ANALYSIS FOR MECHANISM OF SELF-RENEWAL SIGNAL TRANSDUCTION IN EMBRYONIC STEM CELLS

在LIF的存在下，胚胎干细胞的多能表型得以维持。LIF结合由LIF受体β和gp130组成的细胞表面受体复合物，通过该复合物激活MAP激酶和STAT3等多种信号分子。我们报道了gp130的胞内结构域在胚胎干细胞的自我更新中起重要作用。在本研究中，我们研究了gp130参与胚胎干细胞自我更新的信号通路。对负责STAT3激活的gp130细胞质结构域进行突变分析是ES细胞自我更新所必需的，而对SHP2和MAP激酶激活所需要的突变分析则是多余的。接下来，我们构建了一个由STAT3整个区域与雌激素受体配体结合域组成的融合蛋白。该融合蛋白（STAT3ER）在合成配体4-羟基他莫昔芬（4HT）存在下被二聚体激活。当稳定表达STAT3ER的ES细胞在无LIF和饲养细胞存在的4HT条件下培养时，它们保持形态未分化状态，并表达未分化状态特异性标记物（SSEA-1和碱性磷酸酶）。此外，将STAT3ER和4HT维持的胚胎干细胞注入囊胚后，有助于嵌合小鼠的产生。这些结果表明STAT3的激活足以维持胚胎干细胞的多能性。Oct-3/4转录因子在ES细胞和EG细胞中特异性表达。已知它是胚胎干细胞内细胞团形成和维持未分化状态所必需的。可能是LIF或STAT3信号与Oct-3/4协同作用，通过未知因子抑制ES细胞的分化。

The pluripotent phenotype of ES cells is maintained in the presence of LIF.LIF binds to a cell surface receptor complex composed of LIF receptor β and gp130, through which several signaling molecules including MAP kinase and STAT3 are activated. We reported that the intracellular domain of gp130 plays an important role in self-renewal of ES cells. In the present study, we examined the signaling pathway through which gp130 contributes to the self-renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 responsible for STAT3 activation is necessary for self-renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we have constructed a fusion protein composed of the entire region of STAT3 and the ligand binding domain of estrogen receptor. This fusion protein(STAT3ER)was dimerized and activated in the presence of a synthetic ligand 4-hydroxytamoxifen(4HT). When ES cells stably expressing STAT3ER were cultured in the presence of 4HT without LIF and feeder cells, they maintained a morphologically undifferentiated state and expressed undifferentiated state-specific markers(SSEA-1 and alkaline phosphatase). Moreover, ES cells maintained by STAT3ER and 4HT contributed to chimeric mice production when they were injected into blastocysts. These results indicate that STAT3 activation is sufficient to maintain the pluripotency of ES cells. Oct-3/4 transcription factor is expressed in ES cells and EG cells specifically. It is known to be essential for the formation of inner cell mass and for the maintenance of undifferentiated state of ES cells. It is likely that LIF or STAT3 signals inhibit differentiation of ES cells through yet unidentified factor by cooperating with Oct-3/4.