Processing Of Sterol Regulatory Element-binding Proteins Research Articles

Pharmacologic Inhibition of Site 1 Protease Activity Inhibits Sterol Regulatory Element-Binding Protein Processing and Reduces Lipogenic Enzyme Gene Expression and Lipid Synthesis in Cultured Cells and Experimental Animals

Sterol regulatory element-binding proteins (SREBPs) are major transcriptional regulators of cholesterol, fatty acid, and glucose metabolism. Genetic disruption of SREBP activity reduces plasma and liver levels of cholesterol and triglycerides and insulin-stimulated lipogenesis, suggesting that SREBP is a viable target for pharmacological intervention. The proprotein convertase SREBP site 1 protease (S1P) is an important posttranscriptional regulator of SREBP activation. This report demonstrates that 10 microM PF-429242 (Bioorg Med Chem Lett 17:4411-4414, 2007), a recently described reversible, competitive aminopyrrolidineamide inhibitor of S1P, inhibits endogenous SREBP processing in Chinese hamster ovary cells. The same compound also down-regulates the signal from an SRE-luciferase reporter gene in human embryonic kidney 293 cells and the expression of endogenous SREBP target genes in cultured HepG2 cells. In HepG2 cells, PF-429242 inhibited cholesterol synthesis, with an IC(50) of 0.5 microM. In mice treated with PF-429242 for 24 h, the expression of hepatic SREBP target genes was suppressed, and the hepatic rates of cholesterol and fatty acid synthesis were reduced. Taken together, these data establish that small-molecule S1P inhibitors are capable of reducing cholesterol and fatty acid synthesis in vivo and, therefore, represent a potential new class of therapeutic agents for dyslipidemia and for a variety of cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome.

Niemann-Pick type C1 protein influences the delivery of cholesterol to the SREBP: SCAP complex

The proposed role of Niemann-Pick type C1 protein (NPC1) in the delivery of low-density lipoprotein (LDL) cholesterol to the sterol regulatory element binding protein (SREBP):SREBP cleavage activation protein (SCAP) complex in the endoplasmic reticulum has been largely based on indirect studies and remains contentious. The major aim of the present study was to assess whether NPC1 is involved in the delivery of LDL cholesterol to the SREBP:SCAP complex. A cell line stably expressing green fluorescence protein-SCAP was cultured in the presence of U18666A, which can induce a Niemann-Pick type C disease phenotype, in order to locate the SREBP:SCAP complex by fluorescence microscopy. Our major finding was that defective NPC1 caused a delay in the ability of LDL cholesterol to suppress SREBP processing. This was shown in a time-course experiment by the effect of LDL on green fluorescence protein-SCAP movement when cells were treated with pharmacological agents to induce a Niemann-Pick type C disease phenotype. We demonstrated directly by fluorescence microscopy that defective NPC1 causes a delay in LDL cholesterol delivery to the endoplasmic reticulum where SCAP senses cholesterol.

Inability to fully suppress sterol synthesis rates with exogenous sterol in embryonic and extraembyronic fetal tissues

The requirement for cholesterol is greater in developing tissues (fetus, placenta, and yolk sac) as compared to adult tissues. Here, we compared cholesterol-induced suppression of sterol synthesis rates in the adult liver to the fetal liver, fetal body, placenta, and yolk sac of the Golden Syrian hamster. Sterol synthesis rates were suppressed maximally in non-pregnant adult livers when cholesterol concentrations were increased. In contrast, sterol synthesis rates were suppressed only marginally in fetal livers, fetal bodies, placentas, and yolk sacs when cholesterol concentrations were increased. To begin to elucidate the mechanism responsible for the blunted response of sterol synthesis rates in fetal tissues to exogenous cholesterol, the ratio of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) to Insig-1 was measured in these same tissues since the ratio of SCAP to the Insigs can impact SREBP processing. The fetal tissues had anywhere from a 2- to 6-fold greater ratio of SCAP to Insig-1 than did the adult liver, suggesting constitutive processing of the SREBPs. As expected, the level of mature, nuclear SREBP-2 was not different in the fetal tissues with different levels of cholesterol whereas it was different in adult livers. These findings indicate that the suppression of sterol synthesis to exogenous sterol is blunted in developing tissues and the lack of response appears to be mediated at least partly through relative levels of Insigs and SCAP.

Amplification of the gene for SCAP, coupled with Insig-1 deficiency, confers sterol resistance in mutant Chinese hamster ovary cells

The endoplasmic reticulum membrane proteins Insig-1 and Insig-2 limit cholesterol synthesis, in part through their sterol-dependent binding to sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). This binding prevents proteolytic processing of SREBPs, membrane-bound transcription factors that enhance cholesterol synthesis. We report here the characterization of mutant Chinese hamster ovary (CHO) cells, designated SRD-19, that are resistant to 25-hydroxycholesterol, a potent inhibitor of SREBP processing. SRD-19 cells were produced by mutagenesis of Insig-1-deficient SRD-14 cells, followed by selection in high levels of 25-hydroxycholesterol. 25-Hydroxycholesterol fails to suppress SREBP processing in SRD-19, even though they express normal levels of Insig-2. The number of copies of the gene encoding SCAP was found to be increased by 4-fold in SRD-19 cells compared with wild-type CHO cells, leading to the overproduction of SCAP mRNA and protein. Our data indicate that overproduced SCAP saturates the remaining Insig-2 in SRD-19 cells, thus explaining their resistance to 25-hydroxycholesterol. Consistent with this conclusion, regulated SREBP processing is restored in SRD-19 cells upon transfection of plasmids encoding either Insig-1 or Insig-2. These results highlight the importance of SCAP/Insig ratios in normal sterol-regulated processing of SREBPs in cultured cells.

Fatty Acid Supplied as Triglyceride Regulates SRE‐Mediated Gene Expression as Efficiently as Free Fatty Acids

Sterol regulatory element binding proteins (SREBPs) are key transcription proteins that bind to sterol regulatory elements (SRE) of genes essential for cellular cholesterol and fatty acid homeostasis. Polyunsaturated fatty acids (PUFA) strongly inhibit SREBP processing at post-transcriptional levels. We questioned if delivering PUFA as part of a triglyceride (TG) molecule would have similar effects and efficiency as free non-esterified PUFA. CHO cells stably transfected with an SRE-promoter linked to the luciferase reporter gene were incubated for 8-24 h with linoleic acid (LA) complexed to BSA (molar ratios 0.5-4:1), VLDL-sized trilinolein emulsions (TL, 25-200 microg/ml), and chylomicron-sized soy oil emulsions in the presence and absence of apoE. Effects of LA and TL on decreasing SRE-luciferase activity were similar and dose and time dependent. Both TL and LA significantly and rapidly (<or=2-12 h) reduced SRE-mediated gene expression by up to 75%. At equal fatty acid concentrations, SRE inhibition by TL was as effective as LA. ApoE addition increased inhibition by TL. Inhibition of gene expression was highly correlated to cell TG accumulation. We conclude that TG like fatty acids are rapid and efficient modulators of SRE-mediated gene expression.

SREBP-1 regulates the expression of heme oxygenase 1 and the phosphatidylinositol-3 kinase regulatory subunit p55γ

Sterol-regulatory element binding proteins (SREBPs) control the expression of genes involved in fatty acid and cholesterol biosynthesis. Using microarrays, we observed that mature SREBP-1 also induced the expression of genes unrelated to lipid metabolism, such as heme oxygenase 1 (HMOX1), plasma glutathione peroxidase, the phosphatidylinositol-3 kinase regulatory subunit p55 gamma, synaptic vesicle glycoprotein 2A, and COTE1. The expression of these genes was repressed upon addition of sterols, which block endogenous SREBP cleavage, and was induced by the statin drug mevinolin. Stimulation of fibroblasts with platelet-derived growth factor, which activates SREBP-1, had a similar effect. Fasted mice that were refed with a high-carbohydrate diet presented an increased expression of HMOX1 and p55 gamma in the liver. Overall, the transcriptional signature of SREBP-1 in fibroblasts stimulated by growth factors was very similar to that described in liver cells. We analyzed the HMOX1 promoter and found one SREBP binding site of the E-box type, which was required for regulation by SREBP-1a and SREBP-1c but was insensitive to SREBP-2. In conclusion, our data suggest that SREBP-1 regulates the expression of stress response and signaling genes, which could contribute to the metabolic response to insulin and growth factors in various tissues.

Insig-dependent Ubiquitination and Degradation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Stimulated by δ- and γ-Tocotrienols

Sterol-regulated ubiquitination marks 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-determining enzyme in cholesterol synthesis, for endoplasmic reticulum (ER)-associated degradation by 26 S proteasomes. This degradation, which results from sterol-induced binding of reductase to ER membrane proteins called Insigs, contributes to the complex, multivalent feedback regulation of the enzyme. Degradation of HMG-CoA reductase is also stimulated by various forms of vitamin E, a generic term for alpha-, beta-, delta-, and gamma-tocopherols and tocotrienols, which are primarily recognized for their potent antioxidant activity. Here, we show that delta-tocotrienol stimulates ubiquitination and degradation of reductase and blocks processing of sterol regulatory element-binding proteins (SREBPs), another sterol-mediated action of Insigs. The gamma-tocotrienol analog is more selective in enhancing reductase ubiquitination and degradation than blocking SREBP processing. Other forms of vitamin E neither accelerate reductase degradation nor block SREBP processing. In vitro assays indicate that gamma- and delta-tocotrienol trigger reductase ubiquitination directly and do not require further metabolism for activity. Taken together, these results provide a biochemical mechanism for the hypocholesterolemic effects of vitamin E that have been observed in animals and humans.

Insulin stimulates processing of SREBP‐1 but not SREBP‐2 in rat hepatocytes

To determine whether insulin stimulates cleavage of full length sterol-regulatory element binding protein (SREBP) to generate the transcriptionally active N-terminal fragment (nSREBP) in primary rat hepatocytes, adenoviral vectors (Ad-SREBP-1a, Ad-SREBP-1c, and Ad-SREBP-2) were constructed to constitutively express full-length SREBP isoforms in rat hepatocytes. Insulin increased, and dibutyryl (db)-cAMP inhibited, generation of nSREBP-1a and nSREBP-1c from their full-length precursors. Under the same conditions, formation of nSREBP-2 was not stimulated by insulin. Insulin stimulated processing of SREBP-1 within one hour, and the effect was sustained for at least 24 hours. The initial stimulation of SREBP processing by insulin preceded measurable reduction in Insig-2 mRNA levels. An inhibitor of PI3-kinase (LY294002) reduced both acute and sustained stimulation of SREBP-1 processing by insulin. An inhibitor of MAPK activation (PD98059) blocked acute, but not sustained, insulin stimulation of SREBP processing. To determine whether insulin reduces the rate of turnover of the nuclear form of SREBP, rat hepatocytes were also infected with an adenovirus expressing the nuclear form of SREBP-1c. Insulin decreased the rate of turnover of constitutively expressed nSREBP-1c, and this effect of insulin was also inhibited by db-cAMP Supported by grants from the Office of Research and Development, Department of Veterans Affairs, and from the American Heart Association, Southeast Affiliate

Hypercholesterolemia in rats with hepatomas: Increased oxysterols accelerate efflux but do not inhibit biosynthesis of cholesterol

Hypercholesterolemia is an important paraneoplastic syndrome in patients with hepatoma, but the nature of this defect has not yet been identified. We investigated the molecular mechanisms of hypercholesterolemia in a hepatoma-bearing rat model. Buffalo rats were implanted in both flanks with Morris hepatoma 7777 (McA-RH7777) cells. After 4 weeks, tumor weight was 5.5+/-1.7 g, and serum cholesterol level increased from 60+/-2 to 90+/-2 mg/dL. Protein and mRNA expression of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) was markedly higher in tumors than in livers. These increases were associated with activation of liver X receptor alpha (LXRalpha) as a result of the increased tissue oxysterol concentrations. The accumulation of oxysterols in the hepatomas appeared to be caused mainly by the upregulation of cholesterol biosynthesis, despite the increased tissue sterol concentrations. Overexpression of the sterol regulatory element-binding protein (SREBP) processing system relative to sterol concentration contributed to the resistance to sterols in this tumor. In addition, bile acid biosynthesis was inhibited despite the reduced expression of the small heterodimer partner (SHP) and activated LXRalpha, which also appeared to contribute to the accumulation of oxysterols followed by the acceleration of cholesterol efflux. In conclusion, hypercholesterolemia in McA-RH7777 hepatoma-bearing rats was caused by increased cholesterol efflux from tumors as a result of activation of LXRalpha. Overexpression of the SREBP processing system contributed to the activation of LXRalpha by maintaining high oxysterol levels in tissue.

Blockage of the ERK signaling pathway abrogates the SCAP ligand-induced transcriptional activation of the LDL receptor gene in HepG2 cells

Previous studies identified the putative SCAP ligands including compound GW707 as a new class of up-regulators of LDL receptor (LDLR) transcription by activation of the sterol-regulatory element binding proteins (SREBP). These compounds increase LDLR expression in hepatoma cells in vitro and lower plasma LDL-c in hamsters. However, it is unknown, what signaling pathways are utilized by these agents that lead to the activation of LDLR transcription. Here, we report that the ERK signaling cascade is critically involved in GW707-mediated induction of LDLR expression. We show that: a) blocking ERK activation with U0126, the inhibitor of ERK upstream kinase MEK, completely abolishes the inducing effects of GW707 on LDLR promoter activity, LDLR mRNA expression, and DiI-LDL uptake in HepG2 cells; b) treating HepG2 cells with GW707 induces a dose-dependent conversion of SREBP-2 from the 125 kDa precursor form to the 68 kDa activated form and U0126 does not inhibit this cleavage process, but U0126 significantly reduces the total amount of SREBP-2 protein in GW707-treated cells without affecting the expression levels of other proteins involving in SREBP processing; and c) inhibition of ERK signaling pathway has no effects on the promoter activity or mRNA expression of SREBP-2. Collectively, these new findings establish an important role of ERK signaling pathway in SCAP ligand-induced transcription of LDLR and imply that the protein synthesis or turnover rate of SREBP-2 may be regulated by ERK.

Isolation of Sterol-resistant Chinese Hamster Ovary Cells with Genetic Deficiencies in Both Insig-1 and Insig-2

Insig-1 and Insig-2, a pair of endoplasmic reticulum (ER) membrane proteins, mediate feedback control of cholesterol synthesis through their sterol-dependent binding to the following two polytopic ER membrane proteins: sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Sterol-induced binding of Insigs to SCAP prevents the proteolytic processing of SREBPs, membrane-bound transcription factors that enhance the synthesis of cholesterol, by retaining complexes between SCAP and SREBP in the ER. Sterol-induced binding of Insigs to reductase leads to the ubiquitination and ER-associated degradation of the enzyme, thereby slowing a rate-controlling step in cholesterol synthesis. Here we report the isolation of a new line of mutant Chinese hamster ovary cells, designated SRD-15, deficient in both Insig-1 and Insig-2. The SRD-15 cells were produced by gamma-irradiation of Insig-1-deficient SRD-14 cells, followed by selection in high levels of the oxysterol, 25-hydroxycholesterol. Sterols neither inhibit SREBP processing nor promote reductase ubiquitination/degradation in SRD-15 cells. Sterol regulation of SREBP processing and reductase ubiquitination/degradation is fully restored in SRD-15 cells when they are transfected with expression plasmids encoding either Insig-1 or Insig-2. These results demonstrate an absolute requirement for Insig proteins in the regulatory system that mediates lipid homeostasis in animal cells.

Posttranslational processing of SREBP-1 in rat hepatocytes is regulated by insulin and cAMP

Insulin and cAMP have opposing effects on de novo fatty acid synthesis in liver and in cultured hepatocytes mediated by sterol-regulatory element binding protein (SREBP). To determine whether these agents regulate the cleavage of full-length SREBP to generate the transcriptionally active N-terminal fragment (nSREBP) in primary rat hepatocytes, an adenoviral vector (Ad-SREBP-1a) was constructed to constitutively express full-length SREBP-1a. Insulin increased, and dibutyryl (db)-cAMP inhibited, generation of nSREBP-1a from its full-length precursor. Insulin stimulated processing of SREBP-1a within 1 h, and the effect was sustained for at least 24 h. The initial stimulation of SREBP processing by insulin preceded measurable reduction in Insig-2 mRNA levels. Rat hepatocytes were also infected with an adenovirus expressing the nuclear form of SREBP-1c (Ad-nSREBP-1c). Insulin increased the half-life of constitutively expressed nSREBP-1c, and this effect of insulin was also inhibited by db-cAMP.

Effects of 25-Hydroxycholesterol on Cholesterol Esterification and Sterol Regulatory Element-binding Protein Processing Are Dissociable: IMPLICATIONS FOR CHOLESTEROL MOVEMENT TO THE REGULATORY POOL IN THE ENDOPLASMIC RETICULUM

The regulatory pool of cholesterol is located in the endoplasmic reticulum (ER) and is key to how mammalian cells sense and respond to changes in cellular cholesterol levels. The extent of cholesterol esterification by the ER-resident protein, acyl-coenzyme A:cholesterol acyl-transferase (ACAT), has become the standard method for monitoring cholesterol transport to the ER and is assumed to reflect the regulatory pool of ER cholesterol. The oxysterol, 25-hydroxycholesterol (25HC), is thought to trigger intracellular cholesterol transport to the ER. In support of this contention, we confirmed previous reports that 25HC activates cholesterol esterification and is a potent suppressor of the sterol regulatory element-binding protein (SREBP) pathway. Processing of the ER membrane-bound SREBP into a soluble transcription factor is controlled by cholesterol levels in the ER. In this study, we addressed whether or not cholesterol esterification necessarily reflects cholesterol movement to the cholesterol homeostatic machinery in the ER as determined by SREBP processing. We found that three agents that inhibited the ability of 25HC to induce cholesterol esterification (progesterone, nigericin, and monensin) did not have a corresponding effect on 25HC suppression of SREBP processing. Moreover, ACAT inhibition did not alter the sensitivity of SREBP processing to 25HC. Therefore, cholesterol esterification by the ER-resident protein ACAT is dissociable from cholesterol transport to the cholesterol homeostatic machinery in the ER. In light of our results, we question the security of previous work that has inferred cholesterol transport to the ER regulatory pool based solely on cholesterol esterification.

Soy Isoflavones Affect Sterol Regulatory Element Binding Proteins (SREBPs) and SREBP-Regulated Genes in HepG2 Cells

Soy intake reduces cholesterol levels. However, both the identity of the soy component or components that contribute to this reduction and the cellular mechanism producing this reduction are unknown. Soy consists of protein, lipids, fiber, and phytochemicals including isoflavones. We propose that the isoflavone component of soy mediates this effect, at least in part, by affecting cellular sterol homeostasis. We investigated the effects of an isoflavone-containing soy extract and the individual isoflavones on the maturation of the sterol regulatory element binding proteins (SREBP) and the expression of SRE-regulated genes controlling lipid metabolism. We found a corresponding increase in the mature form of SREBP-2 in both soy extract- and isoflavone-treated HepG2 cells, whereas there was no significant change in the levels of SREBP-1. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase protein and HMG CoA synthase mRNA levels also increased. When HepG2 cells were transiently transfected with HMG CoA synthase and LDL receptor reporter plasmids there was an increase in expression in response to soy extract or isoflavone treatment from both of these promoters, but this induction was blunted in the presence of sterols (P < 0.05). The mechanism responsible for this effect may be via a statin-like inhibition of HMG CoA reductase enzyme activity or by enhanced SREBP processing via the SREBP cleavage activating protein. We hypothesize that maturation of SREBP and induction of SRE-regulated genes produce an increase in surface LDL receptor expression that increases the clearance of plasma cholesterol, thus decreasing plasma cholesterol levels.

SREBP transcription factors: master regulators of lipid homeostasis

Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of a range of enzymes required for endogenous cholesterol, fatty acid (FA), triacylglycerol and phospholipid synthesis. The three SREBP isoforms, SREBP-1a, SREBP-1c and SREBP-2, have different roles in lipid synthesis. In vivo studies using transgenic and knockout mice suggest that SREBP-1c is involved in FA synthesis and insulin induced glucose metabolism (particularly in lipogenesis), whereas SREBP-2 is relatively specific to cholesterol synthesis. The SREBP-1a isoform seems to be implicated in both pathways. SREBP transcription factors are synthetized as inactive precursors bound to the endoplasmic reticulum (ER) membranes. Upon activation, the precursor undergoes a sequential two-step cleavage process to release the NH 2-terminal active domain in the nucleus (designated nSREBPs). SREBP processing is mainly controlled by cellular sterol content. When sterol levels decrease, the precursor is cleaved to activate cholesterogenic genes and maintain cholesterol homeostasis. This sterol-sensitive process appears to be a major point of regulation for the SREBP-1a and SREBP-2 isoforms but not for SREBP-1c. Moreover, the SREBP-1c isoform seems to be mainly regulated at the transcriptional level by insulin. The unique regulation and activation properties of each SREBP isoform facilitate the co-ordinate regulation of lipid metabolism; however, further studies are needed to understand the detailed regulation pathways that specifically regulate each SREBP isoform.

Isolation of Mutant Cells Lacking Insig-1 through Selection with SR-12813, an Agent That Stimulates Degradation of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase

Insig-1 and Insig-2 are membrane proteins of the endoplasmic reticulum that regulate lipid metabolism by the following two actions: 1) sterol-induced binding to 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an action that leads to ubiquitination and degradation of the enzyme; and 2) sterol-induced binding to SREBP cleavage-activating protein, an action that blocks the proteolytic processing of sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that enhance the synthesis of cholesterol and fatty acids. Here we report the isolation of a new mutant line of Chinese hamster ovary cells, designated SRD-14, in which Insig-1 mRNA and protein are not produced due to a partial deletion of the INSIG-1 gene. The SRD-14 cells were produced by gamma-irradiation, followed by selection with the 1,1-bisphosphonate ester SR-12813, which mimics sterols in accelerating reductase degradation but does not block SREBP processing. SRD-14 cells fail to respond to sterols by promoting reductase ubiquitination and degradation. The rate at which sterols suppress SREBP processing is significantly slower in SRD-14 cells than wild type CHO-7 cells. Sterol regulation of reductase degradation and SREBP processing is restored when SRD-14 cells are transfected with expression plasmids encoding either Insig-1 or Insig-2. These results provide formal genetic proof for the essential role of Insig-1 in feedback control of lipid synthesis in cultured cells.

Proteolytic Activation of Sterol Regulatory Element-binding Protein Induced by Cellular Stress through Depletion of Insig-1

Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum (ER) that block proteolytic activation of sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that activate synthesis of cholesterol and fatty acids in animal cells. When cellular cholesterol levels are high, Insig proteins bind to SREBP cleavage-activating protein, retaining it in the ER and preventing it from escorting SREBPs to the site of proteolytic activation in the Golgi complex. Here we report that hypotonic stress reverses the sterol-mediated inhibition of SREBP proteolytic activation by reducing the level of Insig-1 but not Insig-2. The reduction of Insig-1, a protein with a rapid turnover rate, results from a general inhibition of protein synthesis mediated by hypotonic stress. Insig-2 is not affected by hypotonic stress because of its slower turnover rate. Inhibition of protein synthesis by hypotonic shock has not been reported previously. Thapsigargin, an activator of the ER stress response, also inhibits protein synthesis and activates proteolysis of SREBP. Such activation also correlates with the disappearance of Insig-1. The current study demonstrates that animal cells, in response to either hypotonic shock or ER stress, can bypass the cholesterol inhibition of SREBP processing, an effect that is attributable to the rapid turnover of Insig-1.

Platelet-derived Growth Factor Stimulates Membrane Lipid Synthesis Through Activation of Phosphatidylinositol 3-Kinase and Sterol Regulatory Element-binding Proteins

We analyzed the transcriptional program elicited by stimulation of normal human fibroblasts with platelet-derived growth factor (PDGF) using cDNA microarrays. 103 significantly regulated transcripts that had not been previously linked to PDGF signaling were identified. Among them, a cluster of genes involved in fatty acid and cholesterol biosynthesis, including stearoyl-CoA desaturase (SCD), fatty acid synthase, and hydroxymethylglutaryl-CoA synthase (HMGCS), was up-regulated by PDGF after 24 h of treatment, and their expression correlated with increased membrane lipid production. These genes are known to be controlled by sterol regulatory element-binding proteins (SREBP). PDGF increased the amount of mature SREBP-1 and regulated the promoters of SCD and HMGCS in an SREBP-dependent manner. In line with these results, blocking SREBP processing by addition of 25-hydroxycholesterol blunted the effects of PDGF on lipogenic enzymes. SREBP activation was dependent on the phosphatidylinositol 3-kinase (PI3K) pathway, as judged from the effects of the inhibitor LY294002 and mutation of the PDGFbeta receptor tyrosines that bind the PI3K adaptor subunit p85. Fibroblast growth factors (FGF-2 and FGF-4) and other growth factors mimicked the effects of PDGF on NIH3T3 and human fibroblasts. In conclusion, our results suggest that growth factors induce membrane lipid synthesis via the activation SREBP and PI3K.

Fatty acids, cholesterol, and sphingolipids: interactive regulators and targets for SREBP processing

Sterol regulatory element-binding protein (SREBPs) have a major role in regulating transcription of many genes involved in lipid biology and pathobiology. In addition to inhibitory effects of cholesterol on cleavage of the precursor of SREBP (pSREBP) to the active mature nuclear form (mSREBP), polyunsaturated fatty acids (PUFA)—but not saturated fatty acids—have a key role in inhibiting formation of the active mSREBP. The ability of PUFA to decrease SREBP-dependent gene transcription is related in part to effects on modulating intracellular cholesterol partitioning and through effects on sphingolipid metabolism. Effects related to sphingolipids are due in part to their higher affinity for free cholesterol, as well as to the ability of PUFA to increase cell sphingomyelinase activity leading to production of ceramide. The inhibitory effects of ceramide on SREBP cleavage are associated with the ability of ceramide to inhibit its own de novo intracellular synthesis, which we hypothesize effects normal SREBP trafficking between the endoplasmic reticulum (ER) and Golgi compartments. Our work indicates that intermediates of sphingomyelin metabolism now join cholesterol and fatty acids as important regulators of SREBP processing, and offer new therapeutic targets for disorders of lipid metabolism including dyslipidemias, steatohepatitis, obesity, and type II diabetes.

Ceramide Synthesis Correlates with the Posttranscriptional Regulation of the Sterol-Regulatory Element-Binding Protein

Sterol-regulatory element-binding proteins (SREBPs) regulate transcription of genes of lipid metabolism. Ceramide decreases transcriptionally active SREBP levels independently of intracellular cholesterol levels. Mechanisms of the ceramide-mediated decrease of SREBP levels were investigated. Experiments were performed in Chinese hamster ovary cells. Inhibition of ceramide synthesis with myriocin, cycloserine, or fumonisin decreases levels of transcriptionally active SREBP and reduces SRE-mediated gene transcription. When ceramide synthesis is increased through exogenous sphingosine or inhibition of sphingosine kinase, SRE-mediated gene transcription is increased. The important role of ceramide synthesis in SRE-mediated gene transcription is confirmed in LY-B cells that do not synthesize ceramide de novo. LY-B cells fail to increase SRE-mediated gene transcription in sterol depletion. Ceramide synthesis correlates with the generation of transcriptionally active SREBP and SRE-mediated gene transcription. Inhibition of ceramide synthesis decreases levels of transcriptionally active SREBP and SRE-mediated gene transcription. It is hypothesized that the process of ongoing ceramide synthesis contributes to the physiological processing of SREBP, perhaps affecting ER-to-Golgi trafficking. Taken together, modification of ceramide synthesis could be a novel target for drug development in the pharmacologic modification of SRE-dependent pathways.