Please use this identifier to cite or link to this item:

The identification of 14-3-3[gamma] in astrocytes and its mechanism in protecting astrocytes from ischemia

Authors Chen, Xiaoqian
Issue Date 2002
Summary Ischemia caused cell injury and death in astrocytes plays a critical role in affecting neuronal death and the recovery of brain functions after ischemia. The present study aims to investigate the molecular mechanisms underlying ischemic injury and death of astrocytes by using primary cultures of astrocytes and an anaerobic chamber-induced in vitro ischemia model. Novel or unknown differentially expressed genes under ischemia were searched for using RAP-PCR techniques. 14-3-3γ gene was identified as one of differentially expressed genes in astrocytes under ischemia. As 14-3-3γ had been previously regarded as brain- and neuron- specific, not in astrocytes and with unknown function, it is interesting to examine its expression in different tissues of mice and also in purified primary cultures of astrocytes. To further understand whether 14-3-3γ has specific functions in the brain, we studied their expression in astrocytes under ischemia. 14-3-3γ was highly expressed in brains at various developmental stages but was scarcely detected in other tissues. The protein expressed in cultures of astrocyte at various ages and was mainly located in the cytoplasm. Both the 14-3-3γ, transcripts and proteins significantly increased in astrocytes during ischemic incubation. Levels of 14-3-3γ transcripts and proteins decreased during post-ischemia. LY294002 and U0126, inhibitors of PI-3 and MAP kinases, respectively, did not suppress the increase of 14-3-3γ protein during ischemia. The level of 14-3-3γ protein remained unchanged in astrocytes after scratch-wound and heat shock treatment. 14-3-3γ protein was found to increase in surviving astrocytes but decrease in apoptotic cells during ischemia. Transfection of 14-3-3γ gene in astrocytes reduced apoptosis under ischemia while transfection of antisense 14-3-3γ gene increased cell death under ischemia. These results suggested a protective role of the induction of 14-3-3γ protein in astrocytes under ischemia. To elucidate the mechanism by which 14-3-3γ protein exerts its protective role, we studied its binding with Bad, which is considered as a proapoptotic member of Bcl-2 family. Bad was up-regulated and was activated in apoptotic astrocytes under ischemia. Transfection of Bad induced apoptosis in cultured astrocytes. Phosphorylation of Bad was specifically increased at Ser 112 but not at Ser 136 and Ser 155 in astrocytes under ischemia. LY294002 and U0126 inhibited phosphorylation of Bad at Ser 112 but not Ser 136 or Ser 155 in astrocytes under ischemia. Reciprocal co-IP demonstrated that the binding of endogenous 14-3-3γ with Bad protein incrcased in astrocytes under ischemia. Moreover, 14-3-3γ bound more p-Bad 112 but not p-Bad 136 or p-Bad 155 in astrocytes under ischemia. Transfection of 14-3-3γ and Bad gene did not alter the viability of astrocytes under ischemia but transfection of antisense 14-3-3γ and Bad gene led to 80% of cell death in astrocytes under ischemia. In addition to Bad, 14-3-3γ also bound with p-Raf 259 and c-Raf-1. Under ischemia, their binding decreased in astrocytes. These results suggested that 14-3-3γ protected astrocytes from ischemic death by specifically interacting with p-Bad 112. Publications and manuscripts that support this study are: 1. Xiao Qian Chen, Yun Zhang, and Albert Cheung Hoi Yu. (2002). Induction of 14-3-3γ Proteins Prevents Apoptotic Death in Cultured Astrocyte under Ischemia by Specific Regulating Bad Signaling at Ser 112. (In preaparation) 2. Xiao Qian Chen, Jianguo Chen, Yun Zhang, Wen Luan Wendy Hsiao and Albert Cheung Hoi Yu. (2002). 14-3-3γ is Up-regulated by Ischemia and Binds to Protein Kinase Raf in Primary Cultures of Astrocyte. (in review) 3. Xiao Qian Chen and Albert Cheung Hoi Yu. (2002). The Association of 14-3-3γ and Actin Plays a Role in Cell Division and Apoptosis in Astrocytes. Biochem. Biophy. Res. Com. 296:657-663 4. Zhongjian Jiang, Yun Zhang, Xiaoqian Chen, Philip Yeung Lam, Hui Yang, Qunyuan Xu, Albert Cheung Hoi Yu. (2002). Apoptosis and Activation of Erk1/2 and Akt in Astrocytes Post-ischemia. Neurochemical Res. (In press) 5. Zhongjian Jiang, Yun Zhang, Xiaoqian Chen, Philip Yeung Lam, Hui Yang, Qunyuan Xu, Albert Cheung Hoi Yu. (2002). Activation of Erk1/2 and Akt in Astrocytes under Ischemia. Biochem. Biophys. Res. Commun. 294:726-733
Note Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2002
Language English
Format Thesis
Access View full-text via DOI
Files in this item:
File Description Size Format
th_redirect.html 341 B HTML
Copyrighted to the author. Reproduction is prohibited without the author’s prior written consent.