Differential Roles of Glycogen Synthase Kinase-3 Isoforms in the Regulation of Transcriptional Activation

Abstract

Glycogen synthase kinase-3 (GSK-3) exists as two structurally similar isoforms, ␣ and ␤, whose activities are negatively regulated by serine phosphorylation but positively controlled by tyrosine phosphorylation. We used GSK-3 isoform-specific small interfering RNAs, dominant negative mutants, and pharmacological inhibitors to search for the differential roles for both GSK-3 isoforms in regulating transcriptional activation in cultured rat cerebral cortical neurons. GSK-3␣ and GSK-3␤ were shown to have differentially regulated transactivation such that GSK-3␣ silencing/inhibition was more robust than GSK-3␤ silencing/inhibition in causing cAMP-responsive element-and NF-B-dependent transactivation. Moreover, protein-DNA array studies identified two novel GSK-3-regulated transcription factors, early growth response 1 and Smad3/4, which were oppositely affected by GSK-3␣ or GSK-3␤ silencing or inhibition. Taken together, our results underscore critical variations in the function and regulation of GSK-3␣ and GSK-3␤. The development of GSK-3 isoform-specific inhibitors is thus crucial for therapeutic intervention of GSK-3-related neuropathological conditions.

The mood stabilizers lithium and valproate selectively activate the promoter IV of brain-derived neurotrophic factor in neurons

“…Under these treatment conditions, lithium induced a concentration-dependent increase in the levels of phospho-GSK-3a Ser21 /b Ser9 (Figure 2d), thus indicating an inhibition of GSK-3 activity. 23,24,27 VPA treatment also markedly increased the levels of histone 3 acetylation with no apparent effects on the levels of HDAC1 isoform (Figure 2e), suggesting the inhibition of HDAC activity.…”

“…Four short interfering RNAs (siRNAs) used for gene silencing of either GSK-3a or GSK-3b were designed and prepared as described previously. 27 Rat HDAC1-targeted siRNA and non-targeting control siRNA were purchased from Dharmacon (Lafayette, CO, USA). Rat primary cortical cultures were cultured for 8-DIV and transfected for 48 h with GSK-3 or HDAC1 siRNA using 8 ml siPORT Amine transfection reagent (Ambion; Austin, TX, USA) in 1 ml of the transfection mixture.…”

“…27 Polyclonal antibodies against yeast acetylated histones 3 and 4 (AcH3 and AcH4), mouse HDAC1 and human HDAC3 were obtained from Upstate (Charlottesville, VA, USA). Polyclonal antibody against phospho-GSK-3a/ b (Ser21/9) was obtained from Cell Signaling Technology (Beverly, MA, USA).…”

“…Glycogen synthase kinase-3 consists of two isoforms, a and b, whose roles in transcriptional modulation are not always identical. 27,31 We next examined the effects of RNAi-mediated GSK-3 gene silencing using siRNAs targeting GSK-3a or -3b isoforms. Cortical neurons at 8-DIV were transfected with 80 nM of a GSK-3 siRNA, specific for either GSK-3a (a-p1269 and a-p1375) or -3b (b-p555 and b-p1093), using siPORT Amine reagent.…”

“…25,26 Emerging evidence suggests that inhibition of GSK-3 and HDAC are responsible, at least in part, for the neuroprotective effects of lithium and VPA, respectively. 27,28 The present study was undertaken to investigate whether BDNF exon IV-containing mRNA and promoter IV activity are increased by lithium or VPA treatment in rat cortical neurons, and to determine the BDNF promoter region that confers the sensitivity to either drug. We also investigated whether inhibiting GSK-3 or HDAC mimics the ability of mood stabilizers to induce BDNF transcription in cortical neurons.…”