Pyrrolidine dithiocarbamate-induced macrophage inflammatory protein-2 gene expression is NF-kappaB-independent but c-Jun-dependent in macrophage cell line RAW 264.7.
Pyrrolidine dithiocarbamate (PDTC) is a stable compound that acts as antioxidant or prooxidant, and is widely used to inhibit the activation of NF-kappaB. PDTC was also reported to activate NF-kappaB depending on its dose and metal ions in PC12 cells. In this work, we demonstrated a working mechanism of PDTC and its effects on the proinflammatory cytokine gene expression in a mouse macrophage cell line, RAW 264.7. PDTC alone induced NF-kappaB-independent MIP-2 promoter activation that can be assessed by transient transfection and confocal image analysis. The involvement of AP-1 transcription factor was noticed by promoter deletion/site-specific mutation analysis and electrophoretic mobility shift assay (EMSA). Among three different mitogen-activated protein kinase (MAPK) pathways tested, only the stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) pathway was significantly activated in RAW 264.7 cells after the stimulation with PDTC. Using pathway-specific inhibitors, we found that the SAPK/JNK pathway is clearly associated with PDTC-induced MIP-2 gene expression. Our experimental results indicate that PDTC-induced proinflammatory cytokine expressions are mediated by SAPK/JNK pathway, which activates AP-1.
Sohn
Wern-Joo
WJ
Institute of Life Science and Biotechnology, College of Science, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul 120-749, Republic of Korea.
Lee
Keun-Wook
KW
Lee
Younghee
Y
Han
Jung Ho
JH
Choe
Yong-Kyoung
YK
Kim
Doo-Sik
DS
Kwon
Hyung-Joo
HJ
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D054426:Chemokine CXCL2; D004252:DNA Mutational Analysis; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects*; D051379:Mice; D018613:Microscopy, Confocal; D015846:Monokines / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013859:Thiocarbamates / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism
D000975:Antioxidants; D054426:Chemokine CXCL2; D005456:Fluorescent Dyes; D015846:Monokines; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D011759:Pyrrolidines; D013859:Thiocarbamates; D018808:Transcription Factor AP-1; C020972:pyrrolidine dithiocarbamic acid; D008156:Luciferases; D016650:Fluorescein-5-isothiocyanate