Regulation of macrophage inflammatory protein-2 gene expression by oxidative stress in rat alveolar macrophages.
Chemokines are important mediators in the development of inflammation. Our previous work demonstrated that an oxidative stress can up-regulate mRNA expression of a CC chemokine macrophage inflammatory protein (MIP)-1alpha in rat alveolar macrophages. In the present study, we further investigate whether an oxidative stress can regulate the gene expression of a related CXC chemokine MIP-2, involved in both neutrophil chemotaxis and activation. A rat alveolar macrophage cell line (NR8383) was exposed to 10 microg/ml bacterial lipopolysaccharide (LPS) and MIP-2 mRNA levels dramatically increased after 4 hr of stimulation. This increase by LPS was attenuated by co-treatment with the antioxidants N-acetylcysteine and dimethylsulphoxide, suggesting that the induction of MIP-2 mRNA is mediated via the generation of reactive oxygen species. To assess directly the role of oxidative stress on regulation of MIP-2 mRNA expression, macrophages were exposed to H2O2. MIP-2 mRNA levels had significantly increased after 1 hr exposure to 0.5 mm H2O2, were maximally increased after 4 hr and decreased after 6 hr. Co-treatment of macrophages with the transcriptional inhibitor actinomycin D eliminated the H2O2-induction of MIP-2 mRNA, implicating a role for transcriptional activation in increased expression of MIP-2. Genomic cloning of the rat MIP-2 gene 5'-flanking region has identified a consensus nuclear factor-kappaB (NF-kappaB) binding site. Gel-mobility shift assays revealed NF-kappaB binding to the MIP-2 promoter/enhancer sequence was induced by H2O2. LPS treatment for 4 hr also significantly activated NF-kappaB binding, which could also be attenuated by pretreatment with N-acetylcysteine at the doses that reduced MIP-2 mRNA expression. The half-life of MIP-2 mRNA transcripts was also increased by H2O2 treatment. These observations indicate that MIP-2 gene expression is subject to both transcriptional and post-transcriptional control in response to an H2O2 oxidative stress.
Shi
M M
MM
Genomic Pathology Laboratory, Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research, Warner-Lambert Company and Department of Pathology, the University of Michigan Medical School, Ann Arbor, MI 48105, USA.
Chong
I
I
Godleski
J J
JJ
Paulauskis
J D
JD
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D018929:Cell Culture Techniques; D002460:Cell Line; D054426:Chemokine CXCL2; D003609:Dactinomycin / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000378:metabolism*; D015846:Monokines / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats
D000975:Antioxidants; D054426:Chemokine CXCL2; D008070:Lipopolysaccharides; D015846:Monokines; D016328:NF-kappa B; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D003609:Dactinomycin; D006861:Hydrogen Peroxide