Cyp1b1 directs Srebp-mediated cholesterol and retinoid synthesis in perinatal liver; Association with retinoic acid activity during fetal development.

BACKGROUND: Cytochrome P450 1b1 (Cyp1b1) deletion and dietary retinol deficiency during pregnancy (GVAD) affect perinatal liver functions regulated by Srebp. Cyp1b1 is not expressed in perinatal liver but appears in the E9.5 embryo, close to sites of retinoic acid (RA) signaling. HYPOTHESIS: Parallel effects of Cyp1b1 and retinol on postnatal Srebp derive from effects in the developing liver or systemic signaling. APPROACH: Cluster postnatal increases in hepatic genes in relation to effects of GVAD or Cyp1b1 deletion. Sort expression changes in relation to genes regulated by Srebp1 and Srebp2.Test these treatments on embryos at E9.5, examining changes at the site of liver initiation. Use in situ hybridization to resolve effects on mRNA distributions of Aldh1a2 and Cyp26a1 (RA homeostasis); Hoxb1 and Pax6 (RA targets). Assess mice lacking Lrat and Rbp4 (DKO mice) that severely limits retinol supply to embryos. RESULTS: At birth, GVAD and Cyp1b1 deletion stimulate gene markers of hepatic stellate cell (HSC) activation but also suppress Hamp. These treatments then selectively prevent the postnatal onset of genes that synthesize cholesterol (Hmgcr, Sqle) and fatty acids (Fasn, Scd1), but also direct cholesterol transport (Ldlr, Pcsk9, Stard4) and retinoid synthesis (Aldh1a1, Rdh11). Extensive support by Cyp1b1 is implicated, but with distinct GVAD interventions for Srebp1 and Srebp2. At E9.5, Cyp1b1 is expressed in the septum transversum mesenchyme (STM) with ß-carotene oxygenase (Bco1) that generates retinaldehyde. STM provides progenitors for the HSC and supports liver expansion. GVAD and Cyp1b1-/- do not affect RA-dependent Hoxb1 and Pax6. In DKO embryos, RA-dependent Cyp26a1 is lost but Hoxb1 is sustained with Cyp1b1 at multiple sites. CONCLUSION: Cyp1b1-/- suppresses genes supported by Srebp. GVAD effects distinguish Srebp1 and Srebp2 mediation. Srebp regulation overlaps appreciably in cholesterol and retinoid homeostasis. Bco1/Cyp1b1 partnership in the STM may contribute to this later liver regulation.

An aberrant SREBP-dependent lipogenic program promotes metastatic prostate cancer.

Lipids, either endogenously synthesized or exogenous, have been linked to human cancer. Here we found that PML is frequently co-deleted with PTEN in metastatic human prostate cancer (CaP). We demonstrated that conditional inactivation of Pml in the mouse prostate morphs indolent Pten-null tumors into lethal metastatic disease. We identified MAPK reactivation, subsequent hyperactivation of an aberrant SREBP prometastatic lipogenic program, and a distinctive lipidomic profile as key characteristic features of metastatic Pml and Pten double-null CaP. Furthermore, targeting SREBP in vivo by fatostatin blocked both tumor growth and distant metastasis. Importantly, a high-fat diet (HFD) induced lipid accumulation in prostate tumors and was sufficient to drive metastasis in a nonmetastatic Pten-null mouse model of CaP, and an SREBP signature was highly enriched in metastatic human CaP. Thus, our findings uncover a prometastatic lipogenic program and lend direct genetic and experimental support to the notion that a Western HFD can promote metastasis.

SREBP-regulated lipid metabolism: convergent physiology - divergent pathophysiology.

Cellular lipid metabolism and homeostasis are controlled by sterol regulatory-element binding proteins (SREBPs). In addition to performing canonical functions in the transcriptional regulation of genes involved in the biosynthesis and uptake of lipids, genome-wide system analyses have revealed that these versatile transcription factors act as important nodes of convergence and divergence within biological signalling networks. Thus, they are involved in myriad physiological and pathophysiological processes, highlighting the importance of lipid metabolism in biology. Changes in cell metabolism and growth are reciprocally linked through SREBPs. Anabolic and growth signalling pathways branch off and connect to multiple steps of SREBP activation and form complex regulatory networks. In addition, SREBPs are implicated in numerous pathogenic processes such as endoplasmic reticulum stress, inflammation, autophagy and apoptosis, and in this way, they contribute to obesity, dyslipidaemia, diabetes mellitus, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, chronic kidney disease, neurodegenerative diseases and cancers. This Review aims to provide a comprehensive understanding of the role of SREBPs in physiology and pathophysiology at the cell, organ and organism levels.

Lipid desaturation - the next step in targeting lipogenesis in cancer?

Metabolic reprogramming is a central feature of transformed cells. Cancer metabolism is now fully back in the focus of cancer research, as the interactions between oncogenic signalling and cellular metabolic processes are uncovered. One aspect of metabolic reprogramming in cancer is alterations in lipid metabolism. In contrast to most untransformed tissues, which satisfy their demand from dietary lipids, cancer cells frequently re-activate de novo lipogenesis. However, compounds targeting fatty acid synthase (FASN), a multiprotein complex integral to lipogenesis, have so far shown limited efficacy in pre-clinical cancer models and to date only one FASN inhibitor has entered clinical trials. Recently, a number of studies have suggested that enhanced production of fatty acids in cancer cells could also increases their dependence on the activity of desaturases, a class of enzymes that insert double bonds into acyl-CoA chains. Targeting desaturase activity could provide a window of opportunity to selectively interfere with the metabolic activity of cancer cells. This review will summarise some key findings that implicate altered lipid metabolism in cancer and investigate the molecular interactions between lipid desaturation and cancer cell survival.

Hepatitis C virus infection activates an innate pathway involving IKK-α in lipogenesis and viral assembly.

Hepatitis C virus (HCV) interacts extensively with host factors to not only establish productive infection but also trigger unique pathological processes. Our recent genome-wide siRNA screen demonstrated that IκB kinase-α (IKK-α) is a crucial host factor for HCV. Here we describe a new nuclear factor κB (NF-κB)-independent and kinase-mediated nuclear function of IKK-α in HCV assembly. HCV, through its 3' untranslated region, interacts with DEAD box polypeptide 3, X-linked (DDX3X) to activate IKK-α, which translocates to the nucleus and induces a CBP/p300-mediated transcriptional program involving sterol regulatory element-binding proteins (SREBPs). This innate pathway induces lipogenic genes and enhances core-associated lipid droplet formation to facilitate viral assembly. Chemical inhibitors of IKK-α suppress HCV infection and IKK-α-induced lipogenesis, offering a proof-of-concept approach for new HCV therapeutic development. Our results show that HCV uses a novel mechanism to exploit intrinsic innate responses and hijack lipid metabolism, which may contribute to high chronicity rates and the pathological hallmark of steatosis in HCV infection.

SREBP: a novel therapeutic target.

Sterol regulatory element-binding proteins (SREBPs) are major transcription factors regulating the biosynthesis of cholesterol, fatty acid, and triglyceride. They control the expression of crucial genes involved in lipogenesis and uptake. In this review, we summarize the processing of SREBPs and their regulation by insulin, cAMP, and vitamin A, and the relationship between miRNA and lipid metabolism. We also discuss the recent functional studies on SREBPs. These discoveries suggest that inhibition of SREBP can be a novel strategy to treat metabolic diseases, such as type II diabetes, insulin resistance, fatty liver, and atherosclerosis.

An anti-PCSK9 antibody reduces LDL-cholesterol on top of a statin and suppresses hepatocyte SREBP-regulated genes.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.

SREBPs: metabolic integrators in physiology and metabolism.

Recent advances have significantly increased our understanding of how sterol regulatory element binding proteins (SREBPs) are regulated at the transcriptional and post-transcriptional levels in response to cellular signaling. The phosphatidyl inositol-3-kinase (PI3K) and SREBP pathways intersect at multiple points, and recent insights demonstrate the importance of tight regulation of the PI3K pathway for regulating SREBPs in the adaptation to fluctuating dietary calorie load in the mammalian liver. In addition, genetic and genome-wide approaches highlight new functions for SREBPs in connecting lipid metabolism with other cellular processes where lipid pathway flux affects physiologic or pathophysiologic adaptation, such as cancer, steatosis, and innate immunity. This review focuses on recent advances and new roles for mammalian SREBPs in physiology and metabolism.

Dysregulation of SREBP2 induces BACE1 expression.

ß-Amyloid hyperproduction has been observed in response to alterations in neuronal intracellular cholesterol storage, efflux, and synthesis, induced in rats by a high-fat diet. It has been suggested that cholesterol homeostasis is altered in Alzheimer's disease resulting in higher ß- and γ-secretase activity. In the current study the neuronal activation status of sterol regulatory element binding protein 2 (SREBP2) as well as its involvement in ß-secretase BACE1 activity was investigated in high-fat fed rats (26% fat and 4% cholesterol for 20 weeks), and in SK-N-BE neuroblastoma cells exposed to 20 µM cholesterol. This work demonstrates that in the brain a hyperlipidic diet is able to induce a hyper-expression of BACE1 and determine an unbalance in cerebral cholesterol homeostasis so that SREBP2 is activated. In addition, we show for the first time the involvement of SREBP2 on expression of BACE1 in SK-N-BE cells exposed to high cholesterol. Although the enhanced risk of Alzheimer's disease in metabolic syndrome is related to several factors, our results suggest that SREBP2, which can be modulated by the impairment of cerebral cholesterol homeostasis, has a direct role on BACE1 expression and may be involved in Alzheimer's disease progression.

The Akt-SREBP nexus: cell signaling meets lipid metabolism.

Phosphatidylinositol 3'-kinase (PI3K) and Akt are signaling kinases involved in cell survival and proliferation. Recent evidence suggests that PI3K/Akt activates the sterol-regulatory element-binding proteins (SREBPs), master transcriptional regulators of lipid metabolism. The precise molecular mechanisms are controversial and differ between SREBP isoforms; proposed mechanisms include increased trafficking and processing of SREBP, reduced degradation, and involvement of the downstream signaling hub, mammalian target of rapamycin complex 1 (mTORC1). In this report, we explore the various mechanistic links between Akt and SREBP. We consider this relationship in diseases where Akt and lipids play crucial roles, including diabetes, viral infections and cancer, suggesting that this Akt-SREBP link provides fresh insights into human health and disease.

Molecular mechanisms of alcoholic fatty liver.

Alcoholic fatty liver is a potentially pathologic condition which can progress to steatohepatitis, fibrosis, and cirrhosis if alcohol consumption is continued. Alcohol exposure may induce fatty liver by increasing NADH/NAD(+) ratio, increasing sterol regulatory element-binding protein-1 (SREBP-1) activity, decreasing peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activity, and increasing complement C3 hepatic levels. Alcohol may increase SREBP-1 activity by decreasing the activities of AMP-activated protein kinase and sirtuin-1. Tumor necrosis factor-alpha (TNF-alpha) produced in response to alcohol exposure may cause fatty liver by up-regulating SREBP-1 activity, whereas betaine and pioglitazone may attenuate fatty liver by down-regulating SREBP-1 activity. PPAR-alpha agonists have potentials to attenuate alcoholic fatty liver. Adiponectin and interleukin-6 may attenuate alcoholic fatty liver by up-regulating PPAR-alpha and insulin signaling pathways while down-regulating SREBP-1 activity and suppressing TNF-alpha production. Recent studies show that paracrine activation of hepatic cannabinoid receptor 1 by hepatic stellate cell-derived endocannabinoids also contributes to the development of alcoholic fatty liver. Furthermore, oxidative modifications and inactivation of the enzymes involved in the mitochondrial and/or peroxisomal beta-oxidation of fatty acids could contribute to fat accumulation in the liver.

Signalling pathways controlling fatty acid desaturation.

Microorganisms, plants and animals regulate the synthesis of unsaturated fatty acids (UFAs) during changing environmental conditions as well as in response to nutrients. Unsaturation of fatty acid chains has important structural roles in cell membranes: a proper ratio of saturated to UFAs contributes to membrane fluidity. Alterations in this ratio have been implicated in various disease states including cardiovascular diseases, immune disorders, cancer and obesity. They are also the major components of triglycerides and intermediates in the synthesis of biologically active molecules such as eicosanoids, which mediates fever, inflammation and neurotransmission. UFAs homeostasis in many organisms is achieved by feedback regulation of fatty acid desaturases gene transcription. Here, we review recently discovered components and mechanisms of the regulatory machinery governing the transcription of fatty acid desaturases in bacteria, yeast and animals.

SREBPs: the crossroads of physiological and pathological lipid homeostasis.

The uptake, biosynthesis and metabolism of cholesterol and other lipids are exquisitely regulated by feedback and feed-forward pathways in organisms ranging from Caenorhabditis elegans to humans. As endoplasmic reticulum (ER) membrane-embedded transcription factors that are activated in the Golgi apparatus, sterol regulatory element-binding proteins (SREBPs) are central to the intracellular surveillance of lipid catabolism and de novo biogenesis. The biosynthesis of SREBP proteins, their migration from the ER to the Golgi compartment, intra-membrane proteolysis, nuclear translocation and trans-activation potential are tightly controlled in vivo. Here we summarize recent studies elucidating the transcriptional and post-transcriptional regulation of SREBP-1c through nutrition and the action of hormones, particularly insulin, and the resulting implications for dyslipidemia of obesity, metabolic syndrome and type 2 diabetes.

Inhibition of fatty acid synthase activity in prostate cancer cells by dutasteride.

PURPOSE: With malignant progression to androgen independence, prostate cancer cells develop resistance to apoptosis and exhibit a variety of gene expression changes, including increased fatty acid synthase (FASN) expression. Increased FASN expression has been shown to correlate with poor prognosis, and correspondingly, the FASN gene has been proposed as a therapeutic target. Because FASN is an androgen regulated gene in the prostate, we have examined the effects of dutasteride on FASN in prostate cancer cells in vitro. Dutasteride is a novel dual inhibitor of the 5 alpha-reductase enzymes and is currently in use both for treatment of benign prostate hyperplasia (BPH) and in the reduction by dutasteride of prostate cancer events (REDUCE) prostate cancer prevention trial. METHODS: Microarray analysis was used to identify genes affected by treatment with dutasteride, followed by real time PCR confirmation. FASN expression at the protein level was examined using Western blotting and immunocytochemistry. Enzymatic activity of FASN was assayed by (14)C-labeled malonyl-CoA incorporation. Viability after dutasteride treatment was assayed by MTS (Promega) and apoptosis via caspase 3/7 by DEVD cleavage assay. RESULTS: We have demonstrated that the 5 alpha-reductase inhibitor dutasteride, at clinically relevant levels, inhibits FASN mRNA, protein expression and enzymatic activity in prostate cancer cells. CONCLUSIONS: This is the first study to examine the effects of clinically relevant levels of dutasteride on prostate cancer cells at the molecular level and specifically, demonstrating the inhibition of FASN in these cells.

Cholesterol, statins and cancer.

1. The link between cholesterol and cardiovascular disease is well-established. Emerging evidence is now forging a tantalizing link between cholesterol and cancer. 2. Results from a number of case-control studies have indicated that the commonly prescribed cholesterol-lowering drugs, the statins, may reduce the risk of certain cancers, although this area certainly remains controversial. 3. Herein, the recent literature examining statins and cancer is reviewed briefly and the relationship between a key cholesterol homeostatic pathway and signalling pathways that are involved in carcinogenesis is discussed. In particular, how the sterol-regulatory element binding protein, Akt and Hedgehog pathways may converge in cancer is reviewed.

Hepatocyte growth factor regulates E box-dependent plasminogen activator inhibitor type 1 gene expression in HepG2 liver cells.

OBJECTIVE: We sought to determine the etiologic mechanism of pleiotropic growth factor, hepatocyte growth factor (HGF), as a regulator of hepatic synthesis of plasminogen activator inhibitor (PAI)-1, the physiological inhibitor of fibrinolysis and a potential inducer of atherothrombosis. METHODS AND RESULTS: HGF increased PAI-1 mRNA expression and PAI-1 protein accumulation in the conditioned media of human liver-derived HepG2 cells, and increased hepatic PAI-1 mRNA expression in vivo in mice. HGF-inducible PAI-1 mRNA was attenuated by U0126, a specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, and genistein, an inhibitor of tyrosine kinase. HGF increased the human PAI-1 promoter (-829 to +36 bp) activity, and deletion and mutation analysis uncovered a functional E box (5'-CACATG-3') at positions -158 to -153 bp. Electrophoretic mobility shift assays demonstrated that this E box binds upstream stimulatory factors (USFs). HGF phosphorylated USFs through MAPK and tyrosine kinase pathways. Co-transfection of USF1 expression vector increased PAI-1 promoter activity. Sterol regulatory element-binding protein-1 attenuated HGF-inducible PAI-1 promoter activity. CONCLUSIONS: Because USFs are involved in the regulation of carbohydrates and lipid metabolism, HGF-mediated PAI-1 production may provide a novel link between atherothrombosis and metabolic derangements. Targeting HGF signaling pathway may modulate the thrombotic risk in high-risk patients.

Androgen regulation of prostasin gene expression is mediated by sterol-regulatory element-binding proteins and SLUG.

BACKGROUND: Prostasin is downregulated in hormone-refractory prostate cancers (HRPC). The mechanisms by which androgens regulate prostasin expression are unclear. METHODS: LNCaP cells were treated with dihydrotestosterone (DHT), and mRNA expression of prostasin, SREBPs, SNAIL, and SLUG was examined by real-time PCR following reverse transcription. A human prostasin promoter was evaluated in HEK-293 cells co-transfected with transcription factor cDNAs. Regulation of endogenous prostasin expression by transfected SREBP-2 or SLUG was evaluated. Expression of SNAIL and SLUG mRNA in DU-145 cells treated with epidermal growth factor (EGF) was examined. RESULTS: Prostasin mRNA expression in LNCaP cells was not responsive to DHT treatment. DHT marginally upregulated mRNA expression of SREBP-1c, SREBP-2, and SNAIL, but not SREBP-1a, while dramatically increased SLUG mRNA expression, in a dose-dependent manner. Co-transfection of prostasin promoter and SREBP cDNA in HEK-293 cells resulted in stimulation of promoter activity at approximately twofold by SREBP-1c, and up to sixfold by SREBP-2; while co-transfection with SNAIL or SLUG cDNA resulted in repression of promoter activity to 43% or 59%, respectively. Co-transfection of the SLUG cDNA negated SREBP-2's stimulation of prostasin promoter in a dose-dependent manner. Transfection of an SREBP-2 cDNA in HEK-293 and DU-145 resulted in upregulation of prostasin while transfection of a SLUG cDNA in LNCaP repressed prostasin expression. EGF upregulated SNAIL and SLUG mRNA in DU-145. CONCLUSIONS: DHT regulates prostasin expression in prostate cells via SREBP stimulation and SLUG repression of prostasin promoter. SLUG is upregulated by DHT and EGF, providing a molecular mechanism by which epithelial cell-specific genes are silenced during prostate cancer development and progression.

Regulación del metabolismo del colesterol y ácidos grasos en el síndrome nefrótico experimental por las proteínas que se unen a los elementos regulatorios de esteroles (SREBP's): efecto de la soya / Metabolism of cholesterol and fatty acids in nephrotic syndrome and its regulation by sterol regulatory element brinding proteins (SREBP's): Effect of soy protein consumption

El síndrome nefrótico (SN) cursa con hiperlipidemia. Se conoce que la biosíntesis del colesterol y de los ácidos grasos es regulada por los factores transcripcionales que se unen a los elementos de respuesta a esteroles (SREBP's). El consumo de proteína de soya disminuye la concentración de estos lípidos, aunque su mecanismo de acción no es del todo conocido. El objetivo de este estudio fue conocer si el consumo de la proteína de soya reduce los niveles de colesterol y triglicéridos a través de una regulación de las SREBP 's. Se estudiaron ratas Wistar macho con SN experimental por 64 días. Se observó que las concentraciones plasmáticos de colesterol y triglicéridos plasmáticos, así como de la proteinuria eran significativamente menores en las ratas alimentadas con proteína de soya que aquellas que consumían caseína. Estos cambios se asociaron con disminución de la expresión del ARNm SREBP 1 y de las enzimas de la síntesis de ácidos grasos. Los análisis por Western Blot revelaron que en los núcleos de hepatocitos obtenidos de ratas alimentadas con proteína de soya hubo menor presencia del factor transcripcional SREBP 1. Los resultados de este estudio indican que el consumo de proteína de soya produce efectos benéficos durante el síndrome nefrótico.

Hyperlipidemia occurs during nephrotic syndrome (NS). It is known that cholesterol and fatty acid biosynthesis is controlled by the transcription factors sterol regulatory element binding proteins (SREBPs). Soy protein consumption reduces the concentration of these lipids, although its mechanism of action is not well known. The aim of the present study was to establish whether soy protein consumption reduces cholesterol and triglycerides levels by regulating of SREBPs. Male Wistar rats with experimental NS were studied for 64 days. The results showed that rats fed with soy protein had significantly lower plasma cholesterol and triglyceride concentrations as well as proteinuria than rats fed with casein diet. These decrements were associated with a decrease in the expression of SREBP 1 and fatty acid biosynthetic enzymes. In addition, Western blot analysis revealed that in nuclear extracts from hepatocytes of rats fed with soy protein, there was a lower concentration of SREBP 1 than in rats fed with casein. The results of this study indicate that consumption of a soy protein diet has beneficial effects on nephrotic syndrome.