1 Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina; and 2 Departamento de Anatomia, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, 21949 - 900 Rio de Janeiro, Rio de Janeiro, Brazil
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, the effect of
thyroid hormone (triiodothyronine, T3) on the secretion of
mitogenic growth factors in astrocytes and C6 glioma cells was
examined. The proliferating activity of T3 could be due, at
least in part, to the astrocyte secretion of acidic and basic
fibroblast growth factor (aFGF and bFGF), tumor necrosis factor-,
and transforming growth factor-
. In contrast, the conditioned medium
(CM) of T3-treated C6 cells was mitogenic to this cell line
only after hyaluronidase digestion, suggesting the impairment of growth
factor mitogenic activity by hyaluronic acid. Furthermore, the presence
of bFGF was significantly greater in the CM of both
T3-treated astrocytes and T3-treated C6 cells
than in the corresponding control CM. These data show that
T3 induces cerebellar astrocytes to secrete mitogenic
growth factors, predominantly bFGF, that could influence astrocyte and neuronal proliferation via autocrine and paracrine pathways.
cell proliferation; cell differentiation; fibroblast growth factors; interleukin-3
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THYROID HORMONE (triiodothyronine, T3) plays an important role in ensuring normal mammalian brain maturation. In the rat, the absence of T3 during the critical first 3 wk of neonatal life results in retardation and disturbance of normal cellular differentiation (29). T3 increases proliferation of stem cells from the central nervous system both in vivo and in vitro (13, 20) and controls the astrocyte number and maturation of Bergmann glia in the cerebellum (4). Even though the regulatory role of T3 in brain development is well established, the cellular and molecular mechanisms by which this hormone produces its biological effects remain elusive.
Astroglial cells are heterogeneous with respect to their ability to respond to T3, including their capacity to secrete different growth factors or related molecules. T3 regulates protein expression in the rat C6 cells (34), which are immortalized glial cells with properties of both astrocytes and oligodendrocytes (39). The growth factors secreted by cerebral hemisphere astrocytes, after T3 stimulation, promote morphological differentiation in these cells derived from both normal and hypothyroid newborn rats. In contrast, T3 induces cerebellar astrocyte secretion of growth factors responsible for cerebellar astrocyte autocrine proliferation instead of morphological changes (21, 35, 36, 37). This effect is accompanied by the reorganization of glial fibrillary acidic protein (GFAP), fibronectin (36), and laminin (7) filaments, thus suggesting a possible additional role of T3 in cerebellar astrocyte adhesion. In addition, the growth factors secreted by cerebellar astrocytes after T3 stimulation modulate neuronal proliferation (11). Hence, the aim of the present study was to identify the mitogenic growth factors secreted by cerebellar astrocytes and rat C6 glioma cells after T3 stimulation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Cell cultures. Primary cultures of astrocytes were prepared from cerebella obtained from 2- to 3-day-old Wistar rats as described (34-37). Cells were grown to semiconfluence (7-8 days) in the presence of Dulbecco's modified Eagle's medium and nutrient mixture F-12 (DMEM-F-12, Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (FCS; Fazenda Pigue, Rio de Janeiro, Brazil). The cultures were incubated at 37°C in a humidified 5% CO2-95% air atmosphere.
The C6 cell line was grown in the presence of DMEM (Sigma) supplemented with 10% FCS. The cultures were incubated at 37°C in a humidified 5% CO2-95% air atmosphere. Cells were harvested with trypsin (0.125%, Sigma) when they reached confluence.Cell treatment and conditioned medium preparation.
After confluence, astrocyte or C6 monolayers were washed three times
with serum-free DMEM-F-12 and treated with 50 nM
3,3',5-triiodo-L-thyronine (T3) in DMEM-F-12 for 3 days, with medium renewed every day
except after the 3rd day. The hormone solution was dissolved in
DMEM-F-12 without FCS. Control cultures were maintained in DMEM-F-12
without FCS. Control and hormone-treated cultures were then maintained for 2 days without medium changes. The conditioned medium (CM) obtained
from control (CCM) and T3-treated cells (T3CM)
was collected on the 2nd day after the end of T3 treatment,
filtered in nitrocellulose membrane (0.22 µm, Millipore), and stored
at 20°C for later use.
Antibodies. Antibody anti-T3 (1:6,000, Sigma) was used in the evaluation of T3 content in T3CM by RIA and biological activity analysis.
For CM neutralization assays, neutralizing antibodies to growth factors were added to the CMs: rabbit anti-fibroblast growth factor-acidic (anti-aFGF, 1:200; Sigma); rabbit anti-fibroblast growth factor-basic (anti-bFGF, 1:450; Sigma); rabbit anti-tumor necrosis factor-
|
Quantitative Analysis of Astrocyte and C6 Number
C6 monolayers were cultured in 96-well culture plates and treated with DMEM-F-12 or 50 nM T3 as described. After 4 days, the monolayers were washed with PBS, trypsinized (0.125%), and counted in a hemocytometer under a Zeiss optic microscope.Astrocytes were cultured in 24-well culture plates and treated with DMEM-F-12 or 50 nM T3 as described. After 4 days, the monolayers were fixed and stained with 1% toluidine blue solution in PBS for 5 min, and the cell number was quantified. At least eight fields were counted per well.
Cell Proliferation Monitoring
MTT assay. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay method has been extensively used to determine glial cell proliferation (5, 36, 37) and as an in vitro test system for T3 action (14). C6 cells were plated (104 cells/well) in 96-well culture plates and cultured for 4 days. Viable cells were quantified by MTT colorimetric assay for mitochondrial dehydrogenase, as previously described (22). Results were expressed in absorbance at 550 nm.
Primary cerebellar astrocyte cultures were prepared in 96-well culture plates as described and were maintained in DMEM-F-12 enriched with 10% FCS for 4 days until the cells reached semiconfluence. After that, the astrocyte cultures were extensively washed with PBS and submitted to the experimental conditions (described in figure legends). After 4 days, viable cells were quantified by MTT colorimetric assay as described.Bromodeoxyuridil incorporation and detection. Alternatively, the cell proliferation was analyzed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. Astrocyte and C6 cultures were incubated for 24 h in the presence of 1 µg/ml of BrdU (Sigma). The cells were fixed with 4% paraformaldehyde for 20 min. Cultures were washed twice with distilled water and then incubated twice in 2 N HCl at 50°C for 15 min. After that, cultures were washed twice with 0.1 M borate buffer for 10 min at room temperature. The cells were then immunoreacted with anti-BrdU antibody (as described in Sytox green nucleic acid stain) and visualized under an epifluorescent Olympus microscope (Olympus, Tokyo, Japan). The mitotic index was measured by counting the percentage of labeled cells in at least eight different fields per coverslip.
Immunofluorescence. The immunofluorescence was performed as described previously (36, 37). After the BrdU procedure, the monolayers were washed with PBS and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. Cells were incubated with 10% normal goat serum (Sigma) in PBS for 1 h and subsequently with rat anti-BrdU antibody (1:500; Accurate Chemical and Scientific, Westbury, NY). The cells were then washed in PBS and incubated with goat anti-rat antibody conjugated with biotin (1:400; Vector Laboratories, Burlingame, CA). The secondary antibody was revealed by incubation with Texas red-streptavidin conjugate according to the manufacturer's instructions (Vector Laboratories). In all cases, no reactivity was observed when the primary antibody was absent. Cell nuclei were stained with diaminophenylindole (DAPI, Sigma). The cultures were mounted on N-propyl-gallate-glycerol and examined under an epifluorescent Olympus microscope.
Sytox green nucleic acid stain. The astrocyte and C6 monolayers were washed with HEPES buffer (5 mM HEPES; 125 mM NaCl; 5.5 mM KCl; 1.0 mM MgCl2 · 6H20, 1.8 mM CaCl2 · 2H20), pH 7.4, and incubated during 10 min with Sytox green nucleic acid stain according to the manufacturer's instructions (Molecular Probes, Eugene, OR). Cells were then washed with PBS and fixed with 4% paraformaldehyde for 20 min. Cell nuclei were stained with DAPI. The cultures were mounted on N-propyl-gallate-glycerol and examined under an epifluorescent Olympus microscope. Eight fields were counted for each well of experiment.
ELISA. The ELISA was performed as previously described (1). The proteins of CCM and T3CM were lyophilized and quantified by Bradford's method (3). MaxiSorb immunoplates were preincubated overnight at 4°C with 2 µg/well of CCM or T3CM protein or with 0.2 to 100 ng/well of recombinant bFGF (rbFGF, GIBCO-BRL, Grand Island, NY) dissolved in PBS. They were washed and subsequently incubated for 1 h at 37°C with PBS containing 0.1% Tween 20 (Sigma) and 2% bovine serum albumin (Sigma), washed again, and reacted with polyclonal antibody against bFGF (1:2,000, Sigma). The immune reaction was quantified by peroxidase-labeled anti-IgG rabbit antibody (Sigma).
C6 CM digestion.
C6 CCM or T3CM was prepared as described in Cell
treatment and conditioned medium preparation. The CMs were
digested with hyaluronidase (100 mU/ml, Sigma) or with hyaluronidase
(100 mU/ml) plus heparitinase III (10 mU/ml, Sigma) for 15 h at
37°C in a humidified 5% CO2-95% air atmosphere as
described (2). The CMs were then sterilized by filtration
in nitrocellulose membrane (0.22 µm, Millipore) and were either used
immediately or stored at 20°C for later use.
Statistical Analysis
Differences between the groups were evaluated by ANOVA and Student's t-test, and the differences were considered significant at P < 0.05. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
T3 Stimulates Cerebellar Astrocyte and C6 Glioma Cell Proliferation
We further investigated the effects of T3 and CM from T3-treated cerebellar astrocyte (T3CM) treatments on proliferation of cerebellar astrocytes and C6 glioma cells. We verified that both T3 and T3CM induced significant astrocyte proliferation by MTT assay or direct cell counting (Table 2). In addition, >95% of the cells were GFAP positive before and after both treatments, attesting to their astrocyte phenotype (36, 37). These results are in agreement with our previous data (36, 37) and suggest that the secretion of mitogenic growth factors may promote the T3 effects on astrocyte proliferation. In our culture conditions, these proliferative effects were not directly mediated by T3, since no residual hormone was detected (by RIA) in the CM of hormone-treated cultures (37). Alternatively, the anti-T3 antibody did not alter the mitogenic effect of T3CM (data not shown). We also verified that T3 induced significant C6 cell proliferation, with the maximum effect obtained at 50 nM (Table 2).
|
To investigate whether astrocyte and C6 population augmentation was due to increased proliferation or survival, we tested the BrdU incorporation. As shown in Table 2, the incorporation of BrdU was at least four times higher in T3-treated and T3CM-treated astrocytes than in control cells, confirming that T3 plus CM stimulates cerebellar astrocyte proliferation instead of cell survival. In addition, we verified that T3-treated C6 cells incorporated 30% more BrdU than the controls. However, T3CM from C6 cells did not promote a significant increase in C6 proliferation (Table 2).
To analyze the proportion of cell mortality in our culture conditions, we performed incubations with Sytox green. As shown in Table 2, the proportion of Sytox green-positive astrocytes and C6 glioma cells was very low in all treatments, indicating that the cell mortality was insignificant in our experiments. In addition, we could observe an increase in DAPI-positive cells in both T3 (61%) and T3CM-treated (69%) astrocytes and in T3-treated (50%) C6 cells when compared with controls (data not shown).
Cerebellar Astrocyte CM Induces Astrocyte Proliferation
To analyze the growth factors involved in astrocyte proliferation, some commercial neutralizing antibodies against growth factors were added to the CMs. We verified that the addition of anti-bFGF to the T3CM promoted the more significant inhibition in astrocyte proliferation (40%); however, anti-aFGF (23%) and anti-TNF-
|
Cerebellar Astrocyte CM Induces C6 Cell Proliferation
To verify whether the soluble factors secreted by cerebellar astrocytes after T3 stimulation are also mitogenic to C6 cells, proliferating experiments with the use of T3CM from cerebellar astrocytes were performed.As shown in Fig. 2, astrocyte
T3CM induced a significant dose-dependent increase in C6
cell proliferation. The proportion of 50% of astrocyte
T3CM was the initial CM concentration necessary to promote
a significant increase in C6 proliferation compared with astrocyte CCM.
This result indicated that the growth factors secreted by cerebellar
astrocyte after thyroid hormone stimulation were also mitogenic to C6
cells.
|
To analyze the mitogenic growth factor(s) involved in C6 proliferation,
CCM and T3CM from astrocytes were incubated with
neutralizing antibodies to known growth factors (Fig. 1B).
We observed significant inhibition in C6 cell proliferation after the
addition of anti-bFGF (35%) or anti-IL-3 (16.5%) to the astrocyte
T3CM compared with CCM (Fig. 1B). Interestingly,
anti-aFGF and anti-TGF- significantly inhibited the mitogenic effect
of only astrocyte T3CM compared with the same CM without
antibody but not compared with the corresponding CCM, suggesting a
basal astrocyte secretion of these growth factors (Fig. 1B).
In addition, C6 cultures treated with astrocyte CCM presented a
significant inhibition of proliferation after the addition of
neutralizing anti-bFGF compared with the same CM without antibody, also
suggesting a basal concentration of bFGF.
C6 Cell CM Effects on C6 Proliferation
The preceding results in this study suggest that the T3 and astrocyte T3CM mitogenic action is similar in both C6 cells and astrocytes. However, T3CM from C6 cells was not effective in promoting C6 cell proliferation (Table 2).Hyaluronic acid is a negatively charged, high-molecular-weight
polysaccharide that forms strikingly viscous solutions
(19) and is overexpressed in tumor cells
(32). In our culture conditions, we observed that the C6
CM was very viscous. We hypothesized that C6 cells secreted a very
large amount of hyaluronic acid, which accumulated in the culture dish,
interfering with growth factor activity. To test this hypothesis, we
digested C6 CM with hyaluronidase or hyaluronidase plus heparitinase
III and observed the recovery of mitogenic activity. As shown in Fig.
3, digested or nondigested C6 CCM had no
effect on C6 proliferation. As expected, nondigested C6
T3CM did not induce C6 cell proliferation; however, a
significant increase in C6 proliferation was observed when
T3CM from C6 cells was digested by hyaluronidase.
|
In addition, anti-bFGF was added to the T3CM from C6 cells previously digested by hyaluronidase, and the proliferative activity was partially abolished (Fig. 3). Heparitinase III digestion also impaired the recovery of the proliferative effect of the C6 T3CM promoted by hyaluronidase (Fig. 3).
Heparin and C6 Proliferation
It has been demonstrated that heparin enhances the activity of the FGFs (31) and that its excess reduces the growth factor action (18). To verify whether these effects could be observed in our experimental model, a heparin concentration curve was performed with C6 cells (Fig. 4). The addition of low concentrations of heparin (0.1, 0.5, 1, or 10 ng/ml) to the 50 nM T3 solution stimulated C6 proliferation, and as expected, the higher concentrations (50 or 70 ng/ml) inhibited it.
|
bFGF Quantification
The aforementioned experiments with growth factors neutralizing antibodies suggest that the bFGF may be the main factor present in the CM from both astrocytes and C6 cells. To quantify this growth factor, we performed ELISA assays (Fig. 5). We verified a significant increase in bFGF production in T3CM of both astrocytes (eightfold) and C6 cells (twofold) compared with the corresponding CCM.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We had previously shown that T3 induces the synthesis and secretion of growth factors by cerebellar astrocytes that mediate the T3 proliferative effect (21, 36, 37). In addition to astrocyte proliferation, these growth factors are also mitogenic to cerebellar neuronal cells (11), suggesting autocrine and paracrine mechanisms underlying the proliferative T3 mode of action.
The main finding of the present study is the demonstration that astrocytes secrete a cocktail of growth factors that mediate the mitogenic effect of T3 on cerebellar astrocytes and C6 cells. BrdU and Sytox green incorporation clearly point to the proliferative activity of T3 and astrocyte T3CM instead of cell survival.
T3 induces cerebellar astrocytes to secrete a combination
of growth factors, among them bFGF, aFGF, TNF-, and IL-3, that are
partially responsible for the astrocyte and C6 cell proliferation. These growth factors may act coordinately. Therefore, bFGF seems to be
the principal mitogenic growth factor secreted by cerebellar astrocytes
after T3 stimulation. However, some antibodies (anti-bFGF, anti-aFGF, and anti-TGF-
) inhibited the astrocyte or C6 cell proliferation in a mechanism independent of the T3 action,
suggesting that the basal secretion of these growth factors by
astrocytes may occur.
It was reported that TNF induces astrocyte proliferation in a time- and
dose-dependent manner, accompanied by downregulation of GFAP mRNA
(24). TNF is also mitogenic to C6 glioma cells, increasing
the expression of TNF type II receptor (16). Recently, TNF- (also known as lymphotoxin-
) was shown to be involved in the
T3 effects on neuronal proliferation (11). In
our experiments, anti-TNF-
markedly reduced astrocyte, but not C6
cell, proliferation. On the other hand, Frei et al. (8)
reported an IL-3-like factor that was produced by astrocyte exposure to
lipopolysaccharide. However, there is a divergence concerning IL-3
production in the central nervous system (17, 25). Our
results could suggest an involvement of IL-3 on the T3 mode
of action, because the anti-IL-3 antibody significantly inhibited C6
cell proliferation without effects on astrocytes. However, this finding
must be further explored. The differential effects of the neutralizing
growth factor antibodies on C6 cells and cerebellar astrocyte
proliferation may be explained by differences in the metabolism of
these two cell types.
The members of the FGF family are found in developing and adult rat brain and are heparin-binding growth factors (27). bFGF affects the proliferation and differentiation of glial precursor cells (38) and, in vitro, stimulates the proliferation and migration of neonatal rat astrocytes (6, 15). The expression of bFGF has been shown to be elevated in tissue specimens from gliomas, meningiomas, and metastatic brain tumors (23), and antisense oligonucleotides to bFGF can inhibit the growth of transformed human astrocytes (10). C6 cells (30) and cerebellar astrocytes (12) have been demonstrated to synthesize bFGF protein. Furthermore, aFGF and bFGF are suggested to regulate both the activation and inactivation of T3 metabolism in brain (28).
It has been reported that the activity of FGF is modulated by extracellular matrix molecules (9, 31) and that the high amounts of negatively charged polysaccharides impairs interaction of growth factors with their receptors on the cell surface (1, 2). It is well known that hyaluronic acid production is high during cell proliferation and migration (33) and in the developing brain (26). In our experiments, the T3CM from C6 cells was ineffective in promoting cell proliferation. The digestion by hyaluronidase recovered its mitogenic activity, which was impaired by anti-bFGF or heparitinase III digestion. These data suggest that T3 stimulates bFGF secretion in C6 cells and large amounts of hyaluronic acid in C6 cultures, interfering with the growth factor mitogenic activity. The digestion of heparan sulfate chains by hyaluronidase activity also affected C6 proliferation, possibly because heparan sulfate chains are crucial to bFGF activity (40) and bind to hyaluronic acid (9). Our results may suggest the occurrence of a bFGF reservoir in the extracellular matrix through proteoglycans and hyaluronic acid in C6 cell cultures after T3 treatment.
Previous studies have shown that heparin increases the affinity of bFGF for its receptor (31) and, in excess, competes for the binding sites within the signaling complex, and receptor transphosphorylation is reduced (18). In our experiments, we could demonstrate the stimulatory and inhibitory effects of heparin on T3-mediated C6 proliferation. Endogenous heparan sulfate may modulate the action of the FGF secreted by T3 stimulation in C6 cultures.
The presence of bFGF in both astrocyte and C6 T3CM was confirmed by ELISA. T3 stimulation increased eight times the bFGF secretion by astrocytes and two times by C6 cells. However, C6 CCM presented a basal concentration of bFGF that may be responsible for the C6 basal proliferation. The lower amount of bFGF in C6 T3CM may explain its lower proliferative effect. Our data strongly suggest that bFGF is the main growth factor that mediates the T3 mitogenic effect in cerebellar astrocytes and C6 cells. At least in C6 cells, its mitogenic activity may be directed by extracellular molecules such as heparan sulfate and/or hyaluronic acid.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the late Prof. Carlos Chagas for constant encouragement while this work was being done. Special thanks to M. C. Fialho de Mello for helpful comments on the manuscript and to Rosenilde C. H. Afonso for technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by the Third World Academy of Sciences, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Financiadora de Estudos e Projetos, Fundo para Pesquisa/Fundaçao Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro, and Progama de Nucleos de Excelencia-Ministerio de Ciencia e Tecnologia.
Address for reprint requests and other correspondence: V. Moura Neto, Instituto de Ciências Biomédicas, Departamento de Anatomia, Universidade Federal do Rio de Janeiro, CCS, Bloco F, Ilha do Fundão, 21949-900 Rio de Janeiro, RJ, Brazil (E-mail: vivaldo{at}anato.ufrj.br).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 November 2000; accepted in final form 25 June 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alvarez-Silva, M,
and
Borojevic R.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) activities in schistosomal liver granulomas are controlled by stroma-associated heparan sulfate proteoglycans.
J Leukoc Biol
59:
435-441,
1996[Abstract].
2.
Alvarez-Silva, M,
Silva LCF,
and
Borojevic R.
Cell membrane-associated proteoglycans mediate extramedullar myeloid proliferation in granulomatous inflammatory reactions to schistosome eggs.
J Cell Sci
104:
477-484,
1993
3.
Bradford, MM.
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1977[ISI].
4.
Clos, J,
Legrand C,
and
Legrand J.
Effects of thyroid state on the formation and early morphological development of Bergman glia in the developing cerebellum.
Dev Neurosci
3:
199-208,
1980[ISI][Medline].
5.
Cookson, MR,
Mead C,
Austwick SM,
and
Pentreath VW.
Use of the MTT assay for estimating toxicity in primary astrocyte and C6 glioma cell cultures.
Toxic in Vitro
9:
39-48,
1995[ISI].
6.
Engele, J,
and
Bohn MC.
Effects of acidic and basic fibroblast growth factors (aFGF, bFGF) on glial precursor cell proliferation: age dependency and brain region specificity.
Dev Biol
152:
363-372,
1992[ISI][Medline].
7.
Farwell, AP,
and
Dubord-Tomasetti SA.
Thyroid hormone regulates the extracellular organization of laminin on astrocytes.
Endocrinology
140:
5014-5021,
1999
8.
Frei, K,
Bodmer S,
Schwerdel C,
and
Fontana A.
Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages.
J Immunol
137:
3521-3527,
1986
9.
Gallagher, JT.
Heparan sulphates as membrane receptors for the fibroblast growth factors.
Eur J Clin Chem Clin Biochem
32:
239-247,
1994[ISI][Medline].
10.
Gately, S,
Soff GA,
and
Brem S.
The potential role of basic fibroblast growth factor in the transformation of cultured primary human fetal astrocytes and the proliferation of human glioma (U-87) cells.
Neurosurgery
37:
1-10,
1995[ISI][Medline].
11.
Gomes, FCA,
Maia CG,
Menezes JRL,
and
Moura Neto V.
Cerebellar astrocytes treated by thyroid hormone induce neuronal proliferation.
Glia
25:
247-255,
1999[ISI][Medline].
12.
Gömez-Pinilla, F,
Lee JW,
and
Cotman CW.
Basic FGF in adult rat brain: cellular distribution and response to entorhial lesion and fimbria-fornix transection.
J Neurosci
12:
345-355,
1992[Abstract].
13.
Gritti, A,
Parati EA,
Cova L,
Frolichsthal P,
Galli R,
Wanke E,
Faravelli L,
Morassutti DJ,
Roisen F,
Nickel DD,
and
Vescovi AL.
Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor.
J Neurosci
16:
1091-1100,
1996[Abstract].
14.
Hohenwarter, O,
Waltenberger A,
and
Katinger H.
An in vitro test system for thyroid hormone action.
Anal Biochem
234:
56-59,
1996[ISI][Medline].
15.
Holland, EC,
and
Varmus HE.
Basic fibroblast growth factor induces cell migration and proliferation after glia-specific gene transfer in mice.
Proc Natl Acad Sci USA
3:
1218-1223,
1998.
16.
Huang, H,
Lung HL,
Leung KN,
and
Tsang D.
Selective induction of tumor necrosis factor receptor type II gene expression by tumor necrosis factor-alpha in C6 glioma cells.
Life Sci
62:
889-896,
1998[ISI][Medline].
17.
Konishi, Y,
Kamegai M,
Takahashi K,
Kunishita T,
and
Tabira T.
Production of interleukin-3 by murine central nervous system neurons.
Neurosci Lett
182:
271-274,
1994[ISI][Medline].
18.
Krufka, A,
Guimond S,
and
Rapraeger AC.
Two hierarchies of FGF-2 signaling in heparin: mitogenic stimulation and high-affinity binding/receptor transphosphorylation.
Biochemistry
35:
11131-11141,
1996[ISI][Medline].
19.
Laurent, TC,
and
Fraser JRE
Hyaluronan.
FASEB J
6:
2397-2404,
1992
20.
Lezoualc'h, F,
Seugnet I,
Monnier AL,
Ghysdael J,
Behr JP,
and
Demeneix BA.
Inhibition of neurogenic precursor proliferation by antisense thyroid hormone receptor oligonucleotides.
J Biol Chem
270:
12100-12108,
1995
21.
Lima, FRS,
Trentin AG,
Rosenthal D,
Chagas C,
and
Moura Neto V.
Thyroid hormone induces protein secretion and morphological changes in astroglial cells with an increase in expression of glial fibrillary acid protein.
J Endocrinol
154:
167-175,
1997[Abstract].
22.
Mosmann, T.
Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
J Immunol Methods
65:
55-63,
1983[ISI][Medline].
23.
Murphy, PR,
and
Knee RS.
Basic fibroblast growth factor binding and processing by human glioma cells.
Mol Cell Endocrinol
114:
193-203,
1995[ISI][Medline].
24.
Oh, YJ,
Markelonis GJ,
and
Oh TH.
Effects of interleukin-1 beta and tumor necrosis factor-alpha on the expression of glial fibrillary acidic protein and transferrin in cultured astrocytes.
Glia
8:
77-86,
1993[ISI][Medline].
25.
Ohno, K,
Suzumura A,
Sawada M,
and
Marunouchi T.
Production of granulocyte-macrophage colony-stimulating factor by cultured astrocytes.
Biochem Biophys Res Commun
169:
719-724,
1990[ISI][Medline].
26.
Oohira, A,
Matsui F,
Matsuda M,
and
Shoji R.
Developmental change in the glycosaminoglycan composition of the rat brain.
J Biol Chem
257:
5821-5826,
1986
27.
Ozawa, K,
Uruno T,
Miyakawa K,
Seo M,
and
Imamura T.
Expression of the fibroblast growth factor family and their receptor family genes during mouse brain development.
Brain Res Mol Brain Res
41:
279-288,
1996[ISI][Medline].
28.
Pallud, S,
Ramauge M,
Gavaret JM,
Lennon AM,
Munsch N,
St Germain DL,
Pierre M,
and
Courtin F.
Regulation of type 3 iodothyronine deiodinase expression in cultured rat astrocytes: role of the Erk cascade.
Endocrinology
140:
2917-2923,
1999
29.
Porterfield, SP,
and
Hendrich CE.
The role of thyroid hormones in prenatal and neonatal neurological developmentcurrent perspectives.
Endocr Rev
14:
94-106,
1993[ISI][Medline].
30.
Powell, PP,
and
Klagsbrun M.
Regulation of basic fibroblast growth factor mRNA expression in rat C6 glioma cells.
Exp Cell Res
209:
224-230,
1993[ISI][Medline].
31.
Roghani, M,
Mansukhani A,
Dell'Era P,
Bellosta P,
Basilico C,
Rifkin DB,
and
Moscatelli D.
Heparin increases the affinity of basic fibroblast growth factor for its receptor but is not required for binding.
J Biol Chem
269:
3976-3984,
1994
32.
Sherman, L,
Sleeman J,
Herrlich P,
and
Ponta H.
Hyaluronate receptors: key players in growth, differentiation, migration and tumor progression.
Curr Opin Cell Biol
6:
726-733,
1994[ISI][Medline].
33.
Toole, BP,
Munaim SL,
Welles S,
and
Knudson CB.
Hyaluronate-cell interactions and growth factor regulation of hyaluronate synthesis during limb development.
Ciba Found Symp
143:
138-145,
1989[ISI][Medline].
34.
Trentin, AG,
and
Alvarez-Silva M.
Thyroid hormone regulates the protein expression in C6 glioma cells.
Braz J Med Biol Res
31:
1281-1284,
1998[ISI][Medline].
35.
Trentin, AG,
Gomes FCA,
Lima FRS,
and
Moura Neto V.
Thyroid hormone induces astrocyte secretion of factors and progressive morphological changes on primary and subcultures astrocytes.
In Vitro Cell Dev Biol Anim
34:
280-282,
1998[ISI][Medline].
36.
Trentin, AG,
and
Moura Neto V.
T3 affects cerebellar astrocyte proliferation, GFAP and fibronectin organization.
Neuroreport
6:
293-296,
1995[ISI][Medline].
37.
Trentin, AG,
Rosenthal D,
and
Moura Neto V.
Thyroid hormone and conditioned medium effects on astroglial cells from hypothyroid and normal rat brain: factor secretion, cell differentiation and proliferation.
J Neurosci Res
41:
409-417,
1995[ISI][Medline].
38.
Vescovi, AL,
Reynolds BA,
Fraser DD,
and
Weiss S.
bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells.
Neuron
11:
951-966,
1993[ISI][Medline].
39.
Volpe, JJ,
Fujimoto K,
Marasa JC,
and
Agrawal HG.
Relation of C-6 glial cells in culture to myelin.
Biochem J
152:
701-703,
1975[ISI][Medline].
40.
Yayon, A,
Klagsbrun M,
Esko JD,
Leder P,
and
Ornitz DM.
Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor.
Cell
22:
841-848,
1991.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |