Clarification of the mechanisms for cell death regulation by DAP3 and CLIPR59, new binding proteins to death receptors

尽管骨肉瘤的化疗和手术治疗有所改进，但仍难以取得令人满意的结果。需要为这些治疗开发新的治疗方式。TRAIL(肿瘤坏死因子相关的凋亡诱导配体)在大多数肿瘤细胞中被认为是一种选择性的凋亡诱导剂，但在正常细胞中不是。因此，TRAIL是治疗肿瘤的一个很好的候选靶点。然而，骨肉瘤细胞对TRAIL诱导的细胞凋亡的敏感性低于其他类型的肿瘤细胞。我们发现LKB1，一种丝氨酸/苏氨酸激酶，在骨和软组织肉瘤细胞中表达，与DAP3相关。我们还证实了DAP3的表达诱导了骨肉瘤细胞的凋亡。此外，LKB1的表达可诱导骨肉瘤细胞的凋亡，且LKB1与DAP3的共表达可强烈诱导骨肉瘤细胞的凋亡。此外，LKB1激酶死亡突变体LKB1(K78M)的表达可抑制DAP3诱导的细胞凋亡。这些结果表明，LKB1在TRAIL诱导骨肉瘤细胞凋亡中起关键作用，与DAP3协同作用。LKB1和DAP3可能是治疗骨肉瘤的关键靶分子。死亡受体(Death Receptor，DR)6是死亡受体家族的最新成员，通过其特异性配体的刺激而诱导细胞凋亡。我们发现ClipR-59与DR6的胞质尾部直接相互作用。在DR6介导的信号中，ClipR-59在诱导细胞凋亡或激活JNK中起着关键作用。更有趣的是，ClipR-59基因表达的降低显著减少了死亡受体刺激介导的细胞凋亡诱导和JNK激活。我们还证实了ClipR-59与ASK1的相互作用，ASK1的主要负性形式可以阻止ClipR-59依赖的细胞凋亡诱导。这些结果表明，ClipR-59在DR信号通路中通过ASK1诱导细胞凋亡和激活JNK起重要作用。

Despite improvements in chemotherapy and surgery in the treatment of osteosarcoma, satisfactory results are still difficult to achieve. New therapeutic modalities need to be developed for these treatments. TRAIL (TNF-related apoptosis inducing ligand) is known as a selective apoptosis inducer in most tumor cells, but not in normal cells. Therefore, TRAIL is a good candidate target for the treatment of tumors. However, sensitivity of osteosarcoma cells to TRAIL-induced apoptosis is lower than that of other types of tumor cells. We found that LKB1, a serine/threonine kinase, expressed in bone and soft tissue sarcoma cells, associated with DAP3. We also demonstrated that expression of DAP3 induced apoptosis in osteosarcoma cells. Furthermore, expression of LKB1 induced apoptosis and co-expression of LKB1 with DAP3 strongly induced apoptosis in osteosarcoma cells. In addition, expression of LKB1 kinase dead mutant, LKB1 (K78M) inhibited DAP3-induced apoptosis in these cells. These results suggest that LKB1 is critical for TRAIL-induced apoptosis induction, cooperating with DAP3 in osteosarcoma cells. It is predicted that LKB1 and DAP3 could be critical target molecules for the treatment of osteosarcomas.Death receptor (DR) 6 is the newest member of death receptor family, which induces apoptosis in response to their specific ligand stimulation. We show that CLIPR-59 directly interacts with the cytoplasmic tail of DR6. CLIPR-59 is shown to be crucial for apoptosis induction or JNK activation in DR6-mediated signal. More interestingly, reduction of CLIPR-59 gene expression significantly reduced apoptosis induction and JNK activation mediated by death receptor stimulation. We also identified the interaction of CLIPR-59 with ASK1 and the dominant negative form of ASK1 could prevent CLIPR-59-dependent apoptosis induction. These data suggested that CLIPR-59 plays an important role for apoptosis induction and JNK activation through ASK1 in DR signal.