Egr-1 TransactivatesBimGene Expression to Promote Neuronal Apoptosis

Abstract

The proapoptotic BH3-only protein Bim is a crucial regulator of neuronal apoptosis. Previous studies have indicated the involvement of the c-Jun, FOXO1/3a, and B/C-Myb transcription factors in the regulation of Bim during neuronal apoptosis. However, the mechanism underlying the transcriptional regulation of Bim in activity deprivation-induced neuronal apoptosis has remained unclear. The present study demonstrates that early growth response 1 (Egr-1), rather than c-Jun, FOXO1/3a, or B/C-Myb, directly transactivates Bim gene expression to mediate apoptosis of rat cerebellar granule neurons. We showed that Egr-1 was sufficient and necessary for neuronal apoptosis. Suppression of Egr-1 activity using dominant-negative mutant or knockdown of Egr-1 using small interfering RNAs led to a decrease in Bim expression, whereas overexpression of Egr-1 resulted in induction of Bim. Deletion and site-directed mutagenesis of the Bim promoter revealed that Bim transcriptional activation depends primarily on a putative Egr-binding sequence between nucleotides Ϫ56 and Ϫ47 upstream of the start site. We also showed that Egr-1 binding to this sequence increased in response to activity deprivation in vitro and in vivo. Moreover, inhibition of Egr-1 binding to the Bim promoter, by mithramycin A and chromomycin A3, reduced the activity deprivation-induced increases in Bim promoter activity and mRNA and protein levels and protected neurons from apoptosis, further supporting the Egr-1-mediated transactivation of Bim. Additionally, Bim overcame the Egr-1 knockdown-mediated inhibition of apoptosis, whereas Bim knockdown impaired the increase in apoptosis induced by Egr-1. These findings establish Bim as an Egr-1 target gene in neurons, uncovering a novel Egr-1/Bim pathway by which activity deprivation induces neuronal apoptosis.

Site-specific Phosphorylation Protects Glycogen Synthase Kinase-3β from Calpain-mediated Truncation of Its N and C Termini

“…CGNs are healthy in serum-free medium containing 25K but undergo apoptosis after switching to 5K medium, as has been described by our group (21,23,24) and others (22,26). A time-course analysis of GSK-3␤ using a polyclonal antibody against amino acids 345-420 of GSK-3␤ (designated the Mid antibody) showed a time-dependent FIGURE 1.…”

“…Cell Culture and Treatments-Rat cerebellar granule neurons (CGNs) were cultured in basal modified Eagle's medium containing 10% fetal bovine serum and 25 mM KCl (25K) as previously described (21,22). Transfections were performed on culture days 5-6 (DIV5-6), and experiments were performed after 7 days (DIV7) of culture.…”

“…For the apoptosis quantification assay, 36 h after transfection, the neurons were switched to 25K medium for 12 h and stained with Hoechst 33258 to visualize nuclear morphology. Apoptotic rates were quantified by scoring the percentage of GFP-positive cells with condensed or fragmented nuclei as described previously (14,21). Cells were counted in an unbiased manner, with at least 500 transfected cells being counted for each group.…”

“…Because of the low transfection efficiency in cultured primary neurons (ϳ1% for differentiated neurons), the protein expression levels in the transfected neurons were impossible to detect by immunoblotting. Therefore, as previously described (21,24), GFP was used to mark the transfected cells, and the relative fluorescence intensity of FLAG immunostaining was employed to indicate FLAG-tagged GSK-3␤ protein levels in the transfected CGNs. At a comparable expression level (Fig.…”

Early Growth Response 1 Induces Epithelial-to-mesenchymal Transition via Snail

“…It is involved in the regulation of cell proliferation, apoptosis, growth arrest, differentiation, transformation, senescence, and cancer development. Egr-1 regulates positively or negatively tumor growth [1,2,17,30,[32][33][34]36]. It acts as a tumor suppressor; however, many new evidences revealed that Egr-1 promotes cancer progression.…”