FGF-1 induces expression of LXRa and production of 25-hydroxycholesterol to upregulate the apoE gene in rat astrocytes

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 apoEHDL 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 a (LXRa) mRNAs in rat astrocytes. Increase of LXRa 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 LXRa, 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 2450 and2320 bp, and the direct repeat 4 (DR4) element in this region (2448 to 2433 bp) was responsible for the activation. Chromatin immunoprecipitation analysis supported that FGF-1 enhanced association of LXRwith the rat apoE promoter. FGF-1 partially activated the apoE promoter even in the presence of anMEK 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-1induced apoE expression in astrocytes depends on LXRa being mediated by both LXRa expression and an LXRa ligand biosynthesis.—Lu, R., J. Ito, N. Iwamoto, T. NishimakiMogami, and S. Yokoyama. FGF-1 induces expression of LXRa and production of 25-hydroxycholesterol to upregulate the apoE gene in rat astrocytes. J. Lipid Res. 2009. 50: 1156–1164. Supplementary key words high density lipoprotein • cholesterol • direct repeat 4 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 (1, 2). ApoE is also synthesized by astrocytes and microglia in the central nervous system (CNS) and forms HDL as a major lipoprotein in CNS (3, 4). 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 (5). ApoE-HDL plays many key roles in maintaining integrity and regeneration of CNS bymediating intercellular lipid transport. ApoE synthesis in glia cells increases during development and after injury of CNS (6–11). We demonstrated that absence of apoE delayed healing of cryo-injury of mouse brain, as evidenced by expression This study was supported by International HDL Award Program, in part by grantsin-aid from The Ministries of Education, Science, Technology, Culture, and Sports, and of Health, Welfare, and Labor of Japan, and by the Program for Promotion of Fundamental Studies in Health Sciences of National Institute of Biomedical Innovation. Rui Lu is supported by Postdoctoral Fellowship for Foreign Researchers from the Japan Society for the Promotion of Science. Manuscript received 17 November2008 and in revised form 12 December 2008 and in re-revised form 18 February 2009. Published, JLR Papers in Press, February 19, 2009. DOI 10.1194/jlr.M800594-JLR200 Abbreviations: apoE, apolipoprotein E; ChIP, chromatin immunoprecipitation; CNS, central nervous system; ERK, extracellular signalregulated kinase; FGF, fibroblast growth factor; FGFR1, FGF receptor 1; LXR, liver X receptor; LXRE, LXR response element; MEK, mitogenactivated protein kinase kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-OH kinase; siRNA, small interfering RNA; SRE, sterol regulatory element; TK, thymidine kinase. 1 To whom correspondence should be addressed. e-mail: [email protected] Copyright © 2009 by the American Society for Biochemistry and Molecular Biology, Inc. 1156 Journal of Lipid Research Volume 50, 2009 This article is available online at http://www.jlr.org by gest, on O cber 4, 2017 w w w .j.org D ow nladed fom of fibroblast growth factor 1 (FGF-1) and subsequent apoE expression in the astrocytes in the peri-injury regions (12). 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 (13). 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 (14). FGF-1 was shown in astrocytes to activate the mitogenactivated 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 (15). It also stimulated transcription of the apoE gene mediated by the receptor, but with an unknown pathway (15). 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 themechanism 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)a, 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 LXRa expression and by the production of its ligand. MATERIALS AND METHODS