Reporter assays using the ؊2.0-kb promoter revealed that this region contains a functional PPAR␥-responsive element. Gel mobility shift and chromatin immunoprecipitation assays showed that endogenous PPAR␥ protein binds to the perilipin promoter. PPAR␥2, an isoform exclusively expressed in adipocytes, was found to be the most potent regulator from among the PPAR family members including PPAR␣ and PPAR␥1. These results make evident the fact that perilipin gene expression in differentiating adipocytes is crucially regulated by PPAR␥2, providing new insights into the adipogenic action of PPAR␥2 and adipose-specific gene expression, as well as potential anti-obesity pharmaceutical agents targeted to a reduction of the perilipin gene product.Adipocytes are the major reservoir of energy stored in the form of triacylglycerol in the body. Triacylglycerol is stored within intracellular lipid droplets covered by a monolayer of phospholipids, free cholesterol, and proteins. In times of energy need, for example, as brought about by activities such as fasting and/or exercise, catecholamines rapidly activate cAMP-dependent protein kinase (PKA), 1 resulting in hydrolysis of triacylglycerol as catalyzed by activated hormone-sensitive lipase.

The destiny and activity of sterol regulatory element-binding proteins (SREBPs) in the nucleus are regulated by modification with ubiquitin, small ubiquitin-like modifier (SUMO), or phosphorus. ERK-dependent phosphorylation causes an increase in their transcriptional activity, whereas SUMO modification halts it. We hypothesized a causal linkage between phosphorylation and sumoylation because their sites are very closely located in SREBP-1 and -2 molecules. When Ser 455 , a phosphorylation site in SREBP-2, was substituted with Ala, this SREBP-2 mutant was more efficiently modified by SUMO-1. On the other hand, substitution of Asp inhibited SUMO conjugation, mimicking phosphoserine. When cells were cultured with insulin-like growth factor-1, sumoylation of SREBP-2 was decreased with an increase in its phosphorylation, but SREBP-2(S455A) was continuously sumoylated. An ERK cascade inhibitor, U0126, inversely augmented SUMO modification of SREBP-2. Insulin-like growth factor-1 treatment stimulated the expression of SREBP target genes such as the low density lipoprotein (LDL) receptor, squalene synthase, and hydroxymethylglutaryl-CoA synthase genes. These results indicate that growth factor-induced phosphorylation of SREBP-2 inhibits sumoylation, thereby facilitating SREBP transcriptional activity. Glutathione S-transferase pulldown assays revealed that wild-type SREBP-2, but not a mutant lacking Lys 464 , interacts with HDAC3 preferentially among the histone deacetylase family members. HDAC3 small interfering RNA induced gene expression of the LDL receptor and thereby augmented fluorescently labeled LDL uptake in HepG2 cells. In summary, growth factors inhibit sumoylation of SREBPs through their phosphorylation, thus avoiding the recruitment of an HDAC3 corepressor complex and stimulating the lipid uptake and synthesis required for cell growth. Sterol regulatory element-binding proteins (SREBPs)2 regulate a wide variety of genes involved in cholesterol and fatty acid synthesis and low density lipoprotein (LDL) uptake (1). SREBPs are synthesized as membrane proteins located on the endoplasmic reticulum (ER) and thereafter are processed to liberate the N-terminal halves that function as transcription factors in the nucleus. The proteolytic processing of SREBPs is highly controlled by the interaction between two ER membrane proteins, the SREBP cleavage-activating protein (SCAP) and insulin-inducing gene (INSIG). Once the content of ER membrane cholesterol increases, SCAP, an SREBP-associated protein that binds cholesterol, induces conformational change and becomes attached to INSIG, thereby remaining on the ER membrane. Because the proteolytic processing occurs on the Golgi membrane where two processing enzymes reside, SREBPs together with SCAP on the ER membrane are never processed, and therefore, cholesterol is a critical determinant of SREBP activation. In contrast, under cholesterol-depleted conditions, an SREBP⅐SCAP complex is transferred to the Golgi apparatus, and thereafter, the proteolytic activation of SREBPs...

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...

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