Heparin-binding Epidermal Growth Factor Protein Research Articles

Abstract P488: FAK Inhibition Reduces Hemangioma Proliferation By Blocking YAP Mediated HBEGF Expression

Hemangiomas are vascular tumors that develop from proliferative endothelial cells (EC). While primarily occurring on the skin of the head, neck, back and chest, hemangiomas can occur within any vascular beds including brain, liver and heart, which can lead to life-threatening complications. Non-surgical treatments are limited, and there are few phases I and II trials underway. Therefore, better understanding of what drives hemangioma proliferation and new therapeutic targets are needed. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that acts as a primary nexus of extracellular signals from integrins and growth factor receptors that sustain EC migration, proliferation, and survival. We observed that genetic and pharmacological inhibition of FAK significantly blocked vascular endothelial growth factor (VEGF)/ fibroblast growth factor (FGF)-induced Matrigel plug angiogenesis in vivo. To identify angiogenic factors regulated by FAK catalytic activity, we used an angiogenesis array in human umbilical vein ECs (HUVECs) and found that FAK inhibition reduced major regulators of angiogenesis including heparin-binding epidermal growth factor (HB-EGF). Further, using immunoblotting and RT-qPCR, we confirmed that FAK inhibition significantly reduced HB-EGF protein and mRNA expression in various mouse, human EC lines and human infantile hemangioma cell line, resulting in reduced EC proliferation. Importantly, HB-EGF mRNA expression was also reduced within in vivo Matrigel plugs of FAK inhibitor treated mice. Mechanistically, FAK binds to and phosphorylates YAP, which enhances HB-EGF expression. Loss of YAP tyrosine phosphorylation following FAK inhibition decreased YAP nuclear localization and protein stability. Since FAK inhibitors are undergoing clinical trials to treat cancer, these results suggest that FAK inhibitors could also prove beneficial in blocking EC proliferation associated with hemangioma.

Melatonin Pretreated Blastocysts along with Calcitonin Administration Improved Implantation by Upregulation of Heparin Binding-Epidermal Growth Factor Expression in Murine Endometrium.

Objective Implantation failure is an obstacle in assisted reproduction techniques (ART). Calcitonin is a molecules involved in uterine receptivity and embryo implantation. Melatonin can promote embryo quality and improve implantation. This study examines the effect of pretreatment of blastocysts with melatonin and calcitonin on heparin binding-epidermal growth factor (HB-EGF) expression in murine endometrium. Materials and Methods In this experimental study, we collected 2-cell embryos from the oviducts of 1.5 day pregnant NMRI mice. Embryos were cultured to the blastocyst in GTM medium with or without 10-9 M melatonin. Pregnant and pseudo-pregnant mice received intraperitoneal (IP) injections of 2 IU calcitonin. After 24 hours, we transferred the cultured blastocysts into the uteri of pseudo-pregnant mice. Two days later, implantation sites were counted and we assessed the levels of HB-EGF mRNA and protein in the uteri of naturally pregnant and pseudo-pregnant mice by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Statistical analysis was performed with one-way ANOVA followed by the Tukey post hoc test. P<0.05 was considered statistically significant. Results Melatonin pretreatment of blastocysts along with calcitonin administration significantly increased HB-EGF mRNA and protein (P<0.001) in the endometrium of pseudo-pregnant mice. Administration of calcitonin in naturally pregnant mice significantly increased HB-EGF mRNA and protein levels (P<0.001). Compared with the control group (2.6 ± 0.5), the average number of implantation sites in the melatonin group (4.6 ± 0.5, P<0.05) and calcitonin group (7 ± 1, P<0.001) significantly increased. There was a significant increase in implantation sites in the combined melatonin and calcitonin group (8.6 ± 0.5, P<0.001). Calcitonin significantly enhanced calcitonin receptor mRNA (P<0.001) and protein (P<0.05) in the uteri of naturally pregnant and pseudo-pregnant mice. ConclusionMelatonin pretreated blastocysts along with calcitonin increased HB-EGF expression in the uteri of pseudo- pregnant mice. Calcitonin administration upregulated HB-EGF in uteri of naturally pregnant mice.

A mouse model of otitis media identifies HB-EGF as a mediator of inflammation-induced mucosal proliferation.

ObjectiveOtitis media is one of the most common pediatric infections. While it is usually treated without difficulty, up to 20% of children may progress to long-term complications that include hearing loss, impaired speech and language development, academic underachievement, and irreversible disease. Hyperplasia of middle ear mucosa contributes to the sequelae of acute otitis media and is of important clinical significance. Understanding the role of growth factors in the mediation of mucosal hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae.MethodsFrom a whole genome gene array analysis of mRNA expression during acute otitis media, we identified growth factors with expression kinetics temporally related to hyperplasia. We then tested these factors for their ability to stimulate mucosal epithelial growth in vitro, and determined protein levels and histological distribution in vivo for active factors.ResultsFrom the gene array, we identified seven candidate growth factors with upregulation of mRNA expression kinetics related to mucosal hyperplasia. Of the seven, only HB-EGF (heparin-binding-epidermal growth factor) induced significant mucosal epithelial hyperplasia in vitro. Subsequent quantification of HB-EGF protein expression in vivo via Western blot analysis confirmed that the protein is highly expressed from 6 hours to 24 hours after bacterial inoculation, while immunohistochemistry revealed production by middle ear epithelial cells and infiltrating lymphocytes.ConclusionOur data suggest an active role for HB-EGF in the hyperplasia of the middle ear mucosal epithelium during otitis media. These results imply that therapies targeting HB-EGF could ameliorate mucosal growth during otitis media, and thereby reduce detrimental sequelae of this childhood disease.

A miR-1207-5p Binding Site Polymorphism Abolishes Regulation of HBEGF and Is Associated with Disease Severity in CFHR5 Nephropathy

Heparin binding epidermal growth factor (HBEGF) is expressed in podocytes and was shown to play a role in glomerular physiology. MicroRNA binding sites on the 3′UTR of HBEGF were predicted using miRWalk algorithm and followed by DNA sequencing in 103 patients diagnosed with mild or severe glomerulopathy. A single nucleotide polymorphism, miRSNP C1936T (rs13385), was identified at the 3′UTR of HBEGF that corresponds to the second base of the hsa-miR-1207-5p seed region. When AB8/13 undifferentiated podocytes were transfected with miRNA mimics of hsa-miR-1207-5p, the HBEGF protein levels were reduced by about 50%. A DNA fragment containing the miRSNP allele-1936C was cloned into the pMIR-Report Luciferase vector and co-transfected with miRNA mimics of hsa-miR-1207-5p into AB8/13 podocytes. In agreement with western blot data, this resulted in reduced luciferase expression demonstrating the ability of hsa-miR-1207-5p to directly regulate HBEGF expression. On the contrary, in the presence of the miRSNP 1936T allele, this regulation was abolished. Collectively, these results demonstrate that variant 1936T of this miRSNP prevents hsa-miR-1207-5p from down-regulating HBEGF in podocytes. We hypothesized that this variant has a functional role as a genetic modifier. To this end, we showed that in a cohort of 78 patients diagnosed with CFHR5 nephropathy (also known as C3-glomerulopathy), inheritance of miRSNP 1936T allele was significantly increased in the group demonstrating progression to chronic renal failure on long follow-up. No similar association was detected in a cohort of patients with thin basement membrane nephropathy. This is the first report associating a miRSNP as genetic modifier to a monogenic renal disorder.

ADAM17 up-regulation in renal transplant dysfunction and non-transplant-related renal fibrosis

Interstitial fibrosis and tubular atrophy (IF/TA) is an important cause of renal function loss and ischaemia-reperfusion (I/R) injury is considered to play an important role in its pathophysiology. The aim of the present study was to investigate the role of a disintegrin and metalloproteinase 17 (ADAM17) in human renal allograft disease and in experimental I/R injury of the kidney. We studied the expression of ADAM17 messenger RNA (mRNA) in IF/TA and control kidneys by reverse transcription-polymerase chain reaction and in situ hybridization. Moreover, we assessed ADAM17-mediated heparin-binding epidermal growth factor (HB-EGF) shedding in immortalized human cells. Finally, we studied the effect of pharmacological ADAM17 inhibition in a model of renal I/R injury in rats. ADAM17 mRNA was up-regulated in IF/TA when compared to control kidneys. In normal kidneys, ADAM17 mRNA was weakly expressed in proximal tubules, peritubular capillaries, glomerular endothelium and parietal epithelium. In IF/TA, tubular, capillary and glomerular ADAM17 expression was strongly enhanced with de novo expression in the mesangium. In interstitial fibrotic lesions, we observed co-localization of ADAM17 with HB-EGF protein. In vitro, inhibition of ADAM17 with TNF484 resulted in a dose-dependent reduction of HB-EGF shedding in phorbol 12-myrisate 13-acetate-stimulated cells and non-stimulated cells. In vivo, ADAM17 inhibition significantly reduced the number of glomerular and interstitial macrophages at Day 4 of reperfusion. In conclusion, HB-EGF co-expresses with ADAM17 in renal interstitial fibrosis, suggesting a potential interaction in IF/TA. Targeting ADAM17 to reduce epidermal growth factor receptor phosphorylation could be a promising way of intervention in human renal disease.

Acute vascular endothelial growth factor expression during hypertrophy is muscle phenotype specific and localizes as a striated pattern within fibres

Skeletal muscle hypertrophy requires the co-ordinated expression of locally acting growth factors that promote myofibre growth and concurrent adaptive changes in the microvasculature. These studies tested the hypothesis that vascular endothelial growth factor (VEGF) and heparin-binding epidermal growth factor (HB-EGF) expression are upregulated during the early stages of compensatory muscle growth induced by chronic functional overload (FO). Bilateral FO of the plantaris and soleus muscles was induced for 3 or 7 days in the hindlimbs of adult female Sprague-Dawley rats (n = 5 per group) and compared with control (non-FO) rats. Relative muscle mass (in mg (kg body weight)(-1)) increased by 18 and 24% after 3 days and by 20 and 33% after 7 days in the plantaris and soleus muscles, respectively. No differences in HB-EGF mRNA or protein were observed in either muscle of FO rats relative to control muscles. The VEGF mRNA was similar in the soleus muscles of FO and control rats, whereas a significant elevation occurred at 3 and 7 days of FO in the plantaris muscle. However, VEGF protein expression after 3 days of FO exhibited a differential response; expression in the soleus muscle decreased 1.6-fold, whereas that in the plantaris muscle increased 1.8-fold compared with the control muscle. After 7 days of FO, VEGF protein remained elevated within the plantaris muscle, but returned to basal levels in the soleus. Robust basal HB-EGF and VEGF protein expression was consistently seen in control muscles. In all groups, immunohistochemistry for VEGF protein displayed a distinct striated expression pattern within myofibres, with considerably less labelling in extracellular spaces. Constitutive expression of HB-EGF and VEGF in control myofibres is consistent with housekeeping roles for these growth factors in skeletal muscle tissue. However, the specific patterns of VEGF expression in these muscles during FO may reflect the chronic changes in neural recruitment between muscles and the co-ordination of angiogenic and/or other hypertrophic responses.

Heparin binding epidermal growth factor in renal ischaemia/reperfusion injury

The epidermal growth factor (EGF) receptor and its ligands are crucially involved in the renal response to ischaemia. We studied the heparin binding-epidermal growth factor (HB-EGF), a major ligand for the EGF receptor, in experimental and human ischaemia/reperfusion injury (IRI). HB-EGF mRNA and protein expression was studied in rat kidneys and cultured human tubular (HK-2) cells that were subjected to IRI and in human donor kidneys during transplantation. The effect of EGF receptor inhibition was investigated in vivo and in vitro. Furthermore, urinary HB-EGF protein excretion was studied after renal transplantation. Finally, HB-EGF KO and WT mice were subjected to IRI to study the role of HB-EGF in renal injury. HB-EGF mRNA was significantly up-regulated in the early phase of IRI in rats, cells, and human donor biopsies. Treatment with PKI-166 reduces macrophage accumulation and interstitial alpha-SMA in the early phase of IRI in rats. In vitro, PKI-166 causes a marked reduction in HB-EGF-induced cellular proliferation. Urinary HB-EGF is increased after transplantation compared with control urines from healthy subjects. HB-EGF KO mice subjected to IRI revealed significantly less morphological damage after IRI, compared with WT mice. We conclude that IRI results in early induction of HB-EGF mRNA and protein in vivo and in vitro. Absence of HB-EGF and inhibition of the EGF receptor in the early phase of IRI has protective effects, suggesting a modulating role for HB-EGF.

Helicobacter pylori Can Induce Heparin-Binding Epidermal Growth Factor Expression via Gastrin and Its Receptor

Both gastrin and Helicobacter pylori have been shown capable of up-regulating gene expression and protein shedding of heparin-binding epidermal growth factor (HB-EGF). Furthermore, the bacteria have previously been shown to induce serum hypergastrinemia in infected individuals. The aim of this work was to assess the extent to which the ability of H. pylori to up-regulate expression of HB-EGF can be attributed to its effect on gastrin. Gastric cells, transfected with either gastrin small interfering RNA or antisense plasmid or the gastrin/cholecystokinin-2 receptor (CCK-2R), were cultured for 24 hours with H. pylori(+/-), a CCK-2R antagonist. Gene expression levels were measured using reverse transcription-PCR, whereas protein changes were measured using ELISA, Western blotting, and immunofluorescence. H. pylori induced significantly higher levels of HB-EGF gene expression and ectodomain shedding in the CCK-2R-transfected cells than the vector control (P < 0.01). Addition of the CCK-2R inhibitor significantly decreased gene and shedding up-regulation. Gastrin down-regulation reduced the effect of the bacteria on HB-EGF gene and protein expression levels. Endogenous gastrin and CCK-2R expression were also found to be significantly up-regulated in all cell lines as a result of exposure to H. pylori (P < 0.02). Gastric mucosal tissue from H. pylori-infected individuals had significantly higher CCK-2R expression levels than noninfected (P < 0.003), and in hypergastrinemic mice, there was an increase in HB-EGF-expressing cells in the gastric mucosa and colocalization of HB-EGF with CCK-2R-positive enterochromaffin-like cells. In conclusion, gastrin and the CCK-2R play significant roles in the induction of HB-EGF gene and protein expression and ectodomain shedding by H. pylori.

Differential distributions of peptides in the epidermal growth factor family and phosphorylation of ErbB1 receptor in adult rat brain

Using two-site enzyme immunoassays, we measured protein levels of epidermal growth factor (EGF), transforming growth factor alpha (TGFα), and heparin-binding epidermal growth factor (HB-EGF) in adult rat brain, and compared them with the phosphorylation levels of their receptor (ErbB1). There were significant variations in the brain distributions of each ErbB1 ligand. Among these ErbB1 ligands, HB-EGF protein levels were higher than those of TGFα and those of EGF were the lowest. TGFα protein was relatively enriched in the midbrain regions, while HB-EGF levels were most abundant in the cerebellum. Protein distributions of the EGF family members were discordant with previously reported mRNA distributions. In addition, there was significant basal ErbB1 phosphorylation detected with the largest amount of activation in the midbrain. These observations suggest that the activation of brain ErbB1 involves post-translational regulation of multiple EGF family members in a region-specific manner.