Heparin-binding Epidermal Growth Factor-like Growth Factor Expression Research Articles

Expressions of vascular endothelial growth factor A, mucin-1, colony-stimulating factor-1, heparinbinding epidermal growth factor-like growth factor, and fibroblast growth factor 2 genes in the female reproductive tracts of women with ectopic pregnancy

Full Title: Expressions of vascular endothelial growth factor A, mucin-1, colony-stimulating factor-1, heparin-binding epidermal growth factor-like growth factor, and fibroblast growth factor 2 genes in the female reproductive tracts of women with ectopic pregnancy: A case-control study
 Background: Ectopic pregnancy (EP) is defined as embryo implantation in a location other than the uterine cavity.
 Objective: We aimed to evaluate the expression of several genes, which may play a role in EP, in the ampulla region of fallopian tubes and endometrial tissue of women with EP.
 Materials and Methods: In this case-control study, 5 women who underwent salpingectomy due to EP, comprised the 5 pseudo-pregnant women as a control group. These participants referred to the Royan Institute, Shariati, and Arash hospital, Tehran, Iran during 2019-2021. We evaluated the expressions of vascular endothelial growth factor A, mucin-1, colony-stimulating factor-1, heparin-binding epidermal growth factor-like growth factor (HBEGF), and fibroblast growth factor 2 genes in the fallopian tube and endometrium of EP cases by real-time polymerase chain reaction using specific primers.
 Results: The vascular endothelial growth factor expression was significantly higher in the ampulla region of the controls. However, no significant differences were observed in endometrial tissue. Assessments of colony-stimulating factor-1 and fibroblast growth factor 2 showed no significant differences between the case and control groups. HBEGF showed significantly higher expression in the ampulla region of EP cases, but no significant difference was observed in HBEGF expression in the endometrial tissues of the study groups. Mucin-1 expression was significantly higher in both study regions of the EP cases.
 Conclusion: Our results have strongly suggested that these genes play important roles in proper implantation, and disruptions in their expression patterns could lead to EP. However, more studies are needed to confirm the current findings.
 Key words: Ectopic pregnancy, Gene expression, Vascular endothelial growth factor A, Mucin-1, Colony-stimulating factor-1, Heparin-binding epidermal growth factor-like growth factor, Fibroblast growth factor 2.

Clinical application study on miR-98–5p as a prognostic biomarker in hepatocellular carcinoma

BackgroundOn the one hand, to investigate the targeted regulation of miR-98–5p on heparin-binding epidermal growth factor-like growth factor (HBEGF) in patients with hepatocellular carcinoma (HCC). On the other hand, elucidate the predictive effect of miR-98–5p combined with magnetic resonance imaging (MRI) data on the clinical prognosis of HCC patients. MethodsSerum samples from 98 HCC patients and 54 healthy subjects were selected in order to detect miR-98–5p as well as HBEGF expression levels via real-time quantitative PCR (RT-qPCR). A Luciferase reporter assay was performed to detect the interaction between miR-98–5p and HBEGF gene. The serum levels of IL-2, TNF-α, TGF-β1 and IFN-γ in HCC patients and in the control group (healthy subjects) were measured by enzyme-linked immunosorbent assay (ELISA). In addition, receiver operator characteristic curve (ROC) was utilized to analyze the predictive ability of miR-98–5p combined with HBEGF for HCC. Finally, the survival curves were used to analyze the effect of HBEGF and miR-98–5p on the survival of patients with HCC. ResultsRT-qPCR results showed that the expression level of miR-98–5p was significantly decreased, while HBEGF expression was significantly increased in the serum of HCC patients compared with the control group. Luciferase reporter assay confirmed that miR-98–5p could target and bind HBEGF. Additionally, according to ELISA, IL-2, TNF-α, and TGF-β1 were significantly increased, while IFN-γ was significantly decreased in the serum of HCC patients compared with the control group. The results of ROC indicated that expressive levels of miR-98–5p and HBEGF had a high diagnostic value for HCC. At the same time, the survival curve results indicated high HBEGF expression and low miR-98–5p expression, suggesting a poor prognosis for HCC patients. ConclusionMiR-98–5p can target the down-regulating HBEGF gene. In addition, miR-98–5p combined with MRI data is of crucial guiding value in assessing the prognosis of patients with HCC in the clinic.

Myeloid- and Epithelial-derived Heparin-Binding Epidermal Growth Factor-like Growth Factor Promotes PulmonaryFibrosis.

Idiopathic pulmonary fibrosis (IPF) is a poorly understood, progressive lethal lung disease with no known cure. In addition to alveolar epithelial cell (AEC) injury and excessive deposition of extracellular matrix proteins, chronic inflammation is a hallmark of IPF. Literature suggests that the persistent inflammation seen in IPF primarily consists of monocytes and macrophages. Recent work demonstrates that monocyte-derived alveolar macrophages (moAMs) drive lung fibrosis, but further characterization of critical moAM cell attributes is necessary. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an important epidermal growth factor receptor ligand that has essential roles in angiogenesis, wound healing, keratinocyte migration, and epithelial-mesenchymal transition. Our past work has shown HB-EGF is a primary marker of profibrotic M2 macrophages, and this study seeks to characterize myeloid-derived HB-EGF and its primary mechanism of action in bleomycin-induced lung fibrosis using Hbegff/f;Lyz2Cre+ mice. Here, we show that patients with IPF andmice with pulmonary fibrosis have increased expression of HB-EGF and that lung macrophages and transitional AECs of mice with pulmonary fibrosis and humans all express HB-EGF. We also show that Hbegff/f;Lyz2Cre+ mice are protected from bleomycin-induced fibrosis and that this protection is likely multifactorial, caused by decreased CCL2-dependent monocyte migration, decreased fibroblast migration, and decreased contribution of HB-EGF from AEC sources when HB-EGF is removed under the Lyz2Cre promoter.

Association of Serum HB-EGF Value and Response to Chemotherapy in Patients with Recurrent Ovarian Cancer.

Many anticancer agents including molecularly-targeted drugs have been developed for ovarian cancer. However, the prognosis of recurrent ovarian cancer remains extremely poor. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is reported as a rational target for ovarian cancer therapy. Moreover, serum HB-EGF expression is recognized as a biomarker in patients with primary ovarian cancer. We analysed serum samples with recurrent ovarian cancer at the Fukuoka University Hospital from April 2009 to March 2014. To assess the clinical significance of serum HB-EGF in recurrent ovarian cancer, the association between serum HB-EGF levels and prognosis in patients with recurrent ovarian cancer was examined using ELISA. Patients with high serum HB-EGF expression showed a significantly poor response to second-line chemotherapeutic agents compared with patients with low HB-EGF levels. HB-EGF expression in serum may be a potential therapeutic indicator for novel HB-EGF-targeted therapy in recurrent ovarian cancer.

YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity.

The role of YAP (Yes associated protein 1 gene) and MRTF-A (myocardin-related transcription factor A), two transcriptional co-activators regulated downstream of GPCRs (G-protein coupled receptors) and RhoA, in growth of glioblastoma cells and in vivo glioblastoma multiforme (GBM) tumor development was explored using human glioblastoma cell lines and tumor initiating cells derived from patient derived xenografts (PDX). Knockdown of these co-activators in GSC-23 PDX cells using shRNA significantly attenuated in vitro self-renewal capability assessed by limiting dilution, oncogene expression and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. In vitro studies used PDX and 1321N1 glioblastoma cells to examine functional responses to sphingosine 1-phosphate (S1P), a GPCR agonist that activates RhoA signaling, demonstrated that YAP signaling was required for cell migration and invasion whereas MRTF-A was required for cell adhesion; both YAP and MRTF-A were required for proliferation. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor gene; TF), a target gene regulated selectively through YAP, blocked cell invasion and migration, whereas knockdown of HBEGF (Heparin binding EGF-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of TF and HBEGF was also selectively decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in preclinical GBM models and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.

Ligand-Independent Epidermal Growth Factor Receptor Overexpression Correlates with Poor Prognosis in Colorectal Cancer.

PurposeMolecular treatments targeting epidermal growth factor receptors (EGFRs) are important strategies for advanced colorectal cancer (CRC). However, clinicopathologic implications of EGFRs and EGFR ligand signaling have not been fully evaluated. We evaluated the expression of EGFR ligands and correlation with their receptors, clinicopathologic factors, and patients’ survival with CRC.Materials and MethodsThe expression of EGFR ligands, including heparin binding epidermal growth factor-like growth factor (HBEGF), transforming growth factor (TGF), betacellulin, and epidermal growth factor (EGF), were evaluated in 331 consecutive CRC samples using mRNA in situ hybridization (ISH). We also evaluated the expression status of EGFR, human epidermal growth factor receptor 2 (HER2), HER3, and HER4 using immunohistochemistry and/or silver ISH.ResultsUnlike low incidences of TGF (38.1%), betacellulin (7.9%), and EGF (2.1%), HBEGF expression was noted in 62.2% of CRC samples. However, the expression of each EGFR ligand did not reveal significant correlations with survival. The combined analyses of EGFR ligands and EGFR expression indicated that the ligands‒/EGFR+ group showed a significant association with the worst disease-free survival (DFS; p=0.018) and overall survival (OS; p=0.005). It was also an independent, unfavorable prognostic factor for DFS (p=0.026) and OS (p=0.007). Additionally, HER4 nuclear expression, regardless of ligand expression, was an independent, favorable prognostic factor for DFS (p=0.034) and OS (p=0.049), by multivariate analysis.ConclusionLigand-independent EGFR overexpression was suggested to have a significant prognostic impact; thus, the expression status of EGFR ligands, in addition to EGFR, might be necessary for predicting patients' outcome in CRC.

Reverse of the resistance to paclitaxel of the heparin binding-epidermal growth factor-like growth factor inhibitor in ovarian cancer

Objective: To investigate the effect and mechanism of CRM197, the heparin binding-epidermal growth factor-like growth factor (HB-EGF) inhibitor, on the reverse of the resistance of ovarian cancer to paclitaxel. Methods: (1)The effect of CRM197 on the 50% inhibitory concentrations (IC(50)) of human ovarian carcinoma cell line A2780 and paclitaxel-resistant ovarian carcinoma cell line A2780/Taxol was tested by methyl thiazolyl tetrazolium (MTT) assay. Western blot was used to detect the effect of CRM197 on the expression of HB-EGF, epidermal growth factor receptor (EGFR) and plasma membrane glycoprotein (P-gp) protein in A2780 and A2780/Taxol cells. Real-time PCR was used to examine the MDR1 mRNA expression in these cells. (2) A2780/Taxol cells were divided into 4 groups, including the cells transfected with empty vector and saline treatment (empty vector group), MDR1 small interference RNA (siRNA) vector and saline treatment (MDR1 siRNA group), empty vector and CRM197 treatment (empty vector+CRM197 group) and MDR1 siRNA vector and CRM197 treatment (MDR1 siRNA+CRM197 group), respectively. Flow cytometry was used to detecte the effect of intracellular rhodomine 123 (Rh123) accumulation, and caspase-3 activity assay was used to test the effect of apoptosis in four groups of A2780/Taxol cells. (3) In experiments in vivo, A2780/Taxol cells were inoculated to nude mouse subcutaneously to determine the EGFR and P-gp protein expression following CRM197 treatment by immunohistochemistry. Results: (1) In vitro, MTT examination showed that the IC(50) of A2780/Taxol cells to paclitaxel in A2780/Taxol+CRM197 group [(6.4±0.3) μmol/L] was significantly lower than the IC(50) in A2780/Taxol group [ (34.1±0.5) μmol/L, P<0.01], and the reveral fold of CRM197 was 5.3. The expression level of HB-EGF protein in A2780/Taxol+CRM197 group (1.44±0.29) was significantly lower than HB-EGF protein in A2780/Taxol group (2.72±0.32), respectively (P<0.05). The expression level of EGFR protein (0.71±0.25) and P-gp protein (0.82±0.19) in A2780/Taxol+CRM197 group was significantly lower than EGFR protein (1.87±0.31) and P-gp protein (1.84±0.27) of A2780/Taxol group (P<0.05). Compared with A2780/Taxol group (1.78±0.27) , MDR1 mRNA was significantly down-regulated in A2780/Taxol+CRM197 group (0.79±0.13, P<0.05). (2) The fluorescence intensity of Rh123 of the A2780/Taxol cells in empty vector group, MDR1 siRNA group,empty vector+CRM197 group, MDR1 siRNA+CRM197 group was 33.4±1.6, 56.3±3.3, 43.5±3.1,100.4±7.4, and the pNA of the A2780/Taxol cells was (11.4±1.2) , (52.8±0.9) , (71.2±3.6) , (82.7±3.8) μmol/L. The expression levels in MDR1 siRNA+CRM197 group were both higher than the expression levels in empty vector+CRM197 group, and the expression levels in empty vector+CRM197 group, MDR1 siRNA group were both higher than the expression levels in empty vector group (P<0.05). (3) In vivo, the expression scores of EGFR protein in A2780/Taxol+CRM197 tumors (4.4±1.4) were lower than that in A2780/Taxol tumors (10.2±3.1, P<0.05). The expression scores of P-gp protein in A2780/Taxol+CRM197 tumors (3.8±1.1) were lower than that in A2780/Taxol tumors (8.8±2.7, P<0.05). Conclusion: CRM197 reverses the resistance of ovarian cancer to paclitaxel by increasing caspase-3 activity to advance apoptosis via EGFR/MDR1/P-gp pathway.

HB-EGF expression as a potential biomarker of acquired middle ear cholesteatoma

Conclusions: The heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays an essential role in the development and invasiveness of cholesteatoma. This study may help to realize the molecular mechanisms underlying the pathogenesis of cholesteatoma and make HB-EGF a promising target for drug intervention of cholesteatoma.Objective: To detect HB-EGF expression in human surgical specimens of acquired middle ear cholesteatoma and analyze its functional role as a regulator of epithelial keratinocytes hyperproliferation.Methods: A total of 34 patients who underwent surgical treatment for middle ear cholesteatoma were recruited in the study. The mRNA and protein expression of HB-EGF in middle ear cholesteatoma tissues and normal postauricular skin tissues was investigated by real-time quantitative reverse-transcription-polymerase chain reaction (RT-qPCR), immunohistochemical staining, and western blot. The correlation between bone resorption degree and HB-EGF expression was also analyzed.Results: On average, compared with normal postauricular skin, expression of HB-EGF mRNA in the cholesteatoma epithelium was significantly elevated 2.41-fold by RT-qPCR, and HB-EGF protein significantly upregulated 2.32-fold by western blot. Positive HB-EGF immunostaining observed in the basal and suprabasal layers of cholesteatoma epithelium was significantly stronger than in normal postauricular skin. Meanwhile, an obviously positive correlation between HB-EGF protein expression and bone resorption degree was discovered.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Abstract 1182: Heparin-binding epidermal growth factor-like growth factor is a pro-differentiating factor in neuroblastoma

Abstract Neuroblastoma is the most common cancer in infancy. Current therapies are only modestly effective; patients with high-risk disease have less than a 50% chance of survival. High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low Schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble proteins, including heparan sulfate proteoglycans (HSPGs), which act to promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent pro-differentiating factor in neuroblastoma. Using microarray analysis, HBEGF mRNA expression is decreased in neuroblastoma compared to benign disease, correlating to an increase in mortality. HBEGF protein is expressed only in stromal compartments of tumor specimens, with tissue from high-stage disease having decreased stroma and HBEGF. Soluble HBEGF increased neuroblast differentiation and decreased proliferation, while HBEGF knockdown decreased neuroblast differentiation. In patient samples, HBEGF expression correlates with SOX10, a neural crest differentiation marker, and the cell cycle inhibitor, CDKN1A. Alternatively, HBEGF negatively correlates with ASCL1, a primitive neuroectodermal marker, and the cell cycle promoter CCND1. HSPGs, including TβRIII, GPC1, GPC3, and SDC3 further promote the differentiating effects of HBEGF treatment by forming a complex with the epidermal growth factor receptor (EGFR) via glycosaminoglycan modifications, leading to activation of the Erk1/2 pathway and upregulation of the inhibitor of DNA binding transcription factor, whose expression correlates with HBEGF in patient samples. Inhibition of EGFR with erlotinib, lapatinib, and gefitinib diminish HBEGF-induced differentiation. These data identify a novel member of the pro-differentiating secretome and support the use of heparan sulfate mimetics in neuroblastoma, while cautioning against the use of EGFR inhibitors for neuroblastoma treatment. Citation Format: Angela L. Gaviglio, Gerard C. Blobe. Heparin-binding epidermal growth factor-like growth factor is a pro-differentiating factor in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1182.

Regulatory T-cell–intrinsic amphiregulin is dispensable for suppressive function

Regulatory T-cell–intrinsic amphiregulin is dispensable for suppressive function

Cetuximab Inhibits T790M-Mediated Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor in a Lung Adenocarcinoma Patient-Derived Xenograft Mouse Model

BackgroundThe epidermal growth factor receptor (EGFR) kinase domain T790M (amino acid substitution at position 790 in EGFR from threonine [T] to methionine [M]) mutation in non–small-cell lung cancer (NSCLC) results in resistance to EGFR tyrosine kinase inhibitors (TKIs). We used a patient-derived tumor xenograft (PDX) model containing an EGFR exon 19 deletion/T790M mutation to assess response to the EGFR-directed antibody cetuximab. Changes in the EGFR signaling pathway and ligand expression after treatment were investigated. MethodsPDX were randomized into control and treatment arms. Pharmacodynamic studies were performed at 2 and 24 hours and at 4 days after a single administration of cetuximab, erlotinib, or dacomitinib. Changes in the EGFR signaling pathway were assessed using Western blot analysis, and baseline mRNA expression of EGFR ligands using microarray analysis. Relative changes after treatment were assessed using quantitative polymerase chain reaction. ResultsThe xenograft showed a dramatic response to cetuximab. A complete reduction of total EGFR and phosphorylated EGFR occurred after cetuximab treatment. The PDX had increased baseline levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF) compared with other PDX models with or without EGFR mutations. Amphiregulin was significantly reduced 2 hours after treatment with cetuximab. Compared with control mice, cetuximab- and EGFR–TKI-treated mice had significantly reduced HB-EGF gene expression at 2 hours, however, by day 4 the level of HB-EGF expression was higher. The effect of cetuximab compared with EGFR TKI on HB-EGF gene expression levels differed significantly at 2 and 24 hours but not at 4 days. ConclusionWe showed a dramatic tumor response with cetuximab in an exon 19 deletion/T790M EGFR mutant lung adenocarcinoma PDX model, which suggests a role for the autocrine feedback loop in the mutant EGFR signaling pathway. Further investigation using cetuximab in NSCLC with T790M mutation is warranted.

Cross-reacting material 197 reverses the resistance to paclitaxel in paclitaxel-resistant human ovarian cancer.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been proven to be a promising chemotherapeutic target for ovarian cancer. Our previous studies have demonstrated that inhibition of HB-EGF by the special inhibitor, cross-reacting material 197 (CRM197), potently inhibits the anti-tumor activity in paclitaxel-resistant ovarian cancer. Here, we found that inhibition of HB-EGF by CRM197 significantly reverses the resistance to paclitaxel in paclitaxel-resistant ovarian carcinoma cell line (A2780/Taxol). A2780/Taxol cells over-expressed HB-EGF and epidermal growth factor receptor (EGFR) and CRM197 notably suppressed the expression of HB-EGF and EGFR. Experiments performed in vitro and in vivo further suggested that CRM197 markedly down-regulated the ATP-binding cassette sub-family B member 1 (ABCB1/MDR1) messenger RNA (mRNA) expression (P = 0.01), plasma membrane glycoprotein (P-gp) protein (P = 0.009), and P-gp-mediated efflux (P = 0.007) through inhibition of nuclear factor-κB (NF-κB) expression, which were classical chemoresistance-related targets with respect to paclitaxel therapy. Meanwhile, inhibition of HB-EGF enhanced caspase-3 activity to induce apoptosis via MDR1 inhibition in A2780/Taxol cells (P = 0.038). Collectively, HB-EGF is a molecular target for the resistance of ovarian cancer to paclitaxel and CRM197 as a HB-EGF-targeted agent might be a chemosensitizing agent for paclitaxel-resistant ovarian carcinoma. Our findings provide novel possible mechanisms for HB-EGF to be a target to restore the chemosensitivity to paclitaxel.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities

We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and osteoarthritis (OA) human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage.

Mechanisms underlying interleukin-22-induced expression of heparin-binding epidermal growth factor-like growth factor in HaCaT cells

Objective To investigate the mechanisms underlying interleukin-22（IL-22）-induced expression of heparin-binding epidermal growth factor-like growth factor（HB-EGF）in HaCaT cells. Methods Some HaCaT cells were divided into several inverention groups treated with IL-22 at concentrations of 12.5, 25, 50, 100 μg/L, respectively and a control group treated with phosphate buffer saline（PBS）. After 24-hour culture, total proteins were extracted from the HaCaT cells, and Western blot was performed to measure the expression of phosphorylated extracellular signal-regulated kinase 1/2（P-ERK1/2）in the mitogen-activated protein kinase（MAPK）-ERK1/2 pathway, as well as phosphorylated-JAK2（P-JAK2）and phosphorylated-signal transducer and activator of transcription 3（P-STAT3）in the JAK2/STAT3 pathway. In a blocking experiment, some HaCaT cells were divided into 4 groups to be treated with PBS, IL-22, PD98059（an inhibitor of MAPK-ERK1/2）combined with IL-22（PD98059 group）, AG490（an inhibitor of JAK2/STAT3）combined with IL-22（AG490 group）, respectively. After 24-hour treatment, total proteins and mRNAs were extracted from the HaCaT cells followed by Western blot and real-time quantitative reverse transcription-PCR for the measurement of protein and mRNA expressions of HB-EGF respectively. Statistical analysis was carried out with the software SPSS 16.0 by one-way analysis of variance（ANOVA）for intergroup comparisons and by Bonferroni's test for multiple comparisons. Results After treatment with IL-22 at the above 4 concentrations, the expressions of P-ERK1/2, P-JAK2 and P-STAT3 in HaCaT cells were all increased compared with the control group（all P< 0.05）. The protein and mRNA expression levels（expressed as the HB-EGF/β-actin ratio and 2-△△Ct respectively）of HB-EGF were both significantly decreased in the PD98059 group and AG490 group than in the IL-22 group（protein: 0.183 ± 0.020 and 0.199 ± 0.011 vs. 0.924 ± 0.032, F= 37.700, 36.400, respectively, both P< 0.05; mRNA: 1.034 ± 0.072 and 0.989 ± 0.038 vs. 1.844 ± 0.135, F= 11.271, 13.429, respectively, both P< 0.05）. Conclusions IL-22 can activate the MAPK-ERK1/2 and JAK2/STAT3 signaling pathways in HaCaT cells, which may contribute to IL-22-induced expression of HB-EGF in HaCaT cells. Key words: Interleukin-22; Fibroblast growth factor 1; Keratinocytes; Mitogen-activated protein kinases; Janus kinase 2; STAT3 transcription factor; Psoriasis; HaCaT cells

Contribution of transcription factor, SP1, to the promotion of HB-EGF expression in defense mechanism against the treatment of irinotecan in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a worst histological subtype than other ovarian malignant tumor. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. The aims of this study were to validate the efficacy of HB-EGF–targeted therapy for OCCC and to identify the transcription factor that contributed to the induction of HB-EGF by SN38 treatment in OCCC cells. HB-EGF was highly expressed in OCCC cells, and an increase of HB-EGF was induced by SN38 which had only antitumor effect among conventional anticancer agents on OCCC. A specific inhibitor of HB-EGF, a cross-reacting material 197 (CRM197), led to a synergistic increase in the number of apoptotic OCCC cells with the treatment of SN38. The luciferase assay with 5′-deletion promoter constructs identified a GC-rich element between −125 and −178 (the distal transcription start site was denoted +1) as a cis-regulatory region, and the treatment of SN38 induced luciferase activity in this region. An in silico and chromatin immunoprecipitation analysis estimated that SP1 bound to the cis-regulatory region of HB-EGF in OCCC cells. Real-time PCR and cell viability assays showed that the transfection of a small interfering RNA targeting SP1 suppressed the expression of HB-EGF induced by SN38, resulting in the enhanced sensitivity of SN38. Taken together, these results indicate that induction of HB-EGF expression contributed to defense mechanism against treatment of SN38 through the transcriptional activity of SP1 in OCCC cells.

Crucial involvement of the CCL3-CCR5 axis-mediated fibroblast accumulation in colitis-associated carcinogenesis in mice

Patients with inflammatory bowel diseases often develop colon carcinoma. Combined treatment of azoxymethane (AOM) and dextran sulfate sodium (DSS) recapitulates colitis-associated cancer in mice. AOM/DSS-induced tumor formation was reduced in CCL3- or its specific receptor, CCR5-deficient mice despite the presence of a massive infiltration of inflammatory cells. However, AOM/DSS-induced type I collagen-positive fibroblast accumulation in the colon was reduced in CCL3- or CCR5-deficient mice. This was associated with depressed expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is expressed mainly by fibroblasts. Moreover in vitro, CCL3 induced fibroblasts to proliferate and to enhance HB-EGF expression. Furthermore, CCR5 blockade reduced tumor formation together with reduced fibroblast accumulation and HB-EGF expression, even when administered after the development of multiple colon tumors. Thus, CCL3-CCR5-mediated fibroblast accumulation may be required, in addition to leukocyte infiltration, to induce full-blown colitis-associated carcinogenesis. Our studies shed light on a therapeutic potential of CCR5 antagonist for patients with colitis-associated cancer.

Heparin‐binding epidermal growth factor‐like growth factor: A hepatic stellate cell proliferation inducer via ErbB receptors

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has a proliferative effect on several types of cells. However, the role of HB-EGF on hepatic stellate cells (HSCs) is not clear. The present study is to investigate the regulatory effects of HB-EGF on HSC proliferation and apoptosis. Activated primary rat HSCs and two HSC cell lines (human LX2 and rat T6) were used in this study. Four inhibitors (CRM197 to HB-EGF, AG1478 to epidermal growth factor receptor [EGFR], PD98059 to mitogen-activated kinase, and LY294002 to phosphatidylinositol 3-kinase) were employed to verify the pathway of HB-EGF on cell proliferation and apoptosis. HB-EGF expression was significantly increased in activated HSCs. HB-EGF increased the expressions of phospho-EGFR and ErbB4 receptors, the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Consequently, HB-EGF stimulated HSC proliferation and suppressed HSC apoptosis. Each individual inhibitor specifically inhibited the correlated receptor or enzyme and inhibited HSC proliferation and induced its apoptosis. HB-EGF promotes HSC proliferation via activation of the EGFR and ErbB4 receptors and, subsequently, via activation of ERK and Akt. Any blockage in the chain obstructs the flow from HB-EGF to HSC proliferation. Therefore, HB-EGF is a potential therapeutic target in liver fibrosis.

Low nitric oxide bioavailability upregulates renal heparin binding EGF-like growth factor expression

Decreased nitric oxide bioavailability plays an important role in the initiation and progression of diabetic nephropathy, but the underlying mechanisms remain unclear. Here, we found that heparin binding epidermal growth factor-like growth factor (HB-EGF) expression levels increased in the kidneys of both endothelial nitric oxide synthase (eNOS) knockout and eNOS knockout diabetic (Lepr db/db) mice as early as 8 weeks of age. Further increases in expression were only seen in eNOS knockout diabetic mice and paralleled the progression of glomerulopathy. HB-EGF expression increased in endothelium, podocytes, and tubular epithelial cells. In cultured glomerular endothelial cells, the nitric oxide synthase inhibitors NG-nitro-L-arginine methyl ester (L-NAME) or L-N5-(1-Iminoethyl) ornithine increased HB-EGF protein expression. Administration of L-NAME dramatically increased renal HB-EGF expression and urinary HB-EGF excretion in diabetic mice. On the other hand, replenishing nitric oxide with sodium nitrate in eNOS knockout diabetic mice reduced urinary HB-EGF excretion and inhibited the progression of diabetic nephropathy. Furthermore, specific deletion of HB-EGF expression in endothelium attenuated renal injury in diabetic eNOS knockout mice. Thus, our results suggest that decreased nitric oxide bioavailability leads to increased HB-EGF expression, which may be an important mediator of the resulting progressive diabetic nephropathy in eNOS knockout diabetic mice.