Sp family of transcription factors is involved in valproic acid-induced expression of Gαi2

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 Galpha(i2) gene promoter, which lacks AP-1-binding motifs. We find that valproic acid-induced expression of Galpha(i2) 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.