Sp Family of Transcription Factors Is Involved in Valproic Acid-induced Expression of Gαi2
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m209430200
ISSN1083-351X
Autores Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoValproic acid-induced gene expression has been attributed to the DNA-binding activity of the transcription factor activator protein 1 (AP-1). Using K562 cells, we have studied valproic acid-induced transcription from the human Gαi2 gene promoter, which lacks AP-1-binding motifs. We find that valproic acid-induced expression of Gαi2 is inhibited by mithramycin A, a compound that interferes with Sp1 binding to GC boxes in DNA. Three Sp1-binding sequences, located at +68/+75, −50/−36, and −92/−85 in the promoter, accounted for about 60% of this transcriptional effect, as judged by transient transfection assays. Electrophoretic mobility shift assays indicated that these sites bind members of the Sp family of transcription factors. Binding to DNA was inhibited by mithramycin A and was greater in nuclear extracts from cells treated with valproic acid than in control cells. Okadaic acid, calyculin A, and fostriecin, which are potent inhibitors of protein phosphatase, suppressed the transcriptional response to valproic acid. This inhibitory effect was not observed when promoter constructs containing mutations in the referenced Sp1-binding sites were used for transfections. In nuclear extracts from cells cultured in the presence of these inhibitors, the binding of Sp1/Sp3 to DNA probes was much less than in control cells. Alkaline phosphatase treatment of nuclear extracts resulted in enhanced binding of Sp proteins to the DNA probes. These results are consistent with the idea that dephosphorylating conditions enhanced Sp binding to the DNA probes as well as Sp-mediated transcription induced by valproic acid. This study demonstrates that the gene expression-inducing effect of valproic acid occurs, in part, through the Sp family of transcription factors. Valproic acid-induced gene expression has been attributed to the DNA-binding activity of the transcription factor activator protein 1 (AP-1). Using K562 cells, we have studied valproic acid-induced transcription from the human Gαi2 gene promoter, which lacks AP-1-binding motifs. We find that valproic acid-induced expression of Gαi2 is inhibited by mithramycin A, a compound that interferes with Sp1 binding to GC boxes in DNA. Three Sp1-binding sequences, located at +68/+75, −50/−36, and −92/−85 in the promoter, accounted for about 60% of this transcriptional effect, as judged by transient transfection assays. Electrophoretic mobility shift assays indicated that these sites bind members of the Sp family of transcription factors. Binding to DNA was inhibited by mithramycin A and was greater in nuclear extracts from cells treated with valproic acid than in control cells. Okadaic acid, calyculin A, and fostriecin, which are potent inhibitors of protein phosphatase, suppressed the transcriptional response to valproic acid. This inhibitory effect was not observed when promoter constructs containing mutations in the referenced Sp1-binding sites were used for transfections. In nuclear extracts from cells cultured in the presence of these inhibitors, the binding of Sp1/Sp3 to DNA probes was much less than in control cells. Alkaline phosphatase treatment of nuclear extracts resulted in enhanced binding of Sp proteins to the DNA probes. These results are consistent with the idea that dephosphorylating conditions enhanced Sp binding to the DNA probes as well as Sp-mediated transcription induced by valproic acid. This study demonstrates that the gene expression-inducing effect of valproic acid occurs, in part, through the Sp family of transcription factors. activator protein 1 β-isoform of CCAAT box enhancer-binding protein isoform 2 of the α-subunit of inhibitory guanine nucleotide-binding regulatory protein electrophoretic mobility shift assay extracellular signal-regulated protein kinase histone deacetylase mitogen-activated protein kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase protein phosphatase isoform 1 and 2A, respectively Sp2, and Sp3, promoter-specific factor-binding proteins 1, 2, and 3 (also called specificity proteins 1, 2, and 3) Valproic acid (2-propylpentanoic acid), a branched short-chain fatty acid, is an anticonvulsant agent that is also clinically effective as a mood stabilizer in the treatment of manic depression (bipolar affective disorder) (1Joffe R.T. Can. J. Psychiatry. 1993; 38 (suppl.): 46-50Google Scholar, 2Mitchell P.B. Aust. NZ J. Psychiatry. 1999; 33 (suppl.): 99-107Crossref Google Scholar, 3Tunnicliff G. J. Physiol. Pharmacol. 1999; 50: 347-365PubMed Google Scholar, 4Sadock B.J. Sadock V.A. Comprehensive Textbook of Psychiatry. Lippincott Williams & Williams, Philadelphia2000: 1284-1440Google Scholar, 5Johannessaen C.U. Neurochem. Int. 2000; 37: 103-110Crossref PubMed Scopus (324) Google Scholar). The biochemical basis for the neurotrophic effects of valproic acid and other mood stabilizers has intrigued investigators for a long time, but at the present time the modes of action of these drugs are not clearly understood (4Sadock B.J. Sadock V.A. Comprehensive Textbook of Psychiatry. Lippincott Williams & Williams, Philadelphia2000: 1284-1440Google Scholar, 6Phiel C.J. Klein P.S. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 789-813Crossref PubMed Scopus (447) Google Scholar, 7Manji H.K. Moore G.J. Chen G. Brit. J. Psychiatry. 2001; 178 Suppl. 41: 107-119Crossref Scopus (128) Google Scholar). Among the effects of valproic acid is an increase in gene expression, an effect that has been demonstrated for the genes for Bcl-2 and growth cone-associated p43 in SH-SY5Y cells (8Yuan P.-X. Huang L.-D. Jiang Y.-M. Gutkind J.S. Manji H.K. J. Biol. Chem. 2001; 276: 31674-31683Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), Tcf/Lef-dependent transcription in 293T cells (9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar), and the tyrosine hydroxylase gene (10Chen G. Yuan P.-X. Jiang Y.-M. Huang L.-D. Manji H.K. J. Neurochem. 1998; 70: 1768-1771Crossref PubMed Scopus (64) Google Scholar, 11Sands S.A. Guerra V. Morilak D.A. Neuropsychopharmocology. 2000; 22: 27-35Crossref PubMed Scopus (47) Google Scholar).Reports that valproic acid increases the expression of genes regulated by the transcription factor AP-11 (12Wlodarczyk B.C. Craig J.C. Bennett G.D. Calvin J.A. Finnell R.H. Teratology. 1996; 54: 284-297Crossref PubMed Scopus (49) Google Scholar, 13Yuan P. Chen G. Manji H.K. J. Neurochem. 1999; 73: 2299-2309Crossref PubMed Scopus (69) Google Scholar, 14Chen G. Yuan P.-X. Jiang Y.-M Huang L.-D. Manji H.K. Brain Res. Mol. Brain Res. 1999; 64: 52-58Crossref PubMed Scopus (93) Google Scholar) have led to the conclusion that the molecular mechanism of valproic acid-induced gene expression is via DNA binding activity of this transcription factor (8Yuan P.-X. Huang L.-D. Jiang Y.-M. Gutkind J.S. Manji H.K. J. Biol. Chem. 2001; 276: 31674-31683Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 14Chen G. Yuan P.-X. Jiang Y.-M Huang L.-D. Manji H.K. Brain Res. Mol. Brain Res. 1999; 64: 52-58Crossref PubMed Scopus (93) Google Scholar). However, whether AP-1 is the only transcription factor that can mediate valproic acid-induced gene expression is by no means resolved. A gene promoter that lacks AP-1 motifs, such as the Gαi2 gene promoter (15Weinstein L.S. Spiegel A.M. Carter A.D. FEBS Lett. 1988; 232: 333-340Crossref PubMed Scopus (43) Google Scholar, 16Weinstein L.S. Kats I. Spiegel A.M. Carter A.D. Mol. Endocrinol. 1990; 4: 958-964Crossref PubMed Scopus (9) Google Scholar, 17Quandt K. Frec K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2421) Google Scholar), provides an excellent paradigm for testing the role of other transcription factors. Using K562 cells, we show, in this study, that valproic acid induces expression of Gαi2 and that it activates transcription from the Gαi2 gene promoter, in part, through the Sp family of transcription factors.DISCUSSIONValproic acid is the active ingredient in divalproex sodium (depakote) that is used clinically in the treatment of epilepsy. In addition to its anticonvulsant activity, valproic acid also exhibits teratogenic effects in humans and animals (9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar, 39Robert E. Guiband P. Lancet. 1982; 2: 937Abstract PubMed Scopus (450) Google Scholar, 40DiLiberti J.H. Fardon P.A. Dennis N.R. Curry C.J. Am. J. Med. Genet. 1984; 19: 473-481Crossref PubMed Scopus (309) Google Scholar, 41Ardinger H.H. Atkin J.F. Blackston R.D. Elsas L.J. Clarren S.J. Livingstone S. Flannery D.B. Pellock J.M. Harrod M.J. Lammer E.J. Am. J. Med. Genet. 1988; 29: 171-185Crossref PubMed Scopus (226) Google Scholar, 42Hout C. Gauthier M. Lebel M. Larbrisseau A. Can. J. Neurol. Sci. 1987; 14: 290-293Crossref PubMed Scopus (30) Google Scholar, 43Martinez-Frias M.L. Am. J. Med. Genet. 1990; 37: 277-282Crossref PubMed Scopus (46) Google Scholar, 44Nau H. Hauck R.S. Ehlers K. Pharmacol. Toxicol. 1991; 69: 310-321Crossref PubMed Scopus (251) Google Scholar). The mechanism by which valproic acid brings about these effects is not known. A recent study involving induction of differentiation in F9 embryocarcinoma cells has suggested that valproic acid induces the expression of peroxisome proliferating activating receptor δ and has associated this induction with the occurrence of valproic acid-induced teratogenicity (45Werling U. Siehler S. Liften M. Nau H. Göttlicher M. Mol. Pharmacol. 2001; 59: 1269-1276Crossref PubMed Scopus (60) Google Scholar). Valproic acid-induced gene expression (8Yuan P.-X. Huang L.-D. Jiang Y.-M. Gutkind J.S. Manji H.K. J. Biol. Chem. 2001; 276: 31674-31683Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar, 10Chen G. Yuan P.-X. Jiang Y.-M. Huang L.-D. Manji H.K. J. Neurochem. 1998; 70: 1768-1771Crossref PubMed Scopus (64) Google Scholar, 11Sands S.A. Guerra V. Morilak D.A. Neuropsychopharmocology. 2000; 22: 27-35Crossref PubMed Scopus (47) Google Scholar) has been attributed to the DNA-binding activity of the transcription factor AP-1. However, it is unclear whether the gene expression-inducing effect of valproic acid can be attributed solely to its activation of AP-1 binding to DNA. In fact, Phiel et al.(9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar) had noted that valproic acid robustly activated transcription fromRenilla luciferase reporter gene driven by the cytomegalovirus promoter (pRL-CMV), which they indicated does not contain AP-1 sites.In the present study, we have used the human Gαi2 gene promoter, which does not contain AP-1 sequence motifs (15Weinstein L.S. Spiegel A.M. Carter A.D. FEBS Lett. 1988; 232: 333-340Crossref PubMed Scopus (43) Google Scholar, 16Weinstein L.S. Kats I. Spiegel A.M. Carter A.D. Mol. Endocrinol. 1990; 4: 958-964Crossref PubMed Scopus (9) Google Scholar, 17Quandt K. Frec K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2421) Google Scholar), to illustrate involvement of the Sp family of transcription factors in the transcriptional activity of valproic acid. Our results show that in K562 cells, valproic acid-induced transcription from this promoter was inhibited by mithramycin A, a potent inhibitor of Sp1 binding to GC boxes in DNA (23Blume S.W. Snyder R.C. Ray R. Thomas S. Koller C.A. Miller D.M. J. Clin. Invest. 1991; 88: 1613-1621Crossref PubMed Scopus (296) Google Scholar, 24Greenwel P. Inagaki Y. Hu W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 25Lai C.-F. Feng X. Nishimura R. Teitelbaum S.L. Avioli L.V. Ross F.P. Cheng S.-L. J. Biol. Chem. 2000; 275: 36400-36406Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 26Poncelet A.-C. Schnaper H.W. J. Biol. Chem. 2001; 276: 6983-6992Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 27Yoo J. Jeong M.-J. Kwon B.-M. Hur M.-W. Park Y.-M. Han M.Y. J. Biol. Chem. 2002; 277: 11904-11909Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Valproic acid-induced transcription was also substantially depressed when three Sp1-binding sequences in the human Gαi2 gene promoter were mutated. Electrophoretic mobility shift assays indicated that these sequences bind not only Sp1 but also Sp2 and Sp3, three closely related members of the Sp family of transcription factors. Together with the results of our reporter gene assays, these data support the conclusion that one or more members of the Sp family of transcription factors is involved in the transcriptional effect of valproic acid on the human Gαi2gene promoter. This study provides a substantially different perspective from reports (8Yuan P.-X. Huang L.-D. Jiang Y.-M. Gutkind J.S. Manji H.K. J. Biol. Chem. 2001; 276: 31674-31683Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 13Yuan P. Chen G. Manji H.K. J. Neurochem. 1999; 73: 2299-2309Crossref PubMed Scopus (69) Google Scholar) that defined the AP-1 response element as the modality by which valproic acid induces gene expression. The finding that mutations at these critical Sp1 sites from the Gαi2 gene promoter did not completely suppress transcription in our cellular transfection system suggests that other transcription factor(s) besides Sp family members may also be involved in the demonstrated transcriptional effect of valproic acid.Histone deacetylase (HDAC) inhibitors are increasingly gaining attention in cancer research (46Archer S.Y. Meng S. Shei A. Hodin R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6791-6796Crossref PubMed Scopus (497) Google Scholar, 47Warrell Jr., R.P. He L.-Z. Richon V. Calleja E. Pandolfi P.P. J. Natl. Cancer Inst. 1998; 90: 1621-1625Crossref PubMed Scopus (503) Google Scholar, 48Marks P.A. Richon V.M. Rifkind R.A. J. Natl. Cancer Inst. 2000; 92: 1210-1216Crossref PubMed Scopus (1074) Google Scholar, 49Göttlicher M. Minucci S. Zhu P. Krämer O.H. Schimpf A. Giavara S. Sleeman J.P. Coco F.L. Nervi C. Pelicci G. Heinzel T. EMBO J. 2001; 20: 6969-6978Crossref PubMed Scopus (1541) Google Scholar, 50Hinnebusch B.F. Meng S. Wu J.T. Archer S.Y. Hodin R.A. J. Nutr. 2002; 132: 1012-1017Crossref PubMed Scopus (386) Google Scholar) because of their ability to inhibit cell proliferation. Valproic acid is a new addition to this list and was shown recently to inhibit histone deacetylase inXenopus and human embryonic kidney (293T) cells (9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar). Göttlicher et al. (49Göttlicher M. Minucci S. Zhu P. Krämer O.H. Schimpf A. Giavara S. Sleeman J.P. Coco F.L. Nervi C. Pelicci G. Heinzel T. EMBO J. 2001; 20: 6969-6978Crossref PubMed Scopus (1541) Google Scholar) also demonstrated that valproic acid is a powerful inhibitor of HDAC, relieves HDAC-dependent transcriptional repression, and causes hyperacetylation of histones in vivo and in cultured cells. Taken together with the studies of Doetzlhofer et al. (22Doetzlhofer A. Rotheneder H. Lagger G. Koranda M. Kurtev V. Brosch G. Wintersberger E. Seiser C. Mol. Cell. Biol. 1999; 19: 5504-5511Crossref PubMed Scopus (355) Google Scholar) that show that inhibition of HDAC by HDAC inhibitors releases an inhibitory constraint on Sp1, making it possible for this transcription factor to associate with other accessory proteins to effect transcription, our present study provides an important dimension to further understanding of mechanisms underlying valproic acid-induced gene transcription.Valproic acid-induced transcription demonstrated in this study was sensitive to three powerful inhibitors of PP1 and PP2A (i.e.okadaic acid, calyculin A, and fostriecin), indicating that it requires the action of protein phosphatase. These chemical inhibitor studies, however, do not precisely pinpoint which protein phosphatase may be involved. That the transcriptional action of Sp1 can be influenced by its phosphorylation state has been amply demonstrated (31Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 32Armstrong S.A. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 33Rohlff C. Ahmad S. Borellini F. Lei J. Glazer R.I. J. Biol. Chem. 1997; 272: 21137-21141Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 52Cuisset L. Tichonicky L. Jaffray P. Delpech M. J. Biol. Chem. 1997; 272: 24148-24158Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 53Garcia A. Cereghini S. Sontag E. J. Biol. Chem. 2000; 275: 9385-9389Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In this context, it should be noted further that Lacroix et al. (34Lacroix I. Lipcey C. Imbert J. Kahn-Perlès B. J. Biol. Chem. 2002; 277: 9598-9605Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) recently demonstrated, in interleukin-2-treated T lymphoma cell line Kit225, that PP2A but not PP1 dephosphorylates Sp1, resulting in increased binding of Sp1 to DNA as well as up-regulation of its transcriptional activity with respect to the human immunodeficiency virus type 1 long terminal repeat promoter as well as a chloramphenicol acetyltransferase reporter gene under the control of six tandem Sp1-binding sites from SV40 early promoter (SV40-tkCAT). Also, Li et al. (54Li J. Park S.W. Loh H.H. Wei L.-N. J. Biol. Chem. 2002; 277: 39967-39972Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) recently showed in studies with mouse embryonal carcinoma P19 cells that retinoic acid-induced transcription from the mouse κ-opioid receptor gene promoter is mediated by increased binding of dephosphorylated Sp1 to GC box elements in that promoter. We can infer from their studies and from our protein phosphatase experiments (Figs. Figure 7, Figure 8, Figure 9) that dephosphorylated Sp1/Sp3 mediated valproic acid-induced transcription from the Gαi2 gene promoter observed in our present study. Besides Sp1, not much is known about post-translational modification(s) that might influence the transcriptional activity of Sp family of transcription factors. Our present work suggests that, like Sp1, phosphorylation state of Sp3 might influence its DNA binding/transcriptional activity. Interestingly, two research groups (55Ross S. Best J.L. Zon L.I. Gill G. Mol. Cell. 2002; 10: 831-842Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 56Sapetschnig A. Rischitor G. Braun H. Doll A. Schergaut M. Melchior F. Suske G. EMBO J. 2002; 21: 5206-5215Crossref PubMed Scopus (224) Google Scholar) showed recently that another post-translational modification (i.e. sumoylation) influences whether Sp3 functions as a repressor or activator of transcription. Valproic acid (2-propylpentanoic acid), a branched short-chain fatty acid, is an anticonvulsant agent that is also clinically effective as a mood stabilizer in the treatment of manic depression (bipolar affective disorder) (1Joffe R.T. Can. J. Psychiatry. 1993; 38 (suppl.): 46-50Google Scholar, 2Mitchell P.B. Aust. NZ J. Psychiatry. 1999; 33 (suppl.): 99-107Crossref Google Scholar, 3Tunnicliff G. J. Physiol. Pharmacol. 1999; 50: 347-365PubMed Google Scholar, 4Sadock B.J. Sadock V.A. Comprehensive Textbook of Psychiatry. Lippincott Williams & Williams, Philadelphia2000: 1284-1440Google Scholar, 5Johannessaen C.U. Neurochem. Int. 2000; 37: 103-110Crossref PubMed Scopus (324) Google Scholar). The biochemical basis for the neurotrophic effects of valproic acid and other mood stabilizers has intrigued investigators for a long time, but at the present time the modes of action of these drugs are not clearly understood (4Sadock B.J. Sadock V.A. Comprehensive Textbook of Psychiatry. Lippincott Williams & Williams, Philadelphia2000: 1284-1440Google Scholar, 6Phiel C.J. Klein P.S. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 789-813Crossref PubMed Scopus (447) Google Scholar, 7Manji H.K. Moore G.J. Chen G. Brit. J. Psychiatry. 2001; 178 Suppl. 41: 107-119Crossref Scopus (128) Google Scholar). Among the effects of valproic acid is an increase in gene expression, an effect that has been demonstrated for the genes for Bcl-2 and growth cone-associated p43 in SH-SY5Y cells (8Yuan P.-X. Huang L.-D. Jiang Y.-M. Gutkind J.S. Manji H.K. J. Biol. Chem. 2001; 276: 31674-31683Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), Tcf/Lef-dependent transcription in 293T cells (9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar), and the tyrosine hydroxylase gene (10Chen G. Yuan P.-X. Jiang Y.-M. Huang L.-D. Manji H.K. J. Neurochem. 1998; 70: 1768-1771Crossref PubMed Scopus (64) Google Scholar, 11Sands S.A. Guerra V. Morilak D.A. Neuropsychopharmocology. 2000; 22: 27-35Crossref PubMed Scopus (47) Google Scholar). Reports that valproic acid increases the expression of genes regulated by the transcription factor AP-11 (12Wlodarczyk B.C. Craig J.C. Bennett G.D. Calvin J.A. Finnell R.H. Teratology. 1996; 54: 284-297Crossref PubMed Scopus (49) Google Scholar, 13Yuan P. Chen G. Manji H.K. J. Neurochem. 1999; 73: 2299-2309Crossref PubMed Scopus (69) Google Scholar, 14Chen G. Yuan P.-X. Jiang Y.-M Huang L.-D. Manji H.K. Brain Res. Mol. Brain Res. 1999; 64: 52-58Crossref PubMed Scopus (93) Google Scholar) have led to the conclusion that the molecular mechanism of valproic acid-induced gene expression is via DNA binding activity of this transcription factor (8Yuan P.-X. Huang L.-D. Jiang Y.-M. Gutkind J.S. Manji H.K. J. Biol. Chem. 2001; 276: 31674-31683Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 14Chen G. Yuan P.-X. Jiang Y.-M Huang L.-D. Manji H.K. Brain Res. Mol. Brain Res. 1999; 64: 52-58Crossref PubMed Scopus (93) Google Scholar). However, whether AP-1 is the only transcription factor that can mediate valproic acid-induced gene expression is by no means resolved. A gene promoter that lacks AP-1 motifs, such as the Gαi2 gene promoter (15Weinstein L.S. Spiegel A.M. Carter A.D. FEBS Lett. 1988; 232: 333-340Crossref PubMed Scopus (43) Google Scholar, 16Weinstein L.S. Kats I. Spiegel A.M. Carter A.D. Mol. Endocrinol. 1990; 4: 958-964Crossref PubMed Scopus (9) Google Scholar, 17Quandt K. Frec K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2421) Google Scholar), provides an excellent paradigm for testing the role of other transcription factors. Using K562 cells, we show, in this study, that valproic acid induces expression of Gαi2 and that it activates transcription from the Gαi2 gene promoter, in part, through the Sp family of transcription factors. DISCUSSIONValproic acid is the active ingredient in divalproex sodium (depakote) that is used clinically in the treatment of epilepsy. In addition to its anticonvulsant activity, valproic acid also exhibits teratogenic effects in humans and animals (9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar, 39Robert E. Guiband P. Lancet. 1982; 2: 937Abstract PubMed Scopus (450) Google Scholar, 40DiLiberti J.H. Fardon P.A. Dennis N.R. Curry C.J. Am. J. Med. Genet. 1984; 19: 473-481Crossref PubMed Scopus (309) Google Scholar, 41Ardinger H.H. Atkin J.F. Blackston R.D. Elsas L.J. Clarren S.J. Livingstone S. Flannery D.B. Pellock J.M. Harrod M.J. Lammer E.J. Am. J. Med. Genet. 1988; 29: 171-185Crossref PubMed Scopus (226) Google Scholar, 42Hout C. Gauthier M. Lebel M. Larbrisseau A. Can. J. Neurol. Sci. 1987; 14: 290-293Crossref PubMed Scopus (30) Google Scholar, 43Martinez-Frias M.L. Am. J. Med. Genet. 1990; 37: 277-282Crossref PubMed Scopus (46) Google Scholar, 44Nau H. Hauck R.S. Ehlers K. Pharmacol. Toxicol. 1991; 69: 310-321Crossref PubMed Scopus (251) Google Scholar). The mechanism by which valproic acid brings about these effects is not known. A recent study involving induction of differentiation in F9 embryocarcinoma cells has suggested that valproic acid induces the expression of peroxisome proliferating activating receptor δ and has associated this induction with the occurrence of valproic acid-induced teratogenicity (45Werling U. Siehler S. Liften M. Nau H. Göttlicher M. Mol. Pharmacol. 2001; 59: 1269-1276Crossref PubMed Scopus (60) Google Scholar). Valproic acid-induced gene expression (8Yuan P.-X. Huang L.-D. Jiang Y.-M. Gutkind J.S. Manji H.K. J. Biol. Chem. 2001; 276: 31674-31683Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar, 10Chen G. Yuan P.-X. Jiang Y.-M. Huang L.-D. Manji H.K. J. Neurochem. 1998; 70: 1768-1771Crossref PubMed Scopus (64) Google Scholar, 11Sands S.A. Guerra V. Morilak D.A. Neuropsychopharmocology. 2000; 22: 27-35Crossref PubMed Scopus (47) Google Scholar) has been attributed to the DNA-binding activity of the transcription factor AP-1. However, it is unclear whether the gene expression-inducing effect of valproic acid can be attributed solely to its activation of AP-1 binding to DNA. In fact, Phiel et al.(9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar) had noted that valproic acid robustly activated transcription fromRenilla luciferase reporter gene driven by the cytomegalovirus promoter (pRL-CMV), which they indicated does not contain AP-1 sites.In the present study, we have used the human Gαi2 gene promoter, which does not contain AP-1 sequence motifs (15Weinstein L.S. Spiegel A.M. Carter A.D. FEBS Lett. 1988; 232: 333-340Crossref PubMed Scopus (43) Google Scholar, 16Weinstein L.S. Kats I. Spiegel A.M. Carter A.D. Mol. Endocrinol. 1990; 4: 958-964Crossref PubMed Scopus (9) Google Scholar, 17Quandt K. Frec K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2421) Google Scholar), to illustrate involvement of the Sp family of transcription factors in the transcriptional activity of valproic acid. Our results show that in K562 cells, valproic acid-induced transcription from this promoter was inhibited by mithramycin A, a potent inhibitor of Sp1 binding to GC boxes in DNA (23Blume S.W. Snyder R.C. Ray R. Thomas S. Koller C.A. Miller D.M. J. Clin. Invest. 1991; 88: 1613-1621Crossref PubMed Scopus (296) Google Scholar, 24Greenwel P. Inagaki Y. Hu W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 25Lai C.-F. Feng X. Nishimura R. Teitelbaum S.L. Avioli L.V. Ross F.P. Cheng S.-L. J. Biol. Chem. 2000; 275: 36400-36406Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 26Poncelet A.-C. Schnaper H.W. J. Biol. Chem. 2001; 276: 6983-6992Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 27Yoo J. Jeong M.-J. Kwon B.-M. Hur M.-W. Park Y.-M. Han M.Y. J. Biol. Chem. 2002; 277: 11904-11909Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Valproic acid-induced transcription was also substantially depressed when three Sp1-binding sequences in the human Gαi2 gene promoter were mutated. Electrophoretic mobility shift assays indicated that these sequences bind not only Sp1 but also Sp2 and Sp3, three closely related members of the Sp family of transcription factors. Together with the results of our reporter gene assays, these data support the conclusion that one or more members of the Sp family of transcription factors is involved in the transcriptional effect of valproic acid on the human Gαi2gene promoter. This study provides a substantially different perspective from reports (8Yuan P.-X. Huang L.-D. Jiang Y.-M. Gutkind J.S. Manji H.K. J. Biol. Chem. 2001; 276: 31674-31683Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 13Yuan P. Chen G. Manji H.K. J. Neurochem. 1999; 73: 2299-2309Crossref PubMed Scopus (69) Google Scholar) that defined the AP-1 response element as the modality by which valproic acid induces gene expression. The finding that mutations at these critical Sp1 sites from the Gαi2 gene promoter did not completely suppress transcription in our cellular transfection system suggests that other transcription factor(s) besides Sp family members may also be involved in the demonstrated transcriptional effect of valproic acid.Histone deacetylase (HDAC) inhibitors are increasingly gaining attention in cancer research (46Archer S.Y. Meng S. Shei A. Hodin R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6791-6796Crossref PubMed Scopus (497) Google Scholar, 47Warrell Jr., R.P. He L.-Z. Richon V. Calleja E. Pandolfi P.P. J. Natl. Cancer Inst. 1998; 90: 1621-1625Crossref PubMed Scopus (503) Google Scholar, 48Marks P.A. Richon V.M. Rifkind R.A. J. Natl. Cancer Inst. 2000; 92: 1210-1216Crossref PubMed Scopus (1074) Google Scholar, 49Göttlicher M. Minucci S. Zhu P. Krämer O.H. Schimpf A. Giavara S. Sleeman J.P. Coco F.L. Nervi C. Pelicci G. Heinzel T. EMBO J. 2001; 20: 6969-6978Crossref PubMed Scopus (1541) Google Scholar, 50Hinnebusch B.F. Meng S. Wu J.T. Archer S.Y. Hodin R.A. J. Nutr. 2002; 132: 1012-1017Crossref PubMed Scopus (386) Google Scholar) because of their ability to inhibit cell proliferation. Valproic acid is a new addition to this list and was shown recently to inhibit histone deacetylase inXenopus and human embryonic kidney (293T) cells (9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar). Göttlicher et al. (49Göttlicher M. Minucci S. Zhu P. Krämer O.H. Schimpf A. Giavara S. Sleeman J.P. Coco F.L. Nervi C. Pelicci G. Heinzel T. EMBO J. 2001; 20: 6969-6978Crossref PubMed Scopus (1541) Google Scholar) also demonstrated that valproic acid is a powerful inhibitor of HDAC, relieves HDAC-dependent transcriptional repression, and causes hyperacetylation of histones in vivo and in cultured cells. Taken together with the studies of Doetzlhofer et al. (22Doetzlhofer A. Rotheneder H. Lagger G. Koranda M. Kurtev V. Brosch G. Wintersberger E. Seiser C. Mol. Cell. Biol. 1999; 19: 5504-5511Crossref PubMed Scopus (355) Google Scholar) that show that inhibition of HDAC by HDAC inhibitors releases an inhibitory constraint on Sp1, making it possible for this transcription factor to associate with other accessory proteins to effect transcription, our present study provides an important dimension to further understanding of mechanisms underlying valproic acid-induced gene transcription.Valproic acid-induced transcription demonstrated in this study was sensitive to three powerful inhibitors of PP1 and PP2A (i.e.okadaic acid, calyculin A, and fostriecin), indicating that it requires the action of protein phosphatase. These chemical inhibitor studies, however, do not precisely pinpoint which protein phosphatase may be involved. That the transcriptional action of Sp1 can be influenced by its phosphorylation state has been amply demonstrated (31Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 32Armstrong S.A. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 33Rohlff C. Ahmad S. Borellini F. Lei J. Glazer R.I. J. Biol. Chem. 1997; 272: 21137-21141Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 52Cuisset L. Tichonicky L. Jaffray P. Delpech M. J. Biol. Chem. 1997; 272: 24148-24158Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 53Garcia A. Cereghini S. Sontag E. J. Biol. Chem. 2000; 275: 9385-9389Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In this context, it should be noted further that Lacroix et al. (34Lacroix I. Lipcey C. Imbert J. Kahn-Perlès B. J. Biol. Chem. 2002; 277: 9598-9605Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) recently demonstrated, in interleukin-2-treated T lymphoma cell line Kit225, that PP2A but not PP1 dephosphorylates Sp1, resulting in increased binding of Sp1 to DNA as well as up-regulation of its transcriptional activity with respect to the human immunodeficiency virus type 1 long terminal repeat promoter as well as a chloramphenicol acetyltransferase reporter gene under the control of six tandem Sp1-binding sites from SV40 early promoter (SV40-tkCAT). Also, Li et al. (54Li J. Park S.W. Loh H.H. Wei L.-N. J. Biol. Chem. 2002; 277: 39967-39972Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) recently showed in studies with mouse embryonal carcinoma P19 cells that retinoic acid-induced transcription from the mouse κ-opioid receptor gene promoter is mediated by increased binding of dephosphorylated Sp1 to GC box elements in that promoter. We can infer from their studies and from our protein phosphatase experiments (Figs. Figure 7, Figure 8, Figure 9) that dephosphorylated Sp1/Sp3 mediated valproic acid-induced transcription from the Gαi2 gene promoter observed in our present study. Besides Sp1, not much is known about post-translational modification(s) that might influence the transcriptional activity of Sp family of transcription factors. Our present work suggests that, like Sp1, phosphorylation state of Sp3 might influence its DNA binding/transcriptional activity. Interestingly, two research groups (55Ross S. Best J.L. Zon L.I. Gill G. Mol. Cell. 2002; 10: 831-842Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 56Sapetschnig A. Rischitor G. Braun H. Doll A. Schergaut M. Melchior F. Suske G. EMBO J. 2002; 21: 5206-5215Crossref PubMed Scopus (224) Google Scholar) showed recently that another post-translational modification (i.e. sumoylation) influences whether Sp3 functions as a repressor or activator of transcription. Valproic acid is the active ingredient in divalproex sodium (depakote) that is used clinically in the treatment of epilepsy. In addition to its anticonvulsant activity, valproic acid also exhibits teratogenic effects in humans and animals (9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar, 39Robert E. Guiband P. Lancet. 1982; 2: 937Abstract PubMed Scopus (450) Google Scholar, 40DiLiberti J.H. Fardon P.A. Dennis N.R. Curry C.J. Am. J. Med. Genet. 1984; 19: 473-481Crossref PubMed Scopus (309) Google Scholar, 41Ardinger H.H. Atkin J.F. Blackston R.D. Elsas L.J. Clarren S.J. Livingstone S. Flannery D.B. Pellock J.M. Harrod M.J. Lammer E.J. Am. J. Med. Genet. 1988; 29: 171-185Crossref PubMed Scopus (226) Google Scholar, 42Hout C. Gauthier M. Lebel M. Larbrisseau A. Can. J. Neurol. Sci. 1987; 14: 290-293Crossref PubMed Scopus (30) Google Scholar, 43Martinez-Frias M.L. Am. J. Med. Genet. 1990; 37: 277-282Crossref PubMed Scopus (46) Google Scholar, 44Nau H. Hauck R.S. Ehlers K. Pharmacol. Toxicol. 1991; 69: 310-321Crossref PubMed Scopus (251) Google Scholar). The mechanism by which valproic acid brings about these effects is not known. A recent study involving induction of differentiation in F9 embryocarcinoma cells has suggested that valproic acid induces the expression of peroxisome proliferating activating receptor δ and has associated this induction with the occurrence of valproic acid-induced teratogenicity (45Werling U. Siehler S. Liften M. Nau H. Göttlicher M. Mol. Pharmacol. 2001; 59: 1269-1276Crossref PubMed Scopus (60) Google Scholar). Valproic acid-induced gene expression (8Yuan P.-X. Huang L.-D. Jiang Y.-M. Gutkind J.S. Manji H.K. J. Biol. Chem. 2001; 276: 31674-31683Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar, 10Chen G. Yuan P.-X. Jiang Y.-M. Huang L.-D. Manji H.K. J. Neurochem. 1998; 70: 1768-1771Crossref PubMed Scopus (64) Google Scholar, 11Sands S.A. Guerra V. Morilak D.A. Neuropsychopharmocology. 2000; 22: 27-35Crossref PubMed Scopus (47) Google Scholar) has been attributed to the DNA-binding activity of the transcription factor AP-1. However, it is unclear whether the gene expression-inducing effect of valproic acid can be attributed solely to its activation of AP-1 binding to DNA. In fact, Phiel et al.(9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar) had noted that valproic acid robustly activated transcription fromRenilla luciferase reporter gene driven by the cytomegalovirus promoter (pRL-CMV), which they indicated does not contain AP-1 sites. In the present study, we have used the human Gαi2 gene promoter, which does not contain AP-1 sequence motifs (15Weinstein L.S. Spiegel A.M. Carter A.D. FEBS Lett. 1988; 232: 333-340Crossref PubMed Scopus (43) Google Scholar, 16Weinstein L.S. Kats I. Spiegel A.M. Carter A.D. Mol. Endocrinol. 1990; 4: 958-964Crossref PubMed Scopus (9) Google Scholar, 17Quandt K. Frec K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2421) Google Scholar), to illustrate involvement of the Sp family of transcription factors in the transcriptional activity of valproic acid. Our results show that in K562 cells, valproic acid-induced transcription from this promoter was inhibited by mithramycin A, a potent inhibitor of Sp1 binding to GC boxes in DNA (23Blume S.W. Snyder R.C. Ray R. Thomas S. Koller C.A. Miller D.M. J. Clin. Invest. 1991; 88: 1613-1621Crossref PubMed Scopus (296) Google Scholar, 24Greenwel P. Inagaki Y. Hu W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 25Lai C.-F. Feng X. Nishimura R. Teitelbaum S.L. Avioli L.V. Ross F.P. Cheng S.-L. J. Biol. Chem. 2000; 275: 36400-36406Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 26Poncelet A.-C. Schnaper H.W. J. Biol. Chem. 2001; 276: 6983-6992Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 27Yoo J. Jeong M.-J. Kwon B.-M. Hur M.-W. Park Y.-M. Han M.Y. J. Biol. Chem. 2002; 277: 11904-11909Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Valproic acid-induced transcription was also substantially depressed when three Sp1-binding sequences in the human Gαi2 gene promoter were mutated. Electrophoretic mobility shift assays indicated that these sequences bind not only Sp1 but also Sp2 and Sp3, three closely related members of the Sp family of transcription factors. Together with the results of our reporter gene assays, these data support the conclusion that one or more members of the Sp family of transcription factors is involved in the transcriptional effect of valproic acid on the human Gαi2gene promoter. This study provides a substantially different perspective from reports (8Yuan P.-X. Huang L.-D. Jiang Y.-M. Gutkind J.S. Manji H.K. J. Biol. Chem. 2001; 276: 31674-31683Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 13Yuan P. Chen G. Manji H.K. J. Neurochem. 1999; 73: 2299-2309Crossref PubMed Scopus (69) Google Scholar) that defined the AP-1 response element as the modality by which valproic acid induces gene expression. The finding that mutations at these critical Sp1 sites from the Gαi2 gene promoter did not completely suppress transcription in our cellular transfection system suggests that other transcription factor(s) besides Sp family members may also be involved in the demonstrated transcriptional effect of valproic acid. Histone deacetylase (HDAC) inhibitors are increasingly gaining attention in cancer research (46Archer S.Y. Meng S. Shei A. Hodin R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6791-6796Crossref PubMed Scopus (497) Google Scholar, 47Warrell Jr., R.P. He L.-Z. Richon V. Calleja E. Pandolfi P.P. J. Natl. Cancer Inst. 1998; 90: 1621-1625Crossref PubMed Scopus (503) Google Scholar, 48Marks P.A. Richon V.M. Rifkind R.A. J. Natl. Cancer Inst. 2000; 92: 1210-1216Crossref PubMed Scopus (1074) Google Scholar, 49Göttlicher M. Minucci S. Zhu P. Krämer O.H. Schimpf A. Giavara S. Sleeman J.P. Coco F.L. Nervi C. Pelicci G. Heinzel T. EMBO J. 2001; 20: 6969-6978Crossref PubMed Scopus (1541) Google Scholar, 50Hinnebusch B.F. Meng S. Wu J.T. Archer S.Y. Hodin R.A. J. Nutr. 2002; 132: 1012-1017Crossref PubMed Scopus (386) Google Scholar) because of their ability to inhibit cell proliferation. Valproic acid is a new addition to this list and was shown recently to inhibit histone deacetylase inXenopus and human embryonic kidney (293T) cells (9Phiel C.J. Zhang F. Huang E.Y. Guenther M.G. Lazar M.A. Klein P.S. J. Biol. Chem. 2001; 276: 36734-36741Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar). Göttlicher et al. (49Göttlicher M. Minucci S. Zhu P. Krämer O.H. Schimpf A. Giavara S. Sleeman J.P. Coco F.L. Nervi C. Pelicci G. Heinzel T. EMBO J. 2001; 20: 6969-6978Crossref PubMed Scopus (1541) Google Scholar) also demonstrated that valproic acid is a powerful inhibitor of HDAC, relieves HDAC-dependent transcriptional repression, and causes hyperacetylation of histones in vivo and in cultured cells. Taken together with the studies of Doetzlhofer et al. (22Doetzlhofer A. Rotheneder H. Lagger G. Koranda M. Kurtev V. Brosch G. Wintersberger E. Seiser C. Mol. Cell. Biol. 1999; 19: 5504-5511Crossref PubMed Scopus (355) Google Scholar) that show that inhibition of HDAC by HDAC inhibitors releases an inhibitory constraint on Sp1, making it possible for this transcription factor to associate with other accessory proteins to effect transcription, our present study provides an important dimension to further understanding of mechanisms underlying valproic acid-induced gene transcription. Valproic acid-induced transcription demonstrated in this study was sensitive to three powerful inhibitors of PP1 and PP2A (i.e.okadaic acid, calyculin A, and fostriecin), indicating that it requires the action of protein phosphatase. These chemical inhibitor studies, however, do not precisely pinpoint which protein phosphatase may be involved. That the transcriptional action of Sp1 can be influenced by its phosphorylation state has been amply demonstrated (31Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 32Armstrong S.A. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 33Rohlff C. Ahmad S. Borellini F. Lei J. Glazer R.I. J. Biol. Chem. 1997; 272: 21137-21141Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 52Cuisset L. Tichonicky L. Jaffray P. Delpech M. J. Biol. Chem. 1997; 272: 24148-24158Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 53Garcia A. Cereghini S. Sontag E. J. Biol. Chem. 2000; 275: 9385-9389Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In this context, it should be noted further that Lacroix et al. (34Lacroix I. Lipcey C. Imbert J. Kahn-Perlès B. J. Biol. Chem. 2002; 277: 9598-9605Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) recently demonstrated, in interleukin-2-treated T lymphoma cell line Kit225, that PP2A but not PP1 dephosphorylates Sp1, resulting in increased binding of Sp1 to DNA as well as up-regulation of its transcriptional activity with respect to the human immunodeficiency virus type 1 long terminal repeat promoter as well as a chloramphenicol acetyltransferase reporter gene under the control of six tandem Sp1-binding sites from SV40 early promoter (SV40-tkCAT). Also, Li et al. (54Li J. Park S.W. Loh H.H. Wei L.-N. J. Biol. Chem. 2002; 277: 39967-39972Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) recently showed in studies with mouse embryonal carcinoma P19 cells that retinoic acid-induced transcription from the mouse κ-opioid receptor gene promoter is mediated by increased binding of dephosphorylated Sp1 to GC box elements in that promoter. We can infer from their studies and from our protein phosphatase experiments (Figs. Figure 7, Figure 8, Figure 9) that dephosphorylated Sp1/Sp3 mediated valproic acid-induced transcription from the Gαi2 gene promoter observed in our present study. Besides Sp1, not much is known about post-translational modification(s) that might influence the transcriptional activity of Sp family of transcription factors. Our present work suggests that, like Sp1, phosphorylation state of Sp3 might influence its DNA binding/transcriptional activity. Interestingly, two research groups (55Ross S. Best J.L. Zon L.I. Gill G. Mol. Cell. 2002; 10: 831-842Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 56Sapetschnig A. Rischitor G. Braun H. Doll A. Schergaut M. Melchior F. Suske G. EMBO J. 2002; 21: 5206-5215Crossref PubMed Scopus (224) Google Scholar) showed recently that another post-translational modification (i.e. sumoylation) influences whether Sp3 functions as a repressor or activator of transcription. We thank Jianqi Yang for very valuable discussions during the performance of this work and for assistance in the electronic preparation of the figures in the manuscript. Dr. Lee Weinstein kindly provided the plasmid pGαi2(−1214/+115)-CAT containing wild-type promoter for the human Gαi2 gene. We are also grateful to Dr. Melanie Cobb for providing us with the expression plasmid harboring the gene for dominant negative ERK2.
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