Journal of Neurochernistry Raven Press, Ltd., New York
0 I990 International Society for Neurochemistry
Effects of Transforming Growth Factor pl on Astroglial Cells in Culture D. Toru-Delbauffe, D. Baghdassarian-Chalaye, J. M. Gavaret, F. Courtin, M. Pomerance, and M. Pierre INSERM U. 96, H6pital Kremlin-Bi&tre, Le Kremlin-Bicetre, France
Abstract: The effects of transforming growth factor P l (TGFPI) on DNA synthesis and functional differentiation of astroglial cells cultured in serum-free medium were investigated. TGFP I diminished and delayed the peak of DNA synthesis induced by serum. TGFP I -treated cells were larger than control cells. This factor delayed the appearance of processbearing cells induced by acidic fibroblast growth factor treatment and also affected the astrocyte-specific enzyme glutamine synthetase (GS), whose accumulation is under hydrocortisone (HC) control. TGFPl inhibited the induction of
G S activity by HC in a dose- and time-dependent manner. Moreover, pretreatment with TGFPl for 4 h maintained the inhibition of G S activity for 16 h after removal of this factor from culture medium. These results suggest that TGFPl may be an important regulator of astrocyte growth and differentiation. Key Words: Astroglial cells-Transforming growth factor 81-DNA synthesis-Glutamine synthetase activity. Toru-Delbauffe D. et al. Effects of transforming growth factor PI on astroglial cells in culture. J. Neurochem. 54, 1056-106 1 (1990).
Transforming growth factor P (TGFP) belongs to a new family of polypeptide factors that are potentially of great interest in the regulation of cell functions (Sporn et al., 1986, 1987; Massaguk, 1987), notably, of the production and organization of extracellular matrices (Rizzino, 1988). There are four forms of TGFP. Although several recent findings suggest that TGFPl and TGFP2, the most studied forms, may differ in their functions in vivo, they have many similar effects in vitro (Sporn et al., 1987). TGFPl was used in the present study. It is now evident that most, if not all, cells produce TGFP, even those of adult tissues, such as brain, heart, and kidney, where there is little mitotic activity (Roberts et al., 1981). Moreover, TGFP receptors have been found in all the cells examined to date (Wakefield et al., 1987). In spite of an exponential increase in research on TGFP, little is known about its effects on CNS cells. In the present study, we have examined the influence of TGFP 1 on the growth and differentiation of one of the major cell types of the brain, the astrocytes, which appear to play a crucial role in brain development and
cell repair (Bhat and Pfeiffer, 1986; Manthorpe et al., 1986). Moreover, these cells probably produce the greatest part of the extracellular matrix components in the CNS (De Vellis et al., 1986). Some of these components are controlled by TGFP in other types of cells (Rizzino, 1988). This work was carried out on primary glial cells enriched in astrocytes and cultured under serum-free culture conditions wherc TGFP actions could be readily determined. The following cell parameters were monitored: DNA synthesis, morphological appearance, and glutamine synthetase (GS) activity. The accumulation of this enzyme is under glucocorticoid control and associated with astrocyte differentiation (De Vellis et al., 1986). TGFP was found to modulate negatively DNA synthesis and differentiation of astrocytes in vitro.
Received May 3, 1989; revised manuscript received July 3, 1989; accepted August 15, 1989. Address correspondence and reprint requests to Dr, D. Tom-Delbauffe at INSERM U. 96, 78, rue du General Leclerc, 94275 Le Kremlin-Bic@treCedex, France.
Abbrevin~ionsused; aFGF, acidic fibroblast growth factor; CDM, chemically defined medium; DMEM, Dulbecco's modified Eagle's medium; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; HC, hydrocortisone; TGFP, transforming growth factor p.
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MATERIALS AND METHODS Sprague-Dawley rats were from Iffacredo (L'Arbresle, France). Culture media were from GIBCO (Grand Island, NY, U.S.A.). Fetal calf serum was purchased from Seromed (Berlin, F.R.G.). Human TGFPI was obtained from R & D
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Systems (Minneapolis, MN, U.S.A.). Hydrocortisone (HC) was from Sigma. Acidic fibroblast growth factor (aFGF), purified by heparin-Sepharose affinity chromatography, was a generous gift from Dr. D. Barritault (University Paris XII, Paris, France). [3H]Thymidineand [~-'~C]glutamic acid were obtained from Amersham-France SA. Anion exchange resin (AG 1-X8, acetate form) was from Bio-Rad, France.
to synthesize DNA after 12-14 h. On the other hand, this addition diminished the rate of [3H]thymidineincorporation (40-50% at 20 h). The maximal labeling was also reduced and was obtained only at 24 h. TGFP alone did not modify the basal level in serum-free medium at least for the first 24 h.
Preparation of astrocytic cells
Morphological effect of TGFP Astrocytes grown to confluence in serum-supplemented medium exhibited a flat, polygonal shape and expressed GFAP, which was identified by anti-GFAP antiserum (data not shown). The flattened cells formed monolayer areas, and only some cells possessed short cytoplasmic processes (Morrison and De Vellis, 1981). These morphological characteristics were maintained when the cells were subsequently switched to CDM (Fig. 2A). The following experiments were performed to examine, in CDM, the effect of TGFP without or with aFGF, a growth factor that is known to produce important changes in astrocyte morphology (Weibel et al., 1985; Delaunoy et al., 1988; Gavaret et al., 1989). After a 16-h TGFP treatment (40 pM), most of the cells presented a more enlarged cytoplasm compared with that of control cells (Fig. 2B). When cells were treated with aFGF (25 ng/ml) alone, the onset of morphological change began after an 8-h period of treatment; some astrocytes presented small cell bodies with many long, branching processes (Fig. 2C). The number of process-bearing cells increased continuously with time, and 24 h later, all the cells had formed long processes, which produced a dense network. In contrast, when cells were first preincubated with TGFP for 16 h and then simultaneously exposed to TGFP and aFGF, no major morphological changes were observed after an 8-h exposure; the astrocytes presented enlarged cytoplasms, similar to those of the TGFP-treated cells (Fig. 2D). The onset of the morphological effect of aFGF was observed after a period of -24 h (data not
Primary cultures of glial cells were obtained from the cerebral hemispheres of 2-day-old rats and grown to confluence (10 days) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%fetal calf serum as described previously (Pierre et al., 1986). Cells were washed three times with chemically defined medium (CDM) and then cultured in this medium. The CDM, made from a 1 :1 mixture of DMEM and Ham's F-12 medium containing 4.5 g/L of glucose and 2.7 g/L of sodium bicarbonate, was renewed every day. After 4-5 days in this medium, cells were ready for use. These cultures contained mainly glial fibrillary acidic protein (GFAP)-positive cells, when tested with rabbit antiserum directed against GFAP (the gift of Dr. B. Pessac, CNRS, Ivry, France).
[3H]Thymidineincorporation Glial cells cultured in DMEM supplemented with 10% fetal calf serum were plated in CDM 2-3 days before confluence. After 3-4 days in this medium, quiescent cells were either maintained in CDM as the control or transferred to CDM supplemented with 10%fetal calf serum with or without 40 pM TGFP. Incubation was continued for up to 48 h. [3H]Thymidine (2 pCi/ml) was added for the last 6 h of incubation. The medium was then removed, and the monolayers were rinsed three times with fresh medium. [3H]Thymidine incorporation into DNA was measured according to the method of May et al. (1988).
G S assay and DNA content determination At the end of cell incubation with TGFP (40-100 pM, as indicated in each experiment), MHC, or a combination of both factors, the culture medium was discarded, and the cells were washed three times in cold physiological serum and stored at -80°C until GS assay. For assay, the cells were scraped off and sonically disrupted at 4°C by two 5-s treatments with an MSE sonic oscillator. GS activity was measured by the method of Pishak and Phillips (1979) and expressed as nanomoles of glutamine produced per 30 min per microgram of DNA. The DNA content was measured by the method of Groyer and Robel ( 1 980) with calf thymus DNA as the standard. Protein content was measured by the method of Lowry et al. (195 1) with bovine serum albumin as the standard.
RESULTS Effects of TGFP on DNA synthesis Figure 1 illustrates the effect of TGFPl on DNA synthesis. When cells maintained in CDM were stimulated by addition of serum (lo%), the rate of [3H]thymidine incorporation remained at a constant basal level during the first 12 h of stimulation. After this lag period, the rate increased to reach a maximal value at 20 h, then dropped until 30 h, and was constant between 30 and 48 h. Adding TGFP with serum did not change the lag period, because the cells began also
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FIG. 1. Effects of TGFP on DNA synthesis by astrocytes. Confluent, quiescent astroglial cells were cultured in CDM supplemented with 10% fetal calf serum without (0)or with (A)40 pM TGFP. The basal level was measured in cells incubated in CDM without (0)or with (A) 40 pM TGFP. [3H]Thymidine(2 pCi/ml) was added 6 h before the indicated times. The radioactivity incorporated into DNA was determined as described in Materials and Methods. The results shown are from one of a group of similar experiments.
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FIG. 2. Effects of TGFP on astrocyte morphology shown by phase-contrastmicroscopy: (A) cells in CDM; (B) cells after exposure for 16 h to TGFB in CDM; (C) cells after exposure for 8 h to aFGF in CDM; and (D) cells after pretreatment with TGFP for 16 h and exposure for 8 h to TGFP plus aFGF in CDM. The TGFB concentration was 40 pM, and that of aFGF was 25 ng/ml in all experiments. Bar 50 pm.
shown), a result thus indicating a delayed effect of aFGF in the presence of TGFP. Effects of TGFP on G S induction by HC TGFP added simultaneously with HC. Figure 3, line a, shows the effect of TGFP on GS activity. Astroglial cells were treated for various periods with HC (lop6 M ) alone or HC plus TGFP (40 pM). HC caused a continuous increase in GS activity, which depended on the duration of exposure to glucocorticoid in TGFPuntreated cells. TGFP addition simultaneously with HC did not change the activity of GS for the first 8 h, but then the activity decreased rapidly to a minimal value at 16 h, which was maintained until 24 h. The basal level was not affected by TGFP treatment. TGFP added after induction of GS by HC: time course. Astrocytes were pretreated for 24 h with HC ( M ) alone; TGFP (40 pM) was then added for the indicated times with HC remaining in the medium (Fig. 3, line b). GS activity was inhibited soon after TGFP addition and became minimal after 16 h of treatment; afterwards, it remained stable until 24 h. The inhibition continued if the culture medium containing TGFP plus HC was renewed every day. TGFP added after induction of GS bv HC: doseresponse efect. Figure 4 shows the dose-response relationship for the action of TGFP on HC-induced GS activity. Astrocytes were pretreated for 24 h with HC M ) alone; then TGFP (0-200 p M ) was added 3. Neuruchem., Vol. 54, No. 3, 1990
for 48 h. HC remained in the culture medium. GS activity was already inhibited by 2 pM TGFP. Maximal inhibition was produced by 100 pMTGFP; at this concentration, GS was near the basal activity. The TGFP concentration producing half-maximal inhibition was 10 pM. TGFP pretreatment before HC addition. Figure 5A illustrates the cell response in terms of GS activity to
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FIG. 3. Effect of TGFP on the time course of GS induction by HC. Astroglial cells were untreated(0)or treated with 10-6 M HC alone (0)for the indicated times. For line a, time 0, 40 pM TGFO and HC were added simultaneously (A). For line b, at 24 h, 40 pM TGFB was included in addition to HC (m). GS activity was measured at the times indicated.
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growth in vitro. DNA synthesis stimulated by serum %r was markedly decreased by TGFP addition. This factor
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FIG. 4. Inhibition of HC-stimulated GS activity by TGFP. Cells were preincubated for 24 h with 10-6 M HC (O), rinsed, and incubated with HC plus TGFP (A).GS activity was measured 48 h later. The basal level (0)is also shown.
8 h of treatment with HC ( lop6M ) alone after various times of TGFP pretreatment (100 pM). The inhibitory effect of TGFP was maximal after short pretreatment times (4-12 h); it then dropped and was lost between 16 and 24 h. In the experiment illustrated in Fig. 5B,astrocytes were preincubated for 4 or 24 h with TGFP (100 pM). The cells were then rinsed, and the CDM was supplemented with HC ( M ) . GS activity was measured after various times of cell exposure to HC. TGFP pretreatment for 4 h maintained GS activity at the basal level for 16 h. GS activity was induced by HC only after this period. In contrast, the kinetics of GS induction by HC in 24-h TGFP-pretreated cells were similar to those observed with TGFP-untreated cells. This change of response with TGFP pretreatment time can be explained by the disappearance of TGFP after prolonged incubation with the astrocytes. This is supported by the observation that culture medium from astrocytes preincubated for 24 h with TGFP and added to new cells did not elicite GS inhibition, whereas medium from astrocytes preincubated for shorter times remained functionally active on GS (data not shown). Addition of bovine serum albumin (50 pg or 1 mg/ml) to the culture medium during TGFP incubation did not prevent the loss of the TGFP effect (data not shown). TGFP, which did not change the DNA content, significantly enhanced protein synthesis in all the experiments in which GS activity was tested.
DISCUSSION It has been suggested that TGFP is a bifunctional modulator of cell growth. Several studies have clearly demonstrated that this factor can inhibit or stimulate the proliferation of a wide range of cultured cells (Sporn et al., 1986, 1987). However, no study has shown that TGFP affects CNS cell proliferation. We have determined in this work what type of growth response TGFP does elicit in astrocytes. The data presented here demonstrate that TGFP is a potent inhibitor of astrocyte
both delayed and inhibited the maximal rate of DNA synthesis in astrocytes, similarly to other cell types (Shipley et al., 1985; Ignotz and MassaguC, 1986). It is not excluded that, after 24 h in CDM, TGFP alone promotes a minimal DNA synthesis, as described by Shipley et al. (1985). However, in our hands, TGFP treatment for 1-3 days did not modify the amount of DNA. It is generally believed that the effects of TGFP on cell proliferation are diverse and vary according to the type of cell, production of mitogens, or culture conditions. Nevertheless, the molecular mechanism of TGFP action is not clearly understood. It has been generally suggested that TGFP alone has no effect on DNA synthesis (reviewed by Rizzino, 1988). The inhibitory effects observed on cell proliferation may be due to the factor's ability to counteract the mitogenic effects of other growth factors (Roberts et al., 1985; Sporn et al., 1986).However, TGFP does not interfere with the early events that follow growth factor binding to receptors; these include activation of S6 kinase (Like and Massagut, 1986)and increases in Na+/H+antiport activity and in expression of myc and fos in the nucleus (Sporn et al., 1987). On the other hand, it modulates the cyclic AMP effector system (Morris et al., 1988). Moreover, Madri et al. (1988) have correlated the inhibitory effects of TGFP on endothelial cell proliferation with an increase in extracellular matrix production. In addition to its effects on cell growth, TGFP can also affect the differentiation of a great number of cells in vitro and in vivo (reviewedby Rizzino, 1988).These effects are very confusing, because TGFP can either inhibit or stimulate the differentiation of a given cell type. This action could depend on the state of differ-
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FIG. 5. Effect of TGFP pretreatment on GS activity. A Time course for the inhibition of GS activity by TGFP. Cells were pretreated with 100 pM TGFP for the indicated time, rinsed, and incubated with M HC for 8 h (A).The basal level (0)and GS activity after induction for 8 h with HC but without TGFB pretreatment (0) are also shown. B Effect of TGFP pretreatment on time course of HC induction. Cultures were pretreated with 100 pM TGFP for 4 (m) or 24 (A)h. Cells were rinsed and incubated with M HC for the indicated times. HC induction of GS activity without TGFP pretreatment (0)and the basal level (0)are also shown.
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entiation of the target cell (Seyedin et al., 1986; Massagut et al., 1986; Rosen et al., 1988). The results reported here show that TGFP inhibits the in vitro differentiation of astrocytic cells, as assessed by morphological and biochemical criteria. Cells treated with TGFP presented more enlarged cytoplasm than control cells. On the other hand, TGFP delayed the action of aFGF, which transformed the quiescent cells to more differentiated process-bearing astrocytes. We have also studied GS activity, another marker of astrocytic differentiation. This enzyme is considered to play an important role in glial development and in neuronal-astroglial interactions (De Vellis et al., 1986). HC-induced GS activity was strongly inhibited by TGFP in a dose- and time-dependent manner. The time course of the effect of TGFP pretreatment in astrocytes was different from that produced by this factor in adrenocortical (Feige et al., 1987; Rainey et al., 1988) and Leydig (Lin et al., 1987) cells, where TGFP inhibition of adrenocortical and steroidogenic functions, respectively, appears more slowly, increases with time, and is maintained for 2 2 4 h. This apparent discrepancy in the time course of TGFP inhibition could be due to differences in enzyme turnover rates, in the rate of TGFP loss from the culture medium, or in both. Our observations supported the idea that the effects on morphology and GS activity were reversible, as they are on some activities in other cells (Massagut et al., 1986; Feige et al., 1987). The mechanism of TGFP action on differentiated functions in astrocytes is presently unknown. It could involve the pathways proposed above for the mechanism of TGFP action on DNA synthesis in various systems. Recent studies on differentiation suggest that, at least in some cases, TGFP acts via its effects on extracellular matrices (Rizzino, 1988). It increases the expression of collagens, fibronectin, and proteoglycans (Rizzino, 1988) and also controls that of laminin (Chakrabarty et al., 1988). This point is particularly interesting in approaching the role of TGFP in the brain, because (a) all these components have been identified in extracellular matrices from CNS, (b) laminin, collagens, and proteoglycans are produced by astrocytes in culture (Liesi et al., 1983; Liesi, 1985; Gallo et al., 1987),and (c) it has also been demonstrated that laminin supports neurite outgrowth (Lander et al., 1985; Edgar et al., 1988; Pierce et al., 1988; Schinstine and Cornbrooks, 1988) and axon guidance (Liesi and Silver, 1988). The action of TGFP on extracellular matrix components synthesized by astrocytes is under investigation in our laboratory. In the present work, we have shown that TGFP negatively modulates the growth and differentiation of astrocytes in vitro. This observation suggests that TGFP acts on glial cells in vivo but does not indicate which cells produce TGFP in the CNS. We are now examining the possibility that astrocytes, per se, produce this factor and that this production is regulated by other extracellular signals. J. Neurochem.. Vol. 54, No. 3. 1990
Acknowledgment: We are grateful to Dr. D. Barritault (University Pans XII) for his generous gift of aFGF. We thank Mrs. A. Lefevre and Mr. M. Bahloul for the preparation of this manuscript. This work was supported by a grant from the Association pour la Recherche contre le Cancer.
REFERENCES Bhat S. and Pfeiffer S. E. (1986) Stimulation of oligodendrocytes by extracts from astrocyte enriched cultures. J. Neurosci. Res. 15, 19-27. Chakrabarty S., Tobon A,, Varani J., and Brattain M. G. (1988) Induction of carcinoembryonic antigen secretion and modulation of protein secretion/expression and fibronectin/laminin expression in human colon carcinoma cells by transforming growth factor-& Cancer Res. 48,4059-4064. Delaunoy J. P., Langui D., Ghandour S., Labourdette G., and Sensenbrenner M. (1988) Influence of basic fibroblast growth factor on carbonic anhydrase expression by rat glial cells in primary culture. Int. J. Dev. Natrosci. 6, 129-136. De Vellis J., Wu D. K., and Kumar S. (1986) Enzyme induction and regulation of protein synthesis, in Astrocyfes: Biochemistry, Physiology and Pharmacology of Astrocytes, Vol. 2 (Fedoroff S. and Vernadakis A., eds), pp. 209-237. Academic Press, Orlando, Florida. Edgar D., Timpl R., and Thoenen H. (1988) Structural requirements for the stimulation of neurite outgrowth by two variants oflaminin and their inhibition by antibodies. J. Cell Biol. 106, 12991306. Feige J . J., Cochet C., Rainey W. E., Madani C., and Chambaz E. M. (1987) Type 6 transforming growth factor affects adrenocortical cell-differentiated functions. J. Biol. Chem. 262, 13491 - 13495. Gallo V., Bertolotto A., and Levi G. ( 1 987) The proteoglycan chondroitin sulfate is present in a subpopulation of cultured astrocytes and in their precursors. Dev. Biol. 123,282-285. Gavaret J . M., Matricon C., Pomerance M., Jacquemin C., ToruDelbauffe D., and Pierre M. (1989) Activation of S6 kinase in astroglial cells by aFGF and bFGF. Dev. Brain Res. 45, 77-82. Groyer A. and Robel P. (1980) DNA measurement by mithramycin fluorescence in chromatin solubilized by heparin. Anal. Riochem. 106,262-268. Ignotz R. A. and Massagut J. (1986) Transforming growth factor-0 stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. Biol. Chem. 261, 4337-4345. Lander A. D., Fuji D., and Reichardt L. F. (1985) Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules. J. Cell Biol. 101, 898-9 13. Liesi P. ( 1985) Laminin-immunoreactive glia distinguish regenerative adult CNS systems from nonregenerative ones. EMBO J. 4, 2502-25 I 1. Liesi P. and Silver J. (1988) Is astrocyte laminin involved in axon guidance in the mammalian CNS. Dev. Biol. 130, 774-785. Liesi P., Dahl D., and Vaheri A. (1983) Laminin is produced by early rat astrocytes in primary culture. J. Cell Biol. 96, 920-924. Like B. and MassaguC J. (1986) The antiproliferative effect of type p transforming growth factor occurs at a level distal from receptors for growth-activating factors. J. Biol. Chem. 29, 1342613429. Lin T., Blaisdell J., and Haskell J. F. (1987) Transforming growth factor-0 inhibits Leydig cell steroidogenesis in primary culture. Biochem. Biophys. Res. Commun. 146, 387-394. Lowry 0. H., Rosebrough N. J., Farr A. L., and Randall R. J. (I95 1) Protein measurement with the Folin phenol reagent. J. Bid. Chem. 193,265-275. Madri J. A,, Pratt B. M., and Tucker A. M. (1988) Phenotypic modulation of endothelial cells by transforming growth factor-@depends upon the composition and organization of the extracellular matrix. J. Cell Biol. 106, 1375-1384. Manthorpe M., Rudge J. S., and Varon S. (1986) Astroglial cell con-
EFFECTS OF TGFPI ON ASTROCYTES tributions to neuronal survival and neuritic growth, in Astrocytes: Biochemistry, Physiology and Pharmacology of Astrocytes, Vol. 2 (Fedoroff S. and Vernadakis A,, eds), pp. 31 5-376. Academic Press, Orlando, Florida. MassaguC J. (1987) The TGF-@family of growth and differentiation factors. Cell 49,437-438. Massague J., Cheifetz S., Endo T., and Nadal-Ginard B. (1986) Type @ transforming growth factor is an inhibitor of myogenic differentiation. Proc. Natl. Acad Sci. USA 83, 8206-8210. May J. V., Frost J. P., and Schomberg D. W. (1988) Differential effects of epidermal growth factor, somatomedin-C/insulin-like growth factor I, and transforming growth factor+ on porcine granulose cell deoxyribonucleic acid synthesis and cell proliferation. Endocrinology 123, 168-1 79. Morris J. C. 111, Ranganathan G., Hay I. D., Nelson R. E., and Jiang N. S. (1988) The effects of transforming growth factor-@on growth and differentiationof the continuous rat thyroid follicular cell line, FRTL-5. Endocrinology 123, 1385-1394. Momson R. S. and De Vellis J. (198 1) Growth of purified astrocfles in a chemically defined medium. Proc. Natl. Acad. Sci. USA-78, 7205-7209. Pierce S. T., Bishop A. K., and Thompson J. M. (1988) Developmental patterns of neurite outgrowth from chick embryo spinal cord and retinal neurons on laminin substrates. Dev. Brain Res. 40,2 13-222. Pierre M., Toru-Delbauffe D., Gavaret J. M., Pomerance M.. and Jacquemin C. (1986) Activation of 56 kinase activity in astrocytes by insulin, somatomedin C and TPA. FEBS Lett. 206, 162166. Pishak M. R. and Phillips A. T. (1979) A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts. Anal. Biochem. 94,82-88. Rainey W. E., Viard I., Mason J. I., Cochet C., Chambaz E. M., and Saez J. M. (1988) Effects of transforming growth factor beta on ovine adrenocortical cells. Mol. Cell. Endocrinol. 60, 189-198. Rizzino A. (1988) Transforming growth factor-@:multiple effects on cell differentiation and extracellular matrices. Dev. Biol. 130, 41 1-422.
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Roberts A. B., Anzano M. A., Lamb L. C., Smith J. M., and Sporn M. B. (198 I ) New class of transforming growth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissues. Proc. Null. Acad. Sci. USA 78, 5339-5343. Roberts A. B., Anzano M. A,, Wakefield L. M., Roche N. S., Stern D. F., and Sporn M. B. (1985) Type @ transforming growth factor: a bifunctional regulator of cellular growth. Proc. Natl. Acad. Sci. USA 82, I 19- 123. Rosen D. M., Stempien S. A,, Thompson A. Y., and Seyedin S. M. ( 1988) Transforming growth factor-@modulates the expression of osteoblast and chondroblast phenotypes in vitro. J. Cell. Physiol. 134, 337-346. Schinstine M. and Cornbrooks C. J. (1988) Age-dependent patterns and rates of neurite outgrowth from CNS neurons on Schwann cell-derived basal lamina and laminin substrata. Dev. Brain Res. 43,23-37. Seyedin S. M., Thompson A. Y., Bentz H., Rosen D. M., McPherson J. M., Conti A., Siege1 N. R., Gallupi G. R., and Piez K. A. (1986) Cartilage-inducing factor-A. J. Biol. Chem. 261, 56935695. Shipley G. D., Tucker R. F., and Moses H. L. (1985) Type p transforming growth factor/growth inhibitor stimulates entry of monolayer cultures of AKR-2B cells into S phase after a prolonged prereplicative interval. Proc. Natl. Acad. Sci. USA 82, 4147-41 5 1. Sporn M. B., Roberts A. B., Wakefield L. M., and Assoian R. K. (1986) Transforming growth factor-@:biological function and chemical structure. Science 233, 532-534. Sporn M. B., Roberts A. B., Wakefield L. M., and De Crombrugghe B. (1987) Some recent advances in the chemistry and biology of transforming growth factor-beta. J. CellBiol. 105, 1039-1045. Wakefield L. M., Smith D. M., Masui T., Hams C . C., and Sporn M. B. ( 1987) Distribution and modulation of the cellular receptor for transforming growth factor-beta. J. Cell Biol. 105, 965-975. Weibel M., Pettmann B., Labourdette G., Miehe M., Bock E., and Sensenbrenner M. (1985) Morphological and biochemical maturation of rat astroglial cells grown in a chemically defined medium: influence of an astroglial growth factor. In?. J. Dev. Neurosci. 3, 617-630.
J. Neurochem., Vol. 54, No. 3, 1990
0 I990 International Society for Neurochemistry
Effects of Transforming Growth Factor pl on Astroglial Cells in Culture D. Toru-Delbauffe, D. Baghdassarian-Chalaye, J. M. Gavaret, F. Courtin, M. Pomerance, and M. Pierre INSERM U. 96, H6pital Kremlin-Bi&tre, Le Kremlin-Bicetre, France
Abstract: The effects of transforming growth factor P l (TGFPI) on DNA synthesis and functional differentiation of astroglial cells cultured in serum-free medium were investigated. TGFP I diminished and delayed the peak of DNA synthesis induced by serum. TGFP I -treated cells were larger than control cells. This factor delayed the appearance of processbearing cells induced by acidic fibroblast growth factor treatment and also affected the astrocyte-specific enzyme glutamine synthetase (GS), whose accumulation is under hydrocortisone (HC) control. TGFPl inhibited the induction of
G S activity by HC in a dose- and time-dependent manner. Moreover, pretreatment with TGFPl for 4 h maintained the inhibition of G S activity for 16 h after removal of this factor from culture medium. These results suggest that TGFPl may be an important regulator of astrocyte growth and differentiation. Key Words: Astroglial cells-Transforming growth factor 81-DNA synthesis-Glutamine synthetase activity. Toru-Delbauffe D. et al. Effects of transforming growth factor PI on astroglial cells in culture. J. Neurochem. 54, 1056-106 1 (1990).
Transforming growth factor P (TGFP) belongs to a new family of polypeptide factors that are potentially of great interest in the regulation of cell functions (Sporn et al., 1986, 1987; Massaguk, 1987), notably, of the production and organization of extracellular matrices (Rizzino, 1988). There are four forms of TGFP. Although several recent findings suggest that TGFPl and TGFP2, the most studied forms, may differ in their functions in vivo, they have many similar effects in vitro (Sporn et al., 1987). TGFPl was used in the present study. It is now evident that most, if not all, cells produce TGFP, even those of adult tissues, such as brain, heart, and kidney, where there is little mitotic activity (Roberts et al., 1981). Moreover, TGFP receptors have been found in all the cells examined to date (Wakefield et al., 1987). In spite of an exponential increase in research on TGFP, little is known about its effects on CNS cells. In the present study, we have examined the influence of TGFP 1 on the growth and differentiation of one of the major cell types of the brain, the astrocytes, which appear to play a crucial role in brain development and
cell repair (Bhat and Pfeiffer, 1986; Manthorpe et al., 1986). Moreover, these cells probably produce the greatest part of the extracellular matrix components in the CNS (De Vellis et al., 1986). Some of these components are controlled by TGFP in other types of cells (Rizzino, 1988). This work was carried out on primary glial cells enriched in astrocytes and cultured under serum-free culture conditions wherc TGFP actions could be readily determined. The following cell parameters were monitored: DNA synthesis, morphological appearance, and glutamine synthetase (GS) activity. The accumulation of this enzyme is under glucocorticoid control and associated with astrocyte differentiation (De Vellis et al., 1986). TGFP was found to modulate negatively DNA synthesis and differentiation of astrocytes in vitro.
Received May 3, 1989; revised manuscript received July 3, 1989; accepted August 15, 1989. Address correspondence and reprint requests to Dr, D. Tom-Delbauffe at INSERM U. 96, 78, rue du General Leclerc, 94275 Le Kremlin-Bic@treCedex, France.
Abbrevin~ionsused; aFGF, acidic fibroblast growth factor; CDM, chemically defined medium; DMEM, Dulbecco's modified Eagle's medium; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; HC, hydrocortisone; TGFP, transforming growth factor p.
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MATERIALS AND METHODS Sprague-Dawley rats were from Iffacredo (L'Arbresle, France). Culture media were from GIBCO (Grand Island, NY, U.S.A.). Fetal calf serum was purchased from Seromed (Berlin, F.R.G.). Human TGFPI was obtained from R & D
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Systems (Minneapolis, MN, U.S.A.). Hydrocortisone (HC) was from Sigma. Acidic fibroblast growth factor (aFGF), purified by heparin-Sepharose affinity chromatography, was a generous gift from Dr. D. Barritault (University Paris XII, Paris, France). [3H]Thymidineand [~-'~C]glutamic acid were obtained from Amersham-France SA. Anion exchange resin (AG 1-X8, acetate form) was from Bio-Rad, France.
to synthesize DNA after 12-14 h. On the other hand, this addition diminished the rate of [3H]thymidineincorporation (40-50% at 20 h). The maximal labeling was also reduced and was obtained only at 24 h. TGFP alone did not modify the basal level in serum-free medium at least for the first 24 h.
Preparation of astrocytic cells
Morphological effect of TGFP Astrocytes grown to confluence in serum-supplemented medium exhibited a flat, polygonal shape and expressed GFAP, which was identified by anti-GFAP antiserum (data not shown). The flattened cells formed monolayer areas, and only some cells possessed short cytoplasmic processes (Morrison and De Vellis, 1981). These morphological characteristics were maintained when the cells were subsequently switched to CDM (Fig. 2A). The following experiments were performed to examine, in CDM, the effect of TGFP without or with aFGF, a growth factor that is known to produce important changes in astrocyte morphology (Weibel et al., 1985; Delaunoy et al., 1988; Gavaret et al., 1989). After a 16-h TGFP treatment (40 pM), most of the cells presented a more enlarged cytoplasm compared with that of control cells (Fig. 2B). When cells were treated with aFGF (25 ng/ml) alone, the onset of morphological change began after an 8-h period of treatment; some astrocytes presented small cell bodies with many long, branching processes (Fig. 2C). The number of process-bearing cells increased continuously with time, and 24 h later, all the cells had formed long processes, which produced a dense network. In contrast, when cells were first preincubated with TGFP for 16 h and then simultaneously exposed to TGFP and aFGF, no major morphological changes were observed after an 8-h exposure; the astrocytes presented enlarged cytoplasms, similar to those of the TGFP-treated cells (Fig. 2D). The onset of the morphological effect of aFGF was observed after a period of -24 h (data not
Primary cultures of glial cells were obtained from the cerebral hemispheres of 2-day-old rats and grown to confluence (10 days) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%fetal calf serum as described previously (Pierre et al., 1986). Cells were washed three times with chemically defined medium (CDM) and then cultured in this medium. The CDM, made from a 1 :1 mixture of DMEM and Ham's F-12 medium containing 4.5 g/L of glucose and 2.7 g/L of sodium bicarbonate, was renewed every day. After 4-5 days in this medium, cells were ready for use. These cultures contained mainly glial fibrillary acidic protein (GFAP)-positive cells, when tested with rabbit antiserum directed against GFAP (the gift of Dr. B. Pessac, CNRS, Ivry, France).
[3H]Thymidineincorporation Glial cells cultured in DMEM supplemented with 10% fetal calf serum were plated in CDM 2-3 days before confluence. After 3-4 days in this medium, quiescent cells were either maintained in CDM as the control or transferred to CDM supplemented with 10%fetal calf serum with or without 40 pM TGFP. Incubation was continued for up to 48 h. [3H]Thymidine (2 pCi/ml) was added for the last 6 h of incubation. The medium was then removed, and the monolayers were rinsed three times with fresh medium. [3H]Thymidine incorporation into DNA was measured according to the method of May et al. (1988).
G S assay and DNA content determination At the end of cell incubation with TGFP (40-100 pM, as indicated in each experiment), MHC, or a combination of both factors, the culture medium was discarded, and the cells were washed three times in cold physiological serum and stored at -80°C until GS assay. For assay, the cells were scraped off and sonically disrupted at 4°C by two 5-s treatments with an MSE sonic oscillator. GS activity was measured by the method of Pishak and Phillips (1979) and expressed as nanomoles of glutamine produced per 30 min per microgram of DNA. The DNA content was measured by the method of Groyer and Robel ( 1 980) with calf thymus DNA as the standard. Protein content was measured by the method of Lowry et al. (195 1) with bovine serum albumin as the standard.
RESULTS Effects of TGFP on DNA synthesis Figure 1 illustrates the effect of TGFPl on DNA synthesis. When cells maintained in CDM were stimulated by addition of serum (lo%), the rate of [3H]thymidine incorporation remained at a constant basal level during the first 12 h of stimulation. After this lag period, the rate increased to reach a maximal value at 20 h, then dropped until 30 h, and was constant between 30 and 48 h. Adding TGFP with serum did not change the lag period, because the cells began also
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FIG. 1. Effects of TGFP on DNA synthesis by astrocytes. Confluent, quiescent astroglial cells were cultured in CDM supplemented with 10% fetal calf serum without (0)or with (A)40 pM TGFP. The basal level was measured in cells incubated in CDM without (0)or with (A) 40 pM TGFP. [3H]Thymidine(2 pCi/ml) was added 6 h before the indicated times. The radioactivity incorporated into DNA was determined as described in Materials and Methods. The results shown are from one of a group of similar experiments.
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FIG. 2. Effects of TGFP on astrocyte morphology shown by phase-contrastmicroscopy: (A) cells in CDM; (B) cells after exposure for 16 h to TGFB in CDM; (C) cells after exposure for 8 h to aFGF in CDM; and (D) cells after pretreatment with TGFP for 16 h and exposure for 8 h to TGFP plus aFGF in CDM. The TGFB concentration was 40 pM, and that of aFGF was 25 ng/ml in all experiments. Bar 50 pm.
shown), a result thus indicating a delayed effect of aFGF in the presence of TGFP. Effects of TGFP on G S induction by HC TGFP added simultaneously with HC. Figure 3, line a, shows the effect of TGFP on GS activity. Astroglial cells were treated for various periods with HC (lop6 M ) alone or HC plus TGFP (40 pM). HC caused a continuous increase in GS activity, which depended on the duration of exposure to glucocorticoid in TGFPuntreated cells. TGFP addition simultaneously with HC did not change the activity of GS for the first 8 h, but then the activity decreased rapidly to a minimal value at 16 h, which was maintained until 24 h. The basal level was not affected by TGFP treatment. TGFP added after induction of GS by HC: time course. Astrocytes were pretreated for 24 h with HC ( M ) alone; TGFP (40 pM) was then added for the indicated times with HC remaining in the medium (Fig. 3, line b). GS activity was inhibited soon after TGFP addition and became minimal after 16 h of treatment; afterwards, it remained stable until 24 h. The inhibition continued if the culture medium containing TGFP plus HC was renewed every day. TGFP added after induction of GS bv HC: doseresponse efect. Figure 4 shows the dose-response relationship for the action of TGFP on HC-induced GS activity. Astrocytes were pretreated for 24 h with HC M ) alone; then TGFP (0-200 p M ) was added 3. Neuruchem., Vol. 54, No. 3, 1990
for 48 h. HC remained in the culture medium. GS activity was already inhibited by 2 pM TGFP. Maximal inhibition was produced by 100 pMTGFP; at this concentration, GS was near the basal activity. The TGFP concentration producing half-maximal inhibition was 10 pM. TGFP pretreatment before HC addition. Figure 5A illustrates the cell response in terms of GS activity to
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FIG. 3. Effect of TGFP on the time course of GS induction by HC. Astroglial cells were untreated(0)or treated with 10-6 M HC alone (0)for the indicated times. For line a, time 0, 40 pM TGFO and HC were added simultaneously (A). For line b, at 24 h, 40 pM TGFB was included in addition to HC (m). GS activity was measured at the times indicated.
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growth in vitro. DNA synthesis stimulated by serum %r was markedly decreased by TGFP addition. This factor
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FIG. 4. Inhibition of HC-stimulated GS activity by TGFP. Cells were preincubated for 24 h with 10-6 M HC (O), rinsed, and incubated with HC plus TGFP (A).GS activity was measured 48 h later. The basal level (0)is also shown.
8 h of treatment with HC ( lop6M ) alone after various times of TGFP pretreatment (100 pM). The inhibitory effect of TGFP was maximal after short pretreatment times (4-12 h); it then dropped and was lost between 16 and 24 h. In the experiment illustrated in Fig. 5B,astrocytes were preincubated for 4 or 24 h with TGFP (100 pM). The cells were then rinsed, and the CDM was supplemented with HC ( M ) . GS activity was measured after various times of cell exposure to HC. TGFP pretreatment for 4 h maintained GS activity at the basal level for 16 h. GS activity was induced by HC only after this period. In contrast, the kinetics of GS induction by HC in 24-h TGFP-pretreated cells were similar to those observed with TGFP-untreated cells. This change of response with TGFP pretreatment time can be explained by the disappearance of TGFP after prolonged incubation with the astrocytes. This is supported by the observation that culture medium from astrocytes preincubated for 24 h with TGFP and added to new cells did not elicite GS inhibition, whereas medium from astrocytes preincubated for shorter times remained functionally active on GS (data not shown). Addition of bovine serum albumin (50 pg or 1 mg/ml) to the culture medium during TGFP incubation did not prevent the loss of the TGFP effect (data not shown). TGFP, which did not change the DNA content, significantly enhanced protein synthesis in all the experiments in which GS activity was tested.
DISCUSSION It has been suggested that TGFP is a bifunctional modulator of cell growth. Several studies have clearly demonstrated that this factor can inhibit or stimulate the proliferation of a wide range of cultured cells (Sporn et al., 1986, 1987). However, no study has shown that TGFP affects CNS cell proliferation. We have determined in this work what type of growth response TGFP does elicit in astrocytes. The data presented here demonstrate that TGFP is a potent inhibitor of astrocyte
both delayed and inhibited the maximal rate of DNA synthesis in astrocytes, similarly to other cell types (Shipley et al., 1985; Ignotz and MassaguC, 1986). It is not excluded that, after 24 h in CDM, TGFP alone promotes a minimal DNA synthesis, as described by Shipley et al. (1985). However, in our hands, TGFP treatment for 1-3 days did not modify the amount of DNA. It is generally believed that the effects of TGFP on cell proliferation are diverse and vary according to the type of cell, production of mitogens, or culture conditions. Nevertheless, the molecular mechanism of TGFP action is not clearly understood. It has been generally suggested that TGFP alone has no effect on DNA synthesis (reviewed by Rizzino, 1988). The inhibitory effects observed on cell proliferation may be due to the factor's ability to counteract the mitogenic effects of other growth factors (Roberts et al., 1985; Sporn et al., 1986).However, TGFP does not interfere with the early events that follow growth factor binding to receptors; these include activation of S6 kinase (Like and Massagut, 1986)and increases in Na+/H+antiport activity and in expression of myc and fos in the nucleus (Sporn et al., 1987). On the other hand, it modulates the cyclic AMP effector system (Morris et al., 1988). Moreover, Madri et al. (1988) have correlated the inhibitory effects of TGFP on endothelial cell proliferation with an increase in extracellular matrix production. In addition to its effects on cell growth, TGFP can also affect the differentiation of a great number of cells in vitro and in vivo (reviewedby Rizzino, 1988).These effects are very confusing, because TGFP can either inhibit or stimulate the differentiation of a given cell type. This action could depend on the state of differ-
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FIG. 5. Effect of TGFP pretreatment on GS activity. A Time course for the inhibition of GS activity by TGFP. Cells were pretreated with 100 pM TGFP for the indicated time, rinsed, and incubated with M HC for 8 h (A).The basal level (0)and GS activity after induction for 8 h with HC but without TGFB pretreatment (0) are also shown. B Effect of TGFP pretreatment on time course of HC induction. Cultures were pretreated with 100 pM TGFP for 4 (m) or 24 (A)h. Cells were rinsed and incubated with M HC for the indicated times. HC induction of GS activity without TGFP pretreatment (0)and the basal level (0)are also shown.
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entiation of the target cell (Seyedin et al., 1986; Massagut et al., 1986; Rosen et al., 1988). The results reported here show that TGFP inhibits the in vitro differentiation of astrocytic cells, as assessed by morphological and biochemical criteria. Cells treated with TGFP presented more enlarged cytoplasm than control cells. On the other hand, TGFP delayed the action of aFGF, which transformed the quiescent cells to more differentiated process-bearing astrocytes. We have also studied GS activity, another marker of astrocytic differentiation. This enzyme is considered to play an important role in glial development and in neuronal-astroglial interactions (De Vellis et al., 1986). HC-induced GS activity was strongly inhibited by TGFP in a dose- and time-dependent manner. The time course of the effect of TGFP pretreatment in astrocytes was different from that produced by this factor in adrenocortical (Feige et al., 1987; Rainey et al., 1988) and Leydig (Lin et al., 1987) cells, where TGFP inhibition of adrenocortical and steroidogenic functions, respectively, appears more slowly, increases with time, and is maintained for 2 2 4 h. This apparent discrepancy in the time course of TGFP inhibition could be due to differences in enzyme turnover rates, in the rate of TGFP loss from the culture medium, or in both. Our observations supported the idea that the effects on morphology and GS activity were reversible, as they are on some activities in other cells (Massagut et al., 1986; Feige et al., 1987). The mechanism of TGFP action on differentiated functions in astrocytes is presently unknown. It could involve the pathways proposed above for the mechanism of TGFP action on DNA synthesis in various systems. Recent studies on differentiation suggest that, at least in some cases, TGFP acts via its effects on extracellular matrices (Rizzino, 1988). It increases the expression of collagens, fibronectin, and proteoglycans (Rizzino, 1988) and also controls that of laminin (Chakrabarty et al., 1988). This point is particularly interesting in approaching the role of TGFP in the brain, because (a) all these components have been identified in extracellular matrices from CNS, (b) laminin, collagens, and proteoglycans are produced by astrocytes in culture (Liesi et al., 1983; Liesi, 1985; Gallo et al., 1987),and (c) it has also been demonstrated that laminin supports neurite outgrowth (Lander et al., 1985; Edgar et al., 1988; Pierce et al., 1988; Schinstine and Cornbrooks, 1988) and axon guidance (Liesi and Silver, 1988). The action of TGFP on extracellular matrix components synthesized by astrocytes is under investigation in our laboratory. In the present work, we have shown that TGFP negatively modulates the growth and differentiation of astrocytes in vitro. This observation suggests that TGFP acts on glial cells in vivo but does not indicate which cells produce TGFP in the CNS. We are now examining the possibility that astrocytes, per se, produce this factor and that this production is regulated by other extracellular signals. J. Neurochem.. Vol. 54, No. 3. 1990
Acknowledgment: We are grateful to Dr. D. Barritault (University Pans XII) for his generous gift of aFGF. We thank Mrs. A. Lefevre and Mr. M. Bahloul for the preparation of this manuscript. This work was supported by a grant from the Association pour la Recherche contre le Cancer.
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