GLIA 31193-204 (1990)

Fibroblast Growth Factor Inhibits Epidermal Growth Factor-Induced Responses in Rat Astrocytes -

KENNETH R. HUFF AND WAYNE SCHREIER Division of Neurology, Childrens Hospital of Los Angeles, and University of Southern California School of Medicine, Los Angeles, California 90027

KEY WORDS

Glia, Proliferation, 2 Deoxyglucose, EGF, FGF

ABSTRACT

The signals which regulate the proliferation of astrocytes have relevance to normal developmental processes, transformational loss of growth control, and reactive gliosis present in many brain disease states. We have studied, in primary cultures of rat astrocytes, a sequential interaction of two growth factors, epidermal growth factor (EGF) and fibroblast growth factor (FGF), which may be relevant to the brain in these conditions. EGF is a strong mitogen and stimulator of 2 deoxyglucose (2 DG) transport with no effect on plating of cells, and FGF is a lesser mitogen and 2 DG uptake stimulator. However, when FGF is given to the cells as a pretreatment, FGF strongly inhibits the ability of EGF to stimulate both DNA synthesis and 2 DG uptake. The inhibition of EGF stimulation by FGF is across the EGF dose-response curve, present a t high and low culture densities, and stable for at least 3 days. Specificityis indicated by lack of inhibition by PDGF pretreatment and much less inhibition of fetal calf serum-induced stimulations than EGF-induced stimulation. Cell counts confirmed that the FGF pretreatment also inhibits EGF stimulation of cell division. Because of FGF brain derivation and angiogenic and neurotropic functions, it may serve as a regulator of EGF-astrocyte interactions in processes such as development, gliomatous transformation, and neural regeneration.

INTRODUCTION Astrocytes proliferate under a number of different conditions, including normal brain development, neoplastic transformation, and the process-reactive gliosis. When the brain is injured, astrocytes may change drastically in number (Janeczko, 1988)and morphology in a histologically stereotypic pattern proportional to proximity t o the injury (Friede, 19751,suggesting that gliosis is a basic functional pathway for astrocytes and that local signals may regulate it. The proliferation signals for astrocytes in these circumstances may include growth factors, which also have developmental, oncologic, or reactive implications. Their responses in target tissues may include, besides proliferation, hypertrophy of the cell, increases in protein synthesis, changes in carbohydrate metabolism, rapid uptake of nutrients, and changes in cell structural properties (Varon and Saier, 1975). Growth factors might regulate the meta0 1 9 9 0 Wiley-Liss, Inc.

bolic changes which are part of the gliosis response from a number of different sources. They could be released with blood constituents or produced by inflammatory cells at the site of injury (Giulian and Lachman, 19851, be triggered by axotomy of a nerve cell or nerve cell death (Hatten, 1987), or be produced by glial cells themselves. Injury to rat brain induces gliosis along with peptides or factors which both stimulate astrocyte DNA synthesis and differentiate astrocyte morphology (Giulian and Young, 1986;Nieto-Sampedro et al., 1985). Epidermal growth factor (EGF) is well characterized (Cohen and Carpenter, 19751,and has known relevance to the nervous system both in vivo and in vitro (HerschReceived June 1,1989; accepted January 2, 1990. Address reprint requests to Kenneth R. Huff at the address given above Wayne Schreier’s present address is Department ofhatomy, UCLA School of Medicine, Los Angeles, CA 90027.

194

HUFF AND SCHREIER

man et al., 1983). It is notable that EGF is found in cerebrospinal fluid (Hirata et al., 1982); and both EGF (Fallon and Seroogy, 1984)and the EGF receptor kinase substrate p35 (McKanna and Cohen, 1989)are immunohistologically present in the fetal nervous system (and virtually absent in other fetal tissues). In addition, EGF receptor immunoreactivity has recently been demonstrated in adult rat brain astrocytes as a response to injury (Nieto-Sampedro et al., 1988). EGF-related compounds may be involved in glial transformational loss of growth control either through their own or EGF receptor production (Goustin et al., 1986). The EGF receptor is homologous with the product of the retroviral erb B proto-oncogene, which is highly expressed, amplified, and possibly rearranged in some glial-derived brain tumors (Libermann et al., 1985). EGF stimulates release of corticotropin-releasing hormone in hypothalamic organ culture (Luger et al., 19661, implying a possible neuroendocrine system trophic function. Aggregating cultures of fetal rat telencephalon respond t o EGF with dose-dependent increases in DNA synthesis which are cell cycle dependent (Guentert-Lauber and Honneger, 1985).Cerebellar astrocytes synthesize DNA in response to EGF (Leutz and Schachner, 1981), and purified telencephalic grey matter astrocytes respond with increased DNA synthesis and cell binding to EGF (Simpson et al., 1982). Fibroblast growth factor, another trophic protein, is isolated in two forms from the brain, aFGF and bFGF (Gospodarowicz et al., 19751, and has widespread targets, including effects on repair processes in other tissues (Cuevas et al., 1988). Increased bFGF immunoreactivity is found in association with reactive astroglia at the edges of focal brain wounds (Finklestein et al., 1988), and bFGF message expression is increased in lesioned brain (Logan, 1988). Astrocyte mitogenicity (Pettman et al., 1985), plasminogen activator production (Roaster et al., 1988), and morphologic changes (Perraud et al., 1988)are FGF responses seen in culture. It may be that events in gliosis are controlled sequentially. It would seem likely there are locally acting signals which may serve as modulators or switches for the EGF-induced stimulation of astrocytes. Sequential exposure is necessary for maximal biologic effect in other systems (O'Keefe and Pledger, 1983). Because of these findings in other systems and the growing evidence of EGF relevance to the nervous system, we felt it was important to study further proliferation-related responses to this growth factor signal in cultures of isolated and purified rat forebrain astrocytes, and potential sources of regulation of EGF such as the sequential interaction with another growth factor, FGF, which may be relevant to the biologic responses of astrocytes to brain injury.

were 95% or greater glial fibrillary acidic protein (GFAF') immunohistochemiciilly stained astrocytes. Neonatal Sprague-Dawley rat cerebral cortices were rapidly removed into sterile media, and the meninges were carefully dissected awa:y. The tissue was gently pushed through 210 pm nytex nylon monofilament screen, and the dissociated cells were then filtered through stainless steel screens. The yield per neonatal brain was approximately lo6 cells, and these portions of the pooled cells were then added to untreated plastic T-75 flasks in Dulbecco-Vogt, modification of Eagle's medium (DMEM): Ham's F-12 (1:l) (serum-free media, SFM) with 10%fetal calf serum. After 2 days the flasks were shaken on an orbital shaker for 2 h at 200 rpm, the media changed, and then shaken 3 days at 200 rpm and 2 days at 100 rpm with media changes each day to remove nonadherent cells. When the astrocytes had reached confluency at 10 days they were suspended using 0.25% trypsin for 10 min, washed by centrifugation with complete medium, and plated in 35 mm wells, some of which contained covei*slipsfor immunofluorescent examination of cell markers, at a concentration of lo5 cells/well for 5 more days to again reach confluency (2 X lo6 cells/ml). Rare cu1tui.e~containing fibroblast contamination were discarded. The confluent astrocytes were then washed three times in serum-free media and placed in serum free media for 48 h before their treatment or pretreatment with growth factor. DNA synthesis/proliferatio n was measured by 3Hthymidine incorporation in a 2 h assay. One microcurie of 3H-thymidine was added to the media. After 2 h incubation at 37"C, the cultures were placed on ice, the media aspirated off, and the cultures washed three times with ice-cold phosphate buffered saline (PBS). Cells were harvested in cold PBS by scraping with a Teflon policeman and centrifugation of the suspension. The cells were lysed with 10% trichloroacetic acid; the resulting pellet was digested by heating for 30 min at 70°C in 0.5 ml of 10% perchlo4c acid t o solubilize DNA as described by Simpson et al., 1982. Plating efficiency measurements were done by taking a 200 cell aliquot from the purified astrocytes as they were being harvested and plated from the flasks and dispersing the aliquot in 2 ml of media containing the growth factor being tested in a 35 mm well. After 6 days and one media change at 3 days the media were removed, the cells were fixed and Giemsa stained, and the total number of colonies con1,aining at least two cells were counted for each well. Autoradiography and double immunostaining was done on astrocytes plated on coverslips. The cells were treated for 24 h with EGF 10 ng/ml treatment or serumfree media. The cells were then pulsed for 4 h with 1 pCi/ml of 3H-thymidine. The cell monolayers were then washed successively: three times with SFM, two times with PBS, 24 h with PBS while rotating a t 50 rpm, and two more times with PBS fo1:owed by another time on METHODS AND MATERIALS the rotator for 72 h. The extensive washes were necesAstrocyte cultures were prepared by the method of sary to remove unincorporated 3H-thymidine to reduce McCarthy and De Vellis (19801, producing cells which background autoradiographic grains. The cells were

195

FGF INHIBITS EGF STIMULATION OF ASTROCYTES

then fixed with acetone for 20 min at -20" Centigrade. Each antibody treatment was for 30 min at 37°C and was followed by 3 PBS washes. The first antibody treatment was rabbit anti-cow glial fibrillary acidic protein (GFAP) (l:lOO), and then goat anti-rabbit IgGlinked TRITC (1:50). The coverslips were moved to the darkroom (Kodak No. 2 red filter), dipped in emulsion NTB-2 diluted 1:l in 0.6 M ammonium acetate, and dried for 30 min. They were then placed in the dark at 4°C for 5 days. Afterwards they were immersed in Developer D-19 at room temperature for 5 min, rinsed with tapwater, and rinsed for 30 s in 0.1% acetic acid. They were then immersed in fixer for 5 min and rinsed in tapwater. Finally, they were stained with hematoxylin and eosin and mounted with aquamount. Autoradiographic grains were visualized under white light transmission microscopy and rhodamine staining visualized with ultraviolet microscopy and appropriate filters. 2 Deoxyglucose assays were done using a modification of the procedure of Hollenburg and Cuatrecasas (1975). Astrocytes cultured in 35 mm tissue culture wells were rinsed free of medium with warm PBS containing 0.1%bovine serum albumin (BSA).The cells were preincubated for 10 min at 37°C in 1 ml of PBS containing BSA. Then the test compounds were added together with tritiated 2 deoxyglucose to a final concentration of 0.5 pCYm1. After 30 min at 37°C the uptake

h

was terminated by the addition of 1 ml of ice-cold PBS containing phloretin (30 pglrnl). The cultures were placed on ice, the media aspirated, and the cells washed four times with 5 ml ice-cold PBS. The cells were solubilized with 1m10.5 N NaOH for 24 h and then 1ml 0.5 N HC1 added to neutralize samples. Values for the uptake of 2 deoxyglucosein the presence of phloretin (60 pg/ml) were subtracted from experimental values to correct for diffusion. Timed pregnant rats were obtained from Zivic-Miller, Pittsburgh, PA. Tissue culture flasks were from Falcon Labware, Div. Becton, Dickinson & Co., Oxnard, CA; wells were from Costar, Data Packagmg, Cambridge, MA; and media was from Irvine, Santa Anna, CA. Mouse EGF and NGF, bovine basic FGF, and human platelet-derived growth factor (PDGF) were obtained from Collaborative Research, Boston, MA. The radioactive isotopes were from New England Nuclear. The anti-GFAP was from Dako Co., Santa Barbara, CA; and the IgG-linked TRITC was from Sigma, St. Louis, MO. Photographic materials were from Kodak and histologic materials were from Lerner labs. All other reagents were obtained from Sigma unless otherwise specified. In all assays, duplicate aliquots were assayed for protein and liquid scintillation counting of radioactivity. The data have been expressed in c p d p g protein after correcting for cross-channel counts. Scintillation counts

40

140

4-

0 L

Q.

lo a,

0 '0 E

II

SFM

EGF

FGF

Treatments Fig. 1. Stimulation of 3H-thymidine incorporation (dark bars and left abscissa) and plating efficiency (shaded bars and right abscissa) over control serum free media (SFM) levels by EGF and FGF treatments. Concentrations were 2 ngiml for EGF and 50 ngiml for FGF, and

the treatment time was 24 h. Each bar represents the means of three cultures and the crossed lines at the top of the bars represent 2 standard errors of the mean.

196

HUFF AND SCHREIER

Fig. 2. Combined GFAP irnmunohistochemistry "H-thymidine autoradiography of astrocytes-untreated control cultures (A,C,E) and EGF-treated cultures (B,D,F). A and B are low-power fluorescent rhodamine anti-GFAP-stained micrographs, and C and D are lowpower white-light-illuminated 'H-thymidine autoradiograms of the same corresponding microscopic fields. E and F are high-power fluo-

rescent imrnunohistochemical-stainedmicrographs illuminated with both UV and white light to show autoradiographic grains and rhodamine anti-GFAF' staining (the diffuse filamentous fluorescence throughout the cytoplasm) of the same cell. Bars represent 125 prn for low power and 50 Frn for high powe-.

197

FGF INHIBITS EGF STIMULATION OF ASTROCYTES

were found to be linear over a wide range of concentra- 25 ? 4%. Fetal calf serum was also a greater stimulator tions and channel ratios. Protein assays were done of 3H-thymidine incorporation than the growth factors using Biorad kits, individually, producing a rate of synthesis of 240 t 35 cpmlp.g protein. Figure 2 shows in a combined GFAP immunohistochemical staining and 3H-thymidine autoradiogram RESULTS that the same majority morphologic cell type which Astrocytes in tissue culture respond to both EGF and displays the astrocyte-specific marker is the cell type FGF with an increase in DNA synthesis as measured by that is increasing its DNA synthesis. Low-power autoan increase in the rate of 3H-thymidine incorporation. radiographic micrographs of the cultures suggest that Both growth factors produce an increase which maxi- the EGF-treated cultures have more astrocytes taking mizes at about 18-24 h of treatment. Figure 1shows a up the 3H-thymidine than the control treated cultures comparison of the proliferation response measured on do. The higher-power autoradiographic micrographs the left scale with a response in plating efficiency combined with anti-GFAE' rhodamine fluorescence of measured on the right scale. EGF markedly stimulates the same fields indicates that the GFAP-containing DNA synthesis but has no effect on plating efficiency cells are the stimulated cells in the cultures. when compared t o the control effect of serum-free media Figure 3 illustrates several astrocyte responses to (SFM).FGF has a lesser stimulation of DNA synthesis growth factors. In contrast to Figure 1, the values than EGF but has a relatively greater stimulation of plotted have already had the corresponding serum-free plating efficiency than EGF. The astrocytes cultured media control values subtracted. On different scales, seemed to adhere and begin forming colonies which upper for EGF and lower for FGF, dose responses show eventually become confluent best in the presence of stimulation of 3H-thymidine incorporation measured on fetal calf serum, which achieved an efficiency of the left scale and 3H-2 deoxyglucose uptake measured

EGF Log Concentration (ng/ml)

h

0.4 100 1 -

1 7 - T -

-

+-'

I

P

I

9

0L

!

O L

a

7

0

I

75

t

Q 0 Y

Q)

50

.-c .-U

E

Lz >r

25

I

r "

0 4

10

100

FGF Log Concentration (ng/ml) Fig. 3. Dose-response curves of 3H-thymidine incorporation (circles, measured on the left abscissa) and 3H-2deoxyglucose uptake (triangles, measured on the right abscissa) after 24 h treatment by EGF (solid circles or trian les, doses indicated on the upper log scale) and FGF (open circles andgtriangles, doses indicated on the lower log scale). Each point represents the mean of three cultures with the

appropriate serum-free media control subtracted, and the bars indicate 2 standard errors of the means. The lines are extended beyond the graphed experimental points on the left ends of the curves toward the experimental values for the untreated controls. Zero points cannot be accommodated on the log scale. Thymidine incorporation: -, EGF; O---O, FGF; 2 deoxyglucose, A. . . . .A,EGF; a- -A , FGF.

-

198

HUFF AND SCHREIER

on the right scale. EGF stimulates DNA synthesis in a dose-dependent manner with a maximum effect at 2 ng/ml or about 0.3 nM. FGF also stimulates :3H-thymidine incorporation in astrocytes in a dose-dependent manner, but to a considerably lower level. Accompanying this stimulation of DNA synthesis is a stimulation of 2 deoxyglucose uptake for both EGF and FGF treatments. Again the EGF-induced response is higher than the response to FGF. In order to study the sequential interaction of factors influencing the proliferation of astrocytes, a pretreatment protocol was set up. Because the dose-response curve to FGF was observed to be somewhat biphasic (Fig. 3), we elected to use submaximal doses of FGF for

pretreatment experiments which were still significantly inhibitory. Table 1illustrates an 84% reduction (relative to the corresponding control baselines) in the ability of EGF at two different concentrations to stimulate astrocyte thymidine uptake when they were first pretreated with FGF. In contrast, when there was no growth factor pretreatment arid the two growth factors were given as a concomitant treatment to the astrocytes, the response in DNA synthesis rate stimulation was not inhibited. The inhibitory effect of FGF was also FGF dose dependent. Pigure 4 illustrates that the FGF-pretreatment inhibitory effect was present across the EGF-stimulation dose-response curve. In addition, the FGF-inhibitory

TABLE 1 . Effect of FGF on EGF-stimulated thymidine incorporation by cultured rat astrocytesa

Pretreatment (18 h) Serum Free Media SFM SFM SFM SFM + 80 ng/ml FGF SFM 80 ng/ml FGF SFM t 80 ng/ml FGF

+

SFM SFM + 2 ng/ml EGF SFM f 8 ng/ml EGF SFM + 2 ng EGF 80 ng FGF/ml SFM SFM + 2 ng/ml EGF SFM + 8 ng/ml EGF

+

35

* 12 *

125

T

0

n 6

90 k 23 94 f 11 105 12 27 ?z 7.3 36 k 3.6 48 f 15 and the pretreatment inhibition was significant at P < .(12

+

"EGF treatment effect was significant at P < .01,

h

Thymidine incorpora tion (fmol/pg protein/2 h)

Treatment (24 h)

__

3 3

3 3 3 3

T

/ I

I

a IJ)

100

3 \

E 0

I

,/

75

I

I 0.4

1 EGF Log Concentration (ng/ml)

Fig. 4. FGF pretreatment inhibition of EGF 24 h stimulation of thymidine uptake at high and low culture densities expressed as dose-response curves (control SFM-pretreated cultures, solid circles or triangles; FGF-pretreated cultures, open circles and triangles). Concentration and time of FGF pretreatment was 50 ng/ml and 18 hr, respectively. High-density cultures (solid or open circles) used stan-

dard plating densities of 2 x lo6 celldml, whereas low density cultures (solid or open triangles) used 1/10 the standard plating density. Each point represents the mean of three cultures and bars represent 2 standard errors ofthe mean. High density: -, control; o---o, FGF - A , FGF prepretreatment. Low density: A . ' . . A , control; A treatment.

-

199

FGF INHIBITS EGF STIMULATION OF ASTROCYTES

effect was present in both high- and low-cell-density cultures, even though the EGF-induced stimulation was greater at the higher culture density. The maximum stimulation occurred at the same EGF concentration of 2 ng/ml whether or not cultures were exposed to FGF or high-density conditions. The temporal extent of the FGF inhibitory effect was investigated. Inhibition was maximally present a t 18 h of pretreatment, but there was greater than 50% inhibition still present after 72 h of pretreatment. As was shown in Figures 1 and 3, FGF stimulates thymidine incorporation to a small degree, as measured by a pulse at 24 h, which must occur during the pretreatment period also. However, the greater EGF stimulation ability is truncated during the pretreatment. A comparison of growth factor pretreatment effects on the EGF stimulation of 3H-thymidineuptake was made among three growth factors to which astrocytes are known to have some response, EGF, FGF, and PDGF. The results are seen in Figure 5 (solid bars). PDGF pretreatment consistently had no inhibitory effect on the EGF stimulation, and EGF pretreatment had a greater inhibitory effect than FGF pretreatment. Nerve growth factor (NGF) had no effect on either astrocyte thymidine incorporation or pretreatment inhibition of

-125

EGF stimulation of thymidine incorporation (data not shown).A comparison was also made between the inhibitory pretreatment effects of the growth factors on the DNA synthesis stimulation produced by EGF (left scale) and the inhibitory pretreatment effects of the growth factors on the stimulation produced by fetal calf serum (FCS)(right scale). FCS stimulation of DNA synthesis is also inhibited by pretreatment with FGF and EGF but not PDGF as seen in Figure 5 (hatched bars), but the relative inhibition by EGF and FGF pretreatments of FCS stimulation was not as great as with the inhibition of EGF stimulation of DNA synthesis. The inhibitory effect of FGF on DNA synthesis was also reflected in reductions in EGF-stimulated cell counts as seen in Figure 6. Regardless of pretreatment, the cultures treated with EGF began to show increases in cell number between 3 and 8 days after treatment; however, when the treated groups of cultures were pretreated with FGF there were fewer cells at 8 days than in those not pretreated. In addition to inhibition of the EGF stimulation of DNA synthesis, the FGF pretreatment also inhibited the EGF stimulation of 2 deoxyglucose uptake. Figure 7 illustrates stimulation of 2 deoxyglucose uptake by EGF and FGF treatments individually at 3 and 18 h; EGF

'

8

-

1252

v

C

.-0 -a1 0 0 -

E

.-

fj

75

l

i

E

$

1

I

E

I

I

50

+~ I

I

I

251 LL

c!J

w

I

o

-25

cn 0

I

1

l

1 --

SFM

FGF

EGF

(3

0

LI

PDGF

Pretreatments Fig. 5. Comparison of pretreatment effects on EGF stimulation versus fetal calf serum (FCS) stimulation of thymidine uptake. The solid bars represent thymidine uptake values for EGF-treated cultures and the hatched bars represent values for FCS-treated cultures. The EGF treatment concentration was 2 ng/ml, and the FCS treatment concentration was 10% and treatment time for both was 24 h. The pretreatment protocol was the same as in Figure 4, with the other pretreatment concentrations: EGF 10 ng/ml, and PDGF 5 unitdm1

with 1unit defined by the manufacturer as a concentration adequate to produce half-maximal stimulation in the standard bioassay (Raines and Ross, 1982). The control SFM pretreatment values for both EGF and FCS stimulations were set at 100% stimulation, and the other pretreatments were normalized to these respective standards to compare the relative degree of inhibition. Each bar represents the mean of three cultures, and the crossed lines at the top of each bar represent 2 standard errors of the mean.

200

HUFF AND SCHREIER

A

0

1

2

3

4

5

6

’7

8

Time of Treatment (days) Fig. 6. FGF pretreatment inhibition of EGF-induced stimulation of astrocyte cell division. Pretreatment rotocol was as in Figure 4,and the EGF concentration was 2 ngiml. 8ells were counted following the 18h pretreatment period immediately beginning as time 0 and then at 3 days and 8 days of EGF treatment. Solid or open circles represent values for cultures first receiving no pretreatment, and solid or open triangles represent values for cultures first receiving FGF pretreatment. Solid circles or triangles represent cultures which then received

EGF treatment, and open circles or t r tangles represent cultures which then received control serum free mcdia. Each point represents the mean of counts of cells by phase contrast microscopy of six randomly selected low power ( 1 0 0 )~fields (lpf) in each of three cultures and the bars represent 2 standard errors ofthe mean. M, control pretreated, EGF treated; o---o, control pretreated, control treated; A. ’ . .A, FGF pretreated, EGF treated; A -A , FGF pretreated, control treated.

produced a greater uptake stimulation, which was sustained at 48 h. However, when the astrocytes were pretreated with FGF before being treated with EGF the resulting rate of 2 deoxyglucose uptake was less than either individually, indicating again an FGF pretreatment inhibition of the EGF stimulation. This pretreatment inhibition was also seen across the EGF doseresponse curve as seen in Figure 8. The dose of EGF for maximum 2 deoxyglucose uptake stimulation was comparable to that for maximum DNA synthesis stimulation.

The amount of stimulation of DNA synthesis produced, about fivefold, and the dose of EGF stimulating a maximal increase of thymidine incorporation into astrocyte DNA after 24 h of treatment are comparable to those observed by others (Guentert-Lauber and Honneger, 1985; Leutz and Schachner, 1981; Simpson et al., 1982). The proliferation response produced by EGF treatment was accompanied by a stimulation of 2 deoxyglucose uptake with approximately the same dose-response curve as with DNA synthesis stimulation. The resting rate of deoxyglucose uptake in astrocytes is relatively high and is influenced by potassium concentration, sodium pump activity, and culture age (Brookes and Yarowsky, 1985), all of which remained constant in our experiments. EGF induces increases in the transport of hexose in fibroblasts (Allard et al., 1987)and PC-12 cells (Huff et al., 1981) at comparable concentrations to our present studies. Increases in hexose uptake thus can be added to other trophic responses to EGF of astrocytes in culture (Avola et al., 19881. Hexose transport is increased in proliferating cells and greatly increased in transformed cells (Hale and Weber, 19751,and stimulation of glucose uptake by gowth factors may be a necessary response for cell DNA synthesis in some systems (Hamilton et al., 1985). In vivo, 2 deoxyglucose

DISCUSSION Epidermal growth factor is a potent mitogen for a number of cell types (Gospodarowicz and Moran, 19761, and elicits a number of biochemical responses, all of which are not found in all target cells (Covelli et al., 1972; Ho‘Jenberg and Cuatrecasas, 1975; Huff and Guroff, 1978). Very few neural type cells are known to respond to epidermal growth factor (Herschman et al., 1983). Our data confirm that EGF has a definite mitogenic influence in astrocytes and this effect is separate from effects on plating efficiency of the cells in culture.

--

201

FGF INHIBITS EGF STIMULATION OF ASTROCYTES

EGF3FGF3 E G F I 8 F G F 1 8

EGF48

F G F I 8>EGF48

Treatment Time (hours) Fig. 7. EGF and FGF effects on 2 deoxyglucose uptake. Solid bars represent values for cultures treated with EGF alone for the indicated time periods. The two leftmost hatched bars represent values for cultures treated with FGF alone for the indicated time periods. The far right hatched bar represents values for cultures receiving FGF as an

18 h pretreatment then 48 h EGF treatment. Experimental values have had control SFM-treated culture values subtracted a t each respective time point. Each bar represents the mean ofthree cultures and the crossed lines at the top of each bar represent 2 standard errors of the mean.

i i 0

'/ /' i

0.4

//'

1 EGF L o g Concentration (ng/ml)

Fig. 8. FGF pretreatment inhibition of EGF stimulation of 2 deoxyglucose uptake across the EGF dose-response curve. The pretreatment and treatment protocols were as in Figure 4. Each point represents the mean of three cultures and the bars represent 2 standard errors of the mean. H, control; 0- - - 0, FGF pretreated.

202

HUFF AND SCHREIER

uptake is correlated with gliosis following cortical ablations (Cooper and Thurlow, 1984). Although fibroblast growth factor only mildly stimulated astrocyte DNA synthesis, pretreating the cells with FGF greatly reduced the ability of the cells to respond to stimulation by EGF. Table 1 presents data that indicate a lowering of EGF effects on DNA synthesis by pretreatment of the cells with FGF. This result in our astrocyte culture system, another growth factor modulating the effects of EGF, corroborates results obtained before in the PC-12 system (Huff et al., 1981). In the present case the modulator is FGF and in the previous case the modulator was NGF. FGF pretreatment inhibition was unaffected by cell density effects. Previously, differences have been noted in cell responses in sparse and confluent cultures. Westermark (1976) found less EGF stimulation at higher density in the human glial cell line. Our different results may reflect different types of astrocytes or different culture conditions. The greater EGF stimulation in our cultures at high culture density possibly indicates a relative starvation effect for the EGF at the higher cell density or a synergistic effect from another unknown factor produced in greater quantities at higher densities. Regardless, FGF pretreatment uniformly inhibited the EGF induced stimulation at each dose in both high and low culture density situations. Specificity of both the inhibitory pretreatment and the stimulus being inhibited was demonstrated. As expected, homologous pretreatment with EGF was more effective in reducing or down regulating the EGFinduced DNA synthesis stimulation than the heterologous FGF pretreatment. Although PDGF can stimulate astrocyte proliferation just as FGF can (Besnard, 19871, it did not inhibit EGF-induced stimulation of proliferation as FGF did, however. PDGF does not work through the same mechanism of signal transduction. It does not require tyrosine kinase activity of its receptor for downregulation or cell proliferation (Williams, 1989), whereas EGF does in both cases (Chen et al., 1987; Honneger et al., 1987). In addition, we found relatively much greater inhibition of EGF stimulation by the pretreatments than inhibition of fetal calf serum (FCS) stimulation. The source of mitogenic activity in fetal calf serum, whatever it may be, is thus perhaps not regulated in the same way or to the same degree as the EGF mitogenic activity. A regulation of a smaller portion of that mitogenic activity in fetal calf serum such as that due to EGF-like growth factors may occur as equal EGF and FGF inhibitions. The FGF-pretreatment inhibitory effect was seen not only in DNA synthesis stimulation but also in cell number increases after EGF treatment and in 2 deoxyglucose uptake stimulation. The FGF inhibitory effect on cell numbers after treatment with EGF implicates the complete cell-division cycle. The lag period before the observed EGF-induced stimulation was perhaps due to the quiescent serum-free state of the cultures for 48 hours prior to treatment, noted in other systems (Cosenza et al., 1988). Because direct FGF treatment

stimulates 2 deoxyglucose uptake, its pretreatment inhibition of EGF-induced stimulation likely occurs through the EGF signal transduction pathway directly and not through an involvement of protein kinase C known to inhibit uptake (Pearce et al., 1988). We have shown that EGF, which is found in the brain, is a proliferation signal, and that FGF, a brain product, can turn off the EGF stimulation if the astrocytes are pre-exposed to it. Growth factors and hormones may interact at several levels and control each other’s effects. Possibly FGF pretreatment reduces the response of astrocytes to EGF through some effect on the EGF receptor such as heterologous down regulation. NGF pretreatment of PC-12 cells terminating cell division and producing neuronal differentiation is associated with reduction in the number of EGF receptors and intracellular metabolic responses t o EGF including proliferation (Huff et al., 1981). Alternatively, other changes in the receptor might be induced. PDGF is known to modulate EGF binding through a protein kinase C mechanism changing receptor affinity rather than receptor number (Pimer tel, 1987). Transforming growth factor beta (TGF beta) is known t o inhibit EGFinduced proliferation in cultured astrocytes and is also transduced through protein kinase C translocation (Robertson et al., 19881, but stimulates c-myc protooncogene expression (Fernanriez-Pol et al., 1987). Insulin, insulin-like growth factoe-1, dexamethasone, and thyroid-stimulating hormone affect the EGF receptor kinase or otherwise qualitatively change the receptor (Baker et al., 1978; Corps arid Brown, 1988; Westermarket al., 1985). Astrocyte growth factor mechanisms could theoretically have a part in vivo during development, neoplastic transformation, and in neural regeneration. EGF-like DNA sequences have critical rnorphogenic gene regulatory functions in the embryonic nervous system (Lewin, 1987).FGF has an early presence in the nervous system (Berry et al., 1983) and plays a role in embryonic differentiation (Liu and Nicoll, 1988).Perhaps an additional diffentiation aspect of FGF function might be the reduction of astrocyte EGF mitogenic influences during development. Neoplastic trarsformation of astrocytes could potentially involve EGF and FGF in several ways. Secretion of homologs, overproduction of receptor, and interaction with transforming factors or oncogene products are all possibilities known for EGF (Epenetos et al., 1985; Kamata and Feramisco, 1984; Massague et al., 1984; Stromberg et al., 1987). FGF also may be a member of an oncogene product family (Zhan et al., 1988) and could be a means of glial autocrine regulation of proliferation (Ferrara et al., 1988). In regeneration increasing astrocyte numbers could increase the framework for regenerating axons. On the other hand, FGF interacting with EGF may limit an overproliferation and glial scar formation which could lead to distortion of synaptogenesis and epileptic foci. Perhaps signals such as FGF acting in this manner also prevent the gliosis reaction from evolving into a glioma. Clearly more in vivo studies need to be approached.

203

FGF INHIBITS EGF STIMULATION OF ASTROCYTES

ACKNOWLEDGMENTS

Friede, R.L. (1975) Porencephaly, hydranencephaly, multilocular cystic encephalopathy. In: Developmental Neuropathology. Springer Verlag, New York, pp. 102-122. The first author was supported by an NIH Teacher Giulian, D. and Lachman, L. (1985) Interleukin-1 stimulation of astroglial proliferation after brain injury. Science, 228:497-499. Investigator Development Award grant NS00982, and D. and Young, D. (1986) Brain peptides and glial growth. 11. the work was partially supported by a Childrens Hospi- Giulian, Identification of cells that secrete glia-promoting factors. J . Cell tal Biomedical Research Support Grant. We thank Biol., 102:812-820. L. Ibric for technical assistance with some of the plating Gospodarowicz, D. and Moran, J.S. (1976) Growth factors in mammalian cell culture. Annu. Reu. Biochem., 45531-558. efficiency and autoradiographic experiments. Gospodarowicz, D., Rudland, P., and Lindstrom, J. (1975) Fibroblast growth factor: Its localization, purification, mode of action, and physiologxal significance.Adv. Metub. Disord., 8:330-335. Goustin, AS., Leof, E.B., Shipley, G.D., and Moses, L. (1986) Growth factors and cancer. Cancer Res., 46:1015-1029. Guentert-Lauber, B. and Honegger, P. (1985) Responsiveness of astroREFERENCES cytes in serum-free aggregate cultures to epidermal growth factor: Dependence on the cell cycle and the epidermal growth factor Allard, W.J., Gibbs, E., Witters, L., andlienhard, G. (1987)Theglucose concentration. Deu. Neurosci., 7(5-6):286-95. transporter in human fibroblasts is phosphorylated in response to Hale, A.H. and Weber, M.J. (1975) Hydrolase and serum treatment of phorbol ester but not in response to growth factors. Biochim. Bionormal chick embryo cells: Effects of hexose transport. Cell, phys. Acta, 929:28&295. 5:245-252. Avola, R., Condorelli, D.F., Tur eenoja L , Ingrao, F., Reale, S., Hamilton, J.A., Vairo, G., and Lingelbach, S.R. (19881Activation and Ragusa, N., and Giuffrida Stelya, A.M: (1988) Effect of epidermal proliferation signals in murine macrophages: stimulation of glucose growth factor on the labeling of the various RNA species and of uptake by hemopoietic growth factors and other agents. J . Cell nuclear proteins in primary rat astroglial cell cultures. J . Neurosci. Physiol., 134(3):405-12. Res., 20:54-63. Hatten, M. (1987) Neuronal inhibition of astroglial cell proliferation is Baker, J.B., Barsh, G.S., Carney, D.H. (1978) Dexamethasone modumembrane mediated. J . Cell Biol., 104:1353-1360. lates binding and action of epidermal growth factor in serum free cell Herschman, H.R., Goodman, R., and Chandler, C. (1983) Is epidermal culture. Proc. Natl. Acud. Sci. U.S.A., 75:1882-1886. growth factor a modulator of nervous system function? Birth Defects, Berry, M., Maxwell, W., Logan, A. (1983) Deposition of scar tissue in 19:79-94. the central nervous system. Acta Neurochir. [Suppl.], 32:l-53. Hirata,Y., Uchihashi, M., and Nakajima, H. (1982) Presence ofhuman Besnard, F. (1987) Platelet derived growth factor is a mitogen for glial epidermal growth factor in human cerebrospinal fluid. J . Clin. but not neuronal rat brain cells in vitro. Neurosci. Lett., 73:287-292 Endocrinol. Metab., 55:1174-1 177. Bowen-Pope, D.F., Dicorleto, P.E., and Ross, R. (1983) J . Cell Biol. Hollenberg, M.D. and Cuatrecasas, P. (1975) Insulin and epidermal 96:679-683 growth factor: Human fibroblast receptors related to deoxyribonuBrookes, N. and Yarowsky, P.J. (1985) Determinants of deoxyglucose cleic acid synthesis and amino acid uptake. J . Biol. Chem., ?take in cultured astrocytes: the role of the sodium pump. J. 25013845-3853. eurochem., 44(2):473-9. Honegger, A.M., Dull, T.J., Felder, S.,Van Obberghen, E., Bellot, F., Chen, W.S., Lazar, C.S., Poenie, M., Tsien, R.Y., Gill, G.N., and Szapary, D., Schmidt, A., Ullrich, A,, and Schlessinger, J. (1987) Rosenfeld, M.G. (1987) Requirement for intrinsic protein tyrosine Point mutation at the ATP binding site of EGF receptor abolishes kinase in the immediate and late actions kof the EGF receptor. protein-tyrosine kinase activity and alters cellular routing. Cell, Nature, 328:820-823. 511199-209. Cohen, S . and Carpenter, G. (1975) Human epidermal growth factor: Huff, K. and Guroff,G. (1978) Epidermal growth factor stimulates the Isolation and chemical and biological orooerties. Proc. Nut. Acad. phosphorylation of a specific nuclear protein in chick embryo epider" I . Sci., U S A . , 72:1317-1321. mis. Biochem. Biophys. Res. Commun., 85:464-472. Cooper, R. and Thurlow, G. (1984) 2-deoxy lucose uptake in the Huff, K., End, D., and Guroff, G. (1981) Nerve growth factor-induced thalamus of awake rats after neocortical ahations. Exp. Neurol., alteration in the response of PC12 phenochromo-cytoma cells to 86~261-271. epidermal growth factor. J . Cell Biol., 88:189-198. Cosenza, S.C., Owen, T., Soprano, D., and Soprano, K. (1988)Evidence Janeczko, K. (1988)The proliferative response of astrocytes to injury in that the time of entry into S is determined by events occurring in neonatal rat brain. A combined immunocytochemical and autoradioearly G1. J . Biol.Chem., 263(25):12751-8. graphic study. Brain Res., 456:280-285. Corps, A.N. and Brown, K.D. (1988) Insulin-like growth factor 1 and Kamata, T. and Feramisco. J.R. (1984) Epidermal growth factor stiminsulin reduce epidermal growth factor binding to Swiss 3T3 cells by ulates guanine nucleotide binding activity and phospkorylation of an indirect mechanism that is apparently independent of protein ras oncogene proteins. Nature, 310:147-150. kinase C. FEBS Lett., 233:303-306. Leutz. A. and Schachner. M. (1981) Eoidermal mowth factor stimuCovelli, I., Mozzi, R., Rossi, R., and Frati, L. (1972)Mechanism of action lates DNA-synthesis of astrocytes ih primarycerebellar cultures. of epidermal growth factor. 111. Stimulation of uptake of labelled Cell Tissue Res., 220:393-404. precursors into DNA, RNA, and proteins induced by EGF in isolated Lewin, B. (1987) Genes III. John Wiley and Sons, New York, pp, G R 1 - 7.--. 15 tumor cells. Hormones (Basel), 3:183-191. Cuevas, P., Burgos J., and Baird, A. (1988) Basic fibroblast growth Libermann, T.A., Nusbaum, H., Razon, N., Kris, R., Lax, I., Soreq, H., factor (FGF)promote cartilage repair in vivo. Biochem. Biophys. Res. Whittle, N., Waterfield, M., Ullrich, A,, and Schlessinger, J. (1985) Commun., 156:611-618. Amplification, enhanced expression and possible rearrangement of Epenetos, A.A., Courtenay-Luck,N., Pickering, D., Hooker, G., Durbin, EGF receptor gene in primary human brain tumours of glial origin. H., Lavender, J., and McKenzie, C.G. (1985)Antibody guided irradiNature, 313:144-147. ation of brain glioma by arterial infusion of radioactive monoclonal Liu, L. and Nicoll, C.S. (1988) Evidence for a role of basic fibroblast antibody against e idermal growth factor receptor and blood group A growth factor in rat embryonic growth and differentiation. Endocriantigen. Brit. MedJJ., 290:1463-1466. nology, 123:2027-2031. Fallon, J.H. and Seroogy, K.B. (1984) Epidermal growth factor immu- Logan, A. (1988) Elevation of acidic fibroblast growth factor mRNA in noreactive material in the central nervous system: Location and lesioned rat brain. Mol. Cell. Endocrinol., 58:275-278. development. Science, 224: 1107-1109. Luger, A,, Calogero, A,, Kalogeras, K., Gallucci, W., Gold, P., Loriaux, Fernandez-Pol, J.A., Talkad, V.D., Klos, D.J., and Hamilton, P.D. D., and Chorousos, G.P. (1966) Interaction of epidermal growth (1987)Suppression ofthe EGF-dependent induction ofC-MYCprotofactor with the hypothalamic-pituitary- adrenal axis: potential physoncogene expression by transforming growth factor B in a human iologic relevance. J . Clitz. Endocrinol. Metab., 66(2):334-337. breast carcinoma cell line. Biochem. Biophys. Res. Commun., Massague, J.,Kelly, B., and Mottola, C. 11985)Stimulation by Insulin144:1197-1205. like growth factors is required for cellular transformation by type B Ferrara, N., Ousley, F., and Gospodarowicz, D. (1988) Bovine brain transforming growth factor. J . Biol. Chem., 8:4551-4554. astrocytes express basic fibroblast growth factor, a neurotropic and McCarthy and De Vellis, J . (1980) Preparation of separate astroglial angiogenic mitogen. Bruin Res., 462:223-232. and oligodendroglial cell cultures from rat cerebral tissue. J . Cell Finkelstein, S.P., Apostolides, P.J., Caday, C.G., Prosser, J . , Philips, BWl., 85~890-902. M.F., and Klagsbrun, M. (1988) Increased basic fibroblast growth McKanna, J.A. and Cohen, S.(1989) The EGF receptor kinase subfact,or (bFGF) immunoreactivity a t the site of focal brain wounds. strate p35 in the floor plate of the Embryonic rat CNS. Science, Brain Res. 460:253-259. 24311477-1479. ~

V Y _

~~~~

~~~~~~

204

HUFF AND SCHREIER

Nieto-Sampedro, M., Gomez-Pinilla, F., Knauer, D.J., and Broderick, J.T. (1988) Epidermal growth factor receptor immunoreactivity in rat brain astrocytes. Response to injury. Neurosci. Lett., 91:276-282. Nieto-Sampedro,M., Saneto, R., De Vellis, J.,and Cotman, C.W. (1985) The control of glial populations in brain: changes in astrocyte mitogenic and morphogenic factors in response to injury. Brain Res., 343(2):320-328. O'Keefe, E.J. and Pledger, W.J. (1983) A model of cell cycle control: Sequential events regulated by growth factors. Mol. Cell. Endocrinol., 31:167-186. Pearce, B., Morrow, C., and Murphy, S. (1988)Arole for protein kinase C in astrocyte glycogen metabolism. Neurosci. Lett., 90(1-2):191-6. Perraud, F., Labourdette, G., Miehe, M., Loret, C., and Sensenbrenner, M. (1988)Comparison of the morphological effects of acidic and basic fibroblast growth factors on rat astroblasts in culture. J . Neurosci. Res., 2O:l-11. Pettmann, B., Weibel, M., Sensenbrenner, M., and Labourdette, G. (1985) Purification of two astroglial growth factors from bovine brain. FEBS Lett., 189:102-108. Pimentel, E. (1987) Hormones, Growth Factors, and Oncogenes. CRC Press, Boca Raton, Florida, pp. 111-140 and 219-225. Raines, E.W. andRoss, R. (1982)Plateletderivedgrowth factor. I. High yield purification and evidence for multiple forms. J . Bid. Chem., 257:5154-5160. Robertson, P.L., Markovac, J.,Datta, S.C., and Goldstein, G.W. (1988) Transforming Prowth factor beta stimulates Dhosuhoinositol metabolism and tricslocation of protein kinase C Aincdtured astrocytes. Neurosci. Lett., 93(1):107-113.

Rogister, B., Leprince, P., Petterman 1, B., Labourdette, G., Sensenbrenner, M., and Moonen, G. (1988)Brain basic fibroblast growth factor stimulates the release of plasminogen activators by newborn rat cultured astoglial cells. Neurosci. Lett., 91:321-326. Simpson, D.L., Morrison, R., DeVellis, J., andHerschman, H.R. (1982) Epidermal growth factor binding and mitogenic activity on purified populations of cells from the central nervous system. J. Neurosci. Re& 8:453462. Stromber , K , Hudgins, W., Dorman L., Henderson, L., Sowder, R., Sherrelf B.: Mount. C.. and Orth. D.N. (1987) Human brain tumorassociated urinarv high molecular weight transforming mowth factor: A high mole"cul& weight form ouf epidermal gr&&h factor'. Cancer Res., 47: 1190-1 196. Varon, S. and Saier, M. (1975) Culture techniques and glial-neuronal interrelationships in vitro. Exp. Neurol., 4"): Part 2:135-162. Westermark, B. (1976) Density dependent proliferation of human glia cells stimulated by epidermal growt.7 factor. Biochem. Biophys. Res. Commun., 69(2):304-310. Westermark, K., Karlsson, A,, and Wefkermark, B. (1985)Thyrotropin modulates EGF receptor function in porcine thyroid follicle cells. Mol. Cell Endocrinol., 40:17-23. Williams, L.T. (1989) Si a1 transduction by the platelet-derived growth factor rece tor %ience, 243:1564-1570. Zhan, X., Bates, B., Au, X., and Goldfapb, M. (1988) The human FGFd oncogene encodes a novel protein related to fibroblast growth factors. Mol Cell. Biol., 8:3487-3495.