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Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture
Published on: May 10, 2017
Pigment epithelium-derived factor induces pro-survival genes through cyclic AMP-responsive element binding protein
T Yabe1, K Kanemitsu, T Sanagi
1Kitasato Institute for Life Sciences, Kitasato University, 5-9-1, Shirokane, Tokyo 108-8641, Japan. tyabe@lisci.kitasato-u.ac.jp
Abstract:
Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cell neurons (CGCs) against apoptosis induced by K+ and serum deprivation. However, the precise mechanism of this protection remains unknown. We recently reported that the transcription factor nuclear factor kappa B (NF-kappaB) is activated in PEDF-treated CGCs. Although it is well known that NF-kappaB blocks apoptotic cell death through the induction of pro-survival factors, the effects of PEDF on the expression of these factors are not fully understood. In this study, we employed the use of reverse transcriptase-polymerase chain reaction to analyze the gene expression of certain pro-survival genes and found that genes such as c-IAP1, c-IAP2, FLIPs, A1/Bfl-1 and Mn-SOD were induced in PEDF-treated neurons. On the other hand, no induction was observed of the pro-apoptotic Bcl-2 family members Bax and Bid at any time from 3 to 24 h following PEDF addition. Furthermore, phosphorylation of cyclic AMP-responsive element binding protein (CREB) and increment of nuclear cyclic AMP-response element (CRE)-like DNA binding were observed in PEDF-treated CGCs. The anti-apoptotic effect of PEDF was blocked by overexpression of dominant negative CREB or a mutated form of IkappaBalpha. These results suggested that induction of both CRE- and NF-kappaB-dependent genes is required for the observed neuroprotective effects of PEDF on CGCs.
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