Identification of Downstream Signals for Pitx2a

我们先前报道，在HeLa细胞中过表达Pitx2a导致p53和p21/cip1/walf1的积聚，导致细胞周期停滞在G1/G0。然而，目前尚不清楚Pitx2a是如何诱导HeLa细胞中p53和p21蛋白积聚的。在正常细胞和组织中，P53的泛素化和蛋白酶体依赖的降解是由泛素蛋白连接酶MDM2和p300/CBP介导的，从而将P53蛋白维持在适当的水平。在HPV E6蛋白存在的情况下，E6AP(E6相关蛋白)而不是MDM2被用作泛素蛋白连接酶，以靶向肿瘤抑制蛋白P53进行降解。RT-PCR和Northern印迹分析表明，Ptx2a表达24小时后，HeLa细胞中E6和E6AP的表达水平没有受到明显影响，提示Ptx2a诱导HeLa细胞中的P53积聚不是由于抑制HPVE6和E6AP的表达所致。此外，在HeLa细胞中过表达E6AP蛋白并不显著影响Pitx2a诱导的P53积聚。这些结果提示，Pitx2a可能直接阻断E6和E6AP对P53的泛素化和降解。将Pitx2a导入含有高功能MDM2的细胞系，如U2OS，没有导致P53的积聚，提示Pitx2a不影响MDM2介导的P53的降解。免疫共沉淀实验表明，Myc标记的p53和GFP标记的Pitx2a在HeLa细胞中可以相互作用。这种相互作用不需要HPV E6癌蛋白的存在，因为当Pitx2a与P53共转染到HPV阴性的SAO-2细胞时，它们仍然可以与P53结合。缺失了Pitx2a羧基末端的同源结构域或40个氨基酸，就失去了在HeLa细胞中诱导P53积聚的能力。此外，Pitx2a的过表达还导致了E6AP的泛素化和降解。我们的数据表明，Pitx2a通过与P53形成复合体，加速E6AP的泛素化和降解，从而增加P53的稳定性，导致P53的积累。 以往的报道表明，无处不在的NMHC II-B在小鼠体内的消融(B-/B-)会导致大脑和心脏的缺陷，导致E14.5到E18.5之间的死亡。B-/B-小鼠同时存在严重的心脏和大脑缺陷，这增加了这样一种可能性，即在这些器官中发现的至少一些异常可能是其他器官故障的继发性原因，而不是直接由于NMHC II-B的缺失所致。因此，我们利用loxP/Cre策略，以组织特异性的方式特异性地缺失NMHC II-B的外显子II。建立了NMHC II-B外显子II两侧有loxP位点的小鼠品系B：loxP/loxP。NesCre是一种在Nestin启动子控制下携带Cre重组酶表达盒的转基因小鼠，与B：loxP/loxP小鼠杂交，获得了B：loxP/loxP NesCre，它允许NMHC II-B在大脑中去除，但不能在心脏中去除。B：loxP/loxP NesCre小鼠比野生型小鼠小，出现严重的脑积水，表现出异常的神经细胞迁移，没有表现出任何心脏缺陷。B：loxP/loxP小鼠还与一个转基因小鼠品系Alpha-MHC-Cre(MHCCre)杂交，在该品系中，Cre表达框由α-心肌肌球蛋白重链启动子驱动，产生B：loxP/loxP MHCCre小鼠，其中NMHC II-B在心脏中被特异性消融。B：loxP/loxP MHCCre小鼠目前的特征是，但到目前为止已经表现出室间隔缺陷和脑异常。因此，B-/B-小鼠的缺陷似乎是由每个器官中NMHC II-B的缺失直接引起的，而与另一个器官产生的缺陷无关。

We previously reported that over-expression of Pitx2a in HeLa cells resulted in the accumulation of p53 and p21/cip1/walf1, leading to cell cycle arrest at G1/G0. However, it is not known how Pitx2a induced p53 and p21 protein accumulation in HeLa cells. In normal cells and tissues, the ubiquitination and proteasome-dependent degradation of p53 are mediated by ubiquitin protein ligases mdm2 and p300/CBP, maintaining the p53 protein at an appropriate level. In the presence of HPV E6 protein, E6AP (E6-associated protein), but not mdm2, is used as the ubiquitin protein ligase to target the tumor suppressor protein p53 for degradation. However, RT-PCR and northern blot analysis indicated that E6 and the E6AP mRNA levels in HeLa cells were not significantly affected following Ptx2a expression for 24h, suggesting that p53 accumulation induced by Pitx2a in HeLa cells was not due to the repression of HPV E6 and cellular E6AP expression. Furthermore, over-expression of E6AP protein in HeLa cells did not significantly affect the induction of p53 accumulation by Pitx2a. These results suggested that Pitx2a might directly block the ubiquitination and degradation of p53 by E6 and E6AP. Transfection of Pitx2a into cell lines containing high levels of functional mdm2, such as U2OS, did not result in the accumulation of p53, suggesting that Pitx2a did not affect mdm2-mediated p53 degradation. Co-immunoprecipitation experiments indicated that Myc-tagged p53 and GFP-tagged Pitx2a could interact with each other in HeLa cells. This interaction does not require the presence of the HPV E6 oncoprotein, as Pitx2a can still bind to p53 when they were cotransfected into HPV-negative Sao-2 cells. Deletion of either the homeodomain or 40 amino acids in the carboxyl-terminal region of Pitx2a abrogates the ability of Pitx2a to induce p53 accumulation in HeLa cells. In addition, over-expression of Pitx2a caused the ubiquitination and degradation of transfected E6AP. Our data suggest that Pitx2a increases p53 stability by forming a complex with p53 and accelerating the ubiquitination and degradation of E6AP, leading to the accumulation of p53. Previous reports demonstrated that ubiquitous ablation of NMHC II-B in mice (B-/B-) resulted in defects in both the brain and heart, leading to lethality between E14.5 and E18.5. The presence of both severe cardiac and brain defects in B-/B- mice raised the possibility that at least some of the abnormalities found in these organs could be secondary to the malfunction of the other organ, rather than directly due to the absence of NMHC II-B. We, therefore, utilized loxP/Cre strategy to specifically delete exon II of NMHC II-B in a tissue-specific manner. A mouse line, B:loxP/loxP, in which exon II of NMHC II-B was flanked by loxP sites, was generated. NesCre, a transgenic mouse line harboring the Cre recombinase expression cassette under control of the nestin promoter, was crossed to B:loxP/loxP mice to obtain B:loxP/loxP NesCre, which allowed removal of NMHC II-B in the brain, but not the heart. The B:loxP/loxP NesCre mice are smaller than wild-type littermates, develop severe hydrocephalus, and demonstrate abnormal neuronal cell migration without showing any cardiac defects. B:loxP/loxP mice were also crossed to a transgenic mouse line, alpha-MHC-Cre (MHCCre), in which the Cre expression cassette is driven by the alpha-cardiac myosin heavy chain promoter, to generate B:loxP/loxP MHCCre mice, in which NMHC II-B was specifically ablated in the heart. B:loxP/loxP MHCCre mice are presently being characterized, but have demonstrated cardiac ventricular septal defects and no brain abnormalities to date. Thus, the defects in B-/B- mice appear to be directly caused by the loss of NMHC II-B in each organ and are independent of the defects generated in the other organ.