Heparin-binding EGF-like growth factor down regulates proinflammatory cytokine-induced nitric oxide and inducible nitric oxide synthase production in intestinal epithelial cells.
We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) protects intestinal epithelial cells (IEC) from necrosis and apoptosis in vitro and from intestinal ischemia/reperfusion injury in vivo; however, the mechanisms of HB-EGF cytoprotection are unclear. Overproduction of iNOS and NO have been implicated in the pathogenesis of several forms of ischemia/reperfusion injury. We therefore studied whether HB-EGF could down-regulate proinflammatory cytokine-induced iNOS and NO production in intestinal epithelial cells in vitro. DLD-1 human intestinal epithelial cells were exposed to the proinflammatory cytokines interleukin-1beta (IL-1beta) (20 ng/ml) and interferon-gamma (IFN-gamma) (10 ng/ml) to stimulate iNOS induction and NO production. Cells were treated with HB-EGF (0-100 ng/ml) either before or with cytokine exposure, and cells and supernatants were harvested 24 and 48 h later. Accumulated NO was measured in supernatants by chemiluminescence. Total RNA was extracted from cell lysates for iNOS mRNA quantification using real-time reverse transcription-polymerase chain reaction (RT-PCR), and total protein was extracted from cell lysates for detection of iNOS protein. HB-EGF significantly decreased cytokine-induced NO production in a dose dependent manner, and NO reduction was associated with iNOS suppression at both the mRNA and protein levels. While cytokine exposure resulted in a significant increase in iNOS mRNA expression in these cells (109 plus minus 9 fold), HB-EGF reduced iNOS expression by 5.7-fold (P < 0.05). These results suggest that HB-EGF may exert its cytoprotective effects, in part, by down-regulating iNOS and NO production, and provides further rationale for additional testing of the effects of HB-EGF in the treatment of intestinal ischemia/reperfusion injury in vivo.
Lara-Marquez
Maria L
ML
Department of Pediatric Surgery, The Ohio State University and Children's Hospital, 700 Children's Drive, Columbus, OH 43205, USA.
Mehta
Veela
V
Michalsky
Marc P
MP
Fleming
James B
JB
Besner
Gail E
GE
D000906:Antibodies / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D000906:Antibodies; D016207:Cytokines; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D009569:Nitric Oxide; D004815:Epidermal Growth Factor; D006493:Heparin; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II