SREBP-1 Mediates Activation of the Low Density Lipoprotein Receptor Promoter by Insulin and Insulin-like Growth Factor-I

Streicher, Rüdiger; Kotzka, Jörg; Müller‐Wieland, Dirk; Siemeister, Gerhard; Munck, Martina; Avci, Haluk; Krone, Wilhelm

doi:10.1074/jbc.271.12.7128

Cited by 145 publications

(107 citation statements)

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“…Plasmids and Molecular Cloning-Construction of the LDL receptor (LDLR) promoter reporter gene plasmid containing a functional intact sterol regulatory element (sre)-1 flanked by two SP1 elements (phLDL4-luc) was described previously (10). The N-terminal domain of human SREBP-2 (amino acids 1-468) was inserted in-frame to glutathione S-transferase (GST) into pGEX-3X (Amersham Biosciences) to generate GST-fusion proteins (9).…”

Section: Methodsmentioning

confidence: 99%

“…Western blot analysis of prepared cell extracts revealed that wild type-related phosphorylation was significantly reduced in single-mutated SREBP-2-NT and completely abolished in the double-mutant. Previously, we have shown that the stimulatory effect of insulin on LDL receptor promoter is mediated by SREBPs (9,10). To prove that the insulininduced activation of LDL receptor promoter is coupled to the identified phosphorylation sites in SREBP-2, we performed promoter reporter gene assays.…”

Section: Identification Of Ser-432 and Ser-455 As Erk-mapk-specific Pmentioning

confidence: 99%

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Insulin-activated Erk-mitogen-activated Protein Kinases Phosphorylate Sterol Regulatory Element-binding Protein-2 at Serine Residues 432 and 455 in Vivo

Kotzka

Lehr

Roth

et al. 2004

Journal of Biological Chemistry

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The transcription factor sterol regulatory element binding protein (SREBP)-2 plays a pivotal role in lipid metabolism. Previously, we have shown that the mature form of SREBP-2 is a substrate of Erk-mitogen-activated protein kinases (MAPK). The aim of the present study was to identify Erk-specific phosphorylation sites. Using a protein chemistry approach, we could identify Ser-432 and Ser-455 as major phosphorylation sites. Further characterization by electrophoretic mobility shift assay and promoter reporter gene analyses revealed that phosphorylation does not influence protein/DNA interaction, but enhances trans-activity. In intact cells, SREBP-2 is phosphorylated by insulin, which seems to be related to their bio-responses on low density lipoprotein receptor activity. These results suggest that activation of Erk-MAPK pathways by hormones such as insulin might be related to a novel regulatory principle of SREBP-2.Sterol regulatory element binding proteins (SREBPs) 1 are a family of basic helix-loop-helix transcription factors that are embedded as precursor proteins in the endoplasmic reticulum and nuclear envelope. To date, three SREBP isoforms have been detected: SREBP-1a, SREBP-1c (shorter splicing variant of SREBP-1a), as well as SREBP-2 (1-5). SREBP-2 is a major regulator of cholesterol homeostasis, whereas SREBP-1c regulates predominantly de novo synthesis of fatty acids. In contrast, SREBP-1a seems to influence both lipogenic and cholesterogenic enzymes (6).Activation of SREBPs initiated by cellular cholesterol depletion is mediated by sequential cleavage (7). As a result, the amino-terminal domain of the protein translocates into the nucleus and activates transcription of target genes. Beside this mechanism, which controls the abundance of activated SREBPs in the cell, we have demonstrated that trans-activity of the N-terminal domain of SREBPs is regulated directly by extra cellular stimuli, e.g. by hormones such as insulin (8, 9). Moreover, in these studies, we have shown that the N-terminal domains of SREBP-1a, SREBP-1c, and SREBP-2 are substrates of the extracellular signal-regulated kinase (Erk) subfamily of mitogen-activated protein kinases (MAPK). In this study, we have identified Ser-432 and Ser-455 as the major phosphorylation sites of Erk-MAPK in SREBP-2 using protein chemistry methodology. This phosphorylation has no influence on DNA interaction but affects trans-activity of SREBP-2. Accordingly, in cells, activation of low density lipoprotein (LDL) receptor gene by insulin is coupled to the identified Erk-specific phosphorylation sites in SREBP-2.

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Section: Methodsmentioning

confidence: 99%

Section: Identification Of Ser-432 and Ser-455 As Erk-mapk-specific Pmentioning

confidence: 99%

Insulin-activated Erk-mitogen-activated Protein Kinases Phosphorylate Sterol Regulatory Element-binding Protein-2 at Serine Residues 432 and 455 in Vivo

Kotzka

Lehr

Roth

et al. 2004

Journal of Biological Chemistry

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100

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“…The promoter was cloned into the pGL4. 10[luc2] vector (Promega). A deletion construct and site-directed Abbreviations: LDLR, low-density lipoprotein; LXR, liver X receptor; RXR, retinoid X receptor; SRE, sterol-regulatory element; SREBP, SRE-binding protein; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; DR4, direct repeat 4; LXRE, LXR response element; ABC, ATP-binding cassette; CYP7A, cholesterol 7a-hydoroxylase; LPDS, lipoprotein-deficient serum; 25-HC, 25-hydroxycholesterol; PCR, polymerase chain reaction; DMEM, Dulbecco's Modified Eagle Medium; DMSO, dimethyl sulfoxide; CMV, cytomegalovirus mutations were introduced into the LDLR promoter by PCR methods.…”

Section: Plasmid Constructsmentioning

confidence: 99%

“…We used pGL4. 10[luc2] as a negative control vector and corrected each value of the reporter gene expression as compared to the value of the control vector. These values are expressed as fold activation compared with the control (CMV/DMSO: control vector and DMSO treatment) in each LDLR-reporter construct.…”

Section: Transfections and Luciferase Assaymentioning

confidence: 99%

Identification of human low‐density lipoprotein receptor as a novel target gene regulated by liver X receptor alpha

et al. 2006

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Liver X receptor alpha (LXRa) is a member of the nuclear receptor superfamily that is activated by oxysterols, and plays a pivotal role in regulating the metabolism, transport and uptake of cholesterol. Here, we demonstrate that LXRa also regulates the low-density lipoprotein receptor (LDLR) gene, which mediates the endocytic uptake of LDL cholesterol in the liver. An LXR agonist induced the expression of LDLR in cultured hepatoblastoma cells. Moreover, the LDLR promoter contained an LXR response element that was recognized by LXRa/ RXRa (retinoid X receptor alpha) heterodimers in hepatoblastoma cells. These results suggest a novel pathway whereby LXRa might modulate cholesterol metabolism.

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“…The principal pathway of LDL receptor regulation is controlled by feedback repression of the LDL receptor gene by cholesterol and its metabolites. 41 Recently, it was shown that the expression of LDL receptors by cells in culture is also affected by other components when, for example, cholesterol LDL receptor activity was shown to be increased by incubations of cells with insulin, 42 thyroid hormone, 43 calmodulin inhibitors, 44 tumor necrosis factor, 45 and interleukin-6. 46 However, it is not clear whether the effects of these components on LDL receptor activity are secondary to change in cellular cholesterol content or whether they act as primary effectors.…”

Section: Discussionmentioning

confidence: 99%

Effects of fibrillar C-terminal fragment of cleaved ?1-antitrypsin on cholesterol homeostasis in HepG2 cells

Janciauskiene

Lindgren

1999

Hepatology

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The family of serine proteinase inhibitors (serpins) is a class of proteins that can adopt multiple conformations and exist in various physical forms, some of which are known to be pathogenic. 1,2 Serpins can exist as native (active), cleaved (inactive), latent (inactive, but not cleaved), complexed with target enzyme, and polymerized forms, and it has been suggested that these conformers have not only diverse functional properties at the cellular level and different elimination rates, but also may exhibit new physiological functions. 3,4 ␣ 1 -Antitrypsin (AAT) is the prototypic member of the serpin family and primarily inhibits neutrophil elastase and proteinase 3. 5 AAT is an acute-phase protein, predominantly synthesized by the liver, the concentration of which during the acute-phase processes rises by three-to fourfold that of normal (1.34 mg/mL). 1 It follows that levels of AAT-elastase complexes also increase, a conclusion supported by the observation that, e.g., in acute leukemia patients, AATelastase complex levels are found to be more than 10 times higher than normal. 6 Formation of a stable, covalent complex between AAT and its target enzyme is associated with cleavage of the serpinreactive center loop, generating a 4-kd carboxyl-terminal fragment of 36 residues. This peptide remains non-covalently bound to the cleaved AAT in complex with enzymes and also to the core of cleaved AAT, dissociated from the complex. 3 The peptide can be separated from the cleaved AAT under denaturing conditions in vitro. Joslin et al. showed that proteolytically cleaved AAT binds to receptors on HepG2 cells and that binding is mediated by the same domain within the carboxyl-terminal fragment of AAT as for AAT-enzyme complexes. 7 Results from their studies using AAT-elastase complexes and purified fragments of AAT confirmed that a domain within the 4-kd carboxyl-terminal fragment of AAT is necessary for binding to these serpin-enzyme complex (SEC) receptor(s), and that such a domain is available for receptor binding only when AAT forms a complex with serine proteinase or when it is proteolytically modified. Recently, it was demonstrated that cellular internalization and degradation of antithrombin-thrombin, AAT-elastase, AAT-trypsin, urokinasetype plasminogen activator-plasminogen activator inhibitor-1, and some other SECs, but not native or cleaved forms of serpins, is mediated by the low-density lipoprotein receptorrelated protein 8 and very-low-density lipoprotein receptor. 9 Identification that protease-serpin complexes are recognized by endocytosis receptors of the low-density lipoprotein (LDL) receptor family suggests that those receptors play a role in both lipoprotein metabolism and proteinase regulation during the inflammatory response. In our previous work, we showed that proteolytically cleaved AAT increases LDL binding and uptake by up to 70% in a concentration-and time-dependent manner in HepG2 cells, suggesting a possible Abbreviations: AAT, ␣ 1 -antitrypsin; SEC, serpin-enzyme complex; LDL, low-density lipopr...

show abstract

SREBP-1 Mediates Activation of the Low Density Lipoprotein Receptor Promoter by Insulin and Insulin-like Growth Factor-I

Cited by 145 publications

References 26 publications

Insulin-activated Erk-mitogen-activated Protein Kinases Phosphorylate Sterol Regulatory Element-binding Protein-2 at Serine Residues 432 and 455 in Vivo

Insulin-activated Erk-mitogen-activated Protein Kinases Phosphorylate Sterol Regulatory Element-binding Protein-2 at Serine Residues 432 and 455 in Vivo

Identification of human low‐density lipoprotein receptor as a novel target gene regulated by liver X receptor alpha

Effects of fibrillar C-terminal fragment of cleaved ?1-antitrypsin on cholesterol homeostasis in HepG2 cells

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