Chen Leiling, Tanriover Gamze, Yano Hiroko, Friedlander Robert, Louvi Angeliki, Gunel Murat
Department of Neurosurgery, Yale University School of Medicine, New Haven, Conn 06510, USA.
Stroke. 2009 Apr;40(4):1474-81. doi: 10.1161/STROKEAHA.108.527135. Epub 2009 Feb 26.
Mutations in the Programmed Cell Death 10 (PDCD10) gene cause autosomal dominant familial cerebral cavernous malformations (CCM3). To date, little is known about the function of this gene and its role in disease pathogenesis.
We examined the effects of overexpression of wild-type and 2 human disease-causing variants of PDCD10 on cell death using 3 different methods (TUNEL and MTT assays and caspase-3 activation). We analyzed expression of CCM3, activated caspase-3, and p38 in endothelial cell lines using the serum deprivation model of apoptosis induction. Finally, we assayed the effects of siRNA-mediated inhibition of endogenous PDCD10 expression on cell death in endothelial cell cultures.
Overexpression of wild-type CCM3, but not disease-linked mutant forms, induced apoptosis as confirmed by TUNEL and increased levels of activated caspase-3. Serum starvation of endothelial cells, an inducer of apoptosis, led to increased expression of CCM3 and activation of p38 and ultimately activated caspase-3. siRNA-mediated inhibition of CCM3 expression resulted in decreased levels of p38 and activated caspase-3, and decreased cell death.
CCM3 is both necessary and sufficient to induce apoptosis in vitro in well-defined cell culture systems. Even though it is currently unclear whether this effect on apoptosis is direct or indirect through modulation of cell cycle, these results led to the novel hypothesis that CCM lesions may form as a consequence of aberrant apoptosis, potentially altering the balance between the endothelium and neural cells within the neurovascular unit.
程序性细胞死亡10(PDCD10)基因的突变会导致常染色体显性遗传家族性脑海绵状畸形(CCM3)。迄今为止,对该基因的功能及其在疾病发病机制中的作用了解甚少。
我们使用三种不同方法(TUNEL和MTT分析以及半胱天冬酶-3激活)检测野生型和两种人类致病型PDCD10变体过表达对细胞死亡的影响。我们利用凋亡诱导的血清剥夺模型分析了内皮细胞系中CCM3、活化的半胱天冬酶-3和p38的表达。最后,我们检测了siRNA介导的内源性PDCD10表达抑制对内皮细胞培养物中细胞死亡的影响。
如TUNEL所证实的,野生型CCM3而非与疾病相关的突变形式的过表达诱导了细胞凋亡,并增加了活化的半胱天冬酶-3的水平。内皮细胞的血清饥饿是一种凋亡诱导剂,导致CCM3表达增加、p38激活并最终激活半胱天冬酶-3。siRNA介导的CCM3表达抑制导致p38和活化的半胱天冬酶-3水平降低以及细胞死亡减少。
在明确的细胞培养系统中,CCM3在体外诱导细胞凋亡方面既是必要的也是充分的。尽管目前尚不清楚这种对细胞凋亡的影响是直接的还是通过调节细胞周期间接产生的,但这些结果引出了一个新的假说,即CCM病变可能是异常细胞凋亡的结果,潜在地改变了神经血管单元内内皮细胞和神经细胞之间的平衡。