Abstract
Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1), a counterpart of the human growth-regulated oncogene product (GRO), has been suggested to participate in neutrophil recruitment in an experimental model of gastritis in rat. However, the mechanism(s) involved in regulation of CINC-1 production by the gastric mucosa remains unclear. The aim of this study was to investigate the mechanism(s) of CINC-1 production by rat gastric mucosa in vitro. All experiments were performed using rat normal gastric mucosal cell line (RGM-1). RGM-1s were stimulated with tumor necrosis factor (TNF)-α, and CINC-1 mRNA levels (reverse transcription-polymerase chain reaction) and protein secretion (enzyme-linked immunosorbent assay) were assessed. The production of reactive oxygen species (ROS) and nuclear factor (NF)-κB activation (translocation to the nuclei) in response to TNF-α stimulation was evaluated using fluorescence microscopy in the presence or absence of the inhibitors of mitochondrial electron flow and NF-κB activation. Stimulation of RGM-1 cells with TNF-α resulted in an increase in intracellular oxidative stress, NF-κB translocation to the nuclei, and up-regulation of CINC-1 mRNA and protein, which was prevented by interfering with mitochondria-dependent ROS production and NF-κB activation. Taken together, these findings indicate that CINC-1, a counterpart of the human GRO, production by rat gastric epithelial cells in response to TNF-α stimulation is an oxidant stress-mediated and NF-κB-dependent event.
Footnotes
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This work was supported by Grant-in-Aid for Scientific Research 14570943 (to Y.N.) and 15390178 (to T.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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DOI: 10.1124/jpet.103.062216.
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ABBREVIATIONS. IL-8, interleukin-8; GRO, growth-regulated oncogene product; CINC-1, cytokine-induced neutrophil chemoattractant-1; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; LPS, lipopolysaccharide; RGM-1, rat gastric mucosal cells; PSI, proteasome inhibitor; NF-κB, nuclear factor-κB; PDTC, pyrrolidine dithiocarbamate; DPI, diphenyleneiodonium; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RT, reverse transcription; PCR, polymerase chain reaction ROS, reactive oxygen species; CH2DCF-DA, 5-(and 6-)carboxy-2′,7′-dichlorodihydrofluorescein diacetate; DHR-123, dihydrorhodamine-123.
- Received October 31, 2003.
- Accepted January 23, 2004.
- The American Society for Pharmacology and Experimental Therapeutics