Department of Hematology, Medical Research Institute, Tokyo Women's Medical University, Tokyo, Japan.
Histochem Cell Biol. 2010 Dec;134(6):555-63. doi: 10.1007/s00418-010-0764-0. Epub 2010 Nov 18.
We have shown previously that PI3K/Akt pathway is active after cell differentiation in HL60 cells. In the present study, we have investigated whether additional molecules, such as protein kinase C (PKC), are involved in the regulation, not only of telomerase, but also of leukemia cell differentiation. We show that PKC activates telomerase and is, itself, activated following VD3- or ATRA-induced differentiation of HL60 cells, as was observed for PI3K/Akt. To clarify the significance of PI3K/Akt and PKC pathway activation in leukemia cell differentiation, we examined the active proteins in either the downstream or upstream regulation of these pathways. In conjunction with the activation of Akt or PKC, mTOR and S6K were phosphorylated and the protein expression levels of Rictor were increased, compared with Raptor, following cell differentiation. Silencing by Rictor siRNA resulted in the attenuation of Akt phosphorylation on Ser473 and PKCα/βII phosphorylation, as well as the inhibition of Rictor itself, suggesting that Rictor is an upstream regulator of both Akt and PKC. In addition, in cells induced to differentiate by ATRA or VD3, Nitroblue-tetrazolium (NBT) reduction and esterase activity, were blocked either by LY294002, a PI3K inhibitor, or by BIM, a PKC inhibitor, without affecting cell surface markers such as CD11b or CD14. Intriguingly, the silencing of Rictor by its siRNA also suppressed the reducing ability of NBT following VD3-induced cell differentiation. Taken together, our results show that Rictor associated with mTOR (mTORC2) regulates the activity of both Akt and PKC that are involved in cell functions such as NBT reduction and esterase activity induced by leukemia cell differentiation.
我们之前已经证明，在 HL60 细胞中细胞分化后 PI3K/Akt 通路是活跃的。在本研究中，我们研究了其他分子，如蛋白激酶 C（PKC），是否参与调节，不仅是端粒酶，而且是白血病细胞分化。我们表明 PKC 激活端粒酶，并且自身在 HL60 细胞的 VD3 或 ATRA 诱导分化后被激活，如 PI3K/Akt 观察到的那样。为了阐明 PI3K/Akt 和 PKC 通路激活在白血病细胞分化中的意义，我们检查了这些通路下游或上游调节中的活性蛋白。与 Akt 或 PKC 的激活相结合，mTOR 和 S6K 被磷酸化，并且在细胞分化后，Rictor 的蛋白表达水平增加，与 Raptor 相比。通过 Rictor siRNA 沉默导致 Akt 在 Ser473 上的磷酸化和 PKCα/βII 磷酸化减弱，以及 Rictor 自身的抑制，表明 Rictor 是 Akt 和 PKC 的上游调节剂。此外，在用 ATRA 或 VD3 诱导分化的细胞中，Nitroblue-tetrazolium（NBT）还原和酯酶活性被 PI3K 抑制剂 LY294002 或 PKC 抑制剂 BIM 阻断，而不影响细胞表面标志物如 CD11b 或 CD14。有趣的是，Rictor 的 siRNA 沉默也抑制了 VD3 诱导细胞分化后 NBT 的还原能力。总之，我们的结果表明，与 mTOR（mTORC2）相关的 Rictor 调节 Akt 和 PKC 的活性，这些活性参与细胞功能，如 NBT 还原和酯酶活性诱导的白血病细胞分化。
