- Email: [email protected]
Effect of retinoic acid and ethanol on retinoic acid receptor β and glial fibrillary acidic protein mRNA expression in human astrocytoma cells
Neuroscience Letters 294 (2000) 73±76
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Effect of retinoic acid and ethanol on retinoic acid receptor b and glial ®brillary acidic protein mRNA expression in human astrocytoma cells Mary A. Grummer a, Zeynep N. Salih b, Richard D. Zachman a,* a
Department of Pediatrics, University of Wisconsin-Meriter Perinatal Center, 202 South Park Street, Madison, WI 53715, USA b Christ Hospital and Medical Center, Oakland, IL 60453, USA Accepted 14 September 2000
Abstract This work explores the hypothesis that perturbations caused by ethanol on the regulatory role of retinoids in brain development may be a mechanism involved in the neuropathology of fetal alcohol syndrome. The interaction of ethanol and retinoic acid (RA) on RA receptor (RAR) b and glial ®brillary acidic protein (GFAP) mRNA expression is evaluated. In the U-373 MG astrocytoma, mRNA expression of RAR b was increased and GFAP was decreased by RA. Ethanol decreased the expression of RAR b mRNA, but increased that of GFAP. The RA-stimulated increase in RAR b was not affected by the presence of ethanol. RA prevented the ethanol-induced increase in GFAP mRNA. Cycloheximide abolished only the GFAP response to ethanol. This work shows that an interrelationship between ethanol and RA exists in the astrocyte. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Retinoic acid receptor; Ethanol; Fetal alcohol syndrome; Astrocyte; Glial ®brillary acidic protein
Prenatal exposure of the developing fetus to ethanol often results in fetal alcohol syndrome (FAS), which is de®ned by the presence of prenatal and postnatal growth retardation, craniofacial abnormalities, and other congenital anomalies [14]. Dysfunctions of the central nervous system (CNS) are the most damaging long-term consequences of maternal ethanol consumption [9]. The basic mechanism by which ethanol consumption leads to fetal neurodevelopmental defects remains unknown. Possibly, the regulatory role played by vitamin A in fetal development is adversely affected by ethanol, resulting in the neuropathology of FAS [15]. Ethanol appears to alter the metabolism and function of vitamin A in development. Maternal ethanol consumption in rats alters fetal levels of tissue vitamin A and vitamin A binding proteins [5,15]. Many similarities exist between the organ embryopathy occurring in FAS and fetal abnormalities resulting from vitamin A toxicity and de®ciency [2,15]. Also, ethanol-induced abnormalities in quail embryos, similar to those resulting from vitamin A de®ciency, are prevented by vitamin A [13]. * Corresponding author. Tel.: 11-608-262-6561; fax: 11-608267-6377. E-mail address: [email protected] (R.D. Zachman).
The biological effects of vitamin A on development, growth, and tissue maintenance are mediated through retinoic acid (RA) and another metabolite, 9-cis RA. These signaling molecules are ligands for the nuclear RA receptors (RAR) and retinoid X receptors (RXR), which function as transcription factors that control gene expression throughout development [8,10]. Among many neurologic abnormalities, ethanol induces alterations in glial cell development [9]. A key event in astrocyte differentiation is the onset of expression of glial ®brillary acidic protein (GFAP), whose gene promoter region contains putative response elements for a number of hormones and in¯ammatory mediators that interact to regulate GFAP transcription [6]. Normal astrocyte development and function appear to involve RA, as demonstrated by the presence of nuclear as well as cytosolic retinoid binding proteins in astrocytes [8]. Because of the known relationships between astrocytes and ethanol and between ethanol and RA, it is rational to explore a possible relationship between RA and ethanol in the astrocyte. Previously, the interrelationship between ethanol and the role of retinoids and their receptors as mediators in brain development has been investigated in this laboratory by examining the effect of ethanol on RARs in the developing
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 53 8- X
74
M.A. Grummer et al. / Neuroscience Letters 294 (2000) 73±76
rat embryo and brain [5]. The work reported here extends this work at the cellular level by studying the interaction of ethanol and RA on RAR b and GFAP in an astrocytoma cell line. Although many previous studies have demonstrated RA regulation in continuous cultures of neuroblastoma and embryonal carcinoma cell lines, comparatively little is known in glial cells. U-373 MG human astrocytoma cells (ATCC, Maryland) were grown in minimum essential medium Eagle with Earle's salts, glutamine, and non-essential amino acids, containing 10% fetal bovine serum, 1.5 m/l sodium bicarbonate, 1.0 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were maintained in a humidi®ed atmosphere at 378C and 5% CO2. All work involving all trans-RA was conducted under subdued lighting, and culture ¯asks were kept dark during the treatment period. The concentration of ethanol in the media was measured over time using an alcohol diagnostic kit (Sigma, St. Louis MO). The effect of the protein synthesis inhibitor cycloheximide (CX) was determined by pre-incubating cells with 0.25 mg/ml CX for 4 h, after which cells were treated with RA or ethanol in the presence of CX for 48 h. After a treatment period, total RNA was extracted from cells using Trizol reagent (Gibco BRL, Grand Island, NY). Preparation of gels, transfer to nylon membranes, labeling of probes, and hybridization procedures have been previously described [5]. Filters were quantitated using a phosphorimager (Bio-Rad, Hercules, CA). All data were normalized to corresponding 28S rRNA levels, and treatment values were expressed relative to mean control values. Statistical signi®cance of the difference between control and treatment groups was assessed by one-way analysis of variance (ANOVA) of the ranks. Pairwise comparisons were made using Dunnett's test. To examine treatment interactions, a two-way ANOVA factorial arrangement of treatments was employed. Northern blot analyses of total RNA demonstrate the presence of RAR b and GFAP mRNA in U-373 MG astrocytoma cells (Fig. 1). RAR a mRNA was also present, but did not respond to RA or ethanol treatments (data not shown). No morphological changes or differences in cell growth due to RA or ethanol during the 48 h treatment period were observed.
Fig. 1. Expression of RAR b and glial ®brillary acidic protein (GFAP) mRNAs; one experiment demonstrating control (C) and treatment with 10 26 M retinoic acid (RA) or 150 mM ethanol (ET). Levels of mRNA are normalized to 28S rRNA on same ®lters, shown in lower panel.
The level of RAR b mRNA expression was signi®cantly increased by 48 h treatment with all concentrations of RA from 10 25 to 10 29 M except 10 28 M, which is likely due to the small sample size for this value (Fig. 2). The increase in RAR b mRNA was evident after a 6 h treatment of 10 26 M RA (data not shown). The increase in expression of RAR b mRNA caused by RA in the U-373 MG cell line, although not demonstrated in a recent report [1], is similar to the response in a number of cell types [3]. A signi®cant decrease in expression of RAR b mRNA occurred in the presence of 150 mM ethanol for 48 h (Fig. 3). No ethanol effect resulted from 100 mM ethanol or from shorter treatment periods (data not shown). The ethanolinduced decrease in expression, not previously reported in astrocytes, parallels the response of RAR b to ethanol in rat embryo [5]. The ethanol concentrations used in these experiments fall within the 40±255 mM range of previous astrocyte culture studies [12]. Chronic alcoholics can attain such levels and still be conscious [7]. Since ethanol directly passes from maternal to fetal circulation [9], the range of ethanol used in this work could be present in an ethanol-abused pregnancy. The interaction between RA and ethanol treatments on RAR b mRNA expression, which has not been examined before in any other system, was determined at 10 26 M RA and 150 mM ethanol. A statistically signi®cant interaction between RA and ethanol treatments indicates that the presence of one treatment alters the effect of the other. The lack of signi®cant interaction demonstrates that the response of RAR b to RA is not altered by the presence of ethanol (Fig. 3). It should be considered that the pronounced effect of RA on RAR b expression may overshadow the in¯uence of ethanol.
Fig. 2. Effect of RA dose on expression of RAR b and GFAP mRNA. U-373 MG astrocytoma cells were treated with 10 25 to 10 210 M RA for 48 h. Values are expressed relative to untreated controls (C) and are means ^ SE of 2±4 replicates from three (10 25, 10 27, 10 28, 10 210) or six (10 26, 10 29) experiments. Signi®cant difference from untreated control (P , 0:05) indicated by *.
M.A. Grummer et al. / Neuroscience Letters 294 (2000) 73±76
Fig. 3. The interaction of RA and ethanol (ET) on the expression of RAR b and GFAP mRNA. U-373 MG astrocytoma cells were treated with 10 26 M RA with and without 150 mM ethanol (ET) for 48 h. Values are expressed relative to untreated controls (C) and are means ^ SE of 2±4 replicates from three experiments. Significant difference from untreated control (P , 0:05) indicated by *.
GFAP mRNA expression levels were decreased by 48 h treatment with 10 25 to 10 28 M RA (Fig. 2). This effect was not apparent after 6 h, but was detected after 24 h (data not shown). The signi®cance of the decrease in GFAP due to RA is unknown. In a different cell line, 10 26 M RA caused an increase in GFAP protein [11], but this was not observed until after 5 days of treatment, in contrast to the 48 h maximum treatment in the present studies. Ethanol at 150 mM signi®cantly elevated the level of
Fig. 4. The interaction of cycloheximide (CX) and RA or ethanol (ET) on the expression of RAR b and GFAP mRNA. U-373 MG astrocytoma cells were treated with 0.25 mg/ml CX and/or 10 26 M RA or 150 mM ethanol for 48 h. Values are means ^ SE of three replicates from three experiments. Treatment groups were expressed relative to untreated controls, the value of which is illustrated by the gridline (data not shown). Signi®cant difference from untreated control (P , 0:05) indicated by *.
75
expression of GFAP after 48 h (Fig. 3). Although not shown, 100 mM ethanol also elevated GFAP expression; 6 or 24 h treatments had no effect. The signi®cant interaction between RA and ethanol treatments on GFAP mRNA indicates that the ethanol-induced increase in GFAP mRNA is prevented by the presence of RA (Fig. 3). Experiments were conducted using the protein synthesis inhibitor, CX, to further examine the effects of RA and ethanol on RAR b and GFAP mRNA expression. The alteration in RAR b mRNA resulting from RA was not affected by the presence of CX (Fig. 4). While visualization of the data suggests that CX prevents the effect of ethanol, statistical analyses indicate that protein synthesis is also not required for the ethanol effect. The decrease in GFAP mRNA caused by RA also was not affected by CX (Fig. 4). However, the response of GFAP mRNA to ethanol was abolished by CX, suggesting a mechanism involving translational control. Further study is needed to identify the biological signi®cance of the changes in GFAP expression in response to RA and ethanol. Ethanol exposure during gestation and postnatally during the brain growth spurt period in rats results in transient reactive gliosis, with increased GFAP immunoreactivity and mRNA expression in the cerebellum and cerebral cortex [4]. It is of interest from this work that RA has an opposite effect on astrocyte GFAP expression, and that RA blocks the effect of ethanol on GFAP. Perhaps an interrelationship between ethanol and RA in the astrocyte regulates GFAP expression and thereby has an impact upon the development of the nervous system. This study was supported in part by grants from the March of Dimes Birth Defects Foundation (#6FY-97± 0573), the University of Wisconsin Graduate School (#135±4408) and the Meriter Foundation, Inc. [1] Bouterfa, H., Picht, T., Keb, D., Herbold, C., Noll, E., Black, P.M., Roosen, K. and Tonn, J.C., Retinoids inhibit human glioma cell proliferation and migration in primary cell cultures but not in established cell lines, Neurosurgery, 46 (2000) 419±430. [2] Chen, H., Yang, H.Y., Namkung, M.J. and Juchau, M.R., Interactive dysmorphogenic effects of all-trans-retinol and ethanol on cultured whole rat embryos during organogenesis, Teratology, 54 (1996) 12±19. [3] Clagett-Dame, M. and Plum, L.A., Retinoid-regulated gene expression in neural development, Crit. Rev. Eukaryot. Gene Expr., 7 (1997) 299±342. [4] Fletcher, T.L., Ingraham, C.A. and Morihisa, J.M., Alcoholinduced changes in astrocyte gene expression during central nervous system development: methods and mechanisms, In F.E. Lancaster (Ed.), Alcohol and Glial Cells, National Institute on Alcohol Abuse and Alcoholism Research, Monograph 27, NIH Pub. No. 94±3742, Bethesda, MD, 1994, pp. 103±116. [5] Grummer, M.A. and Zachman, R.D., Prenatal ethanol consumption alters the expression of cellular retinol binding protein and retinoic acid receptor mRNA in fetal rat embryo and brain, Alcohol. Clin. Exp. Res., 19 (1995) 1376±1381.
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M.A. Grummer et al. / Neuroscience Letters 294 (2000) 73±76
[6] Laping, N.J., Teter, B., Nichols, N.R., Rozovsky, I. and Finch, C.E., Glial ®brillary acidic protein: regulation by hormones, cytokines, and growth factors, Brain Pathol., 1 (1994) 259± 275. [7] Lindblad, B. and Olsson, R., Unusually high levels of blood alcohol? J. Am. Med. Assoc., 236 (1976) 1600±1602. [8] Maden, M., Retinoids in neural development, In H. Nau and W.S. Blaner (Eds.), Retinoids: the Biochemical and Molecular Basis of Vitamin A and Retinoid Action, 1999, pp. 399± 442. [9] Phillips, D.E., Effects of alcohol on glial cell development in vivo: morphological studies, In F.E. Lancaster (Ed.), Alcohol and Glial Cells, National Institute on Alcohol Abuse and Alcoholism Research, Monograph 27, NIH Pub. No. 943742, Bethesda, MD, 1994, pp. 69±91. [10] Piedra®ta, F.J. and Pfahl, M., Nuclear retinoid receptors and mechanisms of action, In H. Nau and W.S. Blaner (Eds.), Retinoids: the Biochemical and Molecular Basis of Vitamin A and Retinoid Action, 1999, pp. 153±184.
[11] Rutka, J.T., DeArmond, S.J., Giblin, J., McCulloch, J.R., Wilson, C.B. and Rosenblum, M.L., Effect of retinoids on the proliferation, morphology and expression of glial ®brillary acidic protein of an anaplastic astrocytoma cell line , Int. J. Cancer, 42 (1988) 419±427. [12] Snyder, A.K., Responses of glia to alcohol, In M. Aschner and J.K. Kimelberg (Eds.), The Role of Glia in Neurotoxicity, CRC Press, Boca Raton, FL, 1996, pp. 111±135. [13] Twal, W.O. and Zile, M.H., Retinoic acid reverses ethanolinduced cardiovascular abnormalities in quail embryos, Alcohol. Clin. Exp. Res., 21 (1997) 1137±1143. [14] West, J.R., Chen, W.A. and Pantazis, N.J., Fetal alcohol syndrome: the vulnerability of the developing brain and possible mechanisms of damage, Metab. Brain Disord., 9 (1994) 291±322. [15] Zachman, R.D. and Grummer, M.A., The interaction of ethanol and vitamin A as a potential mechanism for the pathogenesis of fetal alcohol syndrome, Alcohol. Clin. Exp. Res., 22 (1998) 1544±1556.
www.elsevier.com/locate/neulet
Effect of retinoic acid and ethanol on retinoic acid receptor b and glial ®brillary acidic protein mRNA expression in human astrocytoma cells Mary A. Grummer a, Zeynep N. Salih b, Richard D. Zachman a,* a
Department of Pediatrics, University of Wisconsin-Meriter Perinatal Center, 202 South Park Street, Madison, WI 53715, USA b Christ Hospital and Medical Center, Oakland, IL 60453, USA Accepted 14 September 2000
Abstract This work explores the hypothesis that perturbations caused by ethanol on the regulatory role of retinoids in brain development may be a mechanism involved in the neuropathology of fetal alcohol syndrome. The interaction of ethanol and retinoic acid (RA) on RA receptor (RAR) b and glial ®brillary acidic protein (GFAP) mRNA expression is evaluated. In the U-373 MG astrocytoma, mRNA expression of RAR b was increased and GFAP was decreased by RA. Ethanol decreased the expression of RAR b mRNA, but increased that of GFAP. The RA-stimulated increase in RAR b was not affected by the presence of ethanol. RA prevented the ethanol-induced increase in GFAP mRNA. Cycloheximide abolished only the GFAP response to ethanol. This work shows that an interrelationship between ethanol and RA exists in the astrocyte. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Retinoic acid receptor; Ethanol; Fetal alcohol syndrome; Astrocyte; Glial ®brillary acidic protein
Prenatal exposure of the developing fetus to ethanol often results in fetal alcohol syndrome (FAS), which is de®ned by the presence of prenatal and postnatal growth retardation, craniofacial abnormalities, and other congenital anomalies [14]. Dysfunctions of the central nervous system (CNS) are the most damaging long-term consequences of maternal ethanol consumption [9]. The basic mechanism by which ethanol consumption leads to fetal neurodevelopmental defects remains unknown. Possibly, the regulatory role played by vitamin A in fetal development is adversely affected by ethanol, resulting in the neuropathology of FAS [15]. Ethanol appears to alter the metabolism and function of vitamin A in development. Maternal ethanol consumption in rats alters fetal levels of tissue vitamin A and vitamin A binding proteins [5,15]. Many similarities exist between the organ embryopathy occurring in FAS and fetal abnormalities resulting from vitamin A toxicity and de®ciency [2,15]. Also, ethanol-induced abnormalities in quail embryos, similar to those resulting from vitamin A de®ciency, are prevented by vitamin A [13]. * Corresponding author. Tel.: 11-608-262-6561; fax: 11-608267-6377. E-mail address: [email protected] (R.D. Zachman).
The biological effects of vitamin A on development, growth, and tissue maintenance are mediated through retinoic acid (RA) and another metabolite, 9-cis RA. These signaling molecules are ligands for the nuclear RA receptors (RAR) and retinoid X receptors (RXR), which function as transcription factors that control gene expression throughout development [8,10]. Among many neurologic abnormalities, ethanol induces alterations in glial cell development [9]. A key event in astrocyte differentiation is the onset of expression of glial ®brillary acidic protein (GFAP), whose gene promoter region contains putative response elements for a number of hormones and in¯ammatory mediators that interact to regulate GFAP transcription [6]. Normal astrocyte development and function appear to involve RA, as demonstrated by the presence of nuclear as well as cytosolic retinoid binding proteins in astrocytes [8]. Because of the known relationships between astrocytes and ethanol and between ethanol and RA, it is rational to explore a possible relationship between RA and ethanol in the astrocyte. Previously, the interrelationship between ethanol and the role of retinoids and their receptors as mediators in brain development has been investigated in this laboratory by examining the effect of ethanol on RARs in the developing
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 53 8- X
74
M.A. Grummer et al. / Neuroscience Letters 294 (2000) 73±76
rat embryo and brain [5]. The work reported here extends this work at the cellular level by studying the interaction of ethanol and RA on RAR b and GFAP in an astrocytoma cell line. Although many previous studies have demonstrated RA regulation in continuous cultures of neuroblastoma and embryonal carcinoma cell lines, comparatively little is known in glial cells. U-373 MG human astrocytoma cells (ATCC, Maryland) were grown in minimum essential medium Eagle with Earle's salts, glutamine, and non-essential amino acids, containing 10% fetal bovine serum, 1.5 m/l sodium bicarbonate, 1.0 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were maintained in a humidi®ed atmosphere at 378C and 5% CO2. All work involving all trans-RA was conducted under subdued lighting, and culture ¯asks were kept dark during the treatment period. The concentration of ethanol in the media was measured over time using an alcohol diagnostic kit (Sigma, St. Louis MO). The effect of the protein synthesis inhibitor cycloheximide (CX) was determined by pre-incubating cells with 0.25 mg/ml CX for 4 h, after which cells were treated with RA or ethanol in the presence of CX for 48 h. After a treatment period, total RNA was extracted from cells using Trizol reagent (Gibco BRL, Grand Island, NY). Preparation of gels, transfer to nylon membranes, labeling of probes, and hybridization procedures have been previously described [5]. Filters were quantitated using a phosphorimager (Bio-Rad, Hercules, CA). All data were normalized to corresponding 28S rRNA levels, and treatment values were expressed relative to mean control values. Statistical signi®cance of the difference between control and treatment groups was assessed by one-way analysis of variance (ANOVA) of the ranks. Pairwise comparisons were made using Dunnett's test. To examine treatment interactions, a two-way ANOVA factorial arrangement of treatments was employed. Northern blot analyses of total RNA demonstrate the presence of RAR b and GFAP mRNA in U-373 MG astrocytoma cells (Fig. 1). RAR a mRNA was also present, but did not respond to RA or ethanol treatments (data not shown). No morphological changes or differences in cell growth due to RA or ethanol during the 48 h treatment period were observed.
Fig. 1. Expression of RAR b and glial ®brillary acidic protein (GFAP) mRNAs; one experiment demonstrating control (C) and treatment with 10 26 M retinoic acid (RA) or 150 mM ethanol (ET). Levels of mRNA are normalized to 28S rRNA on same ®lters, shown in lower panel.
The level of RAR b mRNA expression was signi®cantly increased by 48 h treatment with all concentrations of RA from 10 25 to 10 29 M except 10 28 M, which is likely due to the small sample size for this value (Fig. 2). The increase in RAR b mRNA was evident after a 6 h treatment of 10 26 M RA (data not shown). The increase in expression of RAR b mRNA caused by RA in the U-373 MG cell line, although not demonstrated in a recent report [1], is similar to the response in a number of cell types [3]. A signi®cant decrease in expression of RAR b mRNA occurred in the presence of 150 mM ethanol for 48 h (Fig. 3). No ethanol effect resulted from 100 mM ethanol or from shorter treatment periods (data not shown). The ethanolinduced decrease in expression, not previously reported in astrocytes, parallels the response of RAR b to ethanol in rat embryo [5]. The ethanol concentrations used in these experiments fall within the 40±255 mM range of previous astrocyte culture studies [12]. Chronic alcoholics can attain such levels and still be conscious [7]. Since ethanol directly passes from maternal to fetal circulation [9], the range of ethanol used in this work could be present in an ethanol-abused pregnancy. The interaction between RA and ethanol treatments on RAR b mRNA expression, which has not been examined before in any other system, was determined at 10 26 M RA and 150 mM ethanol. A statistically signi®cant interaction between RA and ethanol treatments indicates that the presence of one treatment alters the effect of the other. The lack of signi®cant interaction demonstrates that the response of RAR b to RA is not altered by the presence of ethanol (Fig. 3). It should be considered that the pronounced effect of RA on RAR b expression may overshadow the in¯uence of ethanol.
Fig. 2. Effect of RA dose on expression of RAR b and GFAP mRNA. U-373 MG astrocytoma cells were treated with 10 25 to 10 210 M RA for 48 h. Values are expressed relative to untreated controls (C) and are means ^ SE of 2±4 replicates from three (10 25, 10 27, 10 28, 10 210) or six (10 26, 10 29) experiments. Signi®cant difference from untreated control (P , 0:05) indicated by *.
M.A. Grummer et al. / Neuroscience Letters 294 (2000) 73±76
Fig. 3. The interaction of RA and ethanol (ET) on the expression of RAR b and GFAP mRNA. U-373 MG astrocytoma cells were treated with 10 26 M RA with and without 150 mM ethanol (ET) for 48 h. Values are expressed relative to untreated controls (C) and are means ^ SE of 2±4 replicates from three experiments. Significant difference from untreated control (P , 0:05) indicated by *.
GFAP mRNA expression levels were decreased by 48 h treatment with 10 25 to 10 28 M RA (Fig. 2). This effect was not apparent after 6 h, but was detected after 24 h (data not shown). The signi®cance of the decrease in GFAP due to RA is unknown. In a different cell line, 10 26 M RA caused an increase in GFAP protein [11], but this was not observed until after 5 days of treatment, in contrast to the 48 h maximum treatment in the present studies. Ethanol at 150 mM signi®cantly elevated the level of
Fig. 4. The interaction of cycloheximide (CX) and RA or ethanol (ET) on the expression of RAR b and GFAP mRNA. U-373 MG astrocytoma cells were treated with 0.25 mg/ml CX and/or 10 26 M RA or 150 mM ethanol for 48 h. Values are means ^ SE of three replicates from three experiments. Treatment groups were expressed relative to untreated controls, the value of which is illustrated by the gridline (data not shown). Signi®cant difference from untreated control (P , 0:05) indicated by *.
75
expression of GFAP after 48 h (Fig. 3). Although not shown, 100 mM ethanol also elevated GFAP expression; 6 or 24 h treatments had no effect. The signi®cant interaction between RA and ethanol treatments on GFAP mRNA indicates that the ethanol-induced increase in GFAP mRNA is prevented by the presence of RA (Fig. 3). Experiments were conducted using the protein synthesis inhibitor, CX, to further examine the effects of RA and ethanol on RAR b and GFAP mRNA expression. The alteration in RAR b mRNA resulting from RA was not affected by the presence of CX (Fig. 4). While visualization of the data suggests that CX prevents the effect of ethanol, statistical analyses indicate that protein synthesis is also not required for the ethanol effect. The decrease in GFAP mRNA caused by RA also was not affected by CX (Fig. 4). However, the response of GFAP mRNA to ethanol was abolished by CX, suggesting a mechanism involving translational control. Further study is needed to identify the biological signi®cance of the changes in GFAP expression in response to RA and ethanol. Ethanol exposure during gestation and postnatally during the brain growth spurt period in rats results in transient reactive gliosis, with increased GFAP immunoreactivity and mRNA expression in the cerebellum and cerebral cortex [4]. It is of interest from this work that RA has an opposite effect on astrocyte GFAP expression, and that RA blocks the effect of ethanol on GFAP. Perhaps an interrelationship between ethanol and RA in the astrocyte regulates GFAP expression and thereby has an impact upon the development of the nervous system. This study was supported in part by grants from the March of Dimes Birth Defects Foundation (#6FY-97± 0573), the University of Wisconsin Graduate School (#135±4408) and the Meriter Foundation, Inc. [1] Bouterfa, H., Picht, T., Keb, D., Herbold, C., Noll, E., Black, P.M., Roosen, K. and Tonn, J.C., Retinoids inhibit human glioma cell proliferation and migration in primary cell cultures but not in established cell lines, Neurosurgery, 46 (2000) 419±430. [2] Chen, H., Yang, H.Y., Namkung, M.J. and Juchau, M.R., Interactive dysmorphogenic effects of all-trans-retinol and ethanol on cultured whole rat embryos during organogenesis, Teratology, 54 (1996) 12±19. [3] Clagett-Dame, M. and Plum, L.A., Retinoid-regulated gene expression in neural development, Crit. Rev. Eukaryot. Gene Expr., 7 (1997) 299±342. [4] Fletcher, T.L., Ingraham, C.A. and Morihisa, J.M., Alcoholinduced changes in astrocyte gene expression during central nervous system development: methods and mechanisms, In F.E. Lancaster (Ed.), Alcohol and Glial Cells, National Institute on Alcohol Abuse and Alcoholism Research, Monograph 27, NIH Pub. No. 94±3742, Bethesda, MD, 1994, pp. 103±116. [5] Grummer, M.A. and Zachman, R.D., Prenatal ethanol consumption alters the expression of cellular retinol binding protein and retinoic acid receptor mRNA in fetal rat embryo and brain, Alcohol. Clin. Exp. Res., 19 (1995) 1376±1381.
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M.A. Grummer et al. / Neuroscience Letters 294 (2000) 73±76
[6] Laping, N.J., Teter, B., Nichols, N.R., Rozovsky, I. and Finch, C.E., Glial ®brillary acidic protein: regulation by hormones, cytokines, and growth factors, Brain Pathol., 1 (1994) 259± 275. [7] Lindblad, B. and Olsson, R., Unusually high levels of blood alcohol? J. Am. Med. Assoc., 236 (1976) 1600±1602. [8] Maden, M., Retinoids in neural development, In H. Nau and W.S. Blaner (Eds.), Retinoids: the Biochemical and Molecular Basis of Vitamin A and Retinoid Action, 1999, pp. 399± 442. [9] Phillips, D.E., Effects of alcohol on glial cell development in vivo: morphological studies, In F.E. Lancaster (Ed.), Alcohol and Glial Cells, National Institute on Alcohol Abuse and Alcoholism Research, Monograph 27, NIH Pub. No. 943742, Bethesda, MD, 1994, pp. 69±91. [10] Piedra®ta, F.J. and Pfahl, M., Nuclear retinoid receptors and mechanisms of action, In H. Nau and W.S. Blaner (Eds.), Retinoids: the Biochemical and Molecular Basis of Vitamin A and Retinoid Action, 1999, pp. 153±184.
[11] Rutka, J.T., DeArmond, S.J., Giblin, J., McCulloch, J.R., Wilson, C.B. and Rosenblum, M.L., Effect of retinoids on the proliferation, morphology and expression of glial ®brillary acidic protein of an anaplastic astrocytoma cell line , Int. J. Cancer, 42 (1988) 419±427. [12] Snyder, A.K., Responses of glia to alcohol, In M. Aschner and J.K. Kimelberg (Eds.), The Role of Glia in Neurotoxicity, CRC Press, Boca Raton, FL, 1996, pp. 111±135. [13] Twal, W.O. and Zile, M.H., Retinoic acid reverses ethanolinduced cardiovascular abnormalities in quail embryos, Alcohol. Clin. Exp. Res., 21 (1997) 1137±1143. [14] West, J.R., Chen, W.A. and Pantazis, N.J., Fetal alcohol syndrome: the vulnerability of the developing brain and possible mechanisms of damage, Metab. Brain Disord., 9 (1994) 291±322. [15] Zachman, R.D. and Grummer, M.A., The interaction of ethanol and vitamin A as a potential mechanism for the pathogenesis of fetal alcohol syndrome, Alcohol. Clin. Exp. Res., 22 (1998) 1544±1556.