Production of Glycosylated Heparin-Binding EGF-like Growth Factor in HeLa Cells Using Vaccinia Virus

Davis, Karen M.; Brigstock, David R.; Johnson, Philip R.; Crissman-Combs, Melissa A.; McCarthy, Diane W.; Downing, Marc T.; Besner, Gail E.

doi:10.1006/prep.1996.0074

Cited by 30 publications

(14 citation statements)

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“…The next day, cells were replaced with fresh DMEM/F-12 containing 10% fetal bovine serum, and medium replacement was then repeated every other day if applicable. Cells were analyzed by RT-PCR for expression of fibrosis markers after 24-hour treatment with 0–100ng/ml recombinant HB-EGF (22) in the presence or absence of 10 µg/ml Diphtheria toxin mutant CRM197 (BioAcademia, Inc., Ibaraki, Osaka, Japan), which blocks HB-EGF receptor binding and inhibits HB-EGF activity.…”

Section: Methodsmentioning

confidence: 99%

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Huang

Besner

Brigstock

2012

Laboratory Investigation

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Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100mg/kg) was administered by intra-peritoneal injection three times a week for 4 weeks to wild-type HB-EGF+/+ or HB-EGF-null (HB-EGF−/−) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSC isolated from untreated HB-EGF+/+ or HB-EGF−/− mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF+/+ and HB-EGF−/− mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF+/+ mice by 37.6% (p < 0.05) as compared to animals receiving saline alone. HB-EGF−/− mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared to HB-EGF+/+ mice, TAA-stimulated hepatic mRNA levels in HB-EGF−/− mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2-, or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-β1, connective tissue growth factor, or tissue inhibitor of metalloproteinase-1 (p < 0.05). HB-EGF expression was detectable in primary cultured HSC from HB-EGF+/+ mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSC.

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Section: Methodsmentioning

confidence: 99%

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Huang

Besner

Brigstock

2012

Laboratory Investigation

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“…Phospho-stat (serine-727) Ab, IL-1␤, and IFN-␥ were from BioSource International. Recombinant HB-EGF corresponding to aa 74 -148 of the mature protein was produced in Escherichia coli and purified in our laboratory as previously described (28). Abs to NF-B (p65), phospho-and total IB␣ were from Santa Cruz Biotechnologies.…”

Section: Methodsmentioning

confidence: 99%

Heparin-Binding Epidermal Growth Factor-Like Growth Factor Inhibits Cytokine-Induced NF-κB Activation and Nitric Oxide Production via Activation of the Phosphatidylinositol 3-Kinase Pathway

Mehta

Besner

2005

The Journal of Immunology

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NO produced by inducible NO synthase (iNOS) has been implicated in various pathophysiological processes including inflammation. Therefore, inhibitors of NO synthesis or iNOS gene expression have been considered as potential anti-inflammatory agents. We have previously demonstrated that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) decreases proinflammatory cytokine IL-8 and NO production in cytokine-stimulated intestinal epithelial cells by interfering with the NF-κB signaling pathway. However, the upstream signaling mechanisms involved in these responses have not yet been defined. In this report, we show that in intestinal epithelial cells, HB-EGF triggered PI3K-dependent phosphorylation of Akt. Inhibition of PI3K reversed the ability of HB-EGF to block NF-κB activation, expression of iNOS, and NO production. Small interfering RNA of PI3K also reversed the inhibitory effect of HB-EGF on iNOS expression. Alternatively, transient expression of constitutively active PI3K decreased NO production by ∼2-fold more than treatment with HB-EGF alone. This PI3K effect was HB-EGF dependent. Thus, activation of PI3K is essential but not sufficient for decreased NO synthesis. PI3K and HB-EGF act synergistically to decrease NO synthesis. Neither overexpression or inhibition of MEK, Ras, or Akt affected HB-EGF-mediated inhibition of NF-κB activation. These data demonstrate that HB-EGF decreases proinflammatory cytokine-stimulated NF-κB activation and NO production via activation of the PI3K signaling pathway. These results also suggest that inhibition of NF-κB and activation of the PI3K-dependent signaling cascade by HB-EGF may represent key signals responsible for the anti-inflammatory effects of HB-EGF.

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“…All procedures were performed using proper sterile technique. The HB-EGF used in all experiments was a highly purified form of recombinant mature human HB-EGF (corresponding to amino acids 74 -148 of the HB-EGF precursor) which was produced in our laboratory using an Escherichia coli expression system [37].…”

Section: Methodsmentioning

confidence: 99%

Heparin-Binding Epidermal Growth Factor-Like Growth Factor Protects Rat Intestine from Ischemia/Reperfusion Injury

Pillai

Hinman

Luquette

et al. 1999

Journal of Surgical Research

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Production of Glycosylated Heparin-Binding EGF-like Growth Factor in HeLa Cells Using Vaccinia Virus

Cited by 30 publications

References 0 publications

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Heparin-Binding Epidermal Growth Factor-Like Growth Factor Inhibits Cytokine-Induced NF-κB Activation and Nitric Oxide Production via Activation of the Phosphatidylinositol 3-Kinase Pathway

Heparin-Binding Epidermal Growth Factor-Like Growth Factor Protects Rat Intestine from Ischemia/Reperfusion Injury

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