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Chromatin immunoprecipitation analysis of CREB and ATF-2 binding to HMG-CoA reductase promoter in CHO cells cultured in the absence and presence of regulatory sterols. A, a schematic representation of the native HMG-CoA reductase promoter with the heterogeneous transcription initiation sites, TATA element, and binding sites for SREBPs and NF-Y and CREB/ATF is shown. B, an autoradiogram of a polyacrylamide gel that displays the results of the PCR for the HMG-CoA reductase promoter is shown. The primers were designed to hybridize just upstream of the NF-Y site and just downstream of the ATF/CREB site as shown in A. The input chromatin was analyzed in lanes 1 and 2 (1 l of a 1:300 dilution), and 3 l of each resultant immunoprecipitation with the indicated antibodies were also analyzed as indicated. No primary antibody was used for the reactions in lanes 5– 6.
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Sterol regulatory element-binding proteins (SREBPs) activate promoters for key genes of metabolism to keep pace with the cellular demand for lipids. In each SREBP-regulated promoter, at least one ubiquitous co-regulatory factor that binds to a neighboring recognition site is also required for efficient gene induction. Some of these putative co-regu...
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... We next investigated whether key cellular molecules known to be involved in the insulin secretion process are under the control of the CREB transcription factor. The expression of several genes involved in glycolysis such as the rate-limiting enzyme glucokinase 34 , the anaplerotic enzyme pyruvate carboxylase (Pc) 35 and in mitochondrial oxidation process, malate dehydrogenase (Mdh2) 36 , ATP citrate lyase and 3-hydroxyacyl-CoA dehydrogenase (Hadh) 37 are known to respond to CREB. Consistent with this, quantitative real-time PCR (qPCR) performed on RNA isolated from Y1 −/− islets confirmed an increase in mRNA expression of these critical metabolic genes in the absence of Y1 receptor signaling (Fig. 4l). ...
Failure to secrete sufficient quantities of insulin is a pathological feature of type-1 and type-2 diabetes, and also reduces the success of islet cell transplantation. Here we demonstrate that Y1 receptor signaling inhibits insulin release in β-cells, and show that this can be pharmacologically exploited to boost insulin secretion. Transplanting islets with Y1 receptor deficiency accelerates the normalization of hyperglycemia in chemically induced diabetic recipient mice, which can also be achieved by short-term pharmacological blockade of Y1 receptors in transplanted mouse and human islets. Furthermore, treatment of non-obese diabetic mice with a Y1 receptor antagonist delays the onset of diabetes. Mechanistically, Y1 receptor signaling inhibits the production of cAMP in islets, which via CREB mediated pathways results in the down-regulation of several key enzymes in glycolysis and ATP production. Thus, manipulating Y1 receptor signaling in β-cells offers a unique therapeutic opportunity for correcting insulin deficiency as it occurs in the pathological state of type-1 diabetes as well as during islet transplantation.
... A role for the acetyltransferase CREBbinding protein (CBP) has also been described (Oliner et al., 1996). The recruitment of distinct coactivators renders specificity to gene transcription by SREBPs (Bennett and Osborne, 2000;Ngo et al., 2002). ...
Glomerular matrix accumulation is a hallmark of diabetic nephropathy. Recent studies showed that overexpression of the transcription
factor SREBP-1 induces glomerulosclerosis. TGFβ1 is a key profibrotic mediator of glomerulosclerosis, but whether SREBP-1
regulates its effects is unknown. In kidney mesangial cells and in vivo, TGFβ1 activates SREBP-1. This requires SCAP, S1P, and PI3K/Akt signaling, but is independent of Smad3. Activation of the
TGFβ1-responsive reporter plasmid p3TP-lux requires SREBP-1a, but not SREBP-1c, binding to an E-box adjacent to a Smad-binding
element. SREBP-1a overexpression alone activates p3TP-lux. Smad3 is required for SREBP-1a transcriptional activation and TGFβ1
induces association between the two transcription factors. SREBP-1a K333 acetylation by the acetyltransferase CBP is required
for Smad3 association and SREBP-1 transcriptional activity, and is also required for Smad3 transcriptional activity. Thus,
both Smad3 and SREBP-1a activation cooperatively regulate TGFβ transcriptional responses. SREBP-1 inhibition provides a novel
therapeutic strategy for diabetic kidney disease.
... In general, SREBPs are weak transcription factors requiring cooperation with coactivators, most commonly Sp1 (43,44). The recruitment of distinct coactivators renders specificity to gene transcription by SREBPs (4,34). SREBPs have been well described to control the regulation of genes related to lipid synthesis (11). ...
Glomerular matrix accumulation is a hallmark of diabetic nephropathy. Recent studies showed that overexpression of the transcription factor sterol-responsive element-binding protein (SREBP)-1 induces pathology reminiscent of diabetic nephropathy, and SREBP-1 upregulation was observed in diabetic kidneys. We thus studied whether SREBP-1 is activated by high glucose (HG) and mediates its profibrogenic responses. In primary rat mesangial cells, HG activated SREBP-1 by 30 min, seen by the appearance of its cleaved nuclear form (nSREBP-1), EMSA, and by activation of an SREBP-1 response element (SRE)-driven green fluorescent protein construct. Activation was dose dependent and not induced by an osmotic control. Site 1 protease was required, since its inhibition by AEBSF prevented SREBP-1 activation. SCAP, the ER-associated chaperone for SREBP-1, was also necessary since its inhibitor fatostatin also blocked SREBP-1 activation. Signaling through the EGFR/phosphatidylinositol 3-kinase (PI3K) pathway, which we previously showed mediates HG-induced TGF-β1 upregulation, and through RhoA, were upstream of SREBP-1 activation (Wu D, Peng F, Zhang B, Ingram AJ, Gao B, Krepinsky JC. Diabetologia 50: 2008-2018, 2007; Wu D, Peng F, Zhang B, Ingram AJ, Kelly DJ, Gilbert RE, Gao B, Krepinsky JC. J Am Soc Nephrol 20: 554-566, 2009). Fatostatin and AEBSF prevented HG-induced TGF-β1 upregulation by Northern blot analysis, and HG-induced TGF-β1 promoter activation was inhibited by both fatostatin and dominant negative SREBP-1a. Chromatin immunoprecipitation analysis confirmed that HG led to SREBP-1 binding to the TGF-β1 promoter in a region containing a putative SREBP-1 binding site (SRE). Thus HG-induced SREBP-1 activation requires EGFR/PI3K/RhoA signaling and SCAP-mediated transport to the Golgi for its proteolytic cleavage. Activated SREBP-1 binds to the TGF-β promoter, resulting in TGF-β1 upregulation in response to HG. SREBP-1 thus provides a potential novel therapeutic target for the treatment of diabetic nephropathy.
... It was reported that the HMGCR promoter contained a cAMP-responsive element CRE. 21,22 We constructed a recombined luciferase reporter plasmid pGL4-CRE and transfected into L-02 cells. The significant increase in luciferase activity was detected upon TSH or forskolin treatment. ...
... 26 Once CREB has been activated, it interacts efficiently with sterol regulatory element binding protein-2 to stimulate the transcription of the HMGCR gene in the presence of NF-Y. 22 It is conceivable that TSH, through cAMP signal, could induce one or more such regulatory proteins to be actived in promoting reductase gene transcription. The data presented in this work demonstrated that TSH might induce HMGCR transcription at least through the cAMP/PKA/CREB pathway. ...
Elevated thyroid-stimulating hormone (TSH) and hypercholesterolemia commonly coexist, as typically seen in hypothyroidism, but there is no known mechanism directly linking the two. Here, we demonstrated that in liver cells, TSH promoted the expression of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), a rate-limiting enzyme in cholesterol synthesis, by acting on the TSH receptor in hepatocyte membranes and stimulating the cyclic adenosine monophosphate / protein kinase A / cyclic adenosine monophosphate-responsive element binding protein (cAMP/PKA/CREB) signaling system. In thyroidectomized rats, the production of endogenous thyroid hormone was eliminated and endogenous TSH was suppressed through pituitary suppression with constant administration of exogenous thyroid hormone, and hepatic HMGCR expression was increased by administration of exogenous TSH. These results suggested that TSH could up-regulate hepatic HMGCR expression, which indicated a potential mechanism for hypercholesterolemia involving direct action of TSH on the liver.
... What might be the molecular mechanism for this differential response by these promoters? Although the identities of the transcription factors involved in the sterol regulation of Hmgcr promoter still remain incompletely understood, previous studies demonstrated important roles for the sterol regulatory element (SRE-1) SRE binding proteins (SREBPs), CCAAT-binding factor/nuclear factor-Y (CBF/NF-Y) and CREB [65][66][67][68]. Since the BPL-and BPH-Hmgcr did not differ at the SRE-1 or CBF/NF-Y or CRE motifs and the differential expression under the basal conditions was mediated by c-Fos/n-Myc/Max (Fig. 2, 9, 10), the greater sterol-repression (i.e. ...
... SDS-PAGE and Immunoblot Analysis-SL2 nuclear extracts and liver chromatin extracts were analyzed for immunoblotting as described (22,23). The antibodies used were as follows: a polyclonal raised against human CREB (a gift from M. Montminy (24)) pCREB (Cell Signaling, catalog No. 9191), polyclonal antibodies raised against mouse SREBP-1 and SREBP-2 (gifts from J. Horton (13)), polyclonal antibody against TORC2 (gift from Paul Brindle (25)), CBP (Santa Cruz Biotechnology, sc-369), FXR (Santa Cruz Biotechnology, sc-H130), NF-Y A subunit (Rockland, catalog No. 100-401-100), Ac-H3 (Upstate, catalog No. 06-599), and -actin (Sigma, catalog No. A1978). ...
... To test these ideas, we investigated the requirements for SREBP-CREB activation using an SL2-based transfection assay (Fig. 5) where high levels of expression from the HMG-CoA reductase promoter requires the addition of exogenously supplied expression vectors for SREBP and co-regulatory proteins CREB and NF-Y (22). The activity of the HMG-CoA reductase promoter transfected alone was set at 1, and the addition of the expression vector for SREBP did not change this low value, consistent with our previous observations (22). ...
... To test these ideas, we investigated the requirements for SREBP-CREB activation using an SL2-based transfection assay (Fig. 5) where high levels of expression from the HMG-CoA reductase promoter requires the addition of exogenously supplied expression vectors for SREBP and co-regulatory proteins CREB and NF-Y (22). The activity of the HMG-CoA reductase promoter transfected alone was set at 1, and the addition of the expression vector for SREBP did not change this low value, consistent with our previous observations (22). When an expression vector for full-length wild type CREB was included at two different concentrations, a low level of activation was observed. ...
Mice were subjected to different dietary manipulations to selectively alter expression of hepatic sterol regulatory element-binding protein 1 (SREBP-1) or SREBP-2. mRNA levels for key target genes were measured and compared with the direct binding of SREBP-1 and -2 to the associated promoters using isoform specific antibodies in chromatin immunoprecipitation studies. A diet supplemented with Zetia (ezetimibe) and lovastatin increased and decreased nuclear SREBP-2 and SREBP-1, respectively, whereas a fasting/refeeding protocol dramatically altered SREBP-1 but had modest effects on SREBP-2 levels. Binding of both SREBP-1 and -2 increased on promoters for 3-hydroxy-3-methylglutaryl-CoA reductase, fatty-acid synthase, and squalene synthase in livers of Zetia/lovastatin-treated mice despite the decline in total SREBP-1 protein. In contrast, only SREBP-2 binding was increased for the low density lipoprotein receptor promoter. Decreased SREBP-1 binding during fasting and a dramatic increase upon refeeding indicates that the lipogenic "overshoot" for fatty-acid synthase gene expression known to occur during high carbohydrate refeeding can be attributed to a similar overshoot in SREBP-1 binding. SREBP co-regulatory protein recruitment was also increased/decreased in parallel with associated changes in SREBP binding, and there were clear distinctions for different promoters in response to the dietary manipulations. Taken together, these studies reveal that there are alternative molecular mechanisms for activating SREBP target genes in response to the different dietary challenges of Zetia/lovastatin versus fasting/refeeding. This underscores the mechanistic flexibility that has evolved at the individual gene/promoter level to maintain metabolic homeostasis in response to shifting nutritional states and environmental fluctuations.
... We also found increased CREB association with the xCRFb proximal promoter after exposure to shaking/handling stressor, suggesting that the recruitment of CREB is enhanced by exposure to a stressor. Increased association of CREB with the CRF promoter after activation of the cAMP pathway has been observed in cultured cells (55)(56)(57). Shepard et al. (19) used ChIP assay to show that the pCREB signal at the CRF promoter in rat brain increased after exposure to a stressor. ...
Corticotropin-releasing factor (CRF) plays a central role in neuroendocrine, autonomic, immune, and behavioral responses to stressors. We analyzed the proximal promoters of two Xenopus laevis CRF genes and found them to be remarkably conserved with mammalian CRF genes. We found several conserved cis elements in the frog CRF genes including a cAMP response element (CRE), activator protein 1 binding sites, and glucocorticoid response elements. Exposure to a physical stressor caused a rapid elevation in phosphorylated CRE binding protein (CREB; 20 min) and CRF (1 h) in the anterior preoptic area of juvenile frogs. CREB bound to the putative frog CREs in vitro, which was disrupted by point mutations introduced into the CRE. The frog proximal CRF promoters supported basal transcription in transfection assays, and forskolin caused robust transcriptional activation. Mutagenesis of the CRE or overexpression of a dominant-negative CREB reduced forskolin-induced promoter activation. Using electroporation-mediated gene transfer in tadpole brain, we show that the proximal CRF promoters support cAMP or stressor-dependent transcription in vivo, which was abolished by mutation of the CRE. Using chromatin immunoprecipitation, we found that CREB associated with the proximal frog CRF promoter in vivo in a stressor-dependent manner. These data provide strong support for the hypothesis that stressor-induced CRF gene activation in vivo depends on CREB binding to the CRE in the promoter. Our findings show that the basic regulatory elements of the CRF gene responsible for stressor-induced activation arose early in vertebrate evolution and have been maintained by strong positive selection.
... This site was also found to be occupied in vivo in rat liver. CREB is the major factor that binds to this element (38). ...
HMG-CoA reductase (HMGR) catalyzes the rate-controlling step in cholesterol production. This enzyme is highly expressed in the liver, where it is subject to extensive hormonal and dietary regulation. Although much is known about the regulation of the HMGR promoter in cultured cells, this issue has not been directly addressed in liver. The technique of in vivo electroporation was utilized to perform the first functional analysis of the HMGR promoter in live animals. Analysis of a series of deletion constructs showed that deletion of the region containing the cyclic AMP response element (CRE) at -104 to -96 and an NF-Y site at -70 to -65 resulted in marked reduction of promoter activity. Sterol regulation of this promoter was investigated by raising tissue cholesterol levels by feeding cholesterol and by decreasing them through administration of a statin (lovastatin). Using this approach, we found that HMGR promoter constructs were sterol responsive in live animals, adding in vivo relevance to previous findings in cultured cells. We also conclude that in vivo electroporation is a convenient and powerful technique for the analysis of promoter elements in the livers of live animals.
... For glutathione S-transferase-FTF, PCR oligos containing EcoRI sites were used to amplify human FTF from pCI FTF, and the digested product was cloned into EcoRI-digested pGEX-2T. HMG-CoA reductase and LDL receptor promoter luciferase constructs have been described previously (10,11). The CYP8B1 promoter luciferase reporter construct (4) was a gift from Dr. Gregorio Gil (Virginia Medical College). ...
... In our previous studies, we noted that the magnitude of stimulation by sterol depletion or the addition of exogenous SREBP expression constructs was very modest for the HMG-CoA reductase promoter compared with the activation achieved with other SREBP target genes ana- showing the positions of the two putative LRH-1 sites relative to already characterized sites for SREBP, nuclear factor-Y (NF-Y), and cAMP-response-element binding protein (CREB) (10). At the bottom is a lineup of the genomic DNA from the Ϫ300 region of the hamster, human and mouse promoters showing the conservation of this putative LRH-1 site. ...
Cholesterol homeostasis in mammals involves pathways for biosynthesis, cellular uptake, and hepatic conversion to bile acids. Key genes for all three pathways are regulated by negative feedback control. Uptake and biosynthesis are directly regulated by cholesterol through its inhibition of the proteolytic activation of the sterol regulatory element binding proteins. The conversion of cholesterol into bile acids in the liver is regulated through the bile acid-dependent induction of the negatively acting small heterodimer partner nuclear receptor. In this report, we have shown that the small heterodimer partner also directly regulates cholesterol biosynthesis through inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase but has no effect on low density lipoprotein receptor expression. This has significant metabolic significance, as it provides both a mechanism to independently regulate cholesterol synthesis from uptake (an essential regulatory feature known to occur in vivo) and a pathway for direct regulation of cholesterol biosynthesis by bile acids. This latter feature ensures that the early phase of bile acid synthesis (pre-cholesterol) is in metabolic communication with the later stages of the pathway to properly regulate whole pathway flux. This highlights an important regulatory feature that is shared with other key branched, multienzyme pathways, such as glycolysis, where pathway outflow through pyruvate kinase is regulated by the concentration of a key early intermediate, fructose 1,6-bisphosphate.
... Given the overwhelming and invariant occupancy of the CRE in vivo, it seems unlikely that CREB binding is the regulated event in insulin activation. This may differ from sterol regulation in which SREBP binding has been shown to selectively recruit CREB to the promoter in CHO cells (28). Although we previously showed that the CRE was required for insulin activation of this promoter in H4IIE cells (7), in vivo occupancy of this site did not vary in rat liver. ...
Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) protein and mRNA are substantially decreased in diabetic animals and rapidly restored by the administration of insulin. To begin to examine the underlying molecular mechanisms, measurements of transcription by nuclear run-on assays and an investigation of occupancy of the promoter were performed. The rate of transcription was substantially reduced in the diabetic rats and fully restored within 2 h after insulin treatment. In vivo footprinting revealed several areas of protein binding as shown by dimethyl sulfate protection or enhancement. The cAMP-response element was heavily protected in all conditions, including diabetes, feeding of dietary cholesterol, or statin treatment. Striking enhancements in footprints from diabetic animals were visible at -142 and at -161 (in the sterol-response element). Protections at a newly identified NF-Y site at -70/-71 were observed in normal animals and not in diabetics. This NF-Y site was found to be required for efficient HMGR transcription in luciferase assays. CREB-1 was able to bind the HMGR cAMP-response element in vitro and the promoter in vivo. This evidence supports an essential role for cAMP-response element-binding protein in transcription of hepatic HMGR and identifies at least two sites where in vivo occupancy is regulated by insulin.
