A novel role for Oct-2 in the lipopolysaccharide-mediated induction of resistin gene expression in RAW264.7 cells.
Although resistin was first suggested as a possible link between obesity and diabetes, we have demonstrated previously that expression of resistin is induced by LPS (lipopolysaccharide). In the present study, we showed that LPS increased levels of resistin mRNA and promoter activity in murine RAW264.7 macrophages. Investigation of cis-regulatory elements in the mouse resistin promoter required for LPS-mediated induction showed that an Octamer (ATTTGCAT) element, located at -914 to -907, was required for maximal promoter activity in response to LPS stimulation. Co-transfection of RAW264.7 cells with a resistin promoter-luciferase construct and an Oct-1 or Oct-2 expression plasmid (pCG-Oct-1 or pCG-Oct-2) showed that Oct-2, but not Oct-1, activated the resistin promoter upon LPS treatment. Binding of Oct-2 to the Octamer element was demonstrated by supershift DNA-affinity precipitation and chromatin immunoprecipitation assays. Reverse transcription-PCR and Western blot results showed that levels of Oct-2 mRNA and protein were both up-regulated by LPS in RAW264.7 cells. The LPS-induced increase in Oct-2 protein was inhibited by LY294002 (a phosphoinositide 3-kinase inhibitor) post-transcriptionally, and the inhibition also resulted in a lower response of both resistin mRNA and promoter activity to LPS treatment. Moreover, specific knockdown of Oct-2 by RNA interference impaired the LPS-induced increase in resistin mRNA and promoter activity. Together, these results indicate that Oct-2 is involved in the LPS-mediated induction of resistin gene expression in macrophages and suggest that activation of Oct-2 is a part of LPS signalling pathways in macrophages.
Lu
Shao-Chun
SC
Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chang
Shwu-Fen
SF
Chen
Hui-Ling
HL
Chou
Yuan-Yi
YY
Lan
Ya-Hsin
YH
Chuang
Chia-Ying
CY
Yu
Wei-Hsuan
WH
Chen
Chia-Lin
CL
D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D016296:Mutagenesis / Q000235:genetics; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D050813:Octamer Transcription Factor-2 / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D052243:Resistin / Q000235:genetics* / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D014162:Transfection
D002867:Chromones; D008070:Lipopolysaccharides; D009025:Morpholines; D050813:Octamer Transcription Factor-2; D012333:RNA, Messenger; D052243:Resistin; C499623:Retn protein, mouse; D015534:Trans-Activators; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D052247:Nitric Oxide Synthase Type II