Advertisement
J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.M400313-JLR200 on January 1, 2005

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M400313-JLR200v1
46/4/679    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ito, J.-i.
Right arrow Articles by Yokoyama, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ito, J.-i.
Right arrow Articles by Yokoyama, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Journal of Lipid Research, Vol. 46, 679-686, April 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action

Jin-ichi Ito, Yuko Nagayasu, Rui Lu, Alireza Kheirollah, Michi Hayashi and Shinji Yokoyama1

Department of Biochemistry, Cell Biology, and Metabolism, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan

Published, JLR Papers in Press, January 1, 2005. DOI 10.1194/jlr.M400313-JLR200

1 To whom correspondence should be addressed. e-mail: syokoyam{at}med.nagoya-cu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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).

Abbreviations: apoE, apolipoprotein E; DPBS, Dulbecco's phosphate-buffered saline; FCS, fetal calf serum; FGF-1, fibroblast growth factor-1; GFAP, glial fibrillary acidic protein; PI3K, phosphoinositide 3-OH kinase; TBS, salined 0.02 Tris-Hcl buffer

Supplementary key words apolipoprotein E • fibroblast growth factor-1 • high density lipoprotein • brain damage • cholesterol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1–5). Helical apolipoproteins such as apolipoprotein E (apoE), apoA-I, apoD, apoA-IV, and apoJ have been identified in the cerebrospinal fluid as components of HDL, but the main apolipoproteins are apoE and apoA-I (6–10). The phenotype of human brain apoE does not change after liver transplantation, so that brain apoE is mostly produced in the brain (11).

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 (5, 10, 12–16). Astrocytes produce cholesterol-rich HDL with cellular lipid by autologously synthesized apoE (8, 17). They also react with exogenous apoA-I to generate cholesterol-poor HDL through a unique system for intracellular cholesterol transport (18–20). Like plasma HDL, ATP binding cassette transporter A1 supports such production of brain HDL, although other pathways may also function as backup systems (21, 22). 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 (13, 23–31), and this reaction is likely to be involved in the healing process of the injury (32–35).

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 (36, 37). However, some of them are also likely to mediate signals for the migration of brain cells during the developmental integration of the brain (38).

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) (39). 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 (40). We identified a promoter polymorphism of FGF-1 related to risk for Alzheimer's disease (41). 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of fetal rat astrocytes
Astrocytes were prepared from the 17 day fetal brain of Wistar rats according to the method previously described (42). 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) (39).

Synthesis and release of cellular cholesterol
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 (43). The medium was also analyzed by density gradient ultracentrifugation as described previously (17). 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 x 105 g for 48 h. Samples were fractionated and analyzed for cholesterol mass by the enzymatic colorimetric method (44) and for apoE by Western blotting (see below).

Analysis of cell and medium protein by Western blotting
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-BlotTM 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).

Reverse transcription-polymerase chain reaction
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 (39). 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'.

Immunocytostaining of astrocytes
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).

Analysis of signaling pathways
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) {alpha}/Akt antibody (BD Transduction Laboratories) and rabbit anti-phospho-Akt (Thr-308) antibody (Cell Signaling Technology).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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.



View larger version (136K):
[in this window]
[in a new window]
  		Fig. 1. Expression of fibroblast growth factor-1 (FGF-1) mRNA in rat fetal brain cells. A: Microscopic appearance of rat fetal brain cells in primary culture for 3 days, 1 week, and 3 weeks. B: FGF-1 mRNA. Total cellular RNA was prepared with Isogen (Wako Life Science) from brain cells in primary culture for 1 week (1W/P), 3 weeks (3W/P), and 4 weeks (4W/P) and from astrocytes prepared by conventional 1 week primary and 1 week secondary culture (W/W). Total mRNA (5 µg) was reverse-transcribed to cDNA using the SuperScript Preamplification System (Gibco BRL) for 10 min at 25°C, for 50 min at 50°C, and for 15 min at 70°C, and 0.5 µg of cDNA product was amplified using FGF-1 primers (5'-AAGCCCGTCGGTGTCCATGG-3' and 5'-GATGGCACAGTGGATGGGAC-3') for 30 cycles.

 
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).



View larger version (96K):
[in this window]
[in a new window]
  		Fig. 2. Immunostaining of FGF-1 and apolipoprotein E (apoE) in astrocytes. The cells were prepared by 1 week secondary culture after primary culture for 1 week (A, E), 2 weeks (B, F), or 4 weeks (C, D and G, H) of rat fetal brain cells. In the preparations of 4 week primary and 1 week secondary culture, C and G represent the fields in which ordinary astrocytes (type 1) are predominant and D and H represent the fields in which cells with type 2-like appearance are predominant. The cells were immunostained with rabbit anti-rat apoE antibody (A–D) or goat anti-FGF-1 antibody (E–H) as described in Materials and Methods.

 
M/W cells were further analyzed by fluorescence immunostaining to confirm that both FGF-1 and apoE were produced by astrocytes. Figure 3 shows that FGF-1 and apoE were both immunochemically identified in the GFAP-positive cells.



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 3. Immunostaining of M/W cells. A: The cells were immunostained using goat anti-FGF-1 and mouse anti-glial fibrillary acidic protein (GFAP) antibodies and visualized with rhodamine-conjugated donkey anti-goat IgG antibody (Chemicon International) and fluorescein-conjugated horse anti-mouse IgG antibody (Vector Laboratories), respectively. The cells were observed by laser scanning confocal microscopy (LSM5; Zeiss, Jena, Germany). B: The cells were treated in the same manner except that goat anti-rat apoE antibody was used instead of anti-FGF-1 antibody.

 
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 (39).




View larger version (89K):
[in this window]
[in a new window]
  		Fig. 4. FGF-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 stimulatory effects on W/W cells for the release of cholesterol. M/W-CM, M/W cell conditioned medium; CM/IgG, the medium pretreated with normal rabbit IgG-bound protein G-Sepharose; CM/Heparin, heparin-Sepharose CL-6B (Amersham Pharmacia); CM/anti-FGF-1, goat anti-FGF-1 antibody-bound protein G-Sepharose (Amersham Biosciences) at room temperature for 2 h. W/W cells were incubated with or without 500 µl of M/W-CM or pretreated M/W-CM for 24 h and washed, followed by incubation with 20 µCi/ml [3H]acetate in 1 ml of 0.02% BSA/F-10 for 16 h. After washing three times with Dulbecco's phosphate-buffered saline (DPBS), the cells were incubated for 16 h in fresh 0.02% BSA/F-10. The lipid extracted from the conditioned medium was analyzed by TLC to determine radioactivity in cholesterol. Each column represents the average and standard error of triplicate samples.

 
The effect of FGF-1 was further analyzed in the experiments shown in Fig. 5 . Density gradient analysis of the medium revealed an increase of HDL production by astrocytes (W/W cells) when stimulated by FGF-1 (Fig. 5A). There was no shift of the density peak of HDL with this change. Figure 5B demonstrates the distribution of apoE in the density fractions. The increase of apoE by FGF-1 was observed coincidentally with the cholesterol peak.




View larger version (39K):
[in this window]
[in a new window]
  		Fig. 5. Increase of apoE-HDL production by rat astrocytes stimulated by FGF-1. Rat astrocytes were incubated with or without FGF-1 (50 ng/ml) for 24 h and washed, and the conditioned medium was analyzed by density gradient ultracentrifugation after removal of cell debris as described in the text. A: Cholesterol mass was measured as described in the text. Open circles with solid lines indicate the conditioned medium without FGF-1, and closed circles with broken lines represent the conditioned medium with FGF-1. Lines without symbols represent the density of the fractions. B: The same sample fractions were analyzed for apoE by Western blotting as described in the text.

 
The message and protein of FGF-1 in M/W astrocytes were examined. M/W cells expressed a greater level of FGF-1 mRNA than did W/W cells (Fig. 6A) . On the other hand, a similar amount of FGF-1 was detected as a 16.5 kDa protein in the heparin-bound fraction of the extracts of both W/W and M/W cells by Western blotting analysis using an anti-FGF-1 antibody (Fig. 6B). However, a much greater amount of this protein was secreted into the culture medium by M/W cells than by W/W cells, as identified by immunoprecipitation with anti-FGF-1 antibody (Fig. 6C). Furthermore, secretion of apoE by M/W cell was markedly decreased when the cells were exposed to an anti-FGF-1 antibody during the detection period (Fig. 6D). Thus, we concluded that M/W astrocytes produce and secrete FGF-1 and that the secreted FGF-1 stimulates the cells to produce and secrete apoE by an autocrine mechanism.



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 6. Production and secretion of FGF-1 by M/W astrocytes. A: Expression of FGF-1 message in W/W cells and M/W cells. The cells were washed with DPBS three times and cultured in 0.1% BSA/F-10 for 24 h. Total cellular RNA was prepared from the cells, and RT-PCR was carried out using FGF-1 primer pairs with 28 cycles as described for Fig. 1. B: FGF-1 in astrocytes. Three milliliters of cell extract (250 µg of cytosol proteins/ml) was prepared from W/W and M/W astrocytes by sonication of the cells in TBS containing protease inhibitor cocktail (Sigma). The cell extract was mixed with heparin-Sepharose at room temperature for 2 h. The gel was washed three times with TBS containing protease inhibitor cocktail and applied for SDS-PAGE and Western blot analysis using goat anti-FGF-1 antibody. The left two lanes represent standard FGF-1. C: FGF-1 secreted into the conditioned medium of astrocytes. W/W and M/W cells (797 and 1,320 µg of total cell protein, respectively), each in two 10 cm Petri dishes, were metabolically labeled with 500 µCi/0.5 nM [35S]methionine in 7 ml of 0.02% BSA/F-10 without methionine or cysteine for 10 h and washed four times. After the cells were incubated in fresh 0.02% BSA/F-10 for 16 h, the conditioned medium equivalent to 690 µg/cell protein was collected. FGF-1 was immunopurified using goat anti-FGF-1 antibody-bound protein G-Sepharose and analyzed by SDS-PAGE and autoradiography. D: Effect of FGF-1 antibody in the culture medium on apoE secretion by astrocytes. M/W cells were exposed to anti-FGF-1 antibody (Santa Cruz Biotechnology) at the indicated concentration in fresh 10% FCS/F-10 at 3, 5, and 7 days after subculture. The culture medium (equivalent to 45 µg of cell proteins) was collected at the end of the 7th day and analyzed by SDS-PAGE and Western blotting using an anti-rat apoE antibody.

 
Signaling pathways for the stimulation of apoE synthesis by FGF-1 were examined. The increase of apoE secretion by FGF-1 was suppressed by LY294002, a PI3K/Akt inhibitor, but not by U0126, a MEK inhibitor (Fig. 7A) . Phosphorylation of Akt in cytosol was in fact induced by FGF-1 (Fig. 7B). Thus, FGF-1 stimulates apoE synthesis via the PI3K/Akt pathway rather than by the Ras/MEK/Erk pathway.



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 7. Analysis of the signaling pathways. A: The rat astrocytes (W/W cells) were treated with LY294002 (LY; 10 µM) or U0126 (U; 10 µM) in the presence of FGF-1 as described in the text. The conditioned medium was analyzed by Western blotting. M/W-CM, M/W cell conditioned medium. B: The cells were treated with FGF-1 for 5 min, and the cytosol was analyzed for Akt and phosphorylated (Pi) Akt by Western blotting.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We previously reported that FGF-1-like factor is secreted by 4 week primary cultured rat brain cells into the culture medium and enhances the production and secretion of apoE and cholesterol/phospholipid in W/W cells (39). We also discovered in vivo that FGF-1 is produced before the production of apoE in astrocytes after cryoinjury of the mouse brain (40). From these findings, we hypothesized that FGF-1 is one of the trigger factors for the production and release of apoE and apoE-HDL in astrocytes after brain injury and that it is released by astrocytes themselves to carry this reaction in an autocrine manner. In the present study, we further investigated this reaction in vitro to demonstrate that astrocytes are in fact capable of releasing FGF-1 and autostimulating apoE-HDL production.

The results summarized below led us to conclude that M/W astrocytes release FGF-1 and stimulate the production of apoE-HDL by an autocrine reaction. 1) The astrocytes prepared by 1 month primary and 1 week secondary culture of rat fetal brain cells (M/W cells) strongly expressed FGF-1 mRNA and synthesized and released FGF-1 into the conditioned medium, being identified as a heparin binding and anti-FGF-1 antibody-reactive protein of 16.5 kDa. 2) M/W cells themselves actively synthesized and secreted apoE with the cellular lipid to generate a greater amount of apoE-HDL, and this reaction was strongly suppressed by the presence of an anti-FGF-1 antibody in the medium during the culture. The conditioned medium of M/W cells stimulated apoE synthesis in the astrocytes (W/W cells), and this activity was abolished by pretreatment of the medium with an anti-FGF-1 antibody. 3) The cells that produce FGF-1 and apoE were both GFAP-positive. 4) Results from the examination of signaling pathways suggested that FGF-1 stimulates apoE synthesis via PI3K/Akt activation.

Among the several cytokines examined for the stimulation of apoE synthesis and secretion in human astrocytes, epidermal growth factor stimulated apoE secretion, whereas interleukin 1{alpha} and 1ß, interferon {gamma}, and FGF-2 did not (45). In our previous work, we examined the effect of FGF-1, FGF-2, insulin, and interleukin 1ß in rat astrocytes, and only FGF-1 stimulated the synthesis and secretion of apoE and lipid in rat astrocytes (39). FGF-1-like activity was found in the conditioned medium of brain cell culture to stimulate the astrocytes in the same manner. Our finding that FGF-1 is produced in astrocytes before the production of apoE in the brain injury lesion strongly supported the idea of its important role in the healing process. It has been thought that FGF-1 is primarily synthesized by neurons in vivo, but astrocytes are also identified as a potential source (46–52). The present results indicate that astrocytes are the main source of FGF-1 for the stimulation of apoE-HDL production by an autocrine mechanism.

Epidermal growth factor increases apoA-I expression in human hepatoma cells (HepG2) through the Ras-MAP kinase cascade and Sp1 (53). FGF-1 is known to induce signaling through P21ras/Erk (54, 55) and PI3K/Akt (56) in cells, including astrocytes. The present experiments using inhibitors of these pathways preliminarily indicate that apoE synthesis is stimulated by FGF-1 via the PI3K/Akt pathway. It is still unknown how this signaling regulates apoE gene expression. On the other hand, the apoE gene is upregulated by liver X receptor/retinoid X receptor in macrophages/adipocytes (57), fibroblasts (58), and astrocytes (59). Thus, the present results do not exclude the possibility that FGF-1 may indirectly upregulate apoE gene expression via the enhancement of cholesterol metabolism. The involvement of cAMP- and protein kinase C (PKC)-related pathways was also suggested for apoE upregulation (60).

It is puzzling how FGF-1 is released by cells even without a signal peptide. FGF-9 produced in the brain and kidney is also secreted by cells in spite of the lack of a signal peptide (61). As FGF-9 is an N-glycosylated protein, it is thought to be processed and secreted via the Golgi apparatus. The FGF-1-transfected cells release FGF-1 into the culture medium only in heat-shock conditions (62). FGF-1 may thus be secreted by astrocytes under stress to stimulate the secretion of apoE-HDL, such as heat shock, oxidation, and long-term culture, as described in this work. These stress conditions may be related to the brain damage.


   ACKNOWLEDGMENTS
 
This work was supported in part by HDL International Research Awards, by grants-in-aids from the Ministries of Health, Labor, and Welfare of Japan, and by a grant from the Pharmaceuticals and Medical Device Agency.

Manuscript received August 17, 2004


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
  1. Chiba, H., T. Mitamura, S. Fujisawa, A. Ogata, Y. Aimoto, K. Tashiro, and K. Kobayashi. 1991. Apolipoproteins in rat cerebrospinal fluid: a comparison with plasma lipoprotein metabolism and effect of aging. Neurosci. Lett. 133: 207–210.[CrossRef][Medline]

  2. Bjorkhem, I., D. Lutjohann, O. Breuer, A. Sakinis, and A. Wennmalm. 1997. Importance of a novel oxidative mechanism for elimination of brain cholesterol. J. Biol. Chem. 272: 30178–30184.[Abstract/Free Full Text]

  3. Bjorkhem, I., D. Lutjohann, U. Diczfalusy, L. Stahle, G. Ahlborg, and J. Wahren. 1998. Cholesterol homeostasis in human brain: turnover of 24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation. J. Lipid Res. 39: 1594–1600.[Abstract/Free Full Text]

  4. Dietschy, M. J., and D. S. Turley. 2001. Cholesterol metabolism in the brain. Curr. Opin. Lipidol. 12: 105–112.[CrossRef][Medline]

  5. Ito, J., and S. Yokoyama. 2004. Roles of glia cells in cholesterol homeostasis in the brain. Adv. Mol. Cell Biol. 31: 519–534.

  6. Borghini, I., F. Barja, D. Pometta, and W. R. James. 1995. Characterization of subpopulations of lipoprotein particles isolated from human cerebrospinal fluid. Biochim. Biophys. Acta. 1255: 192–200.[Medline]

  7. Amaratunga, A., R. C. Abraham, B. R. Edwards, H. J. Sandell, M. B. Schreiber, and E. R. Fine. 1996. 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. 271: 5628–5632.[Abstract/Free Full Text]

  8. Fagan, M. A., M. D. Holtzman, G. Munson, T. Mathur, D. Schneider, K. L. Chang, S. G. Getz, A. C. Reardon, J. Lukens, A. J. Shah, and J. M. LaDu. 1999. Unique lipoproteins secreted by primary astrocytes from wild type, apoE (–/–), and human apoE transgenic mice. J. Biol. Chem. 274: 30001–30007.[Abstract/Free Full Text]

  9. Koch, S., N. Donarski, K. Goetze, M. Kreckel, J. H. Stuerenburg, C. Buhmann, and U. Beisiegel. 2001. Characterization of four lipoprotein classes in human cerebrospinal fluid. J. Lipid Res. 42: 1143–1151.[Abstract/Free Full Text]

  10. DeMattos, B. R., P. R. Brendza, E. J. Heuser, M. Kierson, R. J. Cirrito, L. Fryer, M. P. Sullivan, M. A. Fagan, X. Han, and M. D. Holtzman. 2001. Purification and characterization of astrocytes-secreted apolipoprotein E and J-containing lipoproteins from wild-type and human apoE transgenic mice. Neurochem. Int. 39: 415–425.[CrossRef][Medline]

  11. Linton, M. F., R. Gish, S. T. Hubl, E. Butler, C. Esquivel, W. L. Bry, J. K. Boyles, M. R. Wardell, and S. G. Young. 1991. Phenotypes of apolipoprotein B and E after liver transplantation. J. Clin. Invest. 88: 270–281.

  12. Pitas, R. E., J. K. Boyles, S. H. Lee, D. Foss, and R. W. Mahley. 1987. Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim. Biophys. Acta. 917: 148–161.[Medline]

  13. Boyles, J. K., R. E. Pitas, E. Wilson, R. W. Mahley, and J. M. Taylor. 1985. Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J. Clin. Invest. 76: 1501–1513.

  14. Nakai, M., T. Kawamata, T. Taniguchi, K. Maeda, and C. Tanaka. 1996. Expression of apolipoprotein E mRNA in rat microglia. Neurosci. Lett. 211: 41–44.[CrossRef][Medline]

  15. LaDu, M. J., S. M. Gilligan, J. R. Lukens, V. G. Cabana, C. A. Reardon, L. J. Van Eldik, and D. M. Holtzman. 1998. Nascent astrocyte particles differ from lipoproteins in CSF. J. Neurochem. 70: 2070–2081.[Medline]

  16. Fujita, C. S., K. Sakuta, R. Tsuchiya, and H. Hamanaka. 1999. Apolipoprotein E is found in astrocytes but not in microglia in the normal mouse brain. Neurosci. Res. 35: 123–133.[CrossRef][Medline]

  17. Ito, J-i., Y. L. Zhang, M. Asai, and S. Yokoyama. 1999. Differential generation of high-density lipoprotein by endogenous and exogenous apolipoproteins in cultured fetal rat astrocytes. J. Neurochem. 72: 2362–2369.[CrossRef][Medline]

  18. Ito, J., Y. Nagayasu, and S. Yokoyama. 2000. Cholesterol-sphingomyelin interaction in membrane and apolipoprotein-mediated cellular cholesterol efflux. J. Lipid Res. 41: 894–904.[Abstract/Free Full Text]

  19. Ito, J., Y. Nagayasu, S. Ueno, and S. Yokoyama. 2002. Apolipoprotein-mediated cellular lipid release requires replenishment of sphingomyelin in a phosphatidylcholine-specific phospholipase C-dependent manner. J. Biol. Chem. 277: 44709–44714.[Abstract/Free Full Text]

  20. Ito, J., Y. Nagayasu, K. Kato, R. Sato, and S. Yokoyama. 2002. Apolipoprotein A-I induces translocation of cholesterol, phospholipid, and caveolin-1 to cytosol in rat astrocytes. J. Biol. Chem. 277: 7929–7935.[Abstract/Free Full Text]

  21. Wahrle, S. E., H. Jiang, M. Parsadanian, J. Legleiter, X. Han, J. D. Fryer, T. Kowalewski, and D. M. Holtzman. 2004. ABCA1 is required for normal central nervous system apoE levels and for lipidation of astrocyte-secreted apoE. J. Biol. Chem. 279: 40987–40993.[Abstract/Free Full Text]

  22. Hirsch-Reinshagen, V., S. Zhou, B. L. Burgess, L. Bernier, S. A. McIsaac, J. Y. Chan, G. H. Tansley, J. S. Cohn, M. R. Hayden, and C. L. Wellington. 2004. Deficiency of ABCA1 impairs apolipoprotein E metabolism in brain. J. Biol. Chem. 279: 41197–41207.[Abstract/Free Full Text]

  23. Muller, H. W., P. J. Gebicke-Harter, D. H. Hangen, and E. M. Shooter. 1985. A specific-37,000-dalton protein that accumulates in regenerating but not in nonregenerating mammalian nerves. Science. 228: 499–501.[Abstract/Free Full Text]

  24. Dawson, P. A., N. Schechter, and D. L. Williams. 1986. Induction of rat E and chicken A-I apolipoproteins and mRNAs during optic nerve degeneration. J. Biol. Chem. 261: 5681–5684.[Abstract/Free Full Text]

  25. Ignatius, M. J., P. J. Gebicke-Harter, J. H. P. Skene, J. W. Schilling, K. H. Weisgraber, R. W. Mahley, and E. M. Shooter. 1986. Expression of apolipoprotein E during nerve degeneration and regeneration. Proc. Natl. Acad. Sci. USA. 83: 1125–1129.[Abstract/Free Full Text]

  26. Snipes, G. J., C. B. McGuire, J. J. Norden, and J. A. Freeman. 1986. Nerve injury stimulates the secretion of apolipoprotein E by nonneuronal cells. Proc. Natl. Acad. Sci. USA. 83: 1130–1134.[Abstract/Free Full Text]

  27. Mahley, R. W. 1988. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science. 240: 622–630.[Abstract/Free Full Text]

  28. Harel, A., M. Fainaru, Z. Shafer, M. Hernandez, A. Cohen, and M. Schwartz. 1989. Optic nerve regeneration in adult fish and apolipoprotein A-I. J. Neurochem. 52: 1218–1228.[CrossRef][Medline]

  29. Graham, D. I., K. Horsburgh, J. A. Nicoll, and G. M. Teasdale. 1999. Apolipoprotein E and the response of the brain to injury. Acta Neurochir. Suppl. 73: 89–92.[Medline]

  30. Haasdijk, E. D., A. Vlug, M. T. Mulder, and D. Jaarsma. 2002. Increased apolipoprotein E expression correlates with the onset of neuronal degeneration in the spinal cord of G93A-SOD1 mice. Neurosci. Lett. 335: 29–33.[CrossRef][Medline]

  31. Aoki, K., T. Uchihara, N. Sanjo, A. Nakamura, K. Ikeda, K. Tsuchiya, and Y. Wakayama. 2003. Increased expression of neuronal apolipoprotein E in human brain with cerebral infarction. Stroke. 34: 875–880.[Abstract/Free Full Text]

  32. Laskowitz, D. T., H. Sheng, R. D. Bart, K. A. Joyner, A. D. Roses, and D. S. Warner. 1997. Apolipoprotein E-deficient mice have increased susceptibility to focal cerebral ischemia. J. Cereb. Blood Flow Metab. 17: 753–758.[Medline]

  33. Laskowitz, D. T., K. Horsburgh, and A. D. Roses. 1998. Apolipoprotein E and the CNS response to injury. J. Cereb. Blood Flow Metab. 18: 465–471.[CrossRef][Medline]

  34. Sheng, H., D. T. Laskowitz, B. Mackensen, M. Kudo, R. D. Pearlstein, and D. S. Warner. 1999. Apolipoprotein E deficiency worsens outcome from global cerebral ischemia in the mouse. Stroke. 30: 1118–1124.[Abstract/Free Full Text]

  35. Ophir, G., S. Meilin, M. Efrati, J. Chapman, D. Karussis, A. Roses, and D. M. Michaelson. 2003. Human apoE3 but not apoE4 rescues impaired astrocyte activation in apoE null mice. Neurobiol. Dis. 12: 56–64.[CrossRef][Medline]

  36. Bu, G., A. E. Maksymovitch, M. J. Nerbonne, and L. A. Schwartz. 1994. Expression and function of the low density lipoprotein receptor-related protein (LRP) in mammalian central neurons. J. Biol. Chem. 269: 18521–18528.[Abstract/Free Full Text]

  37. Nimpf, J., and J. W. Schneider. 2000. From cholesterol transport to signal transduction: low density lipoprotein receptor, very low density lipoprotein receptor, and apolipoprotein E receptor-2. Biochim. Biophys. Acta. 1529: 287–298.[Medline]

  38. Herz, J. 2001. The LDL receptor gene family: (un)expected signal transducers in the brain. Neuron. 29: 571–581.[CrossRef][Medline]

  39. 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.[Medline]

  40. 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.[CrossRef][Medline]

  41. Yamagata, H., Y. Chen, H. Akatsu, K. Kamino, J. Ito, S. Yokoyama, T. Yamamoto, K. Kosaka, T. Miki, and I. Kondo. 2004. Promoter polymorphism in fibroblast growth factor 1 gene increases risk of definite Alzheimer's disease. Biochem. Biophys. Res. Commun. 321: 320–323.[CrossRef][Medline]

  42. Lim, R., K. Misunobu, and W. K. Li. 1973. Maturation-stimulating effect of brain extract and dibutyryl cyclic AMP on dissociated embryonic brain cells in culture. Exp. Cell Res. 79: 243–246.[CrossRef][Medline]

  43. Hara, H., and S. Yokoyama. 1991. Interaction of free apolipoprotein with macrophages: formation of high density lipoprotein-like lipoproteins and reduction of cellular cholesterol. J. Biol. Chem. 266: 3080–3086.[Abstract/Free Full Text]

  44. Abe-Dohmae, S., S. Suzuki, Y. Wada, H. Aburatani, D. E. Vance, and S. Yokoyama. 2000. Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line. Biochemistry. 39: 11092–11099.[CrossRef][Medline]

  45. Baskin, F., M. G. Smith, A. J. Fosmire, and N. R. Rosenberg. 1997. Altered apolipoprotein E secretion in cytokine treated human astrocyte culture. J. Neurol. Sci. 148: 15–18.[CrossRef][Medline]

  46. Tooyama, I., Y. Hara, O. Yasuhara, Y. Oomura, K. Sasaki, T. Muto, K. Suzuki, K. Hanai, and H. Kimura. 1991. Production of antisera to acidic fibroblast growth factor and their application to immunohistochemical study in rat brain. Neuroscience. 40: 769–779.[CrossRef][Medline]

  47. Eckenstein, F. P., G. D. Shipley, and R. Nishi. 1991. Acidic and basic fibroblast growth factors in the neurons system: distribution and differential alteration. J. Neurosci. 11: 412–419.[Abstract]

  48. Tooyama, I., H. Akiyama, L. P. McGeer, Y. Hara, O. Yasuhara, and H. Kimura. 1991. Acidic fibroblast growth factor-like immunoreactivity in brain of Alzheimer patients. Neurosci. Lett. 121: 155–158.[CrossRef][Medline]

  49. Faucheux, B. A., S. Y. Cohen, P. Delaere, A. Tourbah, C. Dupuis, M. P. Hartmenn, J. C. Jeanny, J. J. Hauw, and Y. Courtois. 1992. Glial cell localization of acidic fibroblast growth factor-like immunoreactivity in the optic nerve of young adult and aged mammals. Gerontology. 38: 308–314.[Medline]

  50. Ryken, C. T., C. V. Traynelis, and R. Lim. 1992. Interaction of acidic fibroblast growth factor and transforming growth factor-ß in normal and transformed glia in vitro. J. Neurosurg. 76: 850–855.[Medline]

  51. Bugra, K., and D. Hicks. 1997. Acidic and basic fibroblast growth factor messenger RNA and protein show increased expression in adult compared to developing normal and dystrophic rat retina. J. Mol. Neurosci. 9: 13–25.[CrossRef][Medline]

  52. Miller, L. D., S. Ortrga, O. Bashayan, R. Basch, and C. Basilico. 2000. Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice. Mol. Cell. Biol. 20: 2260–2268.[Abstract/Free Full Text]

  53. Zheng, L. X., S. Matsubara, C. Diao, D. M. Hollenberg, and C. W. N. Wong. 2001. Epidermal growth factor induction of apolipoprotein A-I is mediated by the Ras-MAP kinase cascade and Sp1. J. Biol. Chem. 27: 13822–13829.

  54. Asada, S., Y. Kasuya, H. Hama, T. Masaki, and K. Goto. 1999. Cytodifferentiation potentiates aFGF-induced p21(ras)/Erk signaling pathway in rat cultured astrocytes. Biochem. Biophys. Res. Commun. 260: 441–445.[CrossRef][Medline]

  55. Cailliau, K., E. Browaeys-Poly, and J. P. Vilain. 2001. RasGAP is involved in signal transduction triggered by FGF1 in Xenopus oocytes expressing FGFR1. FEBS Lett. 496: 161–165.[CrossRef][Medline]

  56. Hashimoto, M., Y. Sagara, D. Langford, I. P. Everall, M. Mallory, A. Everson, M. Digicaylioglu, and E. Masliah. 2002. Fibroblast growth factor 1 regulates signaling via the glycogen synthase kinase-3beta pathway. Implications for neuroprotection. J. Biol. Chem. 277: 32985–32991.[Abstract/Free Full Text]

  57. Laffitte, B. A., J. J. Repa, S. B. Joseph, D. C. Wilpitz, H. R. Kast, D. J. Mangelsdorf, and P. Tontonoz. 2001. LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proc. Natl. Acad. Sci. USA. 98: 507–512.[Abstract/Free Full Text]

  58. Quinn, C. M., K. Kagedal, A. Terman, U. Stroikin, U. T. Brunk, W. Jessup, and B. Garner. 2004. Induction of fibroblast apolipoprotein E expression during apoptosis, starvation-induced growth arrest and mitosis. Biochem. J. 378: 753–761.[CrossRef][Medline]

  59. Liang, Y., S. Lin, T. P. Beyer, Y. Zhang, X. Wu, K. R. Bales, R. B. DeMattos, P. C. May, S. D. Li, X. C. Jiang, P. I. Eacho, G. Cao, and S. M. Paul. 2004. A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes. J. Neurochem. 88: 623–634.[CrossRef][Medline]

  60. Cedazo-Minguez, A., U. Hamker, V. Meske, R. W. Veh, R. Hellweg, C. Jacobi, F. Albert, R. F. Cowburn, and T. G. Ohm. 2001. Regulation of apolipoprotein E secretion in rat primary hippocampal astrocyte cultures. Neuroscience. 105: 651–661.[CrossRef][Medline]

  61. Miyamoto, M., K-i. Naruo, C. Seko, S. Matsumoto, T. Kondo, and T. Kurokawa. 1993. Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property. Mol. Cell. Biol. 13: 4251–4259.[Abstract/Free Full Text]

  62. Jackson, A., S. Friedman, X. Zhan, A. K. Engleka, R. Forough, and T. Maciag. 1992. Heat shock induces the release of fibroblast growth factor 1 from NIH 3T3 cells. Proc. Natl. Acad. Sci. USA. 89: 10691–10695.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
R. Lu, J. Ito, N. Iwamoto, T. Nishimaki-Mogami, and S. Yokoyama
FGF-1 induces expression of LXR{alpha} and production of 25-hydroxycholesterol to upregulate the apoE gene in rat astrocytes
J. Lipid Res., June 1, 2009; 50(6): 1156 - 1164.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
Y. Nagayasu, J.-i. Ito, T. Nishida, and S. Yokoyama
Reactivity of Astrocytes to Fibroblast Growth Factor-1 for Biogenesis of Apolipoprotein E-High Density Lipoprotein is Down-regulated by Long-time Secondary Culture
J. Biochem., May 1, 2008; 143(5): 611 - 616.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
J.-i. Ito, 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., September 1, 2007; 48(9): 2020 - 2027.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M400313-JLR200v1
46/4/679    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ito, J.-i.
Right arrow Articles by Yokoyama, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ito, J.-i.
Right arrow Articles by Yokoyama, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today 
Advertisement
spacer
Advertisement
Advertisement