Quercetin up-regulates paraoxonase 1 gene expression via sterol regulatory element binding protein 2 that translocates from the endoplasmic reticulum to the nucleus where it specifically interacts with sterol responsive element-like sequence in paraoxonase 1 promoter in HuH7 liver cells.
We previously showed that quercetin expresses its antiatherogenic effects by up-regulating paraoxonase 1 (PON1) gene and high-density lipoprotein's protective capacity against low-density lipoprotein oxidation. In an attempt to elucidate the mechanism of action of quercetin, we have now determined the effects of quercetin on PON1 gene expression, activity, protein level, nuclear mature sterol regulatory element binding protein 2 (SREBP2) level, and its translocation from the endoplasmic reticulum to nucleus and its interaction with PON1 promoter in human HuH7 liver cells using real-time reverse transcriptase polymerase chain reaction, spectrophotometry, immunoblot, confocal microscopy, and electrophoretic mobility shift assay techniques, respectively. Quercetin (20 micromol/L) treatment increased PON1 messenger RNA by 75% (P < .02), with a concomitant 2-fold (P < .05) increase in PON1 activity accompanied by 60% (P < .01) increase in PON1 protein level. There was parallel to the 1.5- to 2.0-fold increase (P < .05) in mature SREBP2 in the cell nuclei that was verified by increased immunolocalization of the mature SREBP2 (65-kd species) in the nuclei of quercetin-treated cells by confocal microscopy. Evaluation of the binding of biotin-labeled sterol responsive element (SRE)-like element of the PON1 promoter to the nuclear extract from the 24-hour quercetin (20 micromol/L)-treated HuH7 cells by electrophoretic mobility shift assay revealed that the SREBP2 specifically binds to the SRE-like element that was abolished by prior incubation with anti-SREBP2 or significantly decreased by 200-fold molar excess of unlabeled SRE-like sequence. Based on these results, we conclude that quercetin exhibits its antiatherogenic property by eliciting the translocation of the mature SREBP2 from endoplasmic reticulum to the nucleus, where it binds to SRE-like sequence in the PON1 promoter and up-regulates PON1 gene transcription and PON1 activity.
Garige
Mamatha
M
Department of Biochemistry, Molecular Biology and Medicine, The George Washington University, NW, Washington, DC 20052, USA.
Gong
Maokai
M
Varatharajalu
Ravi
R
Lakshman
M Raj
MR
D000704:Analysis of Variance; D043303:Aryldialkylphosphatase / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D018613:Microscopy, Confocal; D011794:Quercetin / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013053:Spectrophotometry; D051782:Sterol Regulatory Element Binding Protein 2 / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D051782:Sterol Regulatory Element Binding Protein 2; D011794:Quercetin; D043303:Aryldialkylphosphatase