Sterol Regulatory Element-binding Protein-2 Interacts with Hepatocyte Nuclear Factor-4 to Enhance Sterol Isomerase Gene Expression in Hepatocytes

Abstract

SREBPs1 are synthesized as membrane-bound precursor proteins. As long as intracellular sterol concentrations are sufficient, SREBPs remain bound to the endoplasmic reticulum and the nuclear envelope. Upon sterol deprivation, the NH 2 -terminal portion containing the basic helix-loop-helix leucine zipper domain is cleaved by a two-step process that occurs mainly in the Golgi apparatus (1). The mature SREBPs are then transported into the nucleus in a ␤-importin-dependent manner (2) and stimulate the transcription of genes involved in cholesterol and fatty acid metabolism. The SREBP family comprises three subtypes: SREBP-1a and SREBP-1c, which are derived from a single gene by alternative promoters and splicing, and SREBP-2, which is derived from a different gene. In addition to the sterol regulatory cleavage system, rapid degradation by the ubiquitin-proteasome pathway and another posttranslational modification, sumoylation, also regulate SREBP activity in the nucleus (3, 4). All of the data reported so far support the notion that SREBP-1c and SREBP-2 mainly regulate the transcription of genes involved in fatty acid synthesis and cholesterol metabolism, respectively, whereas SREBP-1a regulates both (5-7). SREBP-1a is the more potent activator of transcription than SREBP-1c because of a more extensive transactivation region but is expressed at a much lower level than SREBP-1c in most organs.We have identified several SREBP-responsive genes by a PCR subtraction method using a Chinese hamster ovary (CHO) cell line (CHO-487) expressing a nuclear form of human SREBP-1a with a LacSwitch inducible mammalian expression system (8). The benefit of this cell line is that it affords a higher induction of target genes, which is a crucial feature so as not to overlook any candidates, rather than a CHO cell line expressing SREBP-1c, which does not afford such a high induction level. Furthermore, the inducible genes in this cell line likely include those that are SREBP-2-responsive. Through this method we have detected SREBP-responsive genes such as the 3-hydroxy-3-methylglutaryl (HMG) CoA synthase and the stearoyl CoA desaturase-1 genes and further the ATP-citrate lyase gene (8), which catalyzes acetyl CoA synthesis. We also detected insulin-inducing gene-1, which was recently demonstrated to be a novel SCAP-binding protein regulating the proteolytic activation of SREBPs (9, 10). In the course of screening for SREBP-responsive genes, we have cloned and characterized the 5Ј-flanking region of the human SI gene.Cholesterol biosynthesis in mammals requires more than 30 enzymes, and most of this enzymatic activity is under sterolmediated feedback control (11). In the later stage of this pathway, SI catalyzes the conversion of the 8-ene isomer into the 7-ene isomer. In addition to its role in sterol isomerization, SI also functions as a multidrug-binding protein for various drugs, including the Ca 2ϩ antagonist emopamil, the immunosuppressant SR31747A, and the antiestrogen tamoxifen (12-15). Furthermore, recent genetic analyses revea...

Transcriptional regulation of human and murine 17β-hydroxysteroid dehydrogenase type-7 confers its participation in cholesterol biosynthesis

“…All the SREBP-regulated genes so far examined contain either SP1 or NF-Y-binding sites close to the SREBP-binding site within their promoters. Although based on limited published data, genes of postsqualene cholesterol biosynthesis and fatty acid synthesis seem to be regulated by SREBPs and both SP1 and NF-Y in a cholesterol-dependent manner (Xiong et al 2000, Kim et al 2001, Nagai et al 2002, Misawa et al 2003. As shown in our study, this is also the case for human and murine HSD17B7.…”

“…Besides HSD17B7, there are some further genes of postsqualene cholesterol biosynthesis regulated by HNF4. A study addressing transcriptional regulation of the SI gene (SI catalyzes the step after HSD17B7 in this pathway) identified HNF4 as an important factor of cholesterol-dependent regulation (Misawa et al 2003). Due to the weak effect of mutation of HNF4-binding site in the human HSD17B7 promoter it seems likely that HNF4 is used for transcriptional finetuning only.…”

Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α

“…Plasmid Constructs-The reporter plasmids containing the human MTP promoter (Ϫ204 to ϩ33), pMTP204-Luc, and HNF4␣-responsive element-mutated MTP promoter (HNF4␣ B site: AGTTTGGAGTCTG 3 AGTGCGGCCGCTG), pMTP204-HNF4␣-mut-Luc, and expression plasmids for the GAL4 DNA-binding domain (DBD)-HNF4␣ LBD fusion protein (pGAL4 DBD-HNF4␣ LBD) and pFLAG-HNF4␣ were described previously (12,13). A reporter plasmid, pInsig1-Luc, was constructed by inserting a 2.8-kb NheI-HindIII PCR fragment coding the 5Ј-promoter region (Ϫ2782/ϩ84) of mouse insulin-induced gene 1 (Insig1) into the same restriction sites of the pGL4.10 vector.…”

Quercetin Enhances VDR Activity, Leading to Stimulation of Its Target Gene Expression in Caco-2 Cells

“…To construct an artificial reporter plasmid, pGL-P mSpp1 ϫ 3 (which was also provided by Dr. Jun-ichi Nishikawa), a VDR-responsive element of the mouse Spp-1 gene promoter, was repeated three times and inserted into a pGL3 promoter, vector (Promega). A reporter plasmid, pG5Luc, containing five copies of GAL4-binding sites, has been described previously ( 16 ).…”