HNF4alpha is a crucial modulator of the cholesterol-dependent regulation of NPC1L1.
OBJECTIVE
Niemann-Pick C1-like 1 (NPC1L1) has been identified as a target of ezetimibe and found to be responsible for intestinal cholesterol absorption. Although, it was recently demonstrated that sterol responsive element binding protein 2 (SREBP2) is responsible for the cholesterol-dependent down-regulation of NPC1L1, the molecular mechanism of NPC1L1 expression is not fully understood. In the present study, we examined the involvement of hepatocyte nuclear factor 4alpha (HNF4alpha), a key modulator of lipid metabolism, in the transcriptional regulation of human NPC1L1 gene.


METHODS
Reporter gene assays and EMSAs were performed using human NPC1L1 promoter constructs and the effect of siHNF4alpha was examined.


RESULTS
Transfection of SREBP2 induced the transcriptional activities of NPC1L1 and additional transfection of HNF4alpha results in a marked stimulation of the activities. Studies with deletion mutants indicated that important elements are located within 264 nt upstream in the human NPC1L1 promoter. In addition, studies with mutations in putative binding sites of HNF4alpha indicated the existence of binding sites in -209 to -197 and -52 to -40. Moreover, HNF4alpha knockdown resulted in the reduced expression and regulation by cholesterol.


CONCLUSIONS
It is concluded that HNF4alpha plays a crucial role in the expression and regulation of human NPC1L1 gene.
Iwayanagi
Yuki
Y
Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
Takada
Tappei
T
Suzuki
Hiroshi
H
D024506:5' Flanking Region / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D002784:Cholesterol / Q000502:physiology*; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000502:physiology; D005822:Genetic Vectors; D051557:Hepatocyte Nuclear Factor 4 / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D026901:Membrane Transport Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051782:Sterol Regulatory Element Binding Protein 2 / Q000235:genetics / Q000502:physiology; D015533:Transcriptional Activation; D014162:Transfection
C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C410800:NPC1L1 protein, human; D018160:Receptors, Cytoplasmic and Nuclear; C497390:SREBF2 protein, human; D051782:Sterol Regulatory Element Binding Protein 2; D002784:Cholesterol; D008156:Luciferases