FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2111658016> ?p ?o ?g. }

• W2111658016 endingPage "1164" @default.
• W2111658016 startingPage "1156" @default.
• W2111658016 abstract "Fibroblast growth factor 1 (FGF-1) enhances apolipoprotein E (apoE) expression and apoE-HDL biogenesis in autocrine fashion in astrocytes (Ito, J., Y. Nagayasu, R. Lu, A. Kheirollah, M. Hayashi, and S. Yokoyama. Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action. J. Lipid Res. 2005. 46: 679–686) associated with healing of brain injury (Tada,T., J-i. Ito, M. Asai, and S. Yokoyama. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain. Neurochem. Int. 2004. 45: 23–30). FGF-1 stimulates mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) to increase cholesterol biosynthesis and phosphatidylinositol 3-OH kinase (PI3K)/Akt to enhance apoE-HDL secretion (Ito, J., Y. Nagayasu, K. Okumura-Noji, R. Lu, T. Nishida, Y. Miura, K. Asai, A. Kheirollah, S. Nakaya, and S. Yokoyama. Mechanism for FGF-1 to regulate biogenesis of apoE-HDL in astrocytes. J. Lipid Res. 2007. 48: 2020–2027). We investigated the mechanism for FGF-1 to upregulate apoE transcription. FGF-1 increased apoE and liver X receptor α (LXRα) mRNAs in rat astrocytes. Increase of LXRα mRNA was suppressed by inhibition of the FGF-1 receptor-1 and MEK/ERK but not by inhibition of PI3K/Akt. The increases of apoE mRNA and apoE-HDL secretion were both inhibited by downregulation or inhibition of LXRα, while they were partially suppressed by inhibiting cholesterol biosynthesis. We identified the liver X receptor element responsible for activation of the rat apoE promoter by FGF-1 located between −450 and −320 bp, and the direct repeat 4 (DR4) element in this region (−448 to −433 bp) was responsible for the activation. Chromatin immunoprecipitation analysis supported that FGF-1 enhanced association of LXR with the rat apoE promoter. FGF-1 partially activated the apoE promoter even in the presence of an MEK inhibitor that inhibits the FGF-1-mediated enhancement of cholesterol biosynthesis. On the other hand, FGF-1 induced production of 25-hydroxycholesterol by MEK/ERK as an sterol regulatory element-dependent reaction besides cholesterol biosynthesis. We concluded that FGF-1-induced apoE expression in astrocytes depends on LXRα being mediated by both LXRα expression and an LXRα ligand biosynthesis. Fibroblast growth factor 1 (FGF-1) enhances apolipoprotein E (apoE) expression and apoE-HDL biogenesis in autocrine fashion in astrocytes (Ito, J., Y. Nagayasu, R. Lu, A. Kheirollah, M. Hayashi, and S. Yokoyama. Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action. J. Lipid Res. 2005. 46: 679–686) associated with healing of brain injury (Tada,T., J-i. Ito, M. Asai, and S. Yokoyama. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain. Neurochem. Int. 2004. 45: 23–30). FGF-1 stimulates mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) to increase cholesterol biosynthesis and phosphatidylinositol 3-OH kinase (PI3K)/Akt to enhance apoE-HDL secretion (Ito, J., Y. Nagayasu, K. Okumura-Noji, R. Lu, T. Nishida, Y. Miura, K. Asai, A. Kheirollah, S. Nakaya, and S. Yokoyama. Mechanism for FGF-1 to regulate biogenesis of apoE-HDL in astrocytes. J. Lipid Res. 2007. 48: 2020–2027). We investigated the mechanism for FGF-1 to upregulate apoE transcription. FGF-1 increased apoE and liver X receptor α (LXRα) mRNAs in rat astrocytes. Increase of LXRα mRNA was suppressed by inhibition of the FGF-1 receptor-1 and MEK/ERK but not by inhibition of PI3K/Akt. The increases of apoE mRNA and apoE-HDL secretion were both inhibited by downregulation or inhibition of LXRα, while they were partially suppressed by inhibiting cholesterol biosynthesis. We identified the liver X receptor element responsible for activation of the rat apoE promoter by FGF-1 located between −450 and −320 bp, and the direct repeat 4 (DR4) element in this region (−448 to −433 bp) was responsible for the activation. Chromatin immunoprecipitation analysis supported that FGF-1 enhanced association of LXR with the rat apoE promoter. FGF-1 partially activated the apoE promoter even in the presence of an MEK inhibitor that inhibits the FGF-1-mediated enhancement of cholesterol biosynthesis. On the other hand, FGF-1 induced production of 25-hydroxycholesterol by MEK/ERK as an sterol regulatory element-dependent reaction besides cholesterol biosynthesis. We concluded that FGF-1-induced apoE expression in astrocytes depends on LXRα being mediated by both LXRα expression and an LXRα ligand biosynthesis. Apolipoprotein E (apoE) is a glycoprotein composed of 299 amino acids and plays critical roles in lipid metabolism. While most of apolipoproteins are synthesized and secreted primarily by the liver and intestine, apoE is in addition secreted by other cells outside the enterohepatic axis, such as macrophages and adipocytes (1Curtiss L.K. Boisvert W.A. Apolipoprotein E and atherosclerosis.Curr. Opin. Lipidol. 2000; 11: 243-251Crossref PubMed Scopus (196) Google Scholar, 2Mazzone T. Apolipoprotein E secretion by macrophages: its potential physiological functions.Curr. Opin. Lipidol. 1996; 7: 303-307Crossref PubMed Scopus (111) Google Scholar). ApoE is also synthesized by astrocytes and microglia in the central nervous system (CNS) and forms HDL as a major lipoprotein in CNS (3Pitas R.E. Boyles J.K. Lee S.H. Foss D. Mahley R.W. Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins.Biochim. Biophys. Acta. 1987; 917: 148-161Crossref PubMed Scopus (574) Google Scholar, 4Nakai M. Kawamata T. Taniguchi T. Maeda K. Tanaka C. Expression of apolipoprotein E mRNA in rat microglia.Neurosci. Lett. 1996; 211: 41-44Crossref PubMed Scopus (115) Google Scholar). CNS is segregated from systemic circulation by the blood brain barrier and prevented from access to plasma lipoproteins, so that HDL generated in CNS is the exclusive lipid transport system among the CNS cells (5Ito J. Yokoyama S. Roles of glia cells in cholesterol homeostasis in the brain.in: Hertz L. Non-Neural Cells of the Nervous System: Function and Dysfunction. Elsevier, Amsterdam, The Netherlands2004: 519-534Google Scholar). ApoE-HDL plays many key roles in maintaining integrity and regeneration of CNS by mediating intercellular lipid transport. ApoE synthesis in glia cells increases during development and after injury of CNS (6Boyles J.K. Pitas R.E. Wilson E. Mahley R.W. Taylor J.M. Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system.J. Clin. Invest. 1985; 76: 1501-1513Crossref PubMed Scopus (654) Google Scholar, 7Muller H.W. Gebicke-Harter P.J. Hangen D.H. Shooter E.M. A specific-37,000-dalton protein that accumulates in regenerating but not in nonregenerating mammalian nerves.Science. 1985; 228: 499-501Crossref PubMed Scopus (112) Google Scholar, 8Dawson P.A. Schechter N. Williams D.L. Induction of rat E and chicken A-I apolipoproteins and mRNAs during optic nerve degeneration.J. Biol. Chem. 1986; 261: 5681-5684Abstract Full Text PDF PubMed Google Scholar, 9Ignatius M.J. Gebicke-Harter P.J. Skene J.H.P. Schilling J.W. Weisgraber K.H. Mahley R.W. Shooter E.M. Expression of apolipoprotein E during nerve degeneration and regeneration.Proc. Natl. Acad. Sci. USA. 1986; 83: 1125-1129Crossref PubMed Scopus (496) Google Scholar, 10Snipes G.J. McGuire C.B. Norden J.J. Freeman J.A. Nerve injury stimulates the secretion of apolipoprotein E by nonneuronal cells.Proc. Natl. Acad. Sci. USA. 1986; 83: 1130-1134Crossref PubMed Scopus (224) Google Scholar–11Aoki K. Uchihara T. Sanjo N. Nakamura A. Ikeda K. Tsuchiya K. Wakayama Y. Increased expression of neuronal apolipoprotein E in human brain with cerebral infarction.Stroke. 2003; 34: 875-880Crossref PubMed Scopus (87) Google Scholar). We demonstrated that absence of apoE delayed healing of cryo-injury of mouse brain, as evidenced by expression of fibroblast growth factor 1 (FGF-1) and subsequent apoE expression in the astrocytes in the peri-injury regions (12Tada T. Ito J. Asai M. Yokoyama S. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain.Neurochem. Int. 2004; 45: 23-30Crossref PubMed Scopus (24) Google Scholar). Long-term culture of rat astrocytes induced the increase of apoE synthesis and apoE-HDL production and enhancement of cholesterol biosynthesis, in comparison to the astrocytes prepared by a conventional method of 1-week primary and 1-week secondary culture (13Ueno S. Ito J. Nagayasu Y. Furukawa T. Yokoyama S. An acidic fibroblast growth factor-like factor secreted into the brain cell culture medium upregulates apoE synthesis, HDL secretion and cholesterol metabolism in rat astrocytes.Biochim. Biophys. Acta. 2002; 1589: 261-272Crossref PubMed Scopus (28) Google Scholar). FGF-1 was highly expressed and released in these long-cultured cells, and their conditioned medium and FGF-1 both stimulated apoE synthesis, production of apoE-HDL, and cholesterol biosynthesis in the conventionally prepared astrocytes (14Ito J. Nagayasu Y. Lu R. Kheirollah A. Hayashi M. Yokoyama S. Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action.J. Lipid Res. 2005; 46: 679-686Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). FGF-1 was shown in astrocytes to activate the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathway for the increase of cholesterol biosynthesis and the phosphatidylinositide 3-OH kinase (PI3K)/Akt pathway to enhance secretion of apoE/apoE-HDL, being mediated by the receptor (15Ito J. Nagayasu Y. Okumura-Noji K. Lu R. Nishida T. Miura Y. Asai K. Kheirollah A. Nakaya S. Yokoyama S. Mechanism for FGF-1 to regulate biogenesis of apolipoprotein E-high density lipoprotein in astrocytes.J. Lipid Res. 2007; 48: 2020-2027Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). It also stimulated transcription of the apoE gene mediated by the receptor, but with an unknown pathway (15Ito J. Nagayasu Y. Okumura-Noji K. Lu R. Nishida T. Miura Y. Asai K. Kheirollah A. Nakaya S. Yokoyama S. Mechanism for FGF-1 to regulate biogenesis of apolipoprotein E-high density lipoprotein in astrocytes.J. Lipid Res. 2007; 48: 2020-2027Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). To understand the mechanism for FGF-1 to stimulate astrocytes for biogenesis of apoE-HDL in response to brain injury and to expedite its healing process, we further investigated the mechanism for FGF-1 to increase transcription of apoE in astrocytes. FGF-1 was shown to enhance apoE transcription through the increase of interaction of the liver X receptor (LXR)α, a nuclear receptor for oxysterol being involved in sterol homeostasis, with a conserved direct repeat 4 (DR4) sequence in LXR response element (LXRE) present in the apoE promoter. This reaction was mediated by the increase of LXRα expression and by the production of its ligand. Astrocytes were prepared from 17-day fetal brain of Wistar rats according to the method previously described (16Ito J. Zhang Y.L. Asai M. Yokoyama S. Differential generation of high-density lipoprotein by endogenous and exogenous apolipoproteins in cultured fetal rat astrocytes.J. Neurochem. 1999; 72: 2362-2369Crossref PubMed Scopus (65) Google Scholar). The brain cells were cultured in F-10 medium (GIBCO) containing 10% (v/v) fetal calf serum for 1 week as primary culture and for subsequent 1 week as secondary culture. Fibroblasts of BALB/3T3 mouse were obtained from the RIKEN cell bank. The cells were cultured in DF medium (1:1 mixture of DMEM and Ham's F12 medium) with 10% (v/v) fetal calf serum. Total RNA was isolated from the cells using an ISOGEN kit (Nippon Gene). Real-time PCR analysis was performed on an ABI PRISM 7700 sequence detection system using target-specific probes. Those for apoE and LXRα were described elsewhere (14Ito J. Nagayasu Y. Lu R. Kheirollah A. Hayashi M. Yokoyama S. Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action.J. Lipid Res. 2005; 46: 679-686Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 17Hossain M.A. Tsujita M. Gonzalez F.J. Yokoyama S. Effects of fibrate drugs on expression of ABCA1 and HDL biogenesis in hepatocytes.J. Cardiovasc. Pharmacol. 2008; 51: 258-266Crossref PubMed Scopus (50) Google Scholar). For HMG-CoA reductase, SREBP2, cholesterol 24-hydroxylase (CYP46A1), and cholesterol 25-hydroxylase, the probes chosen were 5′-TGCTGCTTTGGCTGTATGTC-3′ and 5′-TGAGCGTGAACAAGAACCAG-3′, 5′-CCAAGAAGAAGGCAGGTGAC-3′ and 5′-GGACCCGCTCTACTTCAGTG-3′, 5′-GTGACTATGGGCGCTGGTAT-3′ and 5′-ATCAGCTGCTCTGCCTTCTC-3′, and 5′-TAGCCTTCTTGGATGTGCTG-3′and 5′-GTGAGTGGACCACGGAAAGT-3′, respectively. Cell lysates or media concentrates (concentrated using the BCA Protein Assay Kit; Pierce) were subjected to 10% SDS-PAGE (50 μg protein/lane) and then transferred to a polyvinylidene difluoride membrane. Bands of apoE were visualized by using the specific antibody (Santa Cruz Biotechnology). Intensity of photo-image was digitalized by scanning using an EPSON GT-X700 and analyzed with Adobe Photoshop software for quantification. Small interfering RNA (siRNA) specific primers for rat LXRα and a scramble control were obtained from Invitrogen and transfected into rat primary astrocytes using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The promoter region of rat apoE gene was prepared by PCR using rat normal liver genomic DNA (18Fung W.P. Howlett G.J. Schreiber G. Structure and expression of the rat apolipoprotein E gene.J. Biol. Chem. 1986; 261: 13777-13783Abstract Full Text PDF PubMed Google Scholar) as a template using primers tailed with KpnI and XhoI (New England Biolabs). Each PCR product was ligated into KpnI/XhoI sites of PGVB Basic vector and confirmed by sequencing. Six reporter gene constructs were assembled with the segments of the apoE promoter from −690, −600, −450, −320, −200, and −135 bp to +9 bp. To obtain a reporter construct with mutation of apoE DR4 element (DR4mu), site-directed mutagenesis was performed to introduce the DR4 at −448 to −433 bp of the −690 to +9 reporter gene using a Quick Change II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and the respective primers, DR4 of forward: 5′-CCGGGGATGGGGAGttaaCACCGTGGCAGAGGAATCACTA-3′, reverse: 5′-TAGTGATTCCTCTGCCACGGTGttaaCTCCCCATCCCCGG-3′ (lowercase letters indicate mutated nucleotides). The plasmid for expression of LXRα and the reporter plasmid containing four tandem repeats of LXREs upstream of the thymidine kinase (TK) promoter and its mutant, LXRE-TK and LXREmut-TK, the promoter plasmid containing sterol regulatory element (SRE), were prepared as previously described (19Suzuki S. Nishimaki-Mogami T. Tamehiro N. Inoue K. Arakawa R. Abe-Dohmae S. Tanaka A.R. Ueda K. Yokoyama S. Verapamil increases the apolipoprotein-mediated release of cellular cholesterol by induction of ABCA1 expression via Liver X receptor-independent mechanism.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 519-525Crossref PubMed Scopus (45) Google Scholar, 20Iwamoto N. Abe-Dohmae S. Sato R. Yokoyama S. ABCA7 expression is regulated by cellular cholesterol through the SREBP2 pathway and associated with phagocytosis.J. Lipid Res. 2006; 47: 1915-1927Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). 3T3 cells were grown to 60–70% confluence in 24-well plates, transfected with 1 μg of plasmid DNA including 0.2 μg LXRα expression vector, 0.77 μg reporter gene construct, and 30 ng hRluc-TK (Promega; for normalization) using Lipofectamine 2000 (Invitrogen). The luciferase activity was measured using a Dual-Luciferase Reporter System (Promega). Chromatin immunoprecipitation (ChIP) assay was conducted as previously described (20Iwamoto N. Abe-Dohmae S. Sato R. Yokoyama S. ABCA7 expression is regulated by cellular cholesterol through the SREBP2 pathway and associated with phagocytosis.J. Lipid Res. 2006; 47: 1915-1927Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Briefly, rat primary astrocytes were fixed with 1% formaldehyde. Cross-linked adducts were resuspended and sonicated to yield DNA fragments of 200 to 1,200 bp. Immunoprecipitation was performed using anti-LXRα antibody (Perceus Proteomics). Mouse normal IgG (Santa Cruz Biotechnology) was used as a control for nonspecific binding. Protein-bound, immunoprecipitated DNA was reverse cross-linked at 65°C overnight and then purified using a PCR purification Kit (Sigma-Aldrich). Ten microliters from a 50-μl DNA extraction volume was used for PCR amplification (33 cycles). The set of primers forward, 5′-CAGAGCTAACAAGTAACACA-3′, and reverse, 5′-AAAAGGGCTGGAGGCTTAAA-3′, was used to amplify the region on the promoter of the apoE gene. Cholesterol biosynthesis in astrocytes was estimated by incubating astrocytes with 3H-acetate as described elsewhere (15Ito J. Nagayasu Y. Okumura-Noji K. Lu R. Nishida T. Miura Y. Asai K. Kheirollah A. Nakaya S. Yokoyama S. Mechanism for FGF-1 to regulate biogenesis of apolipoprotein E-high density lipoprotein in astrocytes.J. Lipid Res. 2007; 48: 2020-2027Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Lipid was extracted from the cells and analyzed by TLC. For evaluation of production of 25-hydroxycholesterol, the cells were labeled with 14C-cholesterol conjugated with 2% cyclodextrin for 30 min at 37°C, washed with PBS three times, and incubated in 0.1% BSA-F-10 in the presence of FGF-1, U0126, or compactin for 16 h. Lipid was extracted and analyzed by TLC to detect the count in 25-hydroxycholesterol. Efficacy of the lipid extraction procedure (16Ito J. Zhang Y.L. Asai M. Yokoyama S. Differential generation of high-density lipoprotein by endogenous and exogenous apolipoproteins in cultured fetal rat astrocytes.J. Neurochem. 1999; 72: 2362-2369Crossref PubMed Scopus (65) Google Scholar) was 88 ± 5% estimated by the recovery of 14C-cholesterol exogenously added. FGF-1, TO901317 (an LXR agonist), PD173074, LY294002, and U1026 (inhibitors of the FGF receptor 1 (FGFR1), PI3K, and MEK1/2, respectively) were all purchased from Sigma-Aldrich. SB203580 and SP600125, inhibitors of P38 mitogen-activated protein kinase and stress-activated protein kinase/c-Jun NH2-terminal kinase, respectively, were also obtained from Sigma-Aldrich. Astrocytes were stimulated by FGF-1 at a confluent stage, and the levels of mRNA were measured for LXRα and apoE in time-dependent manners (Fig. 1A). The both messages increased by the FGF-1 treatment up to 12 h in parallel, by 3.5-fold for LXRα and by 7.5-fold for apoE mRNA. LXRα mRNA further increased at least for 24 h by the incubation, while apoE mRNA reached plateau at 12 h. FGF-1 enhanced expression of ABCA1 mRNA being consistent with the increase of LXRα but did not influence expression of LXRβ (Fig. 1B). Fig. 1C shows the increases of apoE mRNA and LXRα mRNA expressions by FGF-1 and their almost complete suppression by an FGFR1 inhibitor PD173074. An MEK/ERK pathway inhibitor, U0126, partially suppressed expression of apoE and LXRα mRNAs. The inhibition beyond the level of control without exogenous FGF-1 may indicate the presence of the basal autocrine activation by endogenous FGF-1 (14Ito J. Nagayasu Y. Lu R. Kheirollah A. Hayashi M. Yokoyama S. Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action.J. Lipid Res. 2005; 46: 679-686Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). A PI3K/Akt pathway inhibitor LY294002 that inhibits apoE secretion (15Ito J. Nagayasu Y. Okumura-Noji K. Lu R. Nishida T. Miura Y. Asai K. Kheirollah A. Nakaya S. Yokoyama S. Mechanism for FGF-1 to regulate biogenesis of apolipoprotein E-high density lipoprotein in astrocytes.J. Lipid Res. 2007; 48: 2020-2027Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) did not influence the expression of either mRNA. As the apoE gene transcription was reportedly regulated by LXRs (21Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kast H.R. Mangelsdorf D.J. Tontonoz P. LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes.Proc. Natl. Acad. Sci. USA. 2001; 98: 507-512Crossref PubMed Scopus (573) Google Scholar, 22Whitney K.D. Watson M.A. Collins J.L. Benson W.G. Stone T.M. Numerick M.J. Tippin T.K. Wilson J.G. Winegar D.A. Kliewer S.A. Regulation of cholesterol homeostasis by the liver X receptors in the central nervous system.Mol. Endocrinol. 2002; 16: 1378-1385Crossref PubMed Scopus (0) Google Scholar), we examined whether the FGF-1-induced expression of apoE depends on LXR. Rat astrocytes were transfected with LXRα siRNA to reduce its mRNA expression by 80% and examined for the reactivity of apoE expression by FGF-1. Expression of apoE was reduced by the siRNA treatment estimated as the mRNA and protein level, regardless of the presence of FGF-1, and this was reflected by the decrease in cellular and secreted apoE protein (Fig. 2A). Dependency of the FGF-1-induced apoE expression on LXR was also demonstrated by inhibition of LXR by arachidonic acid, a competitive but not highly specific inhibitor of LXRα (23Ou J. Tu H. Shan B. Luk A. DeBose-Boyd R.A. Bashmakov Y. Goldstein J.L. Brown M.S. Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR.Proc. Natl. Acad. Sci. USA. 2001; 98: 6027-6032Crossref PubMed Scopus (406) Google Scholar), shown as a decrease of cellular and secreted apoE (Fig. 2B). LXRα is activated by oxysterol that generally increases in parallel with cellular cholesterol, so that the effect of inhibition of cholesterol biosynthesis was examined. The cells were treated with compactin, an HMG-CoA reductase inhibitor, and the FGF-1-induced increase of LXRα mRNA was decreased by overall 62% (85% of the increase by exogenous FGF-1) and that of apoE mRNA was by 74% (Fig. 3). Thus, upregulation of the apoE gene transcription in astrocytes is associated with the increase of LXRα, both being regulated by cellular sterol biosynthesis.Fig. 3Effects of inhibition of cholesterol biosynthesis on the increase of expression of LXRα and apoE, as well as on secretion of apoE. Rat primary astrocytes were pretreated with 5 μM compactin for 2 h, and the cells were treated with 50 ng/ml FGF-1 for 16 h. Expression of mRNA of apoE and LXRα was determined by real-time PCR, and cellular and secreted apoE were analyzed by Western blotting. Data represent the average ± SD of three measurements. * P < 0.05 and ** P < 0.01 to compactin (−). Numbers indicate density of the bands relative to control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate molecular mechanism for FGF-1 to upregulate apoE expression, the promoter of the apoE gene was analyzed by using luciferase reporter assay. Six reporter genes were constructed to find a specific region(s) responsible for the FGF-1 induced activation, between −690 and +9 bp of the apoE promoter, −600 and 9 bp, −450 and +9 bp, −320and 9 bp, −200 bp and 9 bp, and −135 bp and +9 bp (Fig. 4A). The activity of the reporter genes was examined for the response to the FGF-1 treatment as well as to an LXR agonist, TO901317, in the transiently transfected 3T3 cells under overexpression of LXRα. As shown in Fig. 4B, FGF-1 and TO901317 activated the reporter genes as far as they contain the region −450 bp or its upstream, and this response was lost with the gene of −320 bp. We thus assumed that the region between −450 and −320 bp is responsible for activation by LXR and FGF-1. DR4 sequence was identified as AGTTCACCGTGGCAGA (−448 to −433 bp) in this region, so that mutation was introduced to this sequence as TTAACACCGTGGCAGA of the −600 to +9 bp construct (LXREmut) (Fig. 4A), and its activity was examined. Responses of the reporter genes to FGF-1 and to TO901317 both disappeared by introducing this mutation (Fig. 4C). To confirm that this sequence is generally responsible for activation by FGF-1, the reporter genes were constructed with the heterologous promoter of TK that contains the same DR4 sequence. The promoter was activated by FGF-1, and activation was abolished by introducing mutation into the DR4 in this assay system (Fig. 4D). Treatment of the cells of the reporter assay system with an LXR antagonist, arachidonate, decreased the activation by FGF-1 (Fig. 4E) and so did compactin (data not shown). We concluded from these results that one LXRE exit of −448 to −433 bp of the apoE promoter is responsible for increase of the apoE gene transcription by FGF-1 being dependent on LXR.Fig. 4The results of the reporter gene assays to identify the element responsible for activation by FGF-1 in the apoE promoter or in the heterologous TK promoter that contains 4 × DR4s. The reporter gene constructs were cotransfected with the expression vector for LXRα in 3T3 cells. After 24-h transfection, the cells were incubated with 50 ng/ml FGF-1 or 5 μM T0901317, an agonist for LXRα for 16 h. A: Diagram of the reporter gene constructs for the rat apoE promoter. Numbers indicate of the nucleotide positions, and location of DR4 sequence is indicated by gray boxes. Nucleotide sequence of the segment that contains DR4 and mutations introduced are indicated as lowercase letters for the corresponding nucleotide positions. B: The results with the apoE promoter constructs with various lengths as listed in A. The baseline activity of each reporter gene was 1.22 for −600 bp, 0.72 for −450 bp, 1.44 for −320 bp, 1.67 for −200 bp, and 1.46 for −135 bp, relative to that of −600 bp, respectively. C: The apoE promoter construct of −600 bp and its DR4 mutant (LXREmut) defined in Fig. 4A were used in luciferase assay with TO901317 and FGF-1. D: The results with the TK promoter and its mutant genes, by activation with TO901317 and FGF-1. E: The effects of an LXRα inhibitor, arachidonate, on activation by TO901317 and FGF-1 of the apoE promoter construct of −600 bp. Incubation was carried out as described in the text. Data represent the average ± SD of three measurements/samples. * P < 0.01 to the respective control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that FGF-1 enhances association of LXR with LXRE of the endogenous apoE promoter in astrocytes, ChIP assay was performed in rat primary astrocytes using specific antibody against rat LXRα (Fig. 5). Increase of the association was demonstrated by an LXR agonist TO901317 as well as by FGF-1. The experiments above demonstrated that FGF-1 activates LXR to enhance expression of the apoE gene through its interaction with the LXRE. Figure 6demonstrates production of LXR ligands by FGF-1. Cholesterol biosynthesis is increased by FGF-1 (Fig. 6A) in association with increase of expression of the HMG-CoA reductase gene (Fig. 6B). This increase seems to be mediated by activation of the SRE and SRE binding protein system because FGF-1 activated the SRE-luciferase assay system (Fig. 6C). Cholesterol 25-hydroxylase is known to be activated by the SRE system (24Russell D.W. Oxysterol biosynthetic enzymes.Biochim. Biophys. Acta. 2000; 1529: 126-135Crossref PubMed Scopus (309) Google Scholar), and its expression was indeed increased by FGF-1 and by compactin to a less extent, while expression of cholesterol 24-hydroxylase that is regulated by LXR (24Russell D.W. Oxysterol biosynthetic enzymes.Biochim. Biophys. Acta. 2000; 1529: 126-135Crossref PubMed Scopus (309) Google Scholar) was not significantly increased by FGF-1 (Fig. 6D, E). Consequently, production of 25-hydroxycholesterol was increased by FGF-1 in astrocytes (Fig. 6F). These effects of FGF-1 were all blocked by an inhibitor of the MEK/ERK pathway, U0126. Thus, FGF-1 at least increases a ligand of LXR, 25-hydroxycholesterol, for the enhancement of the apoE gene expression. We previously demonstrated that FGF-1 enhanced the apoE gene transcription even in the conditions that either the MEK/ERK or PI3K/Akt pathway was inhibited, for enhancement of cholesterol biosynthesis or activation of apoE secretion, respectively (15Ito J. Nagayasu Y. Okumura-Noji K. Lu R. Nishida T. Miura Y. Asai K. Kheirollah A. Nakaya S. Yokoyama S. Mechanism for FGF-1 to regulate biogenesis of apolipoprotein E-high density lipoprotein in astrocytes.J. Lipid Res. 2007; 48: 2020-2027Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The response of the apoE gene promoter to FGF-1 was therefore examined in the presence of those inhibitors to evaluate whether activation of the apoE gene transcription is independent of the related signaling pathways. The response of the reporter genes to FGF-1 was examined in the presence of an FGFR1 inhibitor, PD173074, a PI3K/Akt pathway inhibitor, LY294002, or an MEK/ERK pathway inhibitor, U0126. The effect of FGF-1 on expression of the gene of −450 bp was abolished by inhibition of FGFR1. Inhibition of the PI3K/Akt pathway did not influence the affect of FGF-1. On the other hand, inhibition of the MEK/ERK pathway by U0126 partially suppressed the effect of FGF-1 (Fig. 7A) in the concentration for complete suppression of cholesterol biosynthesis (data not shown), being consiste" @default.
• W2111658016 created "2016-06-24" @default.
• W2111658016 creator A5058832963 @default.
• W2111658016 creator A5069671627 @default.
• W2111658016 creator A5072979416 @default.
• W2111658016 creator A5081161282 @default.
• W2111658016 creator A5085340509 @default.
• W2111658016 date "2009-06-01" @default.
• W2111658016 modified "2023-10-16" @default.
• W2111658016 title "FGF-1 induces expression of LXRα and production of 25-hydroxycholesterol to upregulate the apoE gene in rat astrocytes" @default.
• W2111658016 cites W1486975043 @default.
• W2111658016 cites W1551655244 @default.
• W2111658016 cites W1592656278 @default.
• W2111658016 cites W1604658771 @default.
• W2111658016 cites W1965643342 @default.
• W2111658016 cites W1970259339 @default.
• W2111658016 cites W1972163077 @default.
• W2111658016 cites W1985991637 @default.
• W2111658016 cites W1996457468 @default.
• W2111658016 cites W2003798182 @default.
• W2111658016 cites W2016676112 @default.
• W2111658016 cites W2030442022 @default.
• W2111658016 cites W2035883738 @default.
• W2111658016 cites W2041281849 @default.
• W2111658016 cites W2045170820 @default.
• W2111658016 cites W2054059991 @default.
• W2111658016 cites W2054099490 @default.
• W2111658016 cites W2059485664 @default.
• W2111658016 cites W2063968666 @default.
• W2111658016 cites W2066581195 @default.
• W2111658016 cites W2067064610 @default.
• W2111658016 cites W2067450395 @default.
• W2111658016 cites W2074426271 @default.
• W2111658016 cites W2079649676 @default.
• W2111658016 cites W2080305329 @default.
• W2111658016 cites W2081722421 @default.
• W2111658016 cites W2092775227 @default.
• W2111658016 cites W2099543104 @default.
• W2111658016 cites W2105647248 @default.
• W2111658016 cites W2114806174 @default.
• W2111658016 cites W2116824509 @default.
• W2111658016 cites W2117004224 @default.
• W2111658016 cites W2119662556 @default.
• W2111658016 cites W2122892477 @default.
• W2111658016 cites W2132212053 @default.
• W2111658016 cites W2134551213 @default.
• W2111658016 cites W2147413218 @default.
• W2111658016 cites W2147743457 @default.
• W2111658016 cites W2158742055 @default.
• W2111658016 cites W2169812875 @default.
• W2111658016 cites W2319467927 @default.
• W2111658016 doi "https://doi.org/10.1194/jlr.m800594-jlr200" @default.
• W2111658016 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2681397" @default.
• W2111658016 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19229075" @default.
• W2111658016 hasPublicationYear "2009" @default.
• W2111658016 type Work @default.
• W2111658016 sameAs 2111658016 @default.
• W2111658016 citedByCount "13" @default.
• W2111658016 countsByYear W21116580162013 @default.
• W2111658016 countsByYear W21116580162014 @default.
• W2111658016 countsByYear W21116580162015 @default.
• W2111658016 countsByYear W21116580162017 @default.
• W2111658016 countsByYear W21116580162018 @default.
• W2111658016 countsByYear W21116580162020 @default.
• W2111658016 countsByYear W21116580162022 @default.
• W2111658016 crossrefType "journal-article" @default.
• W2111658016 hasAuthorship W2111658016A5058832963 @default.
• W2111658016 hasAuthorship W2111658016A5069671627 @default.
• W2111658016 hasAuthorship W2111658016A5072979416 @default.
• W2111658016 hasAuthorship W2111658016A5081161282 @default.
• W2111658016 hasAuthorship W2111658016A5085340509 @default.
• W2111658016 hasBestOaLocation W21116580161 @default.
• W2111658016 hasConcept C104317684 @default.
• W2111658016 hasConcept C126322002 @default.
• W2111658016 hasConcept C127561419 @default.
• W2111658016 hasConcept C134018914 @default.
• W2111658016 hasConcept C150194340 @default.
• W2111658016 hasConcept C185592680 @default.
• W2111658016 hasConcept C2777542381 @default.
• W2111658016 hasConcept C2779134260 @default.
• W2111658016 hasConcept C529278444 @default.
• W2111658016 hasConcept C55493867 @default.
• W2111658016 hasConcept C57089818 @default.
• W2111658016 hasConcept C71924100 @default.
• W2111658016 hasConcept C86803240 @default.
• W2111658016 hasConcept C95444343 @default.
• W2111658016 hasConceptScore W2111658016C104317684 @default.
• W2111658016 hasConceptScore W2111658016C126322002 @default.
• W2111658016 hasConceptScore W2111658016C127561419 @default.
• W2111658016 hasConceptScore W2111658016C134018914 @default.
• W2111658016 hasConceptScore W2111658016C150194340 @default.
• W2111658016 hasConceptScore W2111658016C185592680 @default.
• W2111658016 hasConceptScore W2111658016C2777542381 @default.
• W2111658016 hasConceptScore W2111658016C2779134260 @default.
• W2111658016 hasConceptScore W2111658016C529278444 @default.
• W2111658016 hasConceptScore W2111658016C55493867 @default.
• W2111658016 hasConceptScore W2111658016C57089818 @default.
• W2111658016 hasConceptScore W2111658016C71924100 @default.

• next