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• W1985991637 abstract "The astrocytes prepared by 1 week secondary culture after 1 month primary culture of rat brain cells (M/W cells) synthesized and secreted apolipoprotein E (apoE) and cholesterol more than the astrocytes prepared by conventional 1 week primary and 1 week secondary culture (W/W cells) (Ueno, S., J. Ito, Y. Nagayasu, T. Furukawa, and S. Yokoyama. 2002. 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. 1589: 261–272). M/W cells also highly expressed fibroblast growth factor-1 (FGF-1) mRNA. FGF-1 was identified in the cell lysate of both cell types, but M/W cells released more of it into the medium. Immunostaining of FGF-1 and apoE revealed that both localized in the cells that produce glial fibrillary acidic protein. The conditioned media of M/W cells and FGF-1 stimulated W/W cells to release apoE and cholesterol to generate more HDL. Pretreatment with a goat anti-FGF-1 antibody or heparin depleted the stimulatory activity of M/W cell-conditioned medium. The presence of the anti-FGF-1 antibody in the medium suppressed apoE secretion by M/W cells. Differential inhibition of signaling pathways suggested that FGF-1 stimulates apoE synthesis via the phosphoinositide 3-OH kinase for PI3K/Akt pathway. Thus, astrocytes release FGF-1, which promotes apoE-HDL production by an autocrine mechanism.These results are consistent with our in vivo observation that astrocytes produce FGF-1 before the increase of apoE in the postinjury lesion of the mouse brain (Tada, T., J. Ito, M. Asai, and S. Yokoyama. 2004. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain. Neurochem. Int. 45: 23–30). The astrocytes prepared by 1 week secondary culture after 1 month primary culture of rat brain cells (M/W cells) synthesized and secreted apolipoprotein E (apoE) and cholesterol more than the astrocytes prepared by conventional 1 week primary and 1 week secondary culture (W/W cells) (Ueno, S., J. Ito, Y. Nagayasu, T. Furukawa, and S. Yokoyama. 2002. 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. 1589: 261–272). M/W cells also highly expressed fibroblast growth factor-1 (FGF-1) mRNA. FGF-1 was identified in the cell lysate of both cell types, but M/W cells released more of it into the medium. Immunostaining of FGF-1 and apoE revealed that both localized in the cells that produce glial fibrillary acidic protein. The conditioned media of M/W cells and FGF-1 stimulated W/W cells to release apoE and cholesterol to generate more HDL. Pretreatment with a goat anti-FGF-1 antibody or heparin depleted the stimulatory activity of M/W cell-conditioned medium. The presence of the anti-FGF-1 antibody in the medium suppressed apoE secretion by M/W cells. Differential inhibition of signaling pathways suggested that FGF-1 stimulates apoE synthesis via the phosphoinositide 3-OH kinase for PI3K/Akt pathway. Thus, astrocytes release FGF-1, which promotes apoE-HDL production by an autocrine mechanism. These results are consistent with our in vivo observation that astrocytes produce FGF-1 before the increase of apoE in the postinjury lesion of the mouse brain (Tada, T., J. Ito, M. Asai, and S. Yokoyama. 2004. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain. Neurochem. Int. 45: 23–30). The brain cells are segregated from lipoproteins in the systemic circulation by the blood-brain barrier, so that cholesterol homeostasis in the brain is dependent on its specific extracellular lipid transport system by apolipoproteins and lipoproteins (1Chiba H. Mitamura T. Fujisawa S. Ogata A. Aimoto Y. Tashiro K. Kobayashi K.K. Apolipoproteins in rat cerebrospinal fluid: a comparison with plasma lipoprotein metabolism and effect of aging.Neurosci. Lett. 1991; 133: 207-210Crossref PubMed Scopus (23) Google Scholar, 2Bjorkhem I. Lutjohann D. Breuer O. Sakinis A. Wennmalm A. Importance of a novel oxidative mechanism for elimination of brain cholesterol.J. Biol. Chem. 1997; 272: 30178-30184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 3Bjorkhem I. Lutjohann D. Diczfalusy U. Stahle L. Ahlborg G. Wahren J.J. Cholesterol homeostasis in human brain: turnover of 24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation.J. 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Apolipoprotein E is synthesized in the retina by Muller glial cells, secreted into the vitreous, and rapidly transported into the optic nerve by retinal ganglion cells.J. Biol. Chem. 1996; 271: 5628-5632Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 8Fagan M.A. Holtzman M.D. Munson G. Mathur T. Schneider D. Chang K.L. Getz S.G. Reardon A.C. Lukens J. Shah A.J. LaDu J.M.M. Unique lipoproteins secreted by primary astrocytes from wild type, apoE (−/−), and human apoE transgenic mice.J. Biol. Chem. 1999; 274: 30001-30007Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 9Koch S. Donarski N. Goetze K. Kreckel M. Stuerenburg J.H. Buhmann C. Beisiegel U.U. Characterization of four lipoprotein classes in human cerebrospinal fluid.J. Lipid Res. 2001; 42: 1143-1151Abstract Full Text Full Text PDF PubMed Google Scholar, 10DeMattos B.R. Brendza P.R. Heuser E.J. Kierson M. Cirrito R.J. Fryer L. Sullivan M.P. Fagan M.A. Han X. Holtzman M.D.D. Purification and characterization of astrocytes-secreted apolipoprotein E and J-containing lipoproteins from wild-type and human apoE transgenic mice.Neurochem. Int. 2001; 39: 415-425Crossref PubMed Scopus (130) Google Scholar). The phenotype of human brain apoE does not change after liver transplantation, so that brain apoE is mostly produced in the brain (11Linton M.F. Gish R. Hubl S.T. Butler E. Esquivel C. Bry W.L. Boyles J.K. Wardell M.R. Young S.G.G. Phenotypes of apolipoprotein B and E after liver transplantation.J. Clin. Invest. 1991; 88: 270-281Crossref PubMed Scopus (285) Google Scholar). It is known that apoE is produced mainly by astrocytes and partly by microglia in the brain, suggesting that astrocytes play an important role in cholesterol homeostasis in the central nervous system (5Ito J. Yokoyama S. Roles of glia cells in cholesterol homeostasis in the brain.Adv. Mol. Cell Biol. 2004; 31: 519-534Crossref Scopus (15) Google Scholar, 10DeMattos B.R. Brendza P.R. Heuser E.J. Kierson M. Cirrito R.J. Fryer L. Sullivan M.P. Fagan M.A. Han X. Holtzman M.D.D. Purification and characterization of astrocytes-secreted apolipoprotein E and J-containing lipoproteins from wild-type and human apoE transgenic mice.Neurochem. Int. 2001; 39: 415-425Crossref PubMed Scopus (130) Google Scholar, 12Pitas R.E. Boyles J.K. Lee S.H. Foss D. Mahley R.W.W. Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins.Biochim. Biophys. Acta. 1987; 917: 148-161Crossref PubMed Scopus (569) Google Scholar, 13Boyles J.K. Pitas R.E. Wilson E. Mahley R.W. Taylor J.M.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 (651) Google Scholar, 14Nakai 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 (114) Google Scholar, 15LaDu M.J. Gilligan S.M. Lukens J.R. Cabana V.G. Reardon C.A. Van Eldik L.J. Holtzman D.M.M. Nascent astrocyte particles differ from lipoproteins in CSF.J. Neurochem. 1998; 70: 2070-2081Crossref PubMed Scopus (242) Google Scholar, 16Fujita C.S. Sakuta K. Tsuchiya R. Hamanaka H. Apolipoprotein E is found in astrocytes but not in microglia in the normal mouse brain.Neurosci. Res. 1999; 35: 123-133Crossref PubMed Scopus (35) Google Scholar). Astrocytes produce cholesterol-rich HDL with cellular lipid by autologously synthesized apoE (8Fagan M.A. Holtzman M.D. Munson G. Mathur T. Schneider D. Chang K.L. Getz S.G. Reardon A.C. Lukens J. Shah A.J. LaDu J.M.M. Unique lipoproteins secreted by primary astrocytes from wild type, apoE (−/−), and human apoE transgenic mice.J. Biol. Chem. 1999; 274: 30001-30007Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 17Ito J-i. 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). They also react with exogenous apoA-I to generate cholesterol-poor HDL through a unique system for intracellular cholesterol transport (18Ito J. Nagayasu Y. Yokoyama S. Cholesterol-sphingomyelin interaction in membrane and apolipoprotein-mediated cellular cholesterol efflux.J. Lipid Res. 2000; 41: 894-904Abstract Full Text Full Text PDF PubMed Google Scholar, 19Ito J. Nagayasu Y. Ueno S. Yokoyama S. Apolipoprotein-mediated cellular lipid release requires replenishment of sphingomyelin in a phosphatidylcholine-specific phospholipase C-dependent manner.J. Biol. Chem. 2002; 277: 44709-44714Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 20Ito J. Nagayasu Y. Kato K. Sato R. Yokoyama S. Apolipoprotein A-I induces translocation of cholesterol, phospholipid, and caveolin-1 to cytosol in rat astrocytes.J. Biol. Chem. 2002; 277: 7929-7935Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Like plasma HDL, ATP binding cassette transporter A1 supports such production of brain HDL, although other pathways may also function as backup systems (21Wahrle S.E. Jiang H. Parsadanian M. Legleiter J. Han X. Fryer J.D. Kowalewski T. Holtzman D.M.M. ABCA1 is required for normal central nervous system apoE levels and for lipidation of astrocyte-secreted apoE.J. Biol. Chem. 2004; 279: 40987-40993Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 22Hirsch-Reinshagen V. Zhou S. Burgess B.L. Bernier L. McIsaac S.A. Chan J.Y. Tansley G.H. Cohn J.S. Hayden M.R. Wellington C.L.L. Deficiency of ABCA1 impairs apolipoprotein E metabolism in brain.J. Biol. Chem. 2004; 279: 41197-41207Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). These lipoproteins are thought to play important roles in intercellular lipid transport in the brain. It has been noted that apoE synthesis is upregulated in the brain during development and after injury (13Boyles J.K. Pitas R.E. Wilson E. Mahley R.W. Taylor J.M.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 (651) Google Scholar, 23Muller 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, 24Dawson 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, 25Ignatius M.J. Gebicke-Harter P.J. Skene J.H.P. Schilling J.W. Weisgraber K.H. Mahley R.W. Shooter E.M.M. Expression of apolipoprotein E during nerve degeneration and regeneration.Proc. Natl. Acad. Sci. USA. 1986; 83: 1125-1129Crossref PubMed Scopus (495) Google Scholar, 26Snipes 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, 27Mahley R.W. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology.Science. 1988; 240: 622-630Crossref PubMed Scopus (3364) Google Scholar, 28Harel A. Fainaru M. Shafer Z. Hernandez M. Cohen A. Schwartz M.M. Optic nerve regeneration in adult fish and apolipoprotein A-I.J. Neurochem. 1989; 52: 1218-1228Crossref PubMed Scopus (53) Google Scholar, 29Graham D.I. Horsburgh K. Nicoll J.A. Teasdale G.M. Apolipoprotein E and the response of the brain to injury.Acta Neurochir. Suppl. 1999; 73: 89-92Crossref PubMed Scopus (64) Google Scholar, 30Haasdijk E.D. Vlug A. Mulder M.T. Jaarsma D. Increased apolipoprotein E expression correlates with the onset of neuronal degeneration in the spinal cord of G93A-SOD1 mice.Neurosci. Lett. 2002; 335: 29-33Crossref PubMed Scopus (27) Google Scholar, 31Aoki K. Uchihara T. Sanjo N. Nakamura A. Ikeda K. Tsuchiya K. Wakayama Y.Y. Increased expression of neuronal apolipoprotein E in human brain with cerebral infarction.Stroke. 2003; 34: 875-880Crossref PubMed Scopus (87) Google Scholar), and this reaction is likely to be involved in the healing process of the injury (32Laskowitz D.T. Sheng H. Bart R.D. Joyner K.A. Roses A.D. Warner D.S.S. Apolipoprotein E-deficient mice have increased susceptibility to focal cerebral ischemia.J. Cereb. Blood Flow Metab. 1997; 17: 753-758Crossref PubMed Scopus (145) Google Scholar, 33Laskowitz D.T. Horsburgh K. Roses A.D. Apolipoprotein E and the CNS response to injury.J. Cereb. Blood Flow Metab. 1998; 18: 465-471Crossref PubMed Scopus (182) Google Scholar, 34Sheng H. Laskowitz D.T. Mackensen B. Kudo M. Pearlstein R.D. Warner D.S.S. Apolipoprotein E deficiency worsens outcome from global cerebral ischemia in the mouse.Stroke. 1999; 30: 1118-1124Crossref PubMed Scopus (103) Google Scholar, 35Ophir G. Meilin S. Efrati M. Chapman J. Karussis D. Roses A. Michaelson D.M.M. Human apoE3 but not apoE4 rescues impaired astrocyte activation in apoE null mice.Neurobiol. Dis. 2003; 12: 56-64Crossref PubMed Scopus (39) Google Scholar). Four types of apoE binding receptor are identified in the brain: very low density lipoprotein receptor, low density lipoprotein receptor, low density lipoprotein receptor-related protein, and apoE receptor-2; thus, apoE is thought to function as a recognition site of lipoproteins for lipid delivery among brain cells (36Bu G. Maksymovitch A.E. Nerbonne M.J. Schwartz L.A. Expression and function of the low density lipoprotein receptor-related protein (LRP) in mammalian central neurons.J. Biol. Chem. 1994; 269: 18521-18528Abstract Full Text PDF PubMed Google Scholar, 37Nimpf J. Schneider J.W. From cholesterol transport to signal transduction: low density lipoprotein receptor, very low density lipoprotein receptor, and apolipoprotein E receptor-2.Biochim. Biophys. Acta. 2000; 1529: 287-298Crossref PubMed Scopus (66) Google Scholar). However, some of them are also likely to mediate signals for the migration of brain cells during the developmental integration of the brain (38Herz J. The LDL receptor gene family: (un)expected signal transducers in the brain.Neuron. 2001; 29: 571-581Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). We reported previously that astrocytes prepared by 1 month primary culture of rat fetal brain cells and subsequent 1 week secondary culture (M/W cells) synthesized and secreted apoE and cholesterol more actively than astrocytes prepared according to the conventional method of 1 week primary and 1 week secondary culture (W/W cells) (39Ueno 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). A fibroblast growth factor-1 (FGF-1)-like factor is secreted by the long-cultured rat fetal brain cells, and their conditioned media stimulated W/W astrocytes for the secretion of apoE. We also found that FGF-1 is produced by astrocytes adjacent to the cryoinjury lesions of mouse brain before the increase of apoE synthesis in vivo (40Tada 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). We identified a promoter polymorphism of FGF-1 related to risk for Alzheimer's disease (41Yamagata H. Chen Y. Akatsu H. Kamino K. Ito J. Yokoyama S. Yamamoto T. Kosaka K. Miki T. Kondo I.I. Promoter polymorphism in fibroblast growth factor 1 gene increases risk of definite Alzheimer's disease.Biochem. Biophys. Res. Commun. 2004; 321: 320-323Crossref PubMed Scopus (24) Google Scholar). Thus, we hypothesize that FGF-1 is a trigger stimulant of apoE synthesis and generation of HDL in the postinjury brain, presumably by an autocrine mechanism. In the present work, we attempted to identify the cells that secrete FGF-1 in the culture system and demonstrate an autocrine mechanism for this factor to stimulate apoE-HDL production. This is an important process to identify the triggering mechanism for the production of apoE and its HDL in the postinjury brain for recovery from damage. Astrocytes were prepared from the 17 day fetal brain of Wistar rats according to the method previously described (42Lim R. Misunobu K. Li W.K. Maturation-stimulating effect of brain extract and dibutyryl cyclic AMP on dissociated embryonic brain cells in culture.Exp. Cell Res. 1973; 79: 243-246Crossref PubMed Scopus (197) Google Scholar). After removal of the meninges, the brain was cut into small pieces and treated with 0.1% trypsin solution in Dulbecco's phosphate-buffered saline (DPBS) containing 0.15% glucose (0.1% trypsin/DPBS/G) for 3 min at room temperature. The cell pellet by centrifugation at 1,000 rpm for 3 min was cultured in F-10 medium containing 10% fetal calf serum (10% FCS/F-10) at 37°C for 4 weeks as a primary culture. After treatment with 0.1% trypsin/DPBS/G containing 1 mM ethylenediaminetetraacetic acid, the cells were cultured in 10% FCS/F-10 for 1 week as a secondary culture (M/W cells). Alternatively, astrocytes were prepared by a conventional method of 1 week primary and subsequent 1 week secondary culture (W/W cells). Both preparations contained 95% astrocytes [glial fibrillary acidic protein (GFAP)-positive], 0.3% oligodendroglias (anti-myelin basic protein-positive), and 3% microglias (ED-1-positive) (39Ueno 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). Rat astrocytes at a confluent stage were washed with DPBS four times and incubated in 0.1% BSA/F-10 for 24 h. The cells were incubated with [3H]acetate (New England Nuclear) in fresh 0.02% BSA/F-10 for certain periods of time. For the lipid-release experiments, the cells were washed three times with cold DPBS and further incubated in fresh 0.02% BSA/F-10 in the presence of 1 mM acetate. Cholesterol was extracted from the cells and the conditioned medium with hexane-isopropanol (3:2, v/v) and chloroform-methanol (2:1, v/v), respectively, and separated by TLC on Silica Gel-60 plates (E. Merck, Darmstadt, Germany). Radioactivity in the cholesterol fraction was counted (43Hara H. Yokoyama S. Interaction of free apolipoprotein with macrophages: formation of high density lipoprotein-like lipoproteins and reduction of cellular cholesterol.J. Biol. Chem. 1991; 266: 3080-3086Abstract Full Text PDF PubMed Google Scholar). The medium was also analyzed by density gradient ultracentrifugation as described previously (17Ito J-i. 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). After removing cell debris by centrifugation, the medium (8 ml) was overlaid on the sucrose solution (d = 1.175; 17 ml) and centrifuged at 1 × 105 g for 48 h. Samples were fractionated and analyzed for cholesterol mass by the enzymatic colorimetric method (44Abe-Dohmae S. Suzuki S. Wada Y. Aburatani H. Vance D.E. Yokoyama S.S. Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line.Biochemistry. 2000; 39: 11092-11099Crossref PubMed Scopus (101) Google Scholar) and for apoE by Western blotting (see below). The cells were harvested with a rubber policeman after washing four times with DPBS. The cell pellet by centrifugation at 1,000 rpm for 10 min was treated with cold and salined 0.02 M Tris-HCl buffer, pH 7.5 (TBS), containing the protease inhibitor cocktail (Sigma) for 10 min with 25 agitations for 10 s every 5 min. The suspension was centrifuged at 3,000 rpm for 10 min for removal of nuclei and cell debris. The supernatant was sonicated and centrifuged at 370,000 g for 30 min to obtain supernatant as a cell protein extract fraction. Cell debris was removed from the conditioned medium by centrifugation at 15,000 rpm for 30 min. Protein in the cell extract or in the conditioned medium was precipitated by 10% trichloroacetate and centrifugation at 15,000 rpm for 20 min, separated by SDS-PAGE, and transferred to a Sequi-Blot™ polyvinylidene difluoride membrane (Bio-Rad). The membrane was immunostained with a goat anti-FGF-1 antibody (Santa Cruz Biotechnology) and a rabbit antibody against rat apoE, a generous gift from Dr. Jean Vance (University of Alberta). Total cellular RNA was extracted from rat astrocytes with Isogen (Wako Life Science) and reverse-transcribed to generate cDNA in a SuperScript Preamplification System (Gibco BRL). The cDNA was subjected to PCR using the DNA probes for rat apoE mRNA and FGF-1 mRNA as described in the previous paper (39Ueno 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). After electrophoresis of the products, an agarose gel was stained with freshly prepared SYBR Gold nucleic acid gel stain solution. The band was detected by an ultraviolet transilluminator (UVP NLM-20E) at 302 nm. The apoE primer pairs were 5′-GCGCACCTCCTCCATCTCCTC-3′ (sense) and 5′-AGGATCTATGCAACCGACTCG-3′ (antisense). The FGF-1 primers were 5′-AAGCCCGTCGGTGTCCATGG-3′ and 5′-GATGGCACAGTGGATGGGAC-3′. Astrocytes on a tissue culture chamber/slide (Mikes Scientific) were washed with DPBS and fixed with 100% methanol at −20°C for 30 min. The cells were treated with 1% Triton X-100 in 0.02 M phosphate buffered saline at room temperature for 2 min after washing with DPBS. The cells were washed with DPBS again, treated with goat anti-FGF-1 antibody, or rabbit anti-rat apoE antibody, at room temperature for 60 min and washed. After incubation with biotin-conjugated anti-goat IgG, or anti-rabbit IgG antibody (Histofine) for 30 min at room temperature, the cells were washed, treated with peroxidase-conjugated streptavidin (Histofine) for 15 min, and then washed. The cells were stained by reaction with 0.01% 3,3′-diaminobenzidine tetrahydrochloride (Dojindo)/0.03% H2O2/0.05 M Tris buffer, pH 7.5, for 5 min at room temperature. Alternatively, M/W astrocytes were fluorescence immunostained after being fixed in organic solution composed of methanol, chloroform, and acetic acid (6:3:1) at −20°C for 3 h. After washing with cold TBS, the cells were reacted with either goat anti-FGF-1 or goat anti-rat apoE antibody (Santa Cruz Biotechnology) and mouse anti-GFAP antibody (BD Transduction Laboratories) in TBS containing 3% donkey serum and 3% horse serum at room temperature for 1 h. The cells were reacted with rhodamine-conjugated donkey anti-goat IgG antibody (Chemicon International) or fluorescein-conjugated horse anti-mouse IgG antibody (Vector Laboratories) in the presence of 3% donkey or 3% horse serum, respectively, at room temperature for 1 h after washing three times with TBS. The cells were observed by laser scanning confocal microscopy (LSM5; Zeiss, Jena, Germany). For the analysis of the FGF-1-initiated signals to stimulate apoE synthesis, rat astrocytes (W/W cells) were washed, replaced with 0.1% BSA/F-10, and incubated for 24 h with FGF-1 (50 ng/ml) in the presence or absence of an inhibitor of phosphoinositide 3-OH kinase (PI3K), for PI3K, LY294002 (10 μM; Calbiochem), or an inhibitor of MEK, U0126 (10 μM; Calbiochem). The cells were further incubated in the same condition in fresh 0.02% BSA/F-10 for 8 h and then in 0.02% BSA/F-10 for 16 h after washing. The conditioned medium was centrifuged at 15,000 rpm for 60 min to remove cell debris, treated with 10% TCA, and centrifuged at 15,000 rpm for 20 min. The pellet was analyzed by SDS-PAGE and Western blotting using rabbit anti-rat apoE antibody. Phosphorylation of Akt by FGF-1 was also examined. After the cells were incubated with FGF-1 (50 ng/ml) in fresh 0.02% BSA/F-10 for 5 min, the cytosol was prepared as a supernatant of the cell treatment in 0.02 M Tris-HCl buffer, pH 7.5, containing protease inhibitor cocktail (Sigma) for 10 min with 10 s of agitation 25 times every 5 min and centrifugation at 90,000 rpm for 30 min. The cytosol protein precipitated with 10% TCA was analyzed by SDS-PAGE and Western blotting using mouse anti-protein kinase B (PKB) α/Akt antibody (BD Transduction Laboratories) and rabbit anti-phospho-Akt (Thr-308) antibody (Cell Signaling Technology). During long-term culture of the rat brain cells, a large number of neurites were identified at 1 week, and astrocytes became predominant after 2–3 weeks, when neurons were hardly identified (Fig. 1A). Expression of the FGF-1 message was not apparent at 1 week primary culture and was markedly increased after 3 weeks (Fig. 1B). These findings indicated that FGF-1 was produced by astrocytes rather than neurons during the long-term primary culture of brain cells. To identify the cells that produce FGF-1 and apoE more specifically, immunostaining was performed for FGF-1 and apoE in various astrocyte preparations by 1 week secondary culture after primary culture of the brain cells for 1, 2, and 4 weeks (Fig. 2). The increase of FGF-1 and apoE was observed in cells prepared after primary culture for both 2 and 4 weeks (Fig. 2A–C, 2E–G). In the astrocyte preparation of 4 week primary and 1 week secondary culture (M/W cells), a group of cells were found with an appearance of “type 2” astrocyte-like cells. Both proteins were also identified in these cells (Fig. 2D, H). M/W cells were further analyzed by fluorescence immunostaining to confirm that both FGF-1 and apoE were produced by astrocytes. Figure 3shows that FGF-1 and apoE were both immunochemically identified in the GFAP-positive cells. M/W cells were examined for the production and secretion of FGF-1. The conditioned media of the brain cell primary culture and of the astrocyte preparations were examined for effects on the astrocytes prepared by a conventional method of 1 week primary and 1 week secondary culture (W/W cells) (Fig. 4). The medium of the primary culture for 2, 3, and 4 weeks and that of M/W cells stimulated apoE secretion from W/W cells (Fig. 4A). The astrocytes after 1 week primary and 4 week secondary culture (W/M cells) also generated the conditioned medium to stimulate apoE secretion. Stimulation of cholesterol release from W/W cells by M/W cell-conditioned medium was neutralized by pretreatment of the medium with anti-FGF-1 antibody-Sepharose and with heparin-Sepharose (Fig. 4B). This finding is consistent with the results with the conditioned medium of long-cultured whole brain cells (39Ueno 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).Fig. 4FGF-1-like activity in conditioned media. A: The effect of the conditioned media of the various brain cells and astrocytes on the secretion of apoE by W/W cells. The conditioned media were prepared from the primary culture for 2 weeks (2W; 356 μg cell protein/ml/well) and 4 weeks (4W; 644 μg/ml/well) of W/W cells (W/W; 142 μg/ml/well) and from M/W cells (M/W; 409 μg/ml/well) and W/M cells prepared by 1 week primary and 4 week secondary culture (W/M; 376 μg/ml/well). W/W cells were incubated with each conditioned medium (500 μl in 1 ml of culture medium) for 24 h. After washing and replacement with fresh 0.02% BSA/F-10 medium, the 16 h cultured medium was analyzed by immunoblotting for apoE. B: Effect of treatment with anti-FGF-1 antibody or heparin of the conditioned medium of M/W cells on its s" @default.
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