. The control Ad.dE1.3-treated mice represented remarkable increases in serum ALT and AST levels and histopathologically severe liver injuries with numerous apoptosis, but a limited number of mitogenic hepatocytes. In contrast, the liver injuries and apoptotic changes were significantly inhibited, but the mitogenic hepatocytes remarkably increased, in both the Ad.HB-EGF- and Ad.HGF-treated mice. More mitogenic hepatocytes and milder injuries were observed in the Ad.HB-EGF-treated mice. HB-EGF has more potent protective and mitogenic effects for hepatocytes than HGF, at least for the present conditions. In vivo hepatic HB-EGF gene transduction is therapeutic for Fas-induced liver injury.

Heparin-binding EGF-like growth factor is a promising target for ovarian cancer therapy.

Ovarian cancer is the most frequent cause of cancer death among all gynecologic cancers. We demonstrate here that lysophosphatidic acid (LPA)-induced ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF) is a critical to tumor formation in ovarian cancer. We found that among the epidermal growth factor receptor (EGFR) family of growth factors, HB-EGF gene expression in cancerous tissues and HB-EGF protein levels in patients' ascites fluid were significantly elevated. The human ovarian cancer cell lines SKOV3 and RMG-1 form tumors in nude mice. Tumor formation of these cells was enhanced by exogenous expression of pro-HB-EGF and completely blocked by pro-HB-EGF gene RNA interference or by CRM197, a specific HB-EGF inhibitor. Transfection with mutant forms of HB-EGF indicated that the release of soluble HB-EGF is essential for tumor formation. LPA, which is constitutively produced by ovarian cancer cells, induced HB-EGF ectodomain shedding in SKOV3 and RMG-1 cells, resulting in the transactivation of EGFR and the downstream kinase extracellular signal-regulated kinase/mitogen-activated protein kinase. LPA-induced transactivation was abrogated by HB-EGF gene RNA interference or by CRM197. Introduction of lipid phosphate phosphohydrolase, which hydrolyzes LPA, decreased the constitutive shedding of HB-EGF, EGFR transactivation, and the tumorigenic potential of SKOV3 and RMG-1 cells. These results indicate that HB-EGF is the primary member of the EGFR family of growth factors expressed in ovarian cancer and that LPA-induced ectodomain shedding of this growth factor is a critical step in tumor formation, making HB-EGF a novel therapeutic target for ovarian cancer.

Heparin-binding EGF-like growth factor gene transcription regulated by Cdx2 in the intestinal epithelium.

Development and differentiation of the intestinal epithelium appear to be regulated by various growth factors. Using cDNA microarrays, we identified heparin-binding EGF-like growth factor (HB-EGF) as one of the genes induced by intestinal-specific transcription factor Cdx2 in an intestinal undifferentiated rat cell line, intestinal epithelial cell (IEC)-6. Both Cdx2 and HB-EGF stimulated cell proliferation and migration, and their effects were inhibited partially by an EGF receptor-specific tyrosine kinase inhibitor, PD-153035. HB-EGF may function as one of the mediators of Cdx2 and may be associated with the proliferation and migration in the intestinal epithelium. The Cdx2 protein can bind to the Cdx2-binding element of the HB-EGF gene. Reporter gene analyses showed that the HB-EGF gene promoter is Cdx2 responsive and that the activity of the promoter in the IEC-6 cells depends on the number of consensus Cdx2-binding site-like sequences. These data indicate that HB-EGF gene expression can be regulated by Cdx2 and serves to mediate the control of Cdx2 of the proliferation and migration of IEC-6 cells.

Cyclic AMP Inhibits Agonist-Induced Heparin-Binding EGF Gene Expression Independently of Effects on p42/p44 MAPK Activation

Heparin-binding EGF-like growth factor (HB-EGF) mRNA levels are increased up to 20-fold in RIE-1 cells by two agonists that act through distinct receptor types. We demonstrated a common requirement for p42/p44 mitogen-activated protein kinase (MAPK) in this response using the selective MAPK kinase (MEK) inhibitor, PD 098059. Agonist-mediated induction of HB-EGF mRNA was markedly suppressed in cells that had been treated with cyclic AMP-elevating agents. In contrast, the activation of p42 MAPK in response to agonists was not affected by raising cellular cyclic AMP levels. We conclude that cyclic AMP negatively regulates the HB-EGF gene, but that the inhibitory action is either independent of the p42/p44 MAPK pathway or the site of action is distal to MAPK activation.

Heparin-Binding EGF-Like Growth Factor Is Up-Regulated in the Obstructed Kidney in a Cell- and Region-Specific Manner and Acts to Inhibit Apoptosis

The expression of certain growth factors in the epidermal growth factor (EGF) family is altered in response to renal injury. Recent studies have demonstrated that heparin binding EGF-like growth factor (HB-EGF) expression may be cytoprotective in response to apoptotic signals. The purpose of this study was to investigate the potential role of HB-EGF in the upper urinary tract following unilateral ureteral obstruction. We present evidence that: i) ureteral obstruction induced cell-specific but transient activation of HB-EGF gene expression; ii) HB-EGF expression in renal epithelial cells increased under conditions where mechanical deformation, such as that caused by hydronephrotic distension, induces apoptosis, but HB-EGF expression did not increase in renal pelvis smooth muscle cells under identical conditions; and iii) enforced expression of HB-EGF served to protect renal epithelial cells from stretch-induced apoptosis. These results suggest a potential mechanism by which the kidney protects itself from apoptosis triggered by urinary tract obstruction.

Regulation of heparin-binding EGF-like growth factor expression by phorbol ester in a human hepatoma-derived cell line

Heparin-binding EGF-like growth factor (HB-EGF) is a recently identified potent mitogen for smooth muscle cells and fibroblasts. HB-EGF has been shown to be an EGF receptor ligand, and also to stimulate epithelial cell growth. A human hepatoma-derived cell line, Mahlavu, was analyzed for the production of HB-EGF mRNA and active HB-EGF protein. It was found that the cell line synthesized very low or undetectable basal level of HB-EGF mRNA. However, the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a rapid and transient rise in HB-EGF mRNA level. HB-EGF in Mahlavu cells appears to be regulated by a protein kinase C (PKC) pathway, since PKC inhibitors, H7, staurosporin, and calphostin C, abrogated the induction of HB-EGF mRNA by TPA. Unlike vascular smooth muscle cells, induction of HB-EGF gene transcription by TPA was blocked completely by incubation with cycloheximide, suggesting that protein synthesis may be a prerequisite for HB-EGF gene transcription in Mahlavu cells. Mahlavu cells were also found to release a bioactive HB-EGF-like protein into conditioned medium which stimulates DNA synthesis in EP170.7 cells. This activity was neutralized by an anti-HB-EGF antibody. These results indicate that HB-EGF gene transcription is regulated via a PKC pathway, resulting in secretion of active HB-EGF into the culture medium of hepatoma-derived Mahlavu cells.

P-168 Role of heparin-binding EGF-like growth factor (HB-EGF) in rat liver regeneration: Cell specificity of HB-EGF gene expression and induction of HGF and TGF-α gene expression by HB-EGF

P-168 Role of heparin-binding EGF-like growth factor (HB-EGF) in rat liver regeneration: Cell specificity of HB-EGF gene expression and induction of HGF and TGF-α gene expression by HB-EGF

Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation

Heparin-binding EGF-like growth factor (HB-EGF) is a newly discovered member of the EGF family of growth factors. HB-EGF can bind to two loci on cell surfaces, heparan sulphate proteoglycans and EGF-receptor (EGF-R), and either one or both of these interactions could play a role in cell-cell interactions. In the rodent, increased endometrial vascular permeability at the site of blastocyst apposition is considered to be an earliest discernible prerequisite event in the process of implantation and this event coincides with the initial attachment reaction between the blastocyst trophectoderm and uterine luminal epithelium. This investigation demonstrates that the HB-EGF gene is expressed in the mouse uterine luminal epithelium surrounding the blastocyst 6-7 hours before the attachment reaction that occurs at 2200-2300 hours on day 4 of pregnancy. It was further demonstrated that this gene is not expressed in the luminal epithelium at the site of the blastocyst apposition during the progesterone-maintained delayed implantation, but is readily induced in the luminal epithelium surrounding an activated blastocyst after termination of the delay by an estrogen injection. In vitro studies showed that HB-EGF induced blastocyst EGF-R autophosphorylation, and promoted blastocyst growth, zona-hatching and trophoblast outgrowth. These results suggest possible interactions between the uterine HB-EGF and blastocyst EGF-R very early in the process of implantation, earlier than any other embryo-uterine interactions defined to date at the molecular level.

Heparin-Binding EGF-like Growth-Factor Is a Potent Mitogen for Rat Hepatocytes

We examined the hepatotrophic activity of heparin-binding EGF-like growth factor (HB-EGF), a recently identified potent mitogen for vascular smooth muscle cells and tibroblasts. HB-EGF stimulated DNA synthesis of rat hepatocytes in primary culture in a dose-dependent manner up to 30 ng/ml. The maximal stimulation by HB-EGF represented more than 80 % of that induced by HGF. In normal rat liver, the transcript of HB-EGF gene was detected in the non-parenchymal cells and very low level in the hepatocytes. In the regenerating liver on the 3rd day after 70% hepatectomy, the HB-EGF mRNA increased in the non-parenchymal cells, suggesting that HB-EGF may contribute to liver regeneration through a paracrine mechanism.

Heparin-binding EGF-like growth factor gene expression is regulated through protein kinase C pathway in a human hepatoma-derived cell line

Heparin-binding EGF-like growth factor gene expression is regulated through protein kinase C pathway in a human hepatoma-derived cell line