- 學位論文
功能性探討一新穎BCL-2家族分子-BLM-s
Functional Characterization of a Novel BCL-2 Family Member-BLM-s
摘要
胚胎發育時期的神經細胞利用BCL-2家族成員來調控其死亡的機制使皮質形態能夠正常的發育。利用mRNA差顯技術，我們發現了一個在中樞神經系統有著大量表現的新穎BCL-2家族成員，並將之命名為BLM-s。由於BLM-s的表現僅侷限於E14-E17這段神經細胞死亡的高峰時期中有絲分裂後期的神經細胞，我們因此對其是否調控此段時期的神經細胞死亡感到興趣。BLM-s 為一個座落在粒腺體及內質網的分子，不論在體內或體外，當我們在細胞過量表達BLM-s時，細胞會呈現細胞凋亡的狀態並導致粒腺體釋放cytochrome c進而活化下游的caspase 3。 如同其他的BCL-2家族之促死亡分子，BLM-s可藉由與特定的促生存分子即BCL-2和MCL-1作用來調控細胞凋亡，我們亦發現其調控之細胞凋亡是仰賴促死亡分子BAX的存在。因此我們認為BLM-s在調控細胞凋亡的過程中，是扮演著像”sensitizer”或“depressor”的角色。利用gamma放射線來模擬造成神經細胞死亡的DNA雙股斷裂刺激後，我們則發現BLM-s會被大量表現並促進神經細胞凋亡。其中的分子機制我們則是利用和啟動子區結合之分子所調控的轉錄活化狀態來做分析，分析結果顯示p53及AP1轉錄因子能夠有效的藉由直接和BLM-s啟動子區的結合來調控BLM-s之轉錄活化狀態。我們並進一步的證明BLM-s能被大量表現於受到gamma放射線導致的DNA雙股斷裂刺激的細胞中，並且此機制是受ATM/p53或JNK/AP1所調控的。利用p53基因缺失的老鼠，我們也發現到BLM-s的表現受到了影響，即使給予DNA雙股斷裂的刺激，也無法再促進BLM-s的表現。總結我們的實驗結果，BLM-s為一個會在神經系統發育過程中表現並促進細胞凋亡的BCL-2家族成員，經由DNA雙股斷裂的刺激，其大量表現所導致的神經細胞凋亡可透過ATM/p53或JNK/AP1的訊息傳導來調控。
並列摘要
Neuronal cell death during embryonic development is critical for cortical morphogenesis and is mainly regulated by members of the BCL-2 family. Utilizing mRNA differential display, we have identified a CNS-enriched, novel BCL-2 family member, which is designated as Blm-s (Bcl-2-like molecule-small transcript). Blm-s expression is restricted to postmitotic migratory neurons at E14-E17 stage, which is the peak of naturally occurring neuronal cell death, we thus wonder whether BLM-s participate in regulating neuronal apoptosis at this stage. In our data, BLM-s localizes at mitochondria and ER, and overexpressing BLM-s both in vivo and in vitro kills cells by apoptosis via mitochondrial cytochrome c release and caspase-3 activation. Like all the other BCL-2 family members, BLM-s mediates apoptosis through its BH domain, and is able to selectively interact with BCL-2 and MCL-1, but not with BAX and BAK. Moreover, BLM-s-induced apoptosis is inhibited by BCL-2, and its pro-apoptotic activity is in a BAX-dependent fashion. This suggests that BLM-s may perform as a “sensitizer or derepressor” during apoptosis. We use γ-irradiation as a DNA double-strand breaks (DSBs) inducer to trigger developmental neuronal apoptosis in developing mouse brains and the finding shows Blm-s is upregulated to potentiate neuronal apoptosis in vulnerable cortical regions. Via promoter binding assays and luciferase assay, we have demonstrated that transcriptional activation of Blm-s requires p53 and AP1 to bind putative p53 and AP1 consensus motifs in the defined Blm-s promoter. In vivo, diminished Blm-s transcripts are observed in the normal or irradiated embryonic cortex of E15.5 p53-null mouse. We further demonstrate that BLM-s induces apoptosis via transcriptional upregulation through both the ATM/p53 and JNK/AP1 signaling pathways in response to DSB stress. Collectively, our study demonstrates that BLM-s is a novel pro-apoptotic BCL-2 family member in the developing nervous system and it can be upregulated in response to DNA damage to potentiate neuronal apoptosis through ATM/p53 and JNK/AP1 signaling pathway.
參考文獻
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