- Email: [email protected]
Lithium stimulates gene expression through the AP-1 transcription factor pathway
Molecular Brain Research 58 Ž1998. 225–230
Short communication
Lithium stimulates gene expression through the AP-1 transcription factor pathway Pei-Xong Yuan, Guang Chen, Li-Dong Huang, Husseini K. Manji
)
Molecular Pathophysiology Program, Department of Psychiatry and BehaÕioral Neurosciences, and Pharmacology, WSU School of Medicine, 5V, DRH, 4201 St. Antoine BlÕd., Detroit, MI 48201, USA Accepted 21 April 1998
Abstract Lithium, a monovalent cation, is the mainstay in the treatment of manic-depressive ŽMDI. illness, but despite extensive research, its mechanism of action remains to be elucidated. Since lithium requires chronic administration for therapeutic efficacy, and because its beneficial effects last well beyond its discontinuation, it has been postulated that lithium may exert major effects at the genomic level. In the present study we found that lithium, at therapeutically relevant concentrations, increases AP-1 DNA binding activity in human SH-SY5Y cells and rat C6 glioma cells. Additionally, in both SY5Y and C6 cells transiently transfected with a reporter gene vector driven by an SV40 promoter, lithium increased the activity of the reporter gene in a time- and concentration-dependent manner. Furthermore, mutations in the AP-1 sites of the reporter gene promoter significantly attenuated lithium’s effects. These data indicate that lithium stimulates gene expression through the AP-1 transcription factor pathway, effects which may play a role in its long-term mood-stabilizing effects. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Lithium; Gene expression; AP-1; GSK-3; Manic depressive; PKC
The discovery of lithium’s efficacy as a mood-stabilizing agent revolutionized the treatment of patients with manic-depressive illness, but despite its role as one of psychiatry’s most important treatments w8,23,24x, the biochemical basis for lithium’s therapeutic actions remains to be fully elucidated w13,21x. Lithium’s beneficial effects requires a lag period for onset of action, and are generally not immediately reversed upon discontinuation; such patterns of effects suggest alterations at the genomic level w11,13,20x. In this context, it is noteworthy that several independent laboratories have demonstrated that lithium, at therapeutically relevant concentrations, regulates not only the expression of fos protooncogene w7,14x, but also AP-1 DNA binding activity w12,22,26,28x. These effects have the potential to regulate patterns of gene expression in critical neuronal circuits w3,6,19x, thereby mediating the drug’s long term stabilization of mood. In the present study, we
) Corresponding author. [email protected]
Fax:
q 1-313-966-7820;
E-mail:
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 1 1 4 - 4
have sought to examine more directly the hypothesis that lithium can regulate gene expression via its effects on AP-1 DNA binding activity. We have found that in neuronal cells transiently transfected with a reporter gene vector driven by an SV40 promoter, lithium increased the activity of the reporter gene in a time- and concentrationdependent manner; furthermore, mutations in the AP-1 sites of the reporter gene promoter markedly attenuated lithium’s effects. Materials. All cell culture medium and reagents, calcium–phosphate transfection system, and lipofectin were obtained from GIBCO BRL ŽGaithersburg, MD.. pGL2control vector was obtained from Promega. QuikChange site directed mutagenesis Kit was obtained from Stratagene ŽLa Jolla, CA.. Thermal cycle sequence kit was obtained from New England BioLabs. Plasmid Mini and midi kits were obtained from Qiagene. w g-32 Px ATP was from Amersham. LiCl and all other chemicals were obtained from Sigma. Cell culture and treatments. Human neuroblastoma SHSY5Y cells and rat C6 glioma cells were cultured and
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P.-X. Yuan et al.r Molecular Brain Research 58 (1998) 225–230
treated with LiCl for AP-1 DNA binding assays using previously described methods w4x. In brief, the cells were cultured in a humidified atmosphere of 95% airr5% CO 2 at 378C. Dulbecco’s modified Eagle’s medium plus 5% fetal bovine serum and Minimum Essential Medium plus 10% fetal bovine serum were used for C6 and SY5Y cell cultures, respectively. Both media were also supplied with 100 IUrml of penicillin and 50 m grml of streptomycin. AP-1 DNA binding assay. The AP-1 DNA binding assay was performed in whole cell extracts using previously described methods w4x. In brief, the consensus oligo ŽAP-1: 5X-CGC TTG ATG ACT CAG CCG GAA-3X . was labeled with w g-32 Px ATP using T4 kinase according to the manufacturers specifications ŽGIBCO BRL.. Free w g-32 Px ATP was separated from labeled oligos using a pushing column from Stratagene. The washed cells were lysed, and the lysates were sonicated for 10 s. and then centrifuged at 14,000 = g for 15 min to remove residual debris. The whole cell extracts were incubated with 32 P labeled oligos at 378C for 15 min. The DNA binding reaction was terminated by the addition of 5 = loading buffer, and the reaction mixtures were then subjected to gel electrophoresis using a 6% DNA retardation gel. The specificity of the DNA binding assay was confirmed by demonstrating that the addition of 50 fold excess of unlabeled AP-1 oligos,
but not mutant AP-1 oligos completely blocked AP-1 binding in whole C6 cell extracts. Furthermore, we confirmed that an antibody against the jun family of proteins ‘supershifted’ the AP-1 binding band, prior to undertaking the lithium studies. Reporter gene vector and mutations. In the pGL2-control vector from Promega, the reporter gene luciferase is driven by an SV40 promoter. Mutations in the TRE element were made using the QuikChange site directed mutagenesis Kit from Stratagene with the primers indicated in Fig. 3. The mutations were confirmed by sequence analysis using the Thermal cycle sequence kit from New England BioLabs. Both pGL2-control and pGL2-mutant plasmids were amplified in JM109 E. coli. Plasmid DNA was purified using the Qiagene kit. In all the experiments using these kits, the manufacturer’s instructions were followed without any modifications. Transient transfection and luciferase activity assay. C6 cells were transfected using the calcium–phosphate transfection system. SH-SY5Y cells were transfected using lipofectin reagents. The transfections were conducted according to the manufacturer’s instructions without any modifications. After the transfection, the cells were incubated in growth medium for an additional 24 h. The cells were then exposed to LiCl Ž1.0 mM., RbCl Ž1.0 mM., or
Fig. 1. Increases in TRE binding activity by lithium. Rat C6 glioma cells ŽA. and human neuroblastoma SH-SY5Y cells ŽB. were incubated in growth media with 1.0 mM of LiCl for 24 h. The cells were then washed with phosphate buffered saline and lysed in the extraction buffer. TRE binding was performed in whole cell extracts. The autoradiograms were analyzed using an image analysis system with NIH 1.55 software. The values are the means " S.E. from three or more experiments.
P.-X. Yuan et al.r Molecular Brain Research 58 (1998) 225–230
KCl Ž1.0 mM.. The expression of the luciferase gene was measured in whole cells lysates using the Promega luciferase assay kit. Statistics. Statistical analysis was performed by analysis of variance, followed by Fisher’s PLSD or Scheffe’s tests. p - 0.05 was considered significant. Data are expressed as the means" S.E. Results and discussion. Incubation of the cells with 1.0 mM of lithium wthe therapeutically relevant concentration in the treatment of manic-depressive illness Žrange 0.5 to 1.2 mM. w8,24x, resulted in a 50% increase in AP-1 DNA binding activity in C6 cells and a 2 fold increase in TRE binding activity in SY5Y cells ŽFig. 1.. We next sought to investigate if the effects of lithium on the DNA binding activity of AP-1 produces any changes in the expression of a gene driven by an AP-1 containing promoter. Alignment of both the SV40 genome and pGL2-control reveals that
227
the promoter of the luciferase gene in pGL control contains a high affinity TRE element Ždiscussed below.. Incubation of both SY5Y cells and C6 glioma cells with lithium produced a concentration- and time-dependent increase in luciferase activity ŽFig. 2.. Statistically significant increases were observed at 24 h in both SY5Y and C6 cells incubated with lithium at concentrations above 0.8 mM; significant increases in luciferase activity were observed in both cell types after 8 h or more when they were incubated with 1 mM lithium ŽFig. 2.. Together, the AP-1 DNA binding data and luciferase data suggest that lithium may stimulate gene expression, at least in part, through the AP-1 transcription factor pathway. To test the specificity of lithium’s effects, SY5Y cells transfected with the luciferase gene were incubated with other alkali earth metals. In striking contrast to the results observed with lithium, incubation of the SY5Y cells to either KCl Ž1 mM. or
Fig. 2. Increases in luciferase activity induced by lithium: time and concentration-dependency. Rat C6 glioma cells ŽA and B. and human neuroblastoma SH-SY5Y cells ŽC and D. were transfected with pGL2-control vectors, and then incubated with lithium at the concentrations indicated in the figure ŽA and C. for 24 h or with lithium Ž1.0 mM. for the times indicated in the figure ŽB and D.. The cells were washed and lysed in the assay buffer. Luciferase activity was assayed using the Promega kit. Values are means" S.E. of three or more experiments.
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Fig. 3. Increases in luciferase activity induced by lithium: cation specificity. Human neuroblastoma SH-SY5Y cells were transfected with pGL2-control vectors, and then incubated with LiCl Ž1 mM., RbCl Ž1 mM., or KCl Ž1 mM. for 24 h. The cells were washed and lysed in the assay buffer. Luciferase activity was assayed using the Promega kit. Values are means"S.E. of three or more experiments.
RbCl Ž1 mM. for 24 h was almost completely without any effect on luciferase activity ŽFig. 3.. We next wished to confirm the role of the AP-1 DNA binding sites in mediating the lithium-induced increase in luciferase activity. Alignment of both the SV40 genome and pGL2-control ŽFig. 4. shows that the promoter of the luciferase gene in pGL2-control contains two SV40 TREs and further that one of them is a high affinity site w4x. It has been shown that the insertion of this AP-1 DNA binding element into a TK-CAT vector results in a several fold increase in CAT expression in response to TPA w1,19x. Additionally, we found that incubation of pGL2-control transfected C6 cells or SY5Y cells with TPA Ž1 m M. for 2 h resulted in 3–4 fold increases in luciferase activity Ždata not shown.. To confirm the role of the AP-1 transcription factor pathway in lithium’s effect on luciferase activity, we
made mutations of these two TRE elements in the promoter region; the mutations were made using site-directed mutagenesis methods with the primers indicated in Fig. 4. The mutations were then confirmed by sequence analysis ŽFig. 5.. The mutations resulted in a 50% decrease in basal luciferase activity. To test that these mutations did in fact abolish AP-1 DNA binding, the cells were incubated with a PKC activator ŽPMA, 1 m M. for 1 h. The mutations of the two TRE elements completely abolished the luciferase response to PKC activation ŽFig. 5., consistent with an elimination of AP-1 DNA binding w1,18x. In the cells transfected with the mutant pGL2, the effects of lithium were markedly attenuated. Thus, lithium only induced a modest 22.34 " 10.75% increase in luciferase activity in the cells transfected with the mutant pGL2, effects which were not significantly different from control cells Ž0 mM lithium. and only 10% of the lithium-induced increase in the cells transfected with the wild type pGL2 ŽFig. 5.. In summary, we have demonstrated that lithium, at therapeutically relevant concentrations, stimulates gene expression through the AP-1 transcription factor pathway in neuronal cells. Thus, the complex effects of lithium on AP-1 DNA binding activity that have been previously demonstrated w12,22,26,28x do in fact produce changes in AP-1 mediated gene expression. Since very similar effects on AP-1 DNA binding w4x and luciferase gene expression w5x have also been observed after incubation with the structurally dissimilar, but clinically efficacious w2x agent, valproic acid w5x, these effects may play a role in the long-term therapeutic effects of these agents. The precise mechanisms by which lithium brings about these changes remains to be fully elucidated. The AP-1 transcription factors are the downstream targets of mitogen activated protein kinases ŽMAPK. pathways w15,16,27x, and several lines of evidence have indicated protein kinase C ŽPKC. activates these pathways w27x. In view of the evidence demonstrating significant effects of lithium on the PKC signaling pathway Žreviewed in Refs. w13,20,21x., it is possible that lithium may increase AP-1 DNA binding activity via the PKC-MAPK pathways. In addition, it is
Fig. 4. Alignment of the pGL2-control promoter region, SV 40 genome and mutation primers. The DNA sequences of pGL2-control ŽNID: g311874. and SV 40 genome ŽNID: 965480. were obtained from GenBank. The TREs are indicated by the top line.
P.-X. Yuan et al.r Molecular Brain Research 58 (1998) 225–230
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Fig. 5. Attenuation of the lithium induced increases in luciferase activity by TRE mutations. The mutations on the TRE sites were made using the primers described in Fig. 3, and the QuikChange site directed mutagenesis Kit. The autoradiograms show the sequences of the two TRE sites in the pGL2-control and the mutant pGL2-control vectors ŽA.. C6 cells were transfected with either pGL2-control or mutant pGL2-control and then incubated with 1 mM PMA for 1 h ŽB. or 1 mM of lithium for 24 h ŽC.. The cells were washed and lysed in the assay buffer. Luciferase activity was assayed using the Promega kit. The values in the bar graphs are the means " S.E. of three or more experiments.
noteworthy that lithium has recently been demonstrated to inhibit the activity of glycogen synthase kinase-3 ŽGSK-3. w9,17,25x. Since GSK-3 phosphorylates c-jun at three sites adjacent to the DNA binding domain thereby reducing AP-1 binding w19,25x, it is also possible that lithium’s effects are mediated Žin part. via its inhibition of GSK-3. Among the many genes in the brain known to be driven by AP-1 containing promoters are those for key enzymes involved in neurotransmitter synthesis, neuropeptides, neurotrophins, and transcription factors w10x; the regulation of some of these genes in critical areas of the brain is currently under investigation w6x.
Acknowledgements We thank D.B. Hawver, PhD, for providing SH-SY5Y cells, and Ms. Celia Knobelsdorf for editorial assistance. This work was supported in part by the Joseph Young Sr.
Foundation and the Theodore and Vada Stanley Foundation.
References w1x P. Angel, M. Imagawa, R. Chiu, B. Stein, R.J. Imbra, H.J. Rahmsdorf, C. Jonat, P. Herrlich, M. Karin, Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor, Cell 49 Ž1987. 729–739. w2x C.L. Bowden, A.M. Brugger, A.C. Swann, J.R. Calabrese, P.G. Janicak, F. Petty, S.C. Dilsaver, J.M. Davis, A.J. Rush, J.G. Small, Efficacy of divalproex vs. lithium and placebo in the treatment of mania, JAMA 271 Ž1994. 918–924. w3x W.J. Boyle, T. Smeal, L.H. Defize, P. Angel, J.R. Woodgett, M. Karin, T. Hunter, Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity, Cell 64 Ž1991. 573–584. w4x G. Chen, P.X. Yuan, D.B. Hawver, W.Z. Potter, H.K. Manji, Increase in AP-1 transcription factor DNA binding activity by valproic acid, Neuropsychopharmacology 16 Ž1997. 238–245.
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w5x G. Chen, P.X. Yuan, H.K. Manji, Regulation of transcription factor DNA binding activity by mood stabilizing agents, Abstracts of the American College of Neuropsychopharmacology Annual Meeting, HI, December 1997. w6x G. Chen, P.X. Yuan, Y.M. Jiang, L.D. Huang, H.K. Manji, Lithium increases tyrosine hydroxylase levels both in vivo and in vitro, J. Neurochem. 70 Ž1998. 1768–1771. w7x M.M. Divish, G. Sheftel, A. Boyle, V.D. Kalasapudi, D.F. Papolos, H.M. Lachman, Differential effect of lithium on fos protooncogene expression mediated by receptor and postreceptor activators of protein kinase C and cyclic adenosine monophosphate: model for its antimanic action, J. Neurosci. Res. 28 Ž1. Ž1991. 40–48. w8x F. Goodwin, K.R. Jamison, Manic-Depressive Illness, Oxford Univ. Press, New York, 1990. w9x C.M. Hedgepeth, L.J. Conrad, J. Zhang, H.C. Huang, V.M.Y. Lee, P.S. Klein, Activation of the Wnt signaling pathway: a molecular mechanism for lithium action, Dev. Biol. 185 Ž1997. 82–91. w10x P. Hughes, M. Dragunow, Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system, Pharmacol. Rev. 47 Ž1995. 133–178. w11x S.E. Hyman, E.J. Nestler, Initiation and adaptation: a paradigm for understanding psychotropic drug action, Am. J. Psychiatry 153 Ž1996. 151–162. w12x R.S. Jope, L. Song, AP-1 and NF-k B stimulated by carbachol in human neuroblastoma SH-SY5Y cells are differentially sensitive to inhibition by lithium, Mol. Brain Res. 50 Ž1997. 171–180. w13x R.S. Jope, M.B. Williams, Lithium and brain signal transduction systems, Biochem. Pharmacol. 77 Ž1994. 429–441. w14x V.D. Kalasapudi, G. Sheftel, M.M. Divish, D.F. Papolos, H.M. Lachman, Lithium augments fos protoonocogene expression in PC12 pheochromocytoma cells: implications for therapeutic action of lithium, Brain Res. 521 Ž1–2. Ž1990. 47–54. w15x M. Karin, The regulation of AP-1 activity by mitogen-activated protein kinases, J. Biol. Chem. 270 Ž1995. 16483–16486. w16x M. Karin, T. Smeal, Control of transcription factors by signal transduction pathways: the beginning of the end, TIBS 17 Ž1992. 418–422.
w17x P.S. Klein, D.A. Melton, A molecular mechanism for the effect of lithium on development, Proc. Natl. Acad. Sci. U.S.A. 93 Ž1996. 8455–8459. w18x W. Lee, P. Mitchell, R. Tjian, Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements, Cell 49 Ž1987. 741–752. w19x A. Lin, T. Smeal, B. Binetruy, T. Deng, J.C. Chambard, M. Karin, Control of AP-1 activity by signal transduction cascades, Adv. Second Messenger Phosphoprotein Res. 28 Ž1993. 255–260. w20x H.K. Manji, R.H. Lenox, Long-term action of lithium: a role for transcriptional and posttranscriptional factors regulated by protein kinase C, Synapse 16 Ž1994. 11–28. w21x H.K. Manji, W.Z. Potter, R.H. Lenox, Signal transduction pathways. Molecular targets for lithium’s actions, Arch. Gen. Psychiatry 52 Ž1995. 531–543. w22x N. Ozaki, D.M. Chuang, Lithium increases transcription factor binding to AP-1 and cyclic AMP-responsive element in cultured neurons and rat brain, J. Neurochem. 69 Ž1997. 2336–2344. w23x A. Reifman, R.J. Wyatt, Lithium: a brake in the rising cost of mental illness, Arch. Gen. Psychiatry 37 Ž1980. 385–388. w24x M. Schou, Clinical aspects of lithium in psychiatry, in: N.J. Birch ŽEd.., Lithium and the Cell, Academic Press, London, 1991, pp. 1–6. w25x V. Stambolic, Ruel, R. Laurent, J.R. Woodgett, Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells, Curr. Biol. 6 Ž1996. 1664–1668. w26x M.T. Unlap, R.S. Jope, Lithium attenuates nerve growth factor-induced activation of AP-1 DNA binding activity in PC12 cells, Neuropsychopharmacology 17 Ž1. Ž1997. 12–17. w27x A.J. Whitmarsh, R.J. Davis, Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways, J. Mol. Med. 74 Ž1996. 589–607. w28x M.B. Williams, R.S. Jope, Circadian variation in rat brain AP-1 DNA binding activity after cholinergic stimulation: modulation by lithium, Psychopharmacology ŽBerl.. 122 Ž4. Ž1995. 363–368.
Short communication
Lithium stimulates gene expression through the AP-1 transcription factor pathway Pei-Xong Yuan, Guang Chen, Li-Dong Huang, Husseini K. Manji
)
Molecular Pathophysiology Program, Department of Psychiatry and BehaÕioral Neurosciences, and Pharmacology, WSU School of Medicine, 5V, DRH, 4201 St. Antoine BlÕd., Detroit, MI 48201, USA Accepted 21 April 1998
Abstract Lithium, a monovalent cation, is the mainstay in the treatment of manic-depressive ŽMDI. illness, but despite extensive research, its mechanism of action remains to be elucidated. Since lithium requires chronic administration for therapeutic efficacy, and because its beneficial effects last well beyond its discontinuation, it has been postulated that lithium may exert major effects at the genomic level. In the present study we found that lithium, at therapeutically relevant concentrations, increases AP-1 DNA binding activity in human SH-SY5Y cells and rat C6 glioma cells. Additionally, in both SY5Y and C6 cells transiently transfected with a reporter gene vector driven by an SV40 promoter, lithium increased the activity of the reporter gene in a time- and concentration-dependent manner. Furthermore, mutations in the AP-1 sites of the reporter gene promoter significantly attenuated lithium’s effects. These data indicate that lithium stimulates gene expression through the AP-1 transcription factor pathway, effects which may play a role in its long-term mood-stabilizing effects. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Lithium; Gene expression; AP-1; GSK-3; Manic depressive; PKC
The discovery of lithium’s efficacy as a mood-stabilizing agent revolutionized the treatment of patients with manic-depressive illness, but despite its role as one of psychiatry’s most important treatments w8,23,24x, the biochemical basis for lithium’s therapeutic actions remains to be fully elucidated w13,21x. Lithium’s beneficial effects requires a lag period for onset of action, and are generally not immediately reversed upon discontinuation; such patterns of effects suggest alterations at the genomic level w11,13,20x. In this context, it is noteworthy that several independent laboratories have demonstrated that lithium, at therapeutically relevant concentrations, regulates not only the expression of fos protooncogene w7,14x, but also AP-1 DNA binding activity w12,22,26,28x. These effects have the potential to regulate patterns of gene expression in critical neuronal circuits w3,6,19x, thereby mediating the drug’s long term stabilization of mood. In the present study, we
) Corresponding author. [email protected]
Fax:
q 1-313-966-7820;
E-mail:
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 1 1 4 - 4
have sought to examine more directly the hypothesis that lithium can regulate gene expression via its effects on AP-1 DNA binding activity. We have found that in neuronal cells transiently transfected with a reporter gene vector driven by an SV40 promoter, lithium increased the activity of the reporter gene in a time- and concentrationdependent manner; furthermore, mutations in the AP-1 sites of the reporter gene promoter markedly attenuated lithium’s effects. Materials. All cell culture medium and reagents, calcium–phosphate transfection system, and lipofectin were obtained from GIBCO BRL ŽGaithersburg, MD.. pGL2control vector was obtained from Promega. QuikChange site directed mutagenesis Kit was obtained from Stratagene ŽLa Jolla, CA.. Thermal cycle sequence kit was obtained from New England BioLabs. Plasmid Mini and midi kits were obtained from Qiagene. w g-32 Px ATP was from Amersham. LiCl and all other chemicals were obtained from Sigma. Cell culture and treatments. Human neuroblastoma SHSY5Y cells and rat C6 glioma cells were cultured and
226
P.-X. Yuan et al.r Molecular Brain Research 58 (1998) 225–230
treated with LiCl for AP-1 DNA binding assays using previously described methods w4x. In brief, the cells were cultured in a humidified atmosphere of 95% airr5% CO 2 at 378C. Dulbecco’s modified Eagle’s medium plus 5% fetal bovine serum and Minimum Essential Medium plus 10% fetal bovine serum were used for C6 and SY5Y cell cultures, respectively. Both media were also supplied with 100 IUrml of penicillin and 50 m grml of streptomycin. AP-1 DNA binding assay. The AP-1 DNA binding assay was performed in whole cell extracts using previously described methods w4x. In brief, the consensus oligo ŽAP-1: 5X-CGC TTG ATG ACT CAG CCG GAA-3X . was labeled with w g-32 Px ATP using T4 kinase according to the manufacturers specifications ŽGIBCO BRL.. Free w g-32 Px ATP was separated from labeled oligos using a pushing column from Stratagene. The washed cells were lysed, and the lysates were sonicated for 10 s. and then centrifuged at 14,000 = g for 15 min to remove residual debris. The whole cell extracts were incubated with 32 P labeled oligos at 378C for 15 min. The DNA binding reaction was terminated by the addition of 5 = loading buffer, and the reaction mixtures were then subjected to gel electrophoresis using a 6% DNA retardation gel. The specificity of the DNA binding assay was confirmed by demonstrating that the addition of 50 fold excess of unlabeled AP-1 oligos,
but not mutant AP-1 oligos completely blocked AP-1 binding in whole C6 cell extracts. Furthermore, we confirmed that an antibody against the jun family of proteins ‘supershifted’ the AP-1 binding band, prior to undertaking the lithium studies. Reporter gene vector and mutations. In the pGL2-control vector from Promega, the reporter gene luciferase is driven by an SV40 promoter. Mutations in the TRE element were made using the QuikChange site directed mutagenesis Kit from Stratagene with the primers indicated in Fig. 3. The mutations were confirmed by sequence analysis using the Thermal cycle sequence kit from New England BioLabs. Both pGL2-control and pGL2-mutant plasmids were amplified in JM109 E. coli. Plasmid DNA was purified using the Qiagene kit. In all the experiments using these kits, the manufacturer’s instructions were followed without any modifications. Transient transfection and luciferase activity assay. C6 cells were transfected using the calcium–phosphate transfection system. SH-SY5Y cells were transfected using lipofectin reagents. The transfections were conducted according to the manufacturer’s instructions without any modifications. After the transfection, the cells were incubated in growth medium for an additional 24 h. The cells were then exposed to LiCl Ž1.0 mM., RbCl Ž1.0 mM., or
Fig. 1. Increases in TRE binding activity by lithium. Rat C6 glioma cells ŽA. and human neuroblastoma SH-SY5Y cells ŽB. were incubated in growth media with 1.0 mM of LiCl for 24 h. The cells were then washed with phosphate buffered saline and lysed in the extraction buffer. TRE binding was performed in whole cell extracts. The autoradiograms were analyzed using an image analysis system with NIH 1.55 software. The values are the means " S.E. from three or more experiments.
P.-X. Yuan et al.r Molecular Brain Research 58 (1998) 225–230
KCl Ž1.0 mM.. The expression of the luciferase gene was measured in whole cells lysates using the Promega luciferase assay kit. Statistics. Statistical analysis was performed by analysis of variance, followed by Fisher’s PLSD or Scheffe’s tests. p - 0.05 was considered significant. Data are expressed as the means" S.E. Results and discussion. Incubation of the cells with 1.0 mM of lithium wthe therapeutically relevant concentration in the treatment of manic-depressive illness Žrange 0.5 to 1.2 mM. w8,24x, resulted in a 50% increase in AP-1 DNA binding activity in C6 cells and a 2 fold increase in TRE binding activity in SY5Y cells ŽFig. 1.. We next sought to investigate if the effects of lithium on the DNA binding activity of AP-1 produces any changes in the expression of a gene driven by an AP-1 containing promoter. Alignment of both the SV40 genome and pGL2-control reveals that
227
the promoter of the luciferase gene in pGL control contains a high affinity TRE element Ždiscussed below.. Incubation of both SY5Y cells and C6 glioma cells with lithium produced a concentration- and time-dependent increase in luciferase activity ŽFig. 2.. Statistically significant increases were observed at 24 h in both SY5Y and C6 cells incubated with lithium at concentrations above 0.8 mM; significant increases in luciferase activity were observed in both cell types after 8 h or more when they were incubated with 1 mM lithium ŽFig. 2.. Together, the AP-1 DNA binding data and luciferase data suggest that lithium may stimulate gene expression, at least in part, through the AP-1 transcription factor pathway. To test the specificity of lithium’s effects, SY5Y cells transfected with the luciferase gene were incubated with other alkali earth metals. In striking contrast to the results observed with lithium, incubation of the SY5Y cells to either KCl Ž1 mM. or
Fig. 2. Increases in luciferase activity induced by lithium: time and concentration-dependency. Rat C6 glioma cells ŽA and B. and human neuroblastoma SH-SY5Y cells ŽC and D. were transfected with pGL2-control vectors, and then incubated with lithium at the concentrations indicated in the figure ŽA and C. for 24 h or with lithium Ž1.0 mM. for the times indicated in the figure ŽB and D.. The cells were washed and lysed in the assay buffer. Luciferase activity was assayed using the Promega kit. Values are means" S.E. of three or more experiments.
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Fig. 3. Increases in luciferase activity induced by lithium: cation specificity. Human neuroblastoma SH-SY5Y cells were transfected with pGL2-control vectors, and then incubated with LiCl Ž1 mM., RbCl Ž1 mM., or KCl Ž1 mM. for 24 h. The cells were washed and lysed in the assay buffer. Luciferase activity was assayed using the Promega kit. Values are means"S.E. of three or more experiments.
RbCl Ž1 mM. for 24 h was almost completely without any effect on luciferase activity ŽFig. 3.. We next wished to confirm the role of the AP-1 DNA binding sites in mediating the lithium-induced increase in luciferase activity. Alignment of both the SV40 genome and pGL2-control ŽFig. 4. shows that the promoter of the luciferase gene in pGL2-control contains two SV40 TREs and further that one of them is a high affinity site w4x. It has been shown that the insertion of this AP-1 DNA binding element into a TK-CAT vector results in a several fold increase in CAT expression in response to TPA w1,19x. Additionally, we found that incubation of pGL2-control transfected C6 cells or SY5Y cells with TPA Ž1 m M. for 2 h resulted in 3–4 fold increases in luciferase activity Ždata not shown.. To confirm the role of the AP-1 transcription factor pathway in lithium’s effect on luciferase activity, we
made mutations of these two TRE elements in the promoter region; the mutations were made using site-directed mutagenesis methods with the primers indicated in Fig. 4. The mutations were then confirmed by sequence analysis ŽFig. 5.. The mutations resulted in a 50% decrease in basal luciferase activity. To test that these mutations did in fact abolish AP-1 DNA binding, the cells were incubated with a PKC activator ŽPMA, 1 m M. for 1 h. The mutations of the two TRE elements completely abolished the luciferase response to PKC activation ŽFig. 5., consistent with an elimination of AP-1 DNA binding w1,18x. In the cells transfected with the mutant pGL2, the effects of lithium were markedly attenuated. Thus, lithium only induced a modest 22.34 " 10.75% increase in luciferase activity in the cells transfected with the mutant pGL2, effects which were not significantly different from control cells Ž0 mM lithium. and only 10% of the lithium-induced increase in the cells transfected with the wild type pGL2 ŽFig. 5.. In summary, we have demonstrated that lithium, at therapeutically relevant concentrations, stimulates gene expression through the AP-1 transcription factor pathway in neuronal cells. Thus, the complex effects of lithium on AP-1 DNA binding activity that have been previously demonstrated w12,22,26,28x do in fact produce changes in AP-1 mediated gene expression. Since very similar effects on AP-1 DNA binding w4x and luciferase gene expression w5x have also been observed after incubation with the structurally dissimilar, but clinically efficacious w2x agent, valproic acid w5x, these effects may play a role in the long-term therapeutic effects of these agents. The precise mechanisms by which lithium brings about these changes remains to be fully elucidated. The AP-1 transcription factors are the downstream targets of mitogen activated protein kinases ŽMAPK. pathways w15,16,27x, and several lines of evidence have indicated protein kinase C ŽPKC. activates these pathways w27x. In view of the evidence demonstrating significant effects of lithium on the PKC signaling pathway Žreviewed in Refs. w13,20,21x., it is possible that lithium may increase AP-1 DNA binding activity via the PKC-MAPK pathways. In addition, it is
Fig. 4. Alignment of the pGL2-control promoter region, SV 40 genome and mutation primers. The DNA sequences of pGL2-control ŽNID: g311874. and SV 40 genome ŽNID: 965480. were obtained from GenBank. The TREs are indicated by the top line.
P.-X. Yuan et al.r Molecular Brain Research 58 (1998) 225–230
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Fig. 5. Attenuation of the lithium induced increases in luciferase activity by TRE mutations. The mutations on the TRE sites were made using the primers described in Fig. 3, and the QuikChange site directed mutagenesis Kit. The autoradiograms show the sequences of the two TRE sites in the pGL2-control and the mutant pGL2-control vectors ŽA.. C6 cells were transfected with either pGL2-control or mutant pGL2-control and then incubated with 1 mM PMA for 1 h ŽB. or 1 mM of lithium for 24 h ŽC.. The cells were washed and lysed in the assay buffer. Luciferase activity was assayed using the Promega kit. The values in the bar graphs are the means " S.E. of three or more experiments.
noteworthy that lithium has recently been demonstrated to inhibit the activity of glycogen synthase kinase-3 ŽGSK-3. w9,17,25x. Since GSK-3 phosphorylates c-jun at three sites adjacent to the DNA binding domain thereby reducing AP-1 binding w19,25x, it is also possible that lithium’s effects are mediated Žin part. via its inhibition of GSK-3. Among the many genes in the brain known to be driven by AP-1 containing promoters are those for key enzymes involved in neurotransmitter synthesis, neuropeptides, neurotrophins, and transcription factors w10x; the regulation of some of these genes in critical areas of the brain is currently under investigation w6x.
Acknowledgements We thank D.B. Hawver, PhD, for providing SH-SY5Y cells, and Ms. Celia Knobelsdorf for editorial assistance. This work was supported in part by the Joseph Young Sr.
Foundation and the Theodore and Vada Stanley Foundation.
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