The roles of sterol regulatory element-binding proteins in the transactivation of the rat ATP citrate-lyase promoter

ATP citrate-lyase (ACL) is a key enzyme supplying acetyl-CoA for fatty acid and cholesterol synthesis. Its expression is drastically up-regulated when an animal is fed a low fat, high carbohydrate diet after prolonged fasting. In this report, we describe the role of sterol regulatory element-binding proteins (SREBPs) in the transactivation of the rat ACL promoter. ACL promoter activity was markedly stimulated by the overexpression of SREBP-1a and, to a lesser extent, by SREBP-2 in Alexander human hepatoma cells. The promoter elements responsive to SREBPs were located within the 55-base pair sequences from -114 to -60. The gel mobility shift assay revealed four SREBP-1a binding sites in this region. Of these four elements, the -102/-94 region, immediately upstream of the inverted Y-box, and the -70/-61 region, just adjacent to Sp1 binding site, played critical roles in SREBPs-mediated stimulation. The mutation in the inverted Y-box and the coexpression of dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by SREBP-1a, suggesting that NF-Y binding is a prerequisite for SREBPs to activate the ACL promoter. However, the multiple Sp1 binding sites did not affect the transactivation of the ACL promoter by SREBPs. The binding affinity of SREBP-1a to SREs of the ACL promoter also was much higher than that of SREBP-2. The transactivation potencies of the chimeric SREBPs, of which the activation domains (70 amino acids of the amino terminus) were derived from the different species of their carboxyl-terminal region, were similar to those of SREBPs corresponding to their carboxyl termini. Therefore, it is suggested that the carboxyl-terminal portions of SREBPs containing DNA binding domains are important in determining their transactivation potencies to a certain promoter.     

Differential mRNA expression of seven genes involved in cholesterol metabolism and transport in the liver of atherosclerosis-susceptible and -resistant Japanese quail strains

Background

Two atherosclerosis-susceptible and -resistant Japanese quail (Coturnix japonica) strains obtained by divergent selection are commonly used as models to study atherosclerosis, but no genetic characterization of their phenotypic differences has been reported so far. Our objective was to examine possible differences in the expression of genes involved in cholesterol metabolism and transport in the liver between these two strains and to evaluate the value of this model to analyze the gene system affecting cholesterol metabolism and transport.

Methods

A factorial study with both strains (atherosclerosis-susceptible versus atherosclerosis-resistant) and two diets (control versus cholesterol) was carried out. The mRNA concentrations of four genes involved in cholesterol biosynthesis (HMGCR, FDFT1, SQLE and DHCR7) and three genes in cholesterol transport (ABCG5, ABCG8 and APOA1) were assayed using real-time quantitative PCR. Plasma lipids were also assayed.

Results

Expression of ABCG5 (control diet) and ABCG8 (regardless of dietary treatment) and expression of HMGCR, FDFT1 and SQLE (regardless of dietary treatment) were significantly higher in the atherosclerosis-resistant than in the atherosclerosis-susceptible strain. Plasma triglyceride and LDL levels, and LDL/HDL ratio were significantly higher in the atherosclerosis-susceptible than in the atherosclerosis-resistant strain fed the cholesterol diet. In the atherosclerosis-susceptible strain, ABCG5 expression regressed significantly and positively on plasma LDL level, whereas DHCR7 and SQLE expression regressed significantly and negatively on plasma triglyceride level.

Conclusions

Our results provide support for the hypothesis that the atherosclerosis-resistant strain metabolizes and excretes cholesterol faster than the atherosclerosis-susceptible strain. We have also demonstrated that these quail strains are a useful model to study cholesterol metabolism and transport in relation with atherosclerosis.     

Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress

The StarD4 and StarD5 proteins share approximately 30% identity, and each is a steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. We previously showed StarD4 expression is sterol-repressed, consistent with regulation by sterol regulatory element-binding proteins (SREBPs), whereas StarD5 is not sterol-regulated. Here we further address the regulation and function of StarD4 and StarD5. Unlike StAR, the START family prototype, StarD4 and StarD5 were not induced by steroidogenic stimuli in Leydig cells. However, StarD4 and StarD5 showed StAR-like activity in a cell culture steroidogenesis assay, indicating cholesterol transfer. In transgenic mice expressing active SREBPs, StarD4 was predominantly activated by SREBP-2 rather than SREBP-1a. The mouse and human StarD4 proximal promoters share approximately 70% identity, including several potential sterol regulatory elements (SREs). Reporters driven by the StarD4 promoter from either species were transfected into NIH-3T3 cells, and reporter activity was highly repressed by sterols. Site-directed mutagenesis of potential SREs identified a conserved functional SRE in the mouse (TCGGTCCAT) and human (TCATTCCAT) promoters. StarD5 was not sterol-repressed via SREBPs nor was it sterol-activated via liver X receptors (LXRs). Even though StarD4 and StarD5 were not LXR targets, their overexpression stimulated LXR reporter activity, suggesting roles in cholesterol metabolism. StarD5 expression increased 3-fold in free cholesterol-loaded macrophages, which activate the endoplasmic reticulum (ER) stress response. When NIH-3T3 cells were treated with agents to induce ER stress, StarD5 expression increased 6-8-fold. Because StarD4 is regulated by sterols via SREBP-2, whereas StarD5 is activated by ER stress, they likely serve distinct functions in cholesterol metabolism.     

Hepatic SREBP-2 and cholesterol biosynthesis are regulated by FoxO3 and Sirt6

Cholesterol homeostasis is crucial for cellular function and organismal health. The key regulator for the cholesterol biosynthesis is sterol-regulatory element binding protein (SREBP)-2. The biochemical process and physiological function of SREBP-2 have been well characterized; however, it is not clear how this gene is epigenetically regulated. Here we have identified sirtuin (Sirt)6 as a critical factor for Srebp2 gene regulation. Hepatic deficiency of Sirt6 in mice leads to elevated cholesterol levels. On the mechanistic level, Sirt6 is recruited by forkhead box O (FoxO)3 to the Srebp2 gene promoter where Sirt6 deacetylates histone H3 at lysines 9 and 56, thereby promoting a repressive chromatin state. Remarkably, Sirt6 or FoxO3 overexpression improves hypercholesterolemia in diet-induced or genetically obese mice. In summary, our data suggest an important role of hepatic Sirt6 and FoxO3 in the regulation of cholesterol homeostasis.     

MicroRNA-33a regulates cholesterol synthesis and cholesterol efflux-related genes in osteoarthritic chondrocytes

IntroductionSeveral studies have shown that osteoarthritis (OA) is strongly associated with metabolism-related disorders, highlighting OA as the fifth component of the metabolic syndrome (MetS). On the basis of our previous findings on dysregulation of cholesterol homeostasis in OA, we were prompted to investigate whether microRNA-33a (miR-33a), one of the master regulators of cholesterol and fatty acid metabolism, plays a key role in OA pathogenesis.MethodsArticular cartilage samples were obtained from 14 patients with primary OA undergoing total knee replacement surgery. Normal cartilage was obtained from nine individuals undergoing fracture repair surgery. Bioinformatics analysis was used to identify miR-33a target genes. miR-33a and sterol regulatory element-binding protein 2 (SREBP-2) expression levels were investigated using real-time PCR, and their expression was also assessed after treatment with transforming growth factor-β1 (TGF-β1) in cultured chondrocytes. Akt phosphorylation after treatment with both TGF-β1 and miR-33a inhibitor or TGF-β1 and miR-33a mimic was assessed by Western blot analysis. Furthermore, we evaluated the effect of miR-33a mimic and miR-33a inhibitor on Smad7, a negative regulator of TGF-β signaling, on cholesterol efflux-related genes, ATP-binding cassette transporter A1 (ABCA1), apolipoprotein A1 (ApoA1) and liver X receptors (LXRα and LXRβ), as well as on matrix metalloproteinase-13 (MMP-13), using real-time PCR.ResultsWe found that the expression of miR-33a and its host gene SREBP-2 was significantly elevated in OA chondrocytes compared with normal chondrocytes. Treatment of cultured chondrocytes with TGF-β1 resulted in increased expression of both miR-33a and SREBP-2, as well as in rapid induction of Akt phosphorylation, whereas TGF-β-induced Akt phosphorylation was enhanced by miR-33a and suppressed by inhibition of miR-33a, as a possible consequence of Smad7 regulation by miR-33a. Moreover, treatment of normal chondrocytes with miR-33a resulted in significantly reduced ABCA1 and ApoA1 mRNA expression levels and significantly elevated MMP-13 expression levels, promoting the OA phenotype, whereas miR-33a’s suppressive effect was reversed using its inhibitor.ConclusionsOur findings suggest, for the first time to our knowledge, that miR-33a regulates cholesterol synthesis through the TGF-β1/Akt/SREBP-2 pathway, as well as cholesterol efflux-related genes ABCA1 and ApoA1, in OA chondrocytes, pointing to its identification as a novel target for ameliorating the OA phenotype.