Pi3kcb links Hippo-YAP and PI3K-AKT signaling pathways to promote cardiomyocyte proliferation and survival [microarray]. Mus musculus

Background—YAP, the nuclear effector of Hippo signaling, regulates cellular growth and survival in multiple organs, including the heart, by interacting with TEAD sequence specific DNA-binding proteins. Recent studies showed that YAP stimulates cardiomyocyte proliferation and survival. However, the direct transcriptional targets through which YAP exerts its effects are poorly defined. Methods and Results—To identify genes directly regulated by YAP in cardiomyocytes, we combined differential gene expression analysis in YAP gain- and loss-of-function with genome-wide identification of YAP bound loci using chromatin immunoprecipitation and high throughput sequencing. This screen identified Pik3cb, encoding p110β, a catalytic subunit of phosphoinositol-3-kinase (PI3K), as a candidate YAP effector that promotes cardiomyocyte proliferation and survival. We validated YAP and TEAD occupancy of a conserved enhancer within the first intron of Pik3cb, and show that this enhancer drives YAP-dependent reporter gene expression. Yap gain- and loss-of-function studies indicated that YAP is necessary and sufficient to activate the PI3K-Akt pathway. Like Yap, Pik3cb gain-of-function stimulated cardiomyocyte proliferation, and Pik3cb knockdown dampened the YAP mitogenic activity. Reciprocally, Yap loss-of-function impaired heart function and reduced cardiomyocyte proliferation and survival, all of which were significantly rescued by AAV-mediated Pik3cb expression. Conclusion—Pik3cb is a crucial direct target of YAP, through which the YAP activates PI3K-AKT pathway and regulates cardiomyocyte proliferation and survival. Overall design: Two groups were involved in this study:TNTcreYapfl_het group and TNTcreYapfl_KO group. Each group contained three biological replicates. Embryo hearts were collected at E12.5 and dissociated. Cardiomyocytes were collected by FACS. The total RNA of cardiomyocytes were isolated for microarray analysis.

背景：YAP（Yes-associated protein）是Hippo信号通路的核效应因子，可通过与序列特异性DNA结合蛋白TEAD相互作用，在包括心脏在内的多种器官中调控细胞生长与存活。近期研究表明，YAP可促进心肌细胞增殖与存活。然而，YAP发挥功能所依赖的直接转录靶标仍尚未明确。
方法与结果：为鉴定心肌细胞中YAP直接调控的基因，我们将YAP功能获得与功能缺失实验中的差异基因表达分析，与通过染色质免疫共沉淀联合高通量测序进行的全基因组YAP结合位点鉴定相结合。本次筛选鉴定出Pik3cb，其编码磷酸肌醇3-激酶（PI3K）的催化亚基p110β，作为候选YAP效应因子，可促进心肌细胞增殖与存活。我们验证了YAP与TEAD在Pik3cb首个内含子内的保守增强子区域的结合，并证实该增强子可驱动YAP依赖性的报告基因表达。YAP功能获得与功能缺失实验表明，YAP对于激活PI3K-Akt通路既是必需的，也是充分的。与YAP的作用一致，Pik3cb功能获得可刺激心肌细胞增殖，而Pik3cb敲低则会削弱YAP的促有丝分裂活性。与之相反，YAP功能缺失会损伤心脏功能并降低心肌细胞增殖与存活能力，上述所有表型均可通过腺相关病毒（AAV）介导的Pik3cb表达得到显著挽救。
结论：Pik3cb是YAP的关键直接靶标，YAP通过Pik3cb激活PI3K-AKT通路，并调控心肌细胞增殖与存活。
总体实验设计：本研究共设置两组样本：TNTcreYapfl_het组与TNTcreYapfl_KO组，每组均包含3次生物学重复。于胚胎发育第12.5天（E12.5）收集胚胎心脏并进行解离，通过荧光激活细胞分选（FACS）分离心肌细胞，提取心肌细胞总RNA用于基因芯片分析。