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Interaction of ethanol with retinol and retinoic acid in RAR β and GAP-43 expression
Neurotoxicology and Teratology 22 (2000) 829 ± 836
Interaction of ethanol with retinol and retinoic acid in RAR b and GAP-43 expression Mary A. Grummer, Richard D. Zachman* Department of Pediatrics, University of Wisconsin, Meriter Perinatal Center, 202 South Park Street, Madison, WI 53715, USA Received 25 October 1999; accepted 2 July 2000
Abstract Fetal ethanol exposure has many detrimental effects on neural development, which possibly occurs through ethanol-induced disruption of the function of vitamin A. In LAN-5 neuroblastoma cells, retinol (10 ÿ 6 M) and retinoic acid (RA; 10 ÿ 5 ± 10 ÿ 6 M) increased RAR b mRNA expression. Ethanol downregulated RAR b levels, even in the presence of retinol. RAR b mRNA expression was decreased by ethanol in the presence of 10 ÿ 6 M RA, but not 10 ÿ 5 M RA. With cycloheximide (CX), RA still stimulated RAR b mRNA, but the effect of ethanol was abolished. The mRNA expression of GAP-43, an important factor in neural development, increased with 10 ÿ 6 M retinol and 10 ÿ 5 ± 10 ÿ 9 M RA. Ethanol decreased GAP-43 mRNA expression in the presence or absence of retinol. Ethanol was without effect on GAP-43 mRNA at 10 ÿ 5 M RA, but did lower the levels at 10 ÿ 6 and 10 ÿ 7 M RA. CX prevented the effects of both RA and ethanol on GAP-43 mRNA. These studies provide support for the hypothesis that retinoid function is altered by ethanol. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Retinoic acid; Retinoic acid receptor; Ethanol; GAP-43; Fetal alcohol syndrome; LAN-5 neuroblastoma cell
Ethanol consumption during pregnancy results in an increase in the incidence of spontaneous abortions, an increase in prenatal mortality, and a number of physical and mental birth defects, which are the manifestations of fetal alcohol syndrome (FAS) [7,46]. The most damaging long-term consequences of maternal ethanol consumption are dysfunctions of the central nervous system (CNS) [47]. Prenatal exposure to ethanol interferes with generation and proliferation of neuronal precursors, reduces neuronal and glial cell numbers and sizes in many CNS structures [29], alters neuronal migration, axonal and dendritic outgrowth [35] and plasma membrane physicochemical properties [26], and directly causes neuronal cell loss [3]. The basic mechanism by which ethanol alters fetal neurodevelopment is unknown. One hypothesis suggests that the regulatory role played by vitamin A in fetal development is disrupted by ethanol, leading to the neuropathology of FAS [11,18,41,56]. Vitamin A is integral for normal neural development, as both excess and deficient levels of retinoic acid (RA), a metabolite of vitamin A,
* Corresponding author. Tel.: +1-608-262-6561; fax: +1-608-267-6377. E-mail address: [email protected] (R.D. Zachman).
result in defects in hindbrain patterning, neurite outgrowth, and neural crest development [10,30,31,52]. RA and 9-cis RA affect the gene expression of many cell products through the mechanism of binding to nuclear RA receptors (RARs) and retinoid X receptors (RXRs), which are members of the steroid hormone superfamily of receptors [38]. The complexity of roles of RA in different systems is mediated by the multiple isoforms of six receptor genes (RARs a, b, g and RXRs a, b, g) which act as heterodimers in DNA binding. The complex spatiotemporal patterns that RARs and RXRs express during development [28,32,34,36], and the embryonic alterations resulting from compound RAR and/or RXR null mutations [24,34,36] indicate that retinoid signaling through the receptors is a key regulator of development. Several observations have demonstrated an interaction of ethanol with vitamin A. First, ethanol alters the metabolism of vitamin A. Ethanol consumption by both adult man and mature nonpregnant animals results in lower liver vitamin A reserves, even under sufficient nutritional conditions [15,16,27], and maternal ethanol consumption alters fetal vitamin A tissue levels and levels of vitamin A binding proteins [17 ± 19]. Secondly, an effect of ethanol on vitamin A in development is suggested by the many similarities
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between the organ embryopathy of FAS and those fetal abnormalities found in both vitamin A toxicity and deficiency [12,56]. Also, treatment of rat embryo cultures with excess vitamin A or ethanol causes similar developmental anomalies [4], and cardiovascular abnormalities resulting from vitamin A deficiency in quail embryos are similar to those resulting from treatment with ethanol [49]. A mechanism through which RA influences neural development may involve the nervous-system-specific growth-associated protein of 43 kDa (GAP-43). This protein, which demonstrates a spatial and temporal pattern of expression during development [23], plays an important role in the development of the nervous system, particularly in the determination of neuronal cell lineage and in neurite differentiation, extension, and synaptogenesis [1,33,48]. The regulation of GAP-43 gene expression is apparently under the control of RA. RA-induced differentiation is accompanied by an increase in GAP-43 mRNA in P19 embryonal carcinoma cells [5] and in GAP-43 protein in neuroblastoma cells [44]. Targeted disruption of the GAP-43 gene in P19 cells altered RA-induced increases in neuronal numbers and decreased initiation of neuritogenesis [33]. Previously, in vivo research from this laboratory has demonstrated changes in RAR expression in the developing rat embryo and brain exposed to ethanol [19]. The objective of this present work was to advance these earlier studies at the cellular level using a single cell line Ð the LAN-5 neuroblastoma. The first hypothesis, that retinoid function is altered by ethanol, was evaluated by determining the effects of retinol/RA and ethanol and their interaction on the expression of RAR mRNA. Secondly, since it is possible that ethanol may alter the regulation by RA of other genes involved in development, the effects of retinoids and ethanol on the expression of one such gene, GAP-43, were also evaluated. 1. Methods 1.1. Cell culture LAN-5 human neuroblastoma cells were grown in Leibovitz's L-15 media containing 10% fetal bovine serum, 10 0 acid mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic (HEPES), penicillin (100 U/ml) and streptomycin (100 mg/ ml). For all experiments, plug seal 75 cm2 flasks were utilized, which control the ethanol concentration within the flask. Cells were maintained in a humidified atmosphere at 37°C in room air. 1.2. Treatments Cells were treated with ethanol and/or retinol or all-trans RA at various concentrations and periods of time. Retinol and RA were dissolved in dimethyl sulfoxide (DMSO); controls with similar levels of DMSO as the retinoid
treatment groups showed no difference in mRNA levels. All works involving retinoids were conducted under subdued lighting, and culture flasks were kept dark during the treatment period. In each experiment, two to four replicate flasks were used for each experimental condition, and experiments were repeated various times. The effect of the protein synthesis inhibitor, cycloheximide (CX), was determined by pre-incubating cells with 0.2 or 0.4 mg/ml CX for 4 h, after which cells were treated with RA or ethanol in the presence of CX for 48 h. 1.3. Isolation of RNA and Northern and data analyses After a treatment period, total RNA was extracted from cells using Trizol Reagent (Gibco BRL, Grand Island, NY) as specified by the manufacturer. Preparation of gels, transfer to nylon membranes, labeling of probes, and hybridization procedures have been previously described [20]. RAR a, RAR b and GAP-43 cDNAs were the generous gifts of P. Chambon (Strasbourg, France) and R. Neve (Belmont, MA), respectively. To correct for RNA levels, filters were also probed with 28S rRNA (Clontech, Palo Alto, CA). 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 significance of the difference between control and treatment groups was assessed by one-way analysis of variance (ANOVA) of the ranks using the ANOVA or General Linear Model procedure of SAS [43]. When significant treatment effects were determined, pairwise comparisons to controls were made using Dunnett's test. In experiments examining effects of retinol/RA plus ethanol, a factorial arrangement of treatments was employed, analyzing for main effects of retinol/RA and ethanol and retinol/ RA by ethanol interaction. Similar analyses were performed in experiments with CX and RA or ethanol, analyzing for main effects of RA (or ethanol) and CX and RA (or ethanol) by CX interaction. 2. Results Northern blot analyses of total RNA extracted from LAN-5 neuroblastoma cells treated with retinol or ethanol demonstrate the presence of RAR b and GAP-43 mRNA (Fig. 1). Retinol (10 ÿ 6 M) increased and ethanol (150 mM) decreased RAR b and GAP-43 mRNA expression after 48-h treatment (Fig. 2). The effects of retinol and ethanol treatments combined were analyzed by a two-way ANOVA with interaction to assess the effect of ethanol on the response of RAR b and GAP-43 mRNA to retinol. A statistically significant interaction between retinol and ethanol treatments indicates that the presence of one treatment alters the effect of the other. A non-significant interaction indicates that the response to one treatment is not affected by the
M.A. Grummer, R.D. Zachman / Neurotoxicology and Teratology 22 (2000) 829±836
Fig. 1. Expression of RAR b and GAP-43; one experiment demonstrating (1) control and treatment with (2) 10 ÿ 6 M retinol or (3) 150 mM ethanol. Levels of mRNA are normalized to 28S rRNA on same filters, shown in lower panel. Extraction, fractionation on gel, transfer to membrane, and hybridization to cDNA probes are described in text. Sizes of the mRNA species are: RAR b Ð 3.4 kb; GAP- 43 Ð 1.6 kb.
presence of the other. For both RAR b and GAP-43 mRNA, the interaction was nonsignificant, demonstrating that the ethanol caused a decrease in the mRNA levels, regardless of whether or not retinol was present. The remaining experiments were conducted using RA, thereby circumventing the step of retinol oxidation. The level of RAR b mRNA expression was significantly increased by treatment with 10 ÿ 5 ±10 ÿ 6 M RA for 48 h (Fig. 3A). The increase in RAR b mRNA was also seen after 24-h treatment with 10 ÿ 6 M RA (data not shown). No earlier time points were evaluated. The expression of RAR b was downregulated by 150 mM ethanol; the responses to 75 and 100 mM ethanol were not significantly lower (Fig. 3B). No differences in cell growth occurred due to RA (or retinol) or ethanol during the 48h treatment period. Morphological changes were observed after 96 h; these were not evaluated since they occurred
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later than the expression data presented here. The response of RAR a mRNA to RA and ethanol was also measured; neither ethanol nor RA affected RAR a in LAN-5 cells (data not shown). The interaction between the effect of RA and ethanol was determined at RA concentrations that caused an increase in RAR b (10 ÿ 5 ±10 ÿ 6 M). At 10 ÿ 5 M RA, the interaction between treatments was significant; i.e., ethanol did not cause a decrease in RAR b mRNA when RA was present (Fig. 4A). However, at 10 ÿ 6 M RA, the interaction was not significant, showing that at this lower level of RA, ethanol did reduce RAR b mRNA expression. The effect of the protein synthesis inhibitor, CX, on RAR b mRNA was determined using 10 ÿ 6 M RA and 150 mM ethanol for 48 h, since these parameters maximized treatment effects on both RAR b and GAP-43 mRNAs (see below). Analyses of RA and CX interactions demonstrated that the stimulation of RAR b mRNA by RA still occurred in the presence of 0.2 and 0.4 mg/ml CX (Fig. 4B). A significant interaction between the effect of ethanol and CX indicated that downregulation of RAR b mRNA expression by ethanol was abolished in the presence of both levels of CX. GAP-43 mRNA expression levels were increased by 48h treatment with 10 ÿ 5 ± 10 ÿ 9 M RA (Fig. 3A). As with RAR b, levels were higher after 24 h of RA treatment (data not shown). Ethanol at both 75 and 150 mM significantly downregulated the expression of GAP-43 (Fig. 3B). The interaction between the effect of RA and ethanol on GAP-43 expression was determined at 10 ÿ 5 ±10 ÿ 7 M RA
Fig. 2. Effect of retinol (ROL) and ethanol (ET) on expression of (A) RAR b and (B) GAP- 43 mRNA. LAN-5 cells were treated with 10 ÿ 6 M retinol and/or 150 mM ethanol for 48 h. Values are expressed relative to untreated controls (Cont) and are means SEM of two replicates from three experiments. Significant difference from control ( p < 0.05) indicated by *. For both RAR b and GAP-43, there is a significant main effect of ROL and of ethanol ( p < 0.05).
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Fig. 3. Dose effect of (A) RA and (B) ethanol on expression of RAR b and GAP mRNA. LAN-5 cells were treated with 10 ÿ 5 ± 10 ÿ 9 M RA or 75, 100, or 150 mM ethanol for 48 h. Values are expressed relative to untreated controls (Cont) and are means SEM of two to three replicates from six experiments (A) and of two to three replicates from three (75 and 100 mM) or nine (150 mM) experiments (B). Significant difference from control ( p < 0.05) indicated by *.
(Fig. 5A). As with RAR b mRNA, the interaction between treatments was significant at 10 ÿ 5 M RA, indicating that ethanol did not affect GAP-43 in the presence of RA. However, at lower RA concentrations (10 ÿ 6 and 10 ÿ 7 M), GAP-43 mRNA was reduced by ethanol.
The response of GAP-43 mRNA to RA and ethanol was also determined in the presence of CX. While RAR b mRNA increased with CX alone (Fig. 4B), both 0.2 and 0.4 mg/ml CX caused a 30% decrease in GAP-43 mRNA (Fig. 5B). A lack of significant interaction between RA and 0.2 mg/ml CX
Fig. 4. (A) The interaction of RA and ethanol (ET) on the expression of RAR b mRNA. LAN-5 cells were treated with 10 ÿ 5 and 10 ÿ 6 M RA with and without 150 mM ethanol for 48 h. Values are expressed relative to untreated controls (Cont) and are means SEM of two to four replicates from four experiments. Significant difference from control ( p < 0.05) indicated by *. At each level of RA, there is a significant main effect of RA and of ethanol ( p < 0.01). At 10 ÿ 5 M RA, the interaction of RA and ethanol is significant ( p = 0.02). (B) The interaction of CX and RA or ET on the expression of RAR b mRNA. LAN-5 cells were treated with 0.2 or 0.4 mg/ml CX and either 10 ÿ 6 M RA or 150 mM ethanol for 48 h. Values are expressed relative to untreated controls and are means SEM of three replicates from two experiments. Significant difference from control ( p < 0.05) indicated by *. At each level of CX, there is a significant main effect of RA and of ethanol ( p < 0.05). Significant interactions include: 0.2 mg/ml CX and RA ( p = 0.03), 0.2 mg/ml CX and ethanol ( p = 0.001), and 0.4 mg/ml CX and ethanol ( p = 0.003).
M.A. Grummer, R.D. Zachman / Neurotoxicology and Teratology 22 (2000) 829±836
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Fig. 5. (A) The interaction of RA and ethanol (ET) on the expression of GAP- 43 mRNA. LAN-5 cells were treated with 10 ÿ 5 ± 10 ÿ 7 M RA with and without 150 mM ethanol for 48 h. Values are expressed relative to untreated controls (Cont) and are means SEM of two to four replicates from four experiments. Significant difference from control ( p < 0.05) indicated by *. At each level of RA, there is a significant main effect of RA and of ethanol ( p < 0.05). At 10 ÿ 5 M RA, the interaction of RA and ethanol is significant ( p = 0.04). (B) The interaction of CX and RA or ethanol on the expression of GAP-43 mRNA. LAN-5 cells were treated with 0.2 or 0.4 mg/ml CX and either 10 ÿ 6 M RA or 150 mM ethanol for 48 h. Values are expressed relative to untreated controls and are means SEM of three replicates from two experiments. Significant difference from control ( p < 0.05) indicated by *. At each level of CX, there is a significant main effect of RA and of ethanol ( p < 0.01). Significant interactions include: 0.4 mg/ml CX and RA ( p = 0.03), 0.2 mg/ml CX and ethanol ( p = 0.03).
indicates that the increase in GAP-43 mRNA caused by RA was not altered by the presence of 0.2 mg/ml CX. However, a significant interaction did result at 0.4 mg/ml CX, demonstrating that the response of GAP-43 mRNA to RA was abolished by CX. As with RAR b, a significant interaction between the effect of ethanol and 0.2 mg/ml CX indicated that the downregulation of GAP-43 mRNA expression caused by ethanol did not occur in the presence CX (Fig. 5B). Although the results appear similar at 0.4 mg/ml CX, the statistical analyses did not demonstrate an interaction of treatments. 3. Discussion It has been well documented that normal neural development requires RA and is disrupted by ethanol. Many different areas of research have supported the hypothesis that a disruption of RA function required for normal development by prenatal exposure to ethanol contributes to some of the neuropathology found in the offspring of mothers who consume ethanol while pregnant. This current work advances previous findings in developing rat embryo and brain exposed to ethanol [19] using the LAN-5 neuroblastoma cell line. Similarities between neuronal precursors and neuroblastoma cells make them useful models for studying neuronal development. The presence of RARs [14,22,45,54], the ability to metabolize retinol [9], and the induction of morphological differentiation by RA [2,6,22] make neuroblastoma cells such as LAN-5 an appropriate model for studying
the interaction of RA and ethanol. The ethanol concentrations of 75 ±150 mM used in these experiments fall within the range of 22± 175 mM ethanol used in previous studies in neuroblastoma cells [29]. In this LAN-5 model, retinol increased RAR b and GAP43 mRNA expression; ethanol decreased their expression levels, even in the presence of retinol. Although the ability of LAN-5 cells to oxidize retinol has been reported [9], the response of RAR b and GAP-43 mRNA to retinol and ethanol, and the effect of ethanol on the retinoid response have not been demonstrated previously. RA increased RAR b mRNA expression, which was specific for RAR b, as RAR a mRNA expression was unaffected. Ethanol did not affect the level of RAR b mRNA in the presence of 10 ÿ 5 M RA, but at 10 ÿ 6 M RA, ethanol did cause a decrease in RAR b mRNA. Thus, it appears that sufficient levels of exogenous RA can overcome the effect of ethanol on RAR b mRNA. When RA levels are lower, the effect of ethanol may become apparent. As a mechanism whereby ethanol may affect vitamin A metabolism, it has been proposed that ethanol competitively inhibits the synthesis of RA from retinol via a form of alcohol dehydrogenase important in the oxidation of retinol to retinaldehyde, which is then oxidized to RA [12]. A decrease in the supply of RA would decrease RAR b expression, since RA induces RAR b through a RARE within the promoter of the RAR b gene [32]. Although the concept is controversial [53], decreasing RA levels by inhibition of its synthesis could be a method whereby ethanol
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treatment leads to downregulation of RAR b mRNA. This hypothesis of ethanol competitively inhibiting retinol oxidation is not suggested by the results reported here. The ethanol-induced decrease in RAR b mRNA expression in the presence of retinol and the lack of an ethanol effect with 10 ÿ 5 M RA could be indicative of inhibited retinol oxidation. However, the finding that ethanol decreases RAR b mRNA when 10 ÿ 6 M RA is added does not support inhibition of RA synthesis as the regulatory step. Another mechanism by which ethanol may affect the RA supply is through increasing RA catabolism. Recently, a novel family of cytochrome P450 proteins was found which is involved in the oxidation of RA [13,42,51]. CYP26 is present in early embryos, is expressed in regions of the brain, and is upregulated upon RA-induced differentiation of embryonic stem cells [42]. The enzyme appears to play an autoregulatory role in controlling RA levels [51]. A major pathway for ethanol oxidation also involves a member of the P450 family, which is induced by ethanol in liver [40] and also in brain [50]. Similar inductive effects of ethanol on the RA-metabolizing CYP26 enzyme could lead to reduced levels of RA. Although this has not been examined to date, an ethanol-induced increase in RA catabolism could account for the inability of ethanol to reduce RAR b mRNA expression when protein synthesis was inhibited by CX, as reported here. These studies have additionally demonstrated that protein synthesis is not required for the increase in RAR b mRNA induced by RA. RA stimulation of RAR b gene expression has been shown to be independent of protein synthesis in many cell types [8,21,37,55]. Of interest is that the increase in RAR b gene expression by RA was greater in the presence of CX. This enhanced expression may be related to the finding that CX blocks the P450-induced catabolism of RA [25], which would increase RA levels. It is clear that GAP-43 has a fundamental role in brain development, and there is ample evidence indicating possible regulation of GAP-43 through RA. Therefore, another objective of this work was to evaluate the effects of retinoids and ethanol on GAP-43 mRNA expression, which have not yet been reported. The RA-induced increase in GAP-43 mRNA expression and the ethanol-induced decrease extend previous findings on GAP-43 protein levels [44]. In this present study, there was no effect of ethanol on GAP-43 mRNA at 10 ÿ 5 M RA, but at 10 ÿ 6 and 10 ÿ 7 M RA, the ethanol effect was apparent, again suggesting that sufficient levels of RA can eliminate the effect of ethanol. For both RAR b and GAP-43, ethanol was able to modify the increase in expression caused by the 10 ÿ 6 ± 10 ÿ 7 M RA. Although these RA levels still had a pronounced effect on RAR b and GAP-43 expression in the presence of ethanol, the degree to which ethanol reduced that expression was the same whether or not RA was present. The significance of the amount of change in mRNA expression occurring is not known at this time. What is apparent is that ethanol alters the normal response to RA.
The effects of CX on the response of GAP-43 mRNA to RA and ethanol have not been examined previously to this report. The observations suggest regulatory mechanisms for GAP-43 expression, especially when considered in conjunction with the responses of RAR b mRNA to CX. The diminished response of GAP-43 mRNA to RA at 0.4 mg/ ml CX suggests that protein synthesis may be required in the response of GAP-43 mRNA to RA. Since the interference in the response to RA by CX occurred even though the level of RAR b mRNA was highly elevated in the presence of RA and CX, the regulatory effect of RA on GAP-43 expression does not appear to be directly through RAR b. As with RAR b, CX prevented the effect of ethanol on GAP-43 mRNA. Thus, these studies indicate that ethanol regulation of RAR b and GAP-43 mRNA expression requires protein synthesis. The exact nature of this step is not known. Multiple proteins and mechanisms may be involved, such as the process of mRNA stabilization, which is a known method of GAP-43 regulation [39]. Since GAP43 mRNA expression does not seem to correlate directly with RAR b levels, it does not seem likely that an ethanolinduced decrease in RAR b directly modifies the response of GAP-43 to ethanol. These studies, taken together, demonstrate in a homogeneous neuroblastoma cell line that ethanol interacts with the effects of retinol and RA on RAR b and GAP-43 mRNA expression. Increased RA catabolism or mechanisms not related to RA levels may be involved. For example, ethanol may affect co-repressor and co-activator factors that interact with the RARs to inhibit or enhance transcriptional activity [38]. In this manner, RARs may indirectly alter the expression of genes such as GAP-43 that play an important role in neural development. The results of this study support the hypothesis that ethanol affects the function of retinoids in development through RARs. However, more work is needed to identify RA-dependent developmental processes that are altered by ethanol to establish a link between vitamin A and FAS.
Acknowledgments The authors would like to thank Peter Crump, Computing and Biometry Consultant, College of Agriculture and Life Sciences, University of Wisconsin, Madison, for his assistance with statistical analyses. This study was supported, in part, by grants from the March of Dimes Birth Defects Foundation (no. 6FY-970573) and the University of Wisconsin Graduate School.
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Interaction of ethanol with retinol and retinoic acid in RAR b and GAP-43 expression Mary A. Grummer, Richard D. Zachman* Department of Pediatrics, University of Wisconsin, Meriter Perinatal Center, 202 South Park Street, Madison, WI 53715, USA Received 25 October 1999; accepted 2 July 2000
Abstract Fetal ethanol exposure has many detrimental effects on neural development, which possibly occurs through ethanol-induced disruption of the function of vitamin A. In LAN-5 neuroblastoma cells, retinol (10 ÿ 6 M) and retinoic acid (RA; 10 ÿ 5 ± 10 ÿ 6 M) increased RAR b mRNA expression. Ethanol downregulated RAR b levels, even in the presence of retinol. RAR b mRNA expression was decreased by ethanol in the presence of 10 ÿ 6 M RA, but not 10 ÿ 5 M RA. With cycloheximide (CX), RA still stimulated RAR b mRNA, but the effect of ethanol was abolished. The mRNA expression of GAP-43, an important factor in neural development, increased with 10 ÿ 6 M retinol and 10 ÿ 5 ± 10 ÿ 9 M RA. Ethanol decreased GAP-43 mRNA expression in the presence or absence of retinol. Ethanol was without effect on GAP-43 mRNA at 10 ÿ 5 M RA, but did lower the levels at 10 ÿ 6 and 10 ÿ 7 M RA. CX prevented the effects of both RA and ethanol on GAP-43 mRNA. These studies provide support for the hypothesis that retinoid function is altered by ethanol. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Retinoic acid; Retinoic acid receptor; Ethanol; GAP-43; Fetal alcohol syndrome; LAN-5 neuroblastoma cell
Ethanol consumption during pregnancy results in an increase in the incidence of spontaneous abortions, an increase in prenatal mortality, and a number of physical and mental birth defects, which are the manifestations of fetal alcohol syndrome (FAS) [7,46]. The most damaging long-term consequences of maternal ethanol consumption are dysfunctions of the central nervous system (CNS) [47]. Prenatal exposure to ethanol interferes with generation and proliferation of neuronal precursors, reduces neuronal and glial cell numbers and sizes in many CNS structures [29], alters neuronal migration, axonal and dendritic outgrowth [35] and plasma membrane physicochemical properties [26], and directly causes neuronal cell loss [3]. The basic mechanism by which ethanol alters fetal neurodevelopment is unknown. One hypothesis suggests that the regulatory role played by vitamin A in fetal development is disrupted by ethanol, leading to the neuropathology of FAS [11,18,41,56]. Vitamin A is integral for normal neural development, as both excess and deficient levels of retinoic acid (RA), a metabolite of vitamin A,
* Corresponding author. Tel.: +1-608-262-6561; fax: +1-608-267-6377. E-mail address: [email protected] (R.D. Zachman).
result in defects in hindbrain patterning, neurite outgrowth, and neural crest development [10,30,31,52]. RA and 9-cis RA affect the gene expression of many cell products through the mechanism of binding to nuclear RA receptors (RARs) and retinoid X receptors (RXRs), which are members of the steroid hormone superfamily of receptors [38]. The complexity of roles of RA in different systems is mediated by the multiple isoforms of six receptor genes (RARs a, b, g and RXRs a, b, g) which act as heterodimers in DNA binding. The complex spatiotemporal patterns that RARs and RXRs express during development [28,32,34,36], and the embryonic alterations resulting from compound RAR and/or RXR null mutations [24,34,36] indicate that retinoid signaling through the receptors is a key regulator of development. Several observations have demonstrated an interaction of ethanol with vitamin A. First, ethanol alters the metabolism of vitamin A. Ethanol consumption by both adult man and mature nonpregnant animals results in lower liver vitamin A reserves, even under sufficient nutritional conditions [15,16,27], and maternal ethanol consumption alters fetal vitamin A tissue levels and levels of vitamin A binding proteins [17 ± 19]. Secondly, an effect of ethanol on vitamin A in development is suggested by the many similarities
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between the organ embryopathy of FAS and those fetal abnormalities found in both vitamin A toxicity and deficiency [12,56]. Also, treatment of rat embryo cultures with excess vitamin A or ethanol causes similar developmental anomalies [4], and cardiovascular abnormalities resulting from vitamin A deficiency in quail embryos are similar to those resulting from treatment with ethanol [49]. A mechanism through which RA influences neural development may involve the nervous-system-specific growth-associated protein of 43 kDa (GAP-43). This protein, which demonstrates a spatial and temporal pattern of expression during development [23], plays an important role in the development of the nervous system, particularly in the determination of neuronal cell lineage and in neurite differentiation, extension, and synaptogenesis [1,33,48]. The regulation of GAP-43 gene expression is apparently under the control of RA. RA-induced differentiation is accompanied by an increase in GAP-43 mRNA in P19 embryonal carcinoma cells [5] and in GAP-43 protein in neuroblastoma cells [44]. Targeted disruption of the GAP-43 gene in P19 cells altered RA-induced increases in neuronal numbers and decreased initiation of neuritogenesis [33]. Previously, in vivo research from this laboratory has demonstrated changes in RAR expression in the developing rat embryo and brain exposed to ethanol [19]. The objective of this present work was to advance these earlier studies at the cellular level using a single cell line Ð the LAN-5 neuroblastoma. The first hypothesis, that retinoid function is altered by ethanol, was evaluated by determining the effects of retinol/RA and ethanol and their interaction on the expression of RAR mRNA. Secondly, since it is possible that ethanol may alter the regulation by RA of other genes involved in development, the effects of retinoids and ethanol on the expression of one such gene, GAP-43, were also evaluated. 1. Methods 1.1. Cell culture LAN-5 human neuroblastoma cells were grown in Leibovitz's L-15 media containing 10% fetal bovine serum, 10 0 acid mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic (HEPES), penicillin (100 U/ml) and streptomycin (100 mg/ ml). For all experiments, plug seal 75 cm2 flasks were utilized, which control the ethanol concentration within the flask. Cells were maintained in a humidified atmosphere at 37°C in room air. 1.2. Treatments Cells were treated with ethanol and/or retinol or all-trans RA at various concentrations and periods of time. Retinol and RA were dissolved in dimethyl sulfoxide (DMSO); controls with similar levels of DMSO as the retinoid
treatment groups showed no difference in mRNA levels. All works involving retinoids were conducted under subdued lighting, and culture flasks were kept dark during the treatment period. In each experiment, two to four replicate flasks were used for each experimental condition, and experiments were repeated various times. The effect of the protein synthesis inhibitor, cycloheximide (CX), was determined by pre-incubating cells with 0.2 or 0.4 mg/ml CX for 4 h, after which cells were treated with RA or ethanol in the presence of CX for 48 h. 1.3. Isolation of RNA and Northern and data analyses After a treatment period, total RNA was extracted from cells using Trizol Reagent (Gibco BRL, Grand Island, NY) as specified by the manufacturer. Preparation of gels, transfer to nylon membranes, labeling of probes, and hybridization procedures have been previously described [20]. RAR a, RAR b and GAP-43 cDNAs were the generous gifts of P. Chambon (Strasbourg, France) and R. Neve (Belmont, MA), respectively. To correct for RNA levels, filters were also probed with 28S rRNA (Clontech, Palo Alto, CA). 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 significance of the difference between control and treatment groups was assessed by one-way analysis of variance (ANOVA) of the ranks using the ANOVA or General Linear Model procedure of SAS [43]. When significant treatment effects were determined, pairwise comparisons to controls were made using Dunnett's test. In experiments examining effects of retinol/RA plus ethanol, a factorial arrangement of treatments was employed, analyzing for main effects of retinol/RA and ethanol and retinol/ RA by ethanol interaction. Similar analyses were performed in experiments with CX and RA or ethanol, analyzing for main effects of RA (or ethanol) and CX and RA (or ethanol) by CX interaction. 2. Results Northern blot analyses of total RNA extracted from LAN-5 neuroblastoma cells treated with retinol or ethanol demonstrate the presence of RAR b and GAP-43 mRNA (Fig. 1). Retinol (10 ÿ 6 M) increased and ethanol (150 mM) decreased RAR b and GAP-43 mRNA expression after 48-h treatment (Fig. 2). The effects of retinol and ethanol treatments combined were analyzed by a two-way ANOVA with interaction to assess the effect of ethanol on the response of RAR b and GAP-43 mRNA to retinol. A statistically significant interaction between retinol and ethanol treatments indicates that the presence of one treatment alters the effect of the other. A non-significant interaction indicates that the response to one treatment is not affected by the
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Fig. 1. Expression of RAR b and GAP-43; one experiment demonstrating (1) control and treatment with (2) 10 ÿ 6 M retinol or (3) 150 mM ethanol. Levels of mRNA are normalized to 28S rRNA on same filters, shown in lower panel. Extraction, fractionation on gel, transfer to membrane, and hybridization to cDNA probes are described in text. Sizes of the mRNA species are: RAR b Ð 3.4 kb; GAP- 43 Ð 1.6 kb.
presence of the other. For both RAR b and GAP-43 mRNA, the interaction was nonsignificant, demonstrating that the ethanol caused a decrease in the mRNA levels, regardless of whether or not retinol was present. The remaining experiments were conducted using RA, thereby circumventing the step of retinol oxidation. The level of RAR b mRNA expression was significantly increased by treatment with 10 ÿ 5 ±10 ÿ 6 M RA for 48 h (Fig. 3A). The increase in RAR b mRNA was also seen after 24-h treatment with 10 ÿ 6 M RA (data not shown). No earlier time points were evaluated. The expression of RAR b was downregulated by 150 mM ethanol; the responses to 75 and 100 mM ethanol were not significantly lower (Fig. 3B). No differences in cell growth occurred due to RA (or retinol) or ethanol during the 48h treatment period. Morphological changes were observed after 96 h; these were not evaluated since they occurred
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later than the expression data presented here. The response of RAR a mRNA to RA and ethanol was also measured; neither ethanol nor RA affected RAR a in LAN-5 cells (data not shown). The interaction between the effect of RA and ethanol was determined at RA concentrations that caused an increase in RAR b (10 ÿ 5 ±10 ÿ 6 M). At 10 ÿ 5 M RA, the interaction between treatments was significant; i.e., ethanol did not cause a decrease in RAR b mRNA when RA was present (Fig. 4A). However, at 10 ÿ 6 M RA, the interaction was not significant, showing that at this lower level of RA, ethanol did reduce RAR b mRNA expression. The effect of the protein synthesis inhibitor, CX, on RAR b mRNA was determined using 10 ÿ 6 M RA and 150 mM ethanol for 48 h, since these parameters maximized treatment effects on both RAR b and GAP-43 mRNAs (see below). Analyses of RA and CX interactions demonstrated that the stimulation of RAR b mRNA by RA still occurred in the presence of 0.2 and 0.4 mg/ml CX (Fig. 4B). A significant interaction between the effect of ethanol and CX indicated that downregulation of RAR b mRNA expression by ethanol was abolished in the presence of both levels of CX. GAP-43 mRNA expression levels were increased by 48h treatment with 10 ÿ 5 ± 10 ÿ 9 M RA (Fig. 3A). As with RAR b, levels were higher after 24 h of RA treatment (data not shown). Ethanol at both 75 and 150 mM significantly downregulated the expression of GAP-43 (Fig. 3B). The interaction between the effect of RA and ethanol on GAP-43 expression was determined at 10 ÿ 5 ±10 ÿ 7 M RA
Fig. 2. Effect of retinol (ROL) and ethanol (ET) on expression of (A) RAR b and (B) GAP- 43 mRNA. LAN-5 cells were treated with 10 ÿ 6 M retinol and/or 150 mM ethanol for 48 h. Values are expressed relative to untreated controls (Cont) and are means SEM of two replicates from three experiments. Significant difference from control ( p < 0.05) indicated by *. For both RAR b and GAP-43, there is a significant main effect of ROL and of ethanol ( p < 0.05).
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Fig. 3. Dose effect of (A) RA and (B) ethanol on expression of RAR b and GAP mRNA. LAN-5 cells were treated with 10 ÿ 5 ± 10 ÿ 9 M RA or 75, 100, or 150 mM ethanol for 48 h. Values are expressed relative to untreated controls (Cont) and are means SEM of two to three replicates from six experiments (A) and of two to three replicates from three (75 and 100 mM) or nine (150 mM) experiments (B). Significant difference from control ( p < 0.05) indicated by *.
(Fig. 5A). As with RAR b mRNA, the interaction between treatments was significant at 10 ÿ 5 M RA, indicating that ethanol did not affect GAP-43 in the presence of RA. However, at lower RA concentrations (10 ÿ 6 and 10 ÿ 7 M), GAP-43 mRNA was reduced by ethanol.
The response of GAP-43 mRNA to RA and ethanol was also determined in the presence of CX. While RAR b mRNA increased with CX alone (Fig. 4B), both 0.2 and 0.4 mg/ml CX caused a 30% decrease in GAP-43 mRNA (Fig. 5B). A lack of significant interaction between RA and 0.2 mg/ml CX
Fig. 4. (A) The interaction of RA and ethanol (ET) on the expression of RAR b mRNA. LAN-5 cells were treated with 10 ÿ 5 and 10 ÿ 6 M RA with and without 150 mM ethanol for 48 h. Values are expressed relative to untreated controls (Cont) and are means SEM of two to four replicates from four experiments. Significant difference from control ( p < 0.05) indicated by *. At each level of RA, there is a significant main effect of RA and of ethanol ( p < 0.01). At 10 ÿ 5 M RA, the interaction of RA and ethanol is significant ( p = 0.02). (B) The interaction of CX and RA or ET on the expression of RAR b mRNA. LAN-5 cells were treated with 0.2 or 0.4 mg/ml CX and either 10 ÿ 6 M RA or 150 mM ethanol for 48 h. Values are expressed relative to untreated controls and are means SEM of three replicates from two experiments. Significant difference from control ( p < 0.05) indicated by *. At each level of CX, there is a significant main effect of RA and of ethanol ( p < 0.05). Significant interactions include: 0.2 mg/ml CX and RA ( p = 0.03), 0.2 mg/ml CX and ethanol ( p = 0.001), and 0.4 mg/ml CX and ethanol ( p = 0.003).
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Fig. 5. (A) The interaction of RA and ethanol (ET) on the expression of GAP- 43 mRNA. LAN-5 cells were treated with 10 ÿ 5 ± 10 ÿ 7 M RA with and without 150 mM ethanol for 48 h. Values are expressed relative to untreated controls (Cont) and are means SEM of two to four replicates from four experiments. Significant difference from control ( p < 0.05) indicated by *. At each level of RA, there is a significant main effect of RA and of ethanol ( p < 0.05). At 10 ÿ 5 M RA, the interaction of RA and ethanol is significant ( p = 0.04). (B) The interaction of CX and RA or ethanol on the expression of GAP-43 mRNA. LAN-5 cells were treated with 0.2 or 0.4 mg/ml CX and either 10 ÿ 6 M RA or 150 mM ethanol for 48 h. Values are expressed relative to untreated controls and are means SEM of three replicates from two experiments. Significant difference from control ( p < 0.05) indicated by *. At each level of CX, there is a significant main effect of RA and of ethanol ( p < 0.01). Significant interactions include: 0.4 mg/ml CX and RA ( p = 0.03), 0.2 mg/ml CX and ethanol ( p = 0.03).
indicates that the increase in GAP-43 mRNA caused by RA was not altered by the presence of 0.2 mg/ml CX. However, a significant interaction did result at 0.4 mg/ml CX, demonstrating that the response of GAP-43 mRNA to RA was abolished by CX. As with RAR b, a significant interaction between the effect of ethanol and 0.2 mg/ml CX indicated that the downregulation of GAP-43 mRNA expression caused by ethanol did not occur in the presence CX (Fig. 5B). Although the results appear similar at 0.4 mg/ml CX, the statistical analyses did not demonstrate an interaction of treatments. 3. Discussion It has been well documented that normal neural development requires RA and is disrupted by ethanol. Many different areas of research have supported the hypothesis that a disruption of RA function required for normal development by prenatal exposure to ethanol contributes to some of the neuropathology found in the offspring of mothers who consume ethanol while pregnant. This current work advances previous findings in developing rat embryo and brain exposed to ethanol [19] using the LAN-5 neuroblastoma cell line. Similarities between neuronal precursors and neuroblastoma cells make them useful models for studying neuronal development. The presence of RARs [14,22,45,54], the ability to metabolize retinol [9], and the induction of morphological differentiation by RA [2,6,22] make neuroblastoma cells such as LAN-5 an appropriate model for studying
the interaction of RA and ethanol. The ethanol concentrations of 75 ±150 mM used in these experiments fall within the range of 22± 175 mM ethanol used in previous studies in neuroblastoma cells [29]. In this LAN-5 model, retinol increased RAR b and GAP43 mRNA expression; ethanol decreased their expression levels, even in the presence of retinol. Although the ability of LAN-5 cells to oxidize retinol has been reported [9], the response of RAR b and GAP-43 mRNA to retinol and ethanol, and the effect of ethanol on the retinoid response have not been demonstrated previously. RA increased RAR b mRNA expression, which was specific for RAR b, as RAR a mRNA expression was unaffected. Ethanol did not affect the level of RAR b mRNA in the presence of 10 ÿ 5 M RA, but at 10 ÿ 6 M RA, ethanol did cause a decrease in RAR b mRNA. Thus, it appears that sufficient levels of exogenous RA can overcome the effect of ethanol on RAR b mRNA. When RA levels are lower, the effect of ethanol may become apparent. As a mechanism whereby ethanol may affect vitamin A metabolism, it has been proposed that ethanol competitively inhibits the synthesis of RA from retinol via a form of alcohol dehydrogenase important in the oxidation of retinol to retinaldehyde, which is then oxidized to RA [12]. A decrease in the supply of RA would decrease RAR b expression, since RA induces RAR b through a RARE within the promoter of the RAR b gene [32]. Although the concept is controversial [53], decreasing RA levels by inhibition of its synthesis could be a method whereby ethanol
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treatment leads to downregulation of RAR b mRNA. This hypothesis of ethanol competitively inhibiting retinol oxidation is not suggested by the results reported here. The ethanol-induced decrease in RAR b mRNA expression in the presence of retinol and the lack of an ethanol effect with 10 ÿ 5 M RA could be indicative of inhibited retinol oxidation. However, the finding that ethanol decreases RAR b mRNA when 10 ÿ 6 M RA is added does not support inhibition of RA synthesis as the regulatory step. Another mechanism by which ethanol may affect the RA supply is through increasing RA catabolism. Recently, a novel family of cytochrome P450 proteins was found which is involved in the oxidation of RA [13,42,51]. CYP26 is present in early embryos, is expressed in regions of the brain, and is upregulated upon RA-induced differentiation of embryonic stem cells [42]. The enzyme appears to play an autoregulatory role in controlling RA levels [51]. A major pathway for ethanol oxidation also involves a member of the P450 family, which is induced by ethanol in liver [40] and also in brain [50]. Similar inductive effects of ethanol on the RA-metabolizing CYP26 enzyme could lead to reduced levels of RA. Although this has not been examined to date, an ethanol-induced increase in RA catabolism could account for the inability of ethanol to reduce RAR b mRNA expression when protein synthesis was inhibited by CX, as reported here. These studies have additionally demonstrated that protein synthesis is not required for the increase in RAR b mRNA induced by RA. RA stimulation of RAR b gene expression has been shown to be independent of protein synthesis in many cell types [8,21,37,55]. Of interest is that the increase in RAR b gene expression by RA was greater in the presence of CX. This enhanced expression may be related to the finding that CX blocks the P450-induced catabolism of RA [25], which would increase RA levels. It is clear that GAP-43 has a fundamental role in brain development, and there is ample evidence indicating possible regulation of GAP-43 through RA. Therefore, another objective of this work was to evaluate the effects of retinoids and ethanol on GAP-43 mRNA expression, which have not yet been reported. The RA-induced increase in GAP-43 mRNA expression and the ethanol-induced decrease extend previous findings on GAP-43 protein levels [44]. In this present study, there was no effect of ethanol on GAP-43 mRNA at 10 ÿ 5 M RA, but at 10 ÿ 6 and 10 ÿ 7 M RA, the ethanol effect was apparent, again suggesting that sufficient levels of RA can eliminate the effect of ethanol. For both RAR b and GAP-43, ethanol was able to modify the increase in expression caused by the 10 ÿ 6 ± 10 ÿ 7 M RA. Although these RA levels still had a pronounced effect on RAR b and GAP-43 expression in the presence of ethanol, the degree to which ethanol reduced that expression was the same whether or not RA was present. The significance of the amount of change in mRNA expression occurring is not known at this time. What is apparent is that ethanol alters the normal response to RA.
The effects of CX on the response of GAP-43 mRNA to RA and ethanol have not been examined previously to this report. The observations suggest regulatory mechanisms for GAP-43 expression, especially when considered in conjunction with the responses of RAR b mRNA to CX. The diminished response of GAP-43 mRNA to RA at 0.4 mg/ ml CX suggests that protein synthesis may be required in the response of GAP-43 mRNA to RA. Since the interference in the response to RA by CX occurred even though the level of RAR b mRNA was highly elevated in the presence of RA and CX, the regulatory effect of RA on GAP-43 expression does not appear to be directly through RAR b. As with RAR b, CX prevented the effect of ethanol on GAP-43 mRNA. Thus, these studies indicate that ethanol regulation of RAR b and GAP-43 mRNA expression requires protein synthesis. The exact nature of this step is not known. Multiple proteins and mechanisms may be involved, such as the process of mRNA stabilization, which is a known method of GAP-43 regulation [39]. Since GAP43 mRNA expression does not seem to correlate directly with RAR b levels, it does not seem likely that an ethanolinduced decrease in RAR b directly modifies the response of GAP-43 to ethanol. These studies, taken together, demonstrate in a homogeneous neuroblastoma cell line that ethanol interacts with the effects of retinol and RA on RAR b and GAP-43 mRNA expression. Increased RA catabolism or mechanisms not related to RA levels may be involved. For example, ethanol may affect co-repressor and co-activator factors that interact with the RARs to inhibit or enhance transcriptional activity [38]. In this manner, RARs may indirectly alter the expression of genes such as GAP-43 that play an important role in neural development. The results of this study support the hypothesis that ethanol affects the function of retinoids in development through RARs. However, more work is needed to identify RA-dependent developmental processes that are altered by ethanol to establish a link between vitamin A and FAS.
Acknowledgments The authors would like to thank Peter Crump, Computing and Biometry Consultant, College of Agriculture and Life Sciences, University of Wisconsin, Madison, for his assistance with statistical analyses. This study was supported, in part, by grants from the March of Dimes Birth Defects Foundation (no. 6FY-970573) and the University of Wisconsin Graduate School.
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