Upregulation of the Neuron-Specific K⁺/Cl⁻Cotransporter Expression by Transcription Factor Early Growth Response 4

Abstract

The expression of the neuron-specific K ϩ /ClϪ cotransporter (KCC2) is restricted to the CNS and is strongly upregulated during neuronal maturation, yielding a low intracellular chloride concentration that is required for fast synaptic inhibition in adult neurons. To elucidate the mechanisms of KCC2 gene regulation, we analyzed the KCC2 (alias Slc12a5) promoter and proximal intron-1 regions and revealed 10 candidate transcription factor binding sites that are highly conserved in mammalian KCC2 genes. Here we focus on one of these factors, early growth response 4 (Egr4), which shows a similar developmental upregulation in CNS neurons as KCC2. KCC2 luciferase reporter constructs containing the Egr4 site (Egr4 KCC2 ) were strongly induced by Egr4 overexpression in neuro-2a neuroblastoma cells and in cultured neurons. Egr4-mediated induction was decreased significantly by point-mutating the Egr4 KCC2 . Insertion of Egr4 KCC2 into the KCC2 basal promoter in the endogenous reverse, but not in the opposite, orientation reestablished Egr4-mediated induction. Electrophoretic mobility shift assay confirmed specific Egr4 binding to Egr4 KCC2 . Interference RNA-mediated knock-down of Egr4 and a dominant-negative isoform of Egr4 significantly inhibited KCC2 reporter induction and endogenous KCC2 expression in cultured neurons. Together, the results indicate an important role for Egr4 in the developmental upregulation of KCC2 gene expression.

Early Growth Response 4 Mediates BDNF Induction of Potassium Chloride Cotransporter 2 Transcription

“…This construct is identical to KCC2(Ϫ309/ϩ42) reporter, except for the two point mutations introduced into the Egr4 binding site. These mutations hinder Egr4 binding and render it unable to conduct Egr4-mediated activation of the KCC2b promoter (Uvarov et al, 2006). The reporter activity of the KCC2(Ϫ309/ϩ42) Egr4mut construct was significantly lower compared with the nonmutated KCC2(Ϫ309/ϩ42) construct, reflecting the influence of endogenous Egr4 (Fig.…”

“…In a previous study, we demonstrated that the transcription factor Egr4 increases KCC2 expression in immature neurons through the Egr4 binding site present in the KCC2b proximal promoter region (Uvarov et al, 2006). To assess whether the Egr4 induced by BDNF is able to activate KCC2 gene transcription, we used a previously characterized luciferase reporter construct KCC2(Ϫ309/ϩ42).…”

“…3A). Details of this construct were described in a previous publication (Uvarov et al, 2006). Egr4 regulates KCC2(Ϫ309/ϩ42) reporter similarly to reporters driven by longer (up to 1.4 kb) KCC2b promoter sequences (Uvarov et al, 2006).…”

“…Details of this construct were described in a previous publication (Uvarov et al, 2006). Egr4 regulates KCC2(Ϫ309/ϩ42) reporter similarly to reporters driven by longer (up to 1.4 kb) KCC2b promoter sequences (Uvarov et al, 2006). Dissociated hippocampal neurons were transfected with the KCC2(Ϫ309/ϩ42) reporter construct at DIV 4, treated with 10 and 50 ng/ml BDNF 2 d after transfection, and lysed 2-4 h after BDNF application.…”

“…Egr4 (or NGFI-C), which displays a neural-specific pattern of expression, is rapidly upregulated by nerve growth factor and neuronal activity (Crosby et al, 1991(Crosby et al, , 1992Honkaniemi and Sharp, 1999; for review, see Beckmann and Wilce, 1997). Recently, we have demonstrated that Egr4 contributes directly to the transcriptional upregulation of KCC2 through the Egr4 binding site present in the proximal promoter of the KCC2b isoform (Uvarov et al, 2006).…”