Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death.
Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death.
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Bax缺失进一步命令小脑颗粒细胞中的细胞死亡途径，并提出了与caspase无关的细胞死亡途径。
DOI：
10.1083/jcb.139.1.205
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发表时间：
1997-10-06
影响因子：
7.8
通讯作者：
Johnson, E M Jr
中科院分区：
文献类型：
--
作者：
Miller, T M;Moulder, K L;Knudson, C M;Creedon, D J;Deshmukh, M;Korsmeyer, S J;Johnson, E M Jr
游离的小脑颗粒细胞在含25mm钾的培养基中维持后，转到含5mm钾的培养基中发生凋亡死亡。Bcl-2家族成员Bax基因缺失的小鼠颗粒细胞在5mm钾环境下不发生凋亡，但在30或100 μM NMDA刺激下发生兴奋性毒性细胞死亡。在切换到5mm K+后的2小时内，野生型和bax缺陷颗粒细胞的葡萄糖摄取均下降至对照的20%以下。在切换到5mm钾后的12小时内，野生型和bax缺陷颗粒细胞的蛋白质合成也迅速下降到对照的50%。野生型和Bax−/−神经元在K+剥夺后均增加了c-jun和caspase 3 （CPP32）的mRNA水平，并增加了c-jun反活化结构域的磷酸化。5mm K+环境下的野生型颗粒细胞增加了devd -氨基甲基香豆素（DEVD-AMC）的裂解，DEVD-AMC是半胱天蛋白酶2、3和7的荧光底物；相反，bax缺陷颗粒细胞不分裂DEVD-AMC。这些结果将BAX置于代谢变化、mRNA水平变化和c-Jun磷酸化增加的下游，而位于半胱天蛋白酶激活的上游，并表明BAX是细胞凋亡所必需的，而不是兴奋性毒性细胞死亡。在野生型细胞中，Boc-Asp-FMK和ZVAD-FMK这两种caspases的一般抑制剂阻断了DEVD-AMC的切割，并阻断了tdt介导的dUTP缺口末端标记（TUNEL）阳性的增加。然而，这些抑制剂在防止细胞死亡方面仅具有边际作用，提示在小脑颗粒细胞BAX下游存在caspase不依赖的死亡途径。
Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 μM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax −/− neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD–aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.