Hsp90alpha recruited by Sp1 is important for transcription of 12(S)-lipoxygenase in A431 cells.
Sp1 is a basic transcriptional factor that binds to the GC-rich region in the promoter of the target gene. It is involved in transcription of numerous genes by recruiting transcriptional factors to the promoters of target genes. In this study, we found in vivo and in vitro that Hsp90alpha was recruited to the GC-rich region of the 12(S)-lipoxygenase promoter through interaction with Sp1 in A431 cells by employing DNA affinity immunoprecipitation assay and chromatin immunoprecipitation assay. When Hsp90alpha was inhibited by geldanamycin (GA, a specific inhibitor of the Hsp90 family) or by siRNA of Hsp90alpha (to block its activity or to knockdown protein levels), respectively, luciferase activity (driven by the 12(S)-lipoxygenase promoter) and both mRNA and protein levels of 12(S)-lipoxygenase were reduced significantly in cells. In addition, the effect of GA was abolished when the Sp1 binding sites of 12(S)-lipoxygenase were mutated in A431 cells. Interestingly, binding of Sp1 to the 12(S)-lipoxygenase promoter was also decreased upon GA treatment in cells. In conclusion, our results indicate that Sp1 interacts with Hsp90alpha to recruit it to the promoter of 12(S)-lipoxygenase and then to regulate gene transcription by modulating the binding ability of Sp1 to promoters.
Hung
Jan-Jong
JJ
Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.
Wu
Chih-Ying
CY
Liao
Pao-Chi
PC
Chang
Wen-Chang
WC
D000595:Amino Acid Sequence; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D016227:Benzoquinones; D001665:Binding Sites; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D047029:Lactams, Macrocyclic; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011809:Quinones / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D004247:DNA; D001092:Arachidonate 12-Lipoxygenase; C001277:geldanamycin