Expression and regulation of macrophage inflammatory protein-2 gene by vanadium in mouse macrophages.
Environmental and occupational exposure to vanadium (V) dusts results in inflammation mainly confined to the respiratory tract. Macrophages apparently play an important role in mediating the inflammation via the production of many chemokines. In the current study, we investigated whether vanadium can regulate the gene expression of a CXC chemokine macrophage inflammatory protein-2 (MIP-2), and to determine the molecular mechanisms controlling MIP-2 gene expression. A mouse macrophage cell line RAW 264.7 was treated with sodium metavanadate (NaVO3) at the dose of 0.5, 5, or 10 microg/mi V. Northern blot analysis showed that induction of MIP-2 mRNA expression was in a dose-dependent manner. To define the time course of the inflammatory response, RAW 264.7 cells were exposed to 5 microg/ml V, MIP-2 mRNA in macrophages increased markedly as early as 1 h after treatment, maximally induced at 4 h and reduced to 2-fold above control levels by 6 and 8 h. The protein levels of MIP-2 in conditioned media, measured by enzyme-linked immunosorbent assay (ELISA), was well correlated with the levels of MIP-2 mRNA following all of the treatments in the study. In addition, the increase in MIP-2 mRNA expression by vanadium was attenuated by co-treatment with the antioxidant N-acetylcysteine (NAC), at the doses of 10 and 20 mM, suggesting that the induction of MIP-2 mRNA is mediated via the generation of reactive oxygen species (ROS). To further investigate transcriptional regulation of the MIP-2 gene expression by vanadium, we performed RNA decay assay by measuring the half-life of MIP-2 mRNA. Co-treatment of macrophages with the transcriptional inhibitor actinomycin D at 5 microg/ml following exposure to 5 microg/ml V for 4 h revealed complete stabilization of vanadium-induced MIP-2 mRNA and no sign of mRNA degradation, at least, for 6 h, in comparison to the half-life of MIP-2 mRNA was approximately 2.5 h by bacterial lipopolysaccharide (LPS) treatment, supporting post-transcriptional stabilization as the predominant role of MIP-2 gene expression. In conclusion, these observations demonstrate that in vitro vanadium can induce MIP-2 mRNA expression, mediating, at least in part, via the production of ROS. In addition, the increase in MIP-2 mRNA level involves, most likely, post-transcriptional control via increased mRNA stability.
Chong
I W
IW
Department of Internal Medicine, Kaohsiung Medical University, Taiwan.
Lin
S R
SR
Hwang
J J
JJ
Huang
M S
MS
Wang
T H
TH
Tsai
M S
MS
Hou
J J
JJ
Paulauskis
J D
JD
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D054426:Chemokine CXCL2; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D016166:Free Radical Scavengers / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D008264:Macrophages / Q000737:chemistry*; D051379:Mice; D015846:Monokines / Q000235:genetics* / Q000378:metabolism; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology; D014639:Vanadium / Q000494:pharmacology*
D054426:Chemokine CXCL2; D016166:Free Radical Scavengers; D015846:Monokines; D012333:RNA, Messenger; D014639:Vanadium; D014638:Vanadates; D000111:Acetylcysteine