SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.

The Non Catalytic Protein ERG28 has a Functional Role in Cholesterol Synthesis and is Coregulated Transcriptionally.

The enzymatic pathway of cholesterol biosynthesis has been well characterized. However, there remain several potential interacting proteins that may play ancillary roles in the regulation of cholesterol production. Here, we identified ERG28 (chromosome 14 open reading frame 1 [C14orf1]), a homologue of the yeast protein Erg28p, as a player in mammalian cholesterol synthesis. ERG28 is conserved from yeast to humans but has been largely overlooked in mammals. Using quantitative RT-PCR, luciferase assays, and publicly available chromatin immunoprecipitation sequencing data, we found that transcription of this gene is driven by the transcription factor SREBP-2, akin to most cholesterol synthesis enzymes, as well as identifying sterol-responsive elements and cofactor binding sites in its proximal promoter. Based on a split luciferase system, ERG28 interacted with itself and two enzymes of cholesterol synthesis (NSDHL and SC4MOL). Huh7 ERG28-KO cell lines were generated, revealing reduced total cholesterol levels in sterol-depleted environments. In addition, radiolabeled metabolic flux assays showed a 60-75% reduction in the rate of cholesterol synthesis in the KO versus wild-type cells, which could be rescued by expression of ectopic ERG28. Unexpectedly, KO of ERG28 also impaired the activation of SREBP-2 under sterol-replete conditions, by a yet-to-be defined mechanism. These results indicate that ERG28 is clearly involved in cholesterol synthesis, although the precise role this noncatalytic protein plays in this complex metabolic pathway remains to be fully elucidated. A deeper understanding of ERG28, and other ancillary proteins of cholesterol synthesis, may help inform therapeutic strategies for diseases associated with aberrant cholesterol metabolism.

Damage response protein 1 (Dap1) functions in the synthesis of carotenoids and sterols in Xanthophyllomyces dendrorhous.

Cytochrome P450s (P450s) are heme-containing proteins involved in several cellular functions, including biosynthesis of steroidal hormones, detoxification of xenobiotic compounds, among others. Damage response protein 1 (Dap1) has been described as a positive regulator of P450s through protein-protein interactions in organisms such as Schizosaccharomyces pombe. Three P450s in the carotenogenic yeast Xanthophyllomyces dendrorhous have thus far been characterized: Cyp51 and Cyp61, which are involved in ergosterol biosynthesis, and CrtS (astaxanthin synthase), which is involved in biosynthesis of the carotenoid astaxanthin. In this work, we describe the X. dendrorhous DAP1 gene, deletion of which affected yeast pigmentation by decreasing the astaxanthin fraction and increasing the ß-carotene (a substrate of CrtS) fraction, which is consistent with the known role of CrtS. We found that the proportion of ergosterol was also decreased in the Δdap1 mutant. However, even though the fractions of the end products of these two pathways (the synthesis of carotenoids and sterols) were decreased in the Δdap1 mutant, the transcript levels of genes from the P450 systems involved were higher than those in the wild-type strain. We demonstrate that Dap1 coimmunoprecipitates with these three P450s, suggesting that Dap1 interacts with these three proteins. We propose that Dap1 regulates the synthesis of astaxanthin and ergosterol in X. dendrorhous, probably by regulating the P450s involved in both biosynthetic pathways at the protein level. This work suggests a new role for Dap1 in the regulation of carotenoid biosynthesis in X. dendrorhous.

HCBP6 Modulates Triglyceride Homeostasis in Hepatocytes Via the SREBP1c/FASN Pathway.

Hypertriglyceridemia leads to liver steatosis, cardiovascular disease, and type 2 diabetes. Although HCBP6 (hepatitis C virus core-binding protein 6) was previously shown to be an HCV (hepatitis C virus) core-binding protein, its biological function remains unclear. Here, we demonstrate that HCBP6 negatively regulates intracellular triglyceride (TG) levels in hepatocytes. We found that bidirectional manipulation of hepatocyte HCBP6 expression by knockdown or overexpression results in increased or decreased TG accumulation, respectively. In addition, HCBP6 mRNA and protein levels exhibited significant time- and dose-dependent increases in a cellular model of lipid-overload hepatic steatosis. Furthermore, TG levels are regulated by HCBP6-sterol regulatory element binding protein 1c (SREBP1c)-mediated fatty acid synthase (FASN) expression. We also demonstrate that HCBP6 mRNA and protein expression is inhibited by microRNA-122 (miR-122), and miR-122 overexpression elicited more robust translational repression of luciferase activity driven by the full 3'-UTR of HCBP6. Taken together, our results provide new evidence that miR-122-regulated HCBP6 functions as a sensor protein to maintain intrahepatocyte TG levels.

ß-caryophyllene enhances the transcriptional upregulation of cholesterol biosynthesis in breast cancer cells.

ß-caryophyllene (BCP) exhibits anti-proliferative properties in cancer cells. Here, we examine the hypothesis that BCP induces membrane remodeling. Our data show that high concentrations of BCP increase membrane permeability of human breast cells (hBrC) causing detachment and cell death. At a sub-lethal concentration of BCP, we show that BCP induces a striking upregulation of genes involved in cholesterol biosynthesis, including the gene that encodes for HMGCoA reductase (HMGCR), the rate-determining step in cholesterol biosynthesis. In addition, stearoyl-CoA desaturase (SCD) is also upregulated which would lead to the enhanced formation of monounsaturated fatty acids, specifically oleate and palmitoleate from stearoyl CoA and palmitoyl CoA, respectively. These fatty acids are major components of membrane phospholipids and cholesterol esters. Together, these data suggest that cells respond to BCP by increasing the synthesis of components found in membranes. These responses could be viewed as a repair mechanism and/or as a mechanism to mount resistance to the cytotoxic effect of BCP. Blocking HMGCR activity enhances the cytotoxicity of BCP, suggesting that BCP may provide an additional therapeutic tool in controlling breast cancer cell growth.