Dimethyl fumarate inhibits dendritic cell maturation via nuclear factor κB (NF-κB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and mitogen stress-activated kinase 1 (MSK1) signaling.

Dimethyl fumarate (DMF) is an effective novel treatment for multiple sclerosis in clinical trials. A reduction of IFN-γ-producing CD4(+) T cells is observed in DMF-treated patients and may contribute to its clinical efficacy. However, the cellular and molecular mechanisms behind this clinical observation are unclear. In this study, we investigated the effects of DMF on dendritic cell (DC) maturation and subsequent DC-mediated T cell responses. We show that DMF inhibits DC maturation by reducing inflammatory cytokine production (IL-12 and IL-6) and the expression of MHC class II, CD80, and CD86. Importantly, this immature DC phenotype generated fewer activated T cells that were characterized by decreased IFN-γ and IL-17 production. Further molecular studies demonstrated that DMF impaired nuclear factor κB (NF-κB) signaling via reduced p65 nuclear translocalization and phosphorylation. NF-κB signaling was further decreased by DMF-mediated suppression of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and its downstream kinase mitogen stress-activated kinase 1 (MSK1). MSK1 suppression resulted in decreased p65 phosphorylation at serine 276 and reduced histone phosphorylation at serine 10. As a consequence, DMF appears to reduce p65 transcriptional activity both directly and indirectly by promoting a silent chromatin environment. Finally, treatment of DCs with the MSK1 inhibitor H89 partially mimicked the effects of DMF on the DC signaling pathway and impaired DC maturation. Taken together, these studies indicate that by suppression of both NF-κB and ERK1/2-MSK1 signaling, DMF inhibits maturation of DCs and subsequently Th1 and Th17 cell differentiation.

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.

HB-EGF stimulates eNOS expression and nitric oxide production and promotes eNOS dependent angiogenesis.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family of ligands that is expressed by many cell types including endothelial cells. We have previously shown that HB-EGF stimulates angiogenesis in vitro in human umbilical vein endothelial cells (HUVEC). Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is an important regulator of angiogenesis. However, the role of HB-EGF in regulation of eNOS has not yet been investigated. Whether HB-EGF-induced endothelial cell migration and vascular network formation are mediated via production of NO from eNOS is also unknown. To address these questions, we stimulated HUVEC with HB-EGF and evaluated the expression of eNOS at the mRNA and protein levels. HB-EGF significantly upregulated expression of eNOS mRNA, stimulated eNOS protein production, and increased NO release from HUVEC. HB-EGF phosphorylated eNOS in a phosphatidylinositol 3-kinase (PI3K) dependent fashion, and stimulated in vitro angiogenesis. eNOS siRNA inhibited HB-EGF-stimulated HUVEC migration in a scratch assay. NG-nitro-L-arginine-methyl-ester (L-NAME) and L-N5-(1-lminoethyl)ornithine,dihydochloride (L-NIO) (specific inhibitors of eNOS) also abolished HB-EGF-induced HUVEC migration and angiogenesis. More importantly, we found that HB-EGF also promotes angiogenesis in vivo in the Marigel plug assay. Lastly, inhibition of the p38 MAPK pathway enhanced HB-EGF-induced EC migration and angiogenesis. We conclude that HB-EGF, through its interaction with EGF receptors (EGFR), stimulates eNOS activation and NO production via a PI3K-dependent pathway. Thus, activation of eNOS appears to be one of the key signaling pathways necessary for HB-EGF mediated angiogenesis. These novel findings highlight an important role for HB-EGF as a regulator of endothelial cell function.

HB-EGF promotes angiogenesis in endothelial cells via PI3-kinase and MAPK signaling pathways.

OBJECTIVE: Heparin-binding EGF-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) superfamily of ligands. It has been implicated as a regulator of angiogenesis. However, the mechanisms by which HB-EGF promotes angiogenesis are unknown. The goal of the present study was to define the pathways by which HB-EGF stimulates angiogenesis in endothelial cells. METHODS: To characterize the angiogenic activity of HB-EGF, we treated human umbilical vein endothelial cells (HUVEC) with HB-EGF and analyzed the effects on cell proliferation, migration and tube formation. Side-by-side assays with EGF were used for comparison. RESULTS: Both HB-EGF and EGF stimulated HUVEC migration in scratch assays and promoted vascular tube formation in 2D-angiogenesis assays, without inducing cell proliferation. HB-EGF- and EGF-induced HUVEC migration and capillary tube formation were dependent upon activation of PI3K, MAPK and eNOS. Importantly, HB-EGF-and EGF-induced tube formation was comparable to, but were independent of tube formation induced by VEGF. CONCLUSIONS: We have demonstrated that HB-EGF and EGF induce angiogenesis via activation of PI3K, MAPK and eNOS in a VEGF-independent fashion. Thus, the role played by HB-EGF in stimulating physiologic processes such as wound healing in vivo may be dependent, in part, on its ability to promote angiogenesis.

Heparin-binding EGF-like growth factor decreases neutrophil-endothelial cell interactions.

BACKGROUND: Hyperadhesiveness of neutrophils (PMN) to vascular endothelial cells (EC) followed by neutrophil transendothelial migration play important roles in the initiation of ischemia/reperfusion (I/R)-mediated injury. We investigated whether the ability of heparin-binding EGF-like growth factor (HB-EGF) to decrease intestinal injury after intestinal I/R is mediated, in part, by its ability to affect PMN-EC interactions and EC junctional integrity. MATERIALS AND METHODS: Human umbilical vein EC monolayers were treated with HB-EGF (100 ng/mL) or phosphate-buffered saline followed by anoxia/reoxygenation (A/R). Simultaneously, labeled human PMN were treated with HB-EGF or phosphate-buffered saline and then co-incubated with EC for determination of PMN-EC adherence and PMN transendothelial migration. EC junctional integrity was also determined. RESULTS: PMN-EC adhesion increased after exposure of EC to A/R compared to EC exposed to normoxia (87% versus 64% binding, P < 0.05, Wilcoxon rank sum test). A/R-induced PMN-EC hyperadherence was significantly decreased by treatment of PMN with HB-EGF compared to nontreated cells (51% versus 87% binding, P < 0.05). HB-EGF significantly decreased PMN transendothelial migration and also augmented EC tight junctional integrity after A/R. CONCLUSIONS: HB-EGF significantly reduces A/R-induced PMN-EC adhesion and PMN transendothelial migration and augments junctional integrity in vitro. Thus, HB-EGF acts not only as a potent cytoprotective agent for the intestine, but as an anti-inflammatory agent as well.

Heparin-binding EGF-like growth factor decreases inflammatory cytokine expression after intestinal ischemia/reperfusion injury.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) injury is believed to be the major initiator of the systemic inflammatory response syndrome. As a result of intestinal I/R, the gut becomes a major source of inflammatory cytokine production. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is cytoprotective after intestinal I/R and down-regulates pro-inflammatory cytokine production in vitro. We now examine the effects of HB-EGF on pro-inflammatory cytokine expression in vivo. MATERIALS AND METHODS: Rats were randomized into three groups: sham-operated, superior mesenteric artery occlusion (SMAO) for 90 min followed by 8 h of reperfusion (I/R), and I/R with intraluminal administration of HB-EGF 25 min after the initiation of ischemia (I/R + HB-EGF). Serum was drawn at 2, 4, 6, and 8 h post reperfusion for determination of cytokine protein levels using a bioplex suspension array system. Additional animals underwent the same ischemic protocol followed by 30 and 60 min of reperfusion with harvesting of ileal mucosa. Ileal pro-inflammatory cytokine gene expression was determined using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for TNF-alpha, IL-6, and IL-1beta. RESULTS: HB-EGF decreased TNF-alpha, IL-6, and IL-1beta serum protein levels at 4, 6, and 8 h after intestinal I/R injury. In addition, HB-EGF decreased local intestinal mucosal mRNA expression of TNF-alpha, IL-6, and IL-1beta 30 and 60 min after intestinal injury. CONCLUSIONS: We conclude that pro-inflammatory cytokine expression is increased both locally and in the systemic circulation after intestinal I/R and that the administration of HB-EGF significantly reduces intestinal I/R-induced pro-inflammatory cytokine expression in vivo.

Tissue distribution and plasma clearance of heparin-binding EGF-like growth factor (HB-EGF) in adult and newborn rats.

Heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, can protect intestinal epithelial cells from various forms of injury in vitro and attenuate intestinal ischemia/reperfusion damage in vivo. With the goal of eventual clinical use of HB-EGF to protect the intestines from injury in neonates, children, and adults, the pharmacokinetics and biodistribution of 125I-labeled HB-EGF were investigated. After intravenous bolus, HB-EGF had a distribution half-life of 0.8 min and an elimination half-life of 26.67 min. After gastric administration, the bioavailability was 7.8%, with a 2.38 h half-life in the absorption phase and an 11.13 h half-life in the elimination phase. After intravenous dosing, most radioactivity was found in the plasma, liver, kidneys, bile, and urine, whereas it was mainly distributed in the gastrointestinal tract after intragastric administration. The degradation of 125I-HB-EGF in plasma from newborn rats was lower than that in adult rats after gastric administration. This supports the feasibility of enteral administration of HB-EGF in the treatment of gastrointestinal diseases, including newborns afflicted with necrotizing enterocolitis.

Heparin-binding epidermal growth factor-like growth factor inhibits cytokine-induced NF-kappa B activation and nitric oxide production via activation of the phosphatidylinositol 3-kinase pathway.

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-kappaB 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-kappaB 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 approximately 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-kappaB activation. These data demonstrate that HB-EGF decreases proinflammatory cytokine-stimulated NF-kappaB activation and NO production via activation of the PI3K signaling pathway. These results also suggest that inhibition of NF-kappaB and activation of the PI3K-dependent signaling cascade by HB-EGF may represent key signals responsible for the anti-inflammatory effects of HB-EGF.

Inhibition of NF-kappa B activation and its target genes by heparin-binding epidermal growth factor-like growth factor.

Many cells upon injury mount extensive, compensatory responses that increase cell survival; however, the intracellular signals that regulate these responses are not completely understood. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated as a cytoprotective agent. We have previously demonstrated that pretreatment of human intestinal epithelial cells with HB-EGF significantly decreased cytokine-induced activation of inducible NO synthase mRNA expression and NO production and protected the cells from apoptosis and necrosis. However, the mechanisms by which HB-EGF exerts these effects are not known. Here we show that cytokine exposure (IL-1beta and IFN-gamma) induced NF-kappaB activation and IL-8 and NO production in DLD-1 cells. Transient expression of a dominant negative form of IkappaBalpha decreased NO production, suggesting that the cytokines stimulated NO production in part through activation of NF-kappaB. HB-EGF dramatically suppressed NF-kappaB activity and IL-8 release and decreased NO production in cells pretreated with HB-EGF. HB-EGF blocked NF-kappaB activation by inhibiting IkappaB kinase activation and IkappaB phosphorylation and degradation, thus interfering with NF-kappaB nuclear translocation, DNA-binding activity, and NF-kappaB-dependent transcriptional activity. The data demonstrate that HB-EGF decreases inflammatory cytokine and NO production by interfering with the NF-kappaB signaling pathway. Inhibition of NF-kappaB may represent one of the mechanisms by which HB-EGF exerts its potent anti-inflammatory and cytoprotective effects.

Heparin-binding EGF-like growth factor (HB-EGF) decreases oxygen free radical production in vitro and in vivo.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to protect intestinal epithelial cells from anoxia/reoxygenation in vitro, and to protect the intestines from ischemia/reperfusion (I/R) injury in vivo. The goal of the present study was to determine whether the cytoprotective effects of HB-EGF were due, in part, to its ability to decrease reactive oxygen species (ROS) production. Human whole blood, polymorphonuclear leukocytes, and monocytes, as well as rat intestinal epithelial cells, were exposed to stimuli designed to produce an oxidative burst in these cells. Treatment of the cells with HB-EGF led to a significant decrease in oxidative burst production. In vivo, total midgut I/R injury in rats led to increased ROS production, which was markedly decreased by HB-EGF treatment. Histochemically, I/R injury led to increased ROS production, which was significantly decreased with HB-EGF treatment. HB-EGF cytoprotection is due, in part, to its ability to decrease ROS production. Future studies will determine the mechanisms by which HB-EGF exerts these effects.