Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatory element binding protein-2

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the subtilases that promotes the internalization and degradation of LDL receptor in liver and thereby controls the level of LDL cholesterol in plasma. Here, we show that the expression of PCSK9 in HepG2 cells is completely dependent on the absence or presence of sterols. The minimal promoter region of the PCSK9 gene contains a sterol-regulatory element (SRE), which makes the transcription of PCSK9 dependent on sterols. Expression of nuclear forms of sterol-regulatory element binding protein-1 (SREBP-1) and SREBP-2 dramatically increased the promoter activity of PCSK9. In vitro-translated nuclear forms of SREBPs showed interactions with SRE, whereas mutations in SRE abolished their binding. In vivo studies in mice showed that Pcsk9 protein and mRNA were decreased significantly by fasting and increased by refeeding. However, supplementation with 2% cholesterol in the diet prevented the increase in Pcsk9. The amounts of Pcsk9 mRNA in livers of refed mice showed correlated regulation by the changes in the nuclear form of Srebp-2. In summary, it is suggested that the expression of PCSK9 is regulated by sterol at the transcriptional level in HepG2 cells and that both SREBP-1 and SREBP-2 can transcriptionally activate PCSK9 via SRE in its proximal promoter region in vitro. However, in vivo, it is suggested that the sterol-dependent regulation of PCSK9 is mediated predominantly by SREBP-2. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the subtilases that promotes the internalization and degradation of LDL receptor in liver and thereby controls the level of LDL cholesterol in plasma. Here, we show that the expression of PCSK9 in HepG2 cells is completely dependent on the absence or presence of sterols. The minimal promoter region of the PCSK9 gene contains a sterol-regulatory element (SRE), which makes the transcription of PCSK9 dependent on sterols. Expression of nuclear forms of sterol-regulatory element binding protein-1 (SREBP-1) and SREBP-2 dramatically increased the promoter activity of PCSK9. In vitro-translated nuclear forms of SREBPs showed interactions with SRE, whereas mutations in SRE abolished their binding. In vivo studies in mice showed that Pcsk9 protein and mRNA were decreased significantly by fasting and increased by refeeding. However, supplementation with 2% cholesterol in the diet prevented the increase in Pcsk9. The amounts of Pcsk9 mRNA in livers of refed mice showed correlated regulation by the changes in the nuclear form of Srebp-2. In summary, it is suggested that the expression of PCSK9 is regulated by sterol at the transcriptional level in HepG2 cells and that both SREBP-1 and SREBP-2 can transcriptionally activate PCSK9 via SRE in its proximal promoter region in vitro. However, in vivo, it is suggested that the sterol-dependent regulation of PCSK9 is mediated predominantly by SREBP-2. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilisin serine proteases of which the gain-of-function mutations cause hypercholesterolemia (1Abifadel M. Varret M. Rabes J-P. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. Villeger L. et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2188) Google Scholar, 2Leren T. Mutations in the PCSK9 gene in Norwegian subjects with autosomal dominant hypercholesterolemia.Clin. Genet. 2004; 65: 419-422Crossref PubMed Scopus (206) Google Scholar, 3Timms K. Wagner S. Samuels M. Forbey K. Goldfine H. Jammulapati S. Skolnick M. Hopkins P. Hunt S. Shattuck D. A mutation in PCSK9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree.Hum. Genet. 2004; 114: 349-353Crossref PubMed Scopus (270) Google Scholar). PCSK9 was independently identified as one of the genes that are regulated by sterol-regulatory element binding proteins (SREBPs) (4Horton J.D. Shah N.A. Warrington J.A. Anderson N.N. Park S.W. Brown M.S. Goldstein J.L. Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes.Proc. Natl. Acad. Sci. USA. 2003; 100: 12027-12032Crossref PubMed Scopus (1085) Google Scholar, 5Maxwell K.N. Soccio R.E. Duncan E.M. Sehayek E. Breslow J.L. Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice.J. Lipid Res. 2003; 44: 2109-2119Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). The SREBPs are members of the basic helix-loop-helix leucine zipper family of transcription factors that regulate the expression of the target genes by binding to the sterol-regulatory elements (SREs) in their promoter regions (6Brown M.S. Goldstein J.L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). Using microarrays hybridized with RNA from livers of mice that either overexpressed nuclear forms of human SREBPs (transgenic model) or lacked SREBP-activating protein (knockout model), PCSK9 was identified as a SREBP target gene. Soon after the first cloning of this gene, with its relationship to neural apoptosis and liver regeneration, studies focused on its relationship with the regulation of cholesterol in plasma. Subsequently, the loss-of-function mutations of PCSK9 have been reported to decrease LDL cholesterol level (7Cohen J. Pertsemlidis A. Kotowski I.K. Graham R. Garcia C.K. Hobbs H.H. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.Nat. Genet. 2005; 37: 161-165Crossref PubMed Scopus (1077) Google Scholar, 8Carmeron J. Holla O.L. Ranheim T. Kulseth M.A. Berge K.E. Leren T.P. Effects of mutations in the PCSK9 gene on the cell surface LDL receptors.Hum. Mol. Genet. 2006; 15: 1551-1558Crossref PubMed Scopus (226) Google Scholar, 9Kotowski I.K. Pertsemlidis A. Luke A. Cooper R.S. Vega G.L. Cohen J.C. Hobbs H.H. A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol.Am. J. Hum. Genet. 2006; 78: 410-412Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar) and reduce the risk of coronary heart disease (10Cohen J.C. Boerwinkle E. Mosley Jr, T.H. Hobbs H.H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.N. Engl. J. Med. 2006; 354: 1264-1272Crossref PubMed Scopus (2428) Google Scholar). The definite evidence a of PCSK9 in LDL metabolism was by a of in vivo of PCSK9 the of low lipoprotein receptor in livers K.N. Breslow J.L. expression of Pcsk9 in mice in a low-density lipoprotein receptor knockout Natl. Acad. Sci. USA. 2004; PubMed Scopus Google Scholar, S.W. J.D. regulation of low lipoprotein receptor protein by convertase subtilisin/kexin type in 2004; Full Text Full Text PDF PubMed Scopus Google whereas the of increased significantly in livers of Pcsk9 knockout mice S. R. Anderson N.N. R.E. J.D. plasma cholesterol and to in mice Natl. Acad. Sci. USA. 2005; PubMed Scopus Google Scholar). The by which PCSK9 that PCSK9 in plasma with protein on the cell surface R. Park S.W. Anderson N.N. R.E. J.D. PCSK9 the of LDL in and in livers of mice.J. 2006; PubMed Scopus Google Scholar) and as a to to target degradation J.D. activity is PCSK9 to reduce low lipoprotein in HepG2 Full Text Full Text PDF PubMed Scopus Google Scholar). studies have focused on the of the regulation of PCSK9, is the transcriptional regulation of PCSK9, its as a gene regulated by et A. H. J. L. A. PCSK9, the gene the convertase neural in 2004; PubMed Scopus Google Scholar) reported that the transcription of PCSK9 was increased by SREBP-2 in human and the of SRE in the promoter region of and human PCSK9 et P. A. M. PCSK9 expression is regulated by via and sterol protein 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) reported that increased PCSK9 via in and as as in vivo, on However, the changes in the nuclear forms of both SREBP-1 and SREBP-2 proteins in with the changes in PCSK9 and promoter activity have been In this we show that the expression of PCSK9 is dependent on the absence or presence of via SRE in the minimal promoter region of the human PCSK9 gene by both SREBP-1 and SREBP-2 in HepG2 we that the transcriptional of PCSK9 by cholesterol is SREBP-2 in were J. A Scholar). was from as by et J. A. Goldstein J.L. Brown M.S. 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A. M. PCSK9 expression is regulated by via and sterol protein 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The SRE in the the to SRE in the a at on the the of which in the promoter the transcription or sterol-dependent regulation of the promoter C. Brown M.S. Goldstein J.L. protein that sterol element of low lipoprotein receptor of the protein and of its target Full Text PDF PubMed Google Scholar, Goldstein J.L. Brown M.S. of activity of sterol element in low lipoprotein receptor Full Text PDF PubMed Google Scholar, C. A. J. Goldstein J.L. Brown M.S. a zipper protein that controls transcription of the low lipoprotein receptor Full Text PDF PubMed Scopus Google Scholar). The binding been reported to in the transcriptional regulation of genes regulation of A a of Natl. Acad. Sci. USA. 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Brown M.S. Goldstein J.L. of sterol element binding proteins on both of the Full Text Full Text PDF PubMed Scopus Google Scholar). The amounts of expression were to in a the amounts the of The amounts of proteins were to to by analysis the expression of nuclear forms of SREBPs dramatically increased the transcription of the PCSK9 promoter in The expression of the nuclear form of the of which is that in the PCSK9 promoter or SREBP-2. The of PCSK9 promoter activity by SREBPs was abolished by the mutation of that the of SRE in the regulation of PCSK9 promoter activity is mediated by both SREBP-1 and SREBP-2 in HepG2 the SREBPs can to the SRE in the PCSK9 we in vitro-translated nuclear forms of and SREBP-2 to the and of the in that and SREBP-2 can to the the by SREBP-2 and by The SRE showed and oligonucleotide the binding of SREBPs to the The were by the of against SREBP-1 or SREBP-2 whereas the of the was by the of the that SRE in the PCSK9 promoter both and SREBP-2 in vitro. the regulation of Pcsk9 in livers of mice that were to of fasting by with a diet 2% cholesterol In livers of the in the proteins of the to the form of Pcsk9. is that the of the form of Pcsk9 in vivo is to by analysis the in this that fasting the mice abolished the expression of Pcsk9 protein and a diet or diet the of Pcsk9 to the level in livers of mice of Pcsk9 expression was by cholesterol in a diet and was mice were refed with a diet with cholesterol In the of in livers of mice showed with the in mice whereas a diet or a diet cholesterol the expression of However, the mice with a diet with cholesterol the of to the level in the mice that the of in vivo the changes of Pcsk9 expression the mice are The amounts of the nuclear form of were the mice were of the supplementation of whereas the nuclear form of showed by cholesterol which correlated with the changes of Pcsk9. the changes in Pcsk9 and mRNA by fasting and RNA was from the livers of mice and was The changes in Pcsk9 mRNA to in Pcsk9 fasting dramatically decreased Pcsk9 Pcsk9 and supplementation of cholesterol the of Pcsk9 mRNA by fasting and refeeding. mRNA was the mice were refed a diet with the changes in the genes in and cholesterol the transcription of which is the of to the of cholesterol on the transcription of gene. The mRNA one of the target genes A direct sterol element binding protein in of A Full Text Full Text PDF PubMed Scopus Google showed by supplementation of The changes of H. M. J. K. K. et protein-1 as a transcription of Full Text Full Text PDF PubMed Scopus Google regulation of regulation of Full Text Full Text PDF PubMed Scopus Google and of and transcription factors transcription of and Full Text Full Text PDF PubMed Scopus Google as target genes were The and showed by whereas in their were by a the transcription of target genes was by cholesterol in livers of data that the regulation of Pcsk9 expression by cholesterol in vivo is at the transcription level and that predominantly this PCSK9 been reported as one of the genes transcription is the of SREBPs (4Horton J.D. Shah N.A. Warrington J.A. Anderson N.N. Park S.W. Brown M.S. Goldstein J.L. Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes.Proc. Natl. Acad. Sci. USA. 2003; 100: 12027-12032Crossref PubMed Scopus (1085) Google Scholar, 5Maxwell K.N. Soccio R.E. Duncan E.M. Sehayek E. Breslow J.L. Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice.J. Lipid Res. 2003; 44: 2109-2119Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). the of PCSK9 in cholesterol it is suggested that the of its expression is SREBP-2 A. H. J. L. A. PCSK9, the gene the convertase neural in 2004; PubMed Scopus Google whereas in mice that were by with a diet showed the of SREBP-1 in the of PCSK9 expression P. A. M. PCSK9 expression is regulated by via and sterol protein 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). regulation of PCSK9 transcription by SREBPs been by the presence of SRE on the promoter region of the PCSK9 gene. However, evidence the regulation of PCSK9 proteins by been In the we the against PCSK9 and the regulation of PCSK9 expression by and The expression PCSK9 protein to HepG2 cells were of sterols. the of was by with a of to J.L. Brown M.S. of the PubMed Scopus Google Scholar, D. J. J. Brown M.S. Goldstein J.L. cloning of a gene a putative of 1997; Full Text Full Text PDF PubMed Scopus Google the of PCSK9 was The increase in PCSK9 by the of was completely by the of in the changes in PCSK9 protein by the changes in its The changes in the nuclear forms of SREBP-2 by were to of PCSK9, the changes in PCSK9 expression the of in this which been reported to increase the transcription of PCSK9 A. H. J. L. A. PCSK9, the gene the convertase neural in 2004; PubMed Scopus Google to increase the expression of PCSK9 protein and mRNA at low of PCSK9 by was by a of that the regulation of PCSK9 expression is dependent on the presence or absence of which are either or and that this regulation is at the transcription The expression of showed changes to PCSK9 in HepG2 cells the that the of PCSK9 is to the degradation of J. Cohen J. Hobbs H. of its in LDL Sci. Full Text Full Text PDF PubMed Scopus Google the in to PCSK9 by in HepG2 cells as a this is that the in this is to show the regulation of and PCSK9 the cells are of the increased expression of the nuclear form of SREBP-2 the expression of both PCSK9 and to a level that the regulation of by the that supplementation of to the cells in the of protein and whereas the expression of PCSK9 and the nuclear form of SREBP-2 completely that of a or in HepG2 cells to the expression of to the transcriptional regulation of PCSK9 by we the region of human PCSK9 and its promoter reported by et A. H. J. L. A. PCSK9, the gene the convertase neural in 2004; PubMed Scopus Google the SRE in the PCSK9 promoter to in the sterol-dependent regulation of PCSK9 transcription in HepG2 Mutations of the SRE in the PCSK9 promoter dramatically the promoter activity in the absence of and the sterol-dependent of the promoter activity was from to The of the SRE in the sterol-dependent regulation of the PCSK9 promoter was by the that the expression of the nuclear forms of or significantly the promoter whereas mutation in SRE in the of by However, it is suggested that the presence of SRE is the sterol-dependent regulation of the PCSK9 in which the SRE its transcriptional that the and the SRE is the transcriptional activity and sterol-dependent regulation of the PCSK9 in this region of PCSK9 a nuclear reported in the liver binding protein of mice and transcription factors activate mutations J. 2003; PubMed Scopus Google Scholar). However, it is nuclear is the transcription of the PCSK9 on the by on this region was in this The binding which activate the expression of the promoter with SREBP-1 L. by sterol protein and in sterol regulation of low lipoprotein receptor Full Text Full Text PDF PubMed Scopus Google were identified to the sterol-dependent regulation of PCSK9 The of SREBPs with the SRE in the PCSK9 promoter was by The in vitro-translated nuclear forms of and SREBP-2 to this SRE on the in SREBP-2 is to the SRE of the PCSK9 promoter is the in vitro-translated nuclear form of SREBP-2 the on in which were with of or in the However, it that the of the the of SREBPs to the SRE in vitro-translated SREBPs were their and to we in vitro-translated proteins to the on the by the The absence of the it to the amounts of in vitro-translated proteins in we the of in vitro-translated proteins with the by analysis regulation of Pcsk9 was in livers of mice the dramatically decreased Pcsk9 protein and and the expression of Pcsk9 in livers of However, a diet with 2% cholesterol the of the decreased expression of Pcsk9 in livers of of Pcsk9 by cholesterol correlated with the decrease in the nuclear form of SREBP-2 by whereas the nuclear form of in livers of mice refed changes in the expression of protein were in the mice refed a The of SREBP-2 in the sterol-dependent regulation of Pcsk9 is by the that decrease the expression of SREBP-1 target as and reported by et P. A. M. PCSK9 expression is regulated by via and sterol protein 2006; Full Text Full Text PDF PubMed Scopus Google in this the of in the regulation of Pcsk9 expression in livers of mice fasting and refeeding. is by that fasting dramatically the expression of Pcsk9 and that a diet completely the expression of Pcsk9. can that both the of and the and the expression of Pcsk9. studies that the expression of PCSK9 is regulated by at the transcriptional level both in and in vivo, and the of sterol-dependent expression of PCSK9 is SREBP-2 in the of regulation of PCSK9 studies to a that the of SREBPs on PCSK9 expression and to the transcription factors The the and The at the of at in the of PCSK9 was by by the with low lipoprotein receptor convertase subtilisin/kexin type 9 sterol-regulatory element sterol-regulatory element binding protein