Glutamatergic stimulation triggers rapid Krüpple-like factor 4 expression in neurons and the overexpression of KLF4 sensitizes neurons to NMDA-induced caspase-3 activity

We report the first demonstration that Krüpple-like factor 4 (KLF4) mRNA is dramatically and rapidly upregulated by NMDA application in primary cortical neuron cultures. We also report that NMDA induced significant and transient upregulation of KLF4 protein expression, in both cortical neuron cultures and native brain slices. The increase of KLF4 mRNA and protein expression in response to NMDA was time-dependent, and required NMDA receptor-mediated Ca2+ influx. In addition, AMPA exposure caused a time-dependent increase in KLF4 mRNA expression, which was also Ca2+-dependent and involved activation of AMPA/kainate receptors and L-type voltage-sensitive calcium channels. To assess the downstream signaling pathways and functions of KLF4 activation, we used lentiviral vectors to induce ectopic overexpression of KLF4 in cultured neurons. KLF4 overexpression induced the activation of caspase-3 after a normally subtoxic dose of NMDA (10 μM). KLF4 overexpression also increased both protein and mRNA levels of the cell cycle protein cyclin D1, but reduced p21Waf1/Cip1 protein levels. After the NMDA treatment, cyclin D1 levels increased after a short delay (4 h), but fell back to control levels after 20 h. The effects of NMDA and KLF4 overexpression on cyclin D1 induction were additive. We conclude that glutamatergic stimulation can trigger rapid elevation of KLF4 mRNA and protein levels, and that the overexpression of KLF4 can regulate neuronal cell cycle proteins and sensitize neurons to NMDA-induced caspase-3 activity.