In a recent study, we found that blocking the protein kinase ataxia telangiectasia mutated (ATM) with the small molecule inhibitor (SMI) KU-55933 can completely abrogate Mn-induced phosphorylation of p53 at serine 15 (p-p53) in human induced pluripotent stem cell (hiPSC)-differentiated striatal neuroprogenitors. However, in the immortalized mouse striatal progenitor cell line STHdh^Q7/Q7, a concentration of KU55933 far exceeding its IC₅₀ for ATM was required to inhibit Mn-induced p-p53. This suggested an alternative signaling system redundant with ATM kinase for activating p53 in this cell line- one that was altered by KU55933 at these higher concentrations (i.e. mTORC1, DNApk, PI3K). To test the hypothesis that one or more of these signaling pathways contributed to Mn-induced p-p53, we utilized a set of SMIs (e.g. NU7441 and LY294002) known to block DNApk, PI3K, and mTORC1 at distinct concentrations. We found that the SMIs inhibit Mn-induced p-p53 expression near the expected IC_50s for PI3K, versus other known targets. We hypothesized that inhibiting PI3K reduces intracellular Mn and thereby decreases activation of p53 by Mn. Using the cellular fura-2 manganese extraction assay (CFMEA), we determined that KU55933/60019, NU7441, and LY294002 (at concentrations near their IC_50s for PI3K) all decrease intracellular Mn (∼50%) after a dual, 24-h Mn and SMI exposure. Many pathways are activated by Mn aside from p-p53, including AKT and mTOR pathways. Thus, we explored the activation of these pathways by Mn in STHdh cells as well as the effects of other pathway inhibitors. p-AKT and p-S6 activation by Mn is almost completely blocked upon addition of NU7441(5 μM) or LY294002(7 μM), supporting PI3K’s upstream role in the AKT/mTOR pathway. We also investigated whether PI3K inhibition blocks Mn uptake in other cell lines. LY294002 exposure did not reduce Mn uptake in ST14A, Neuro2A, HEK293, MEF, or hiPSC-derived neuroprogenitors. Next, we sought to determine whether inhibition of PI3K blocked p53 phosphorylation by directly blocking an unknown PI3K/p53 interaction or indirectly reducing intracellular Mn, decreasing p-p53 expression. In-Cell Western and CFMEA experiments using multiple concentrations of Mn exposures demonstrated that intracellular Mn levels directly correlated with p-p53 expression with or without addition of LY294002. Finally, we examined whether PI3K inhibition was able to block Mn-induced p-p53 activity in hiPSC-derived striatal neuroprogenitors. As expected, LY294002 does not block Mn-induced p-p53 as PI3K inhibition is unable to reduce Mn net uptake in this cell line, suggesting the effect of LY294002 on Mn uptake is relatively specific to the STHdh mouse striatal cell line.

在最近的研究中，我们发现用小分子抑制剂（SMI）KU-55933阻断蛋白激酶共济失调毛细血管扩张突变（ATM）可以完全消除人诱导的多能性中Mn诱导的丝氨酸15（p-p53）上p53的磷酸化。干细胞（hiPSC）分化的纹状体神经祖细胞。然而，在永生化的小鼠纹状体祖细胞STHdh ^{Q7 / Q7中}，KU55933的浓度远远超过其IC ₅₀ATM的抑制作用需要抑制Mn诱导的p-p53。这暗示了一种替代的信号转导系统，该信号转导系统多余于ATM激酶，用于激活该细胞系中的p53，该细胞系在较高浓度下被KU55933改变（即mTORC1，DNApk，PI3K）。为了测试一个或多个这些信号传导途径促成Mn诱导的p-p53的假设，我们利用了一组已知的SMI（例如NU7441和LY294002）以不同的浓度阻断DNApk，PI3K和mTORC1。我们发现，SMIs在预期的IC _50s附近抑制了Mn诱导的p-p53表达。与其他已知目标相对比。我们假设抑制PI3K会降低细胞内Mn，从而降低Mn对p53的激活。使用细胞呋喃2锰提取测定法（CFMEA），我们确定KU55933 / 60019，NU7441和LY294002（浓度接近其IC _50s对于PI3K），在24小时Mn和SMI双重暴露后，所有这些都降低了细胞内Mn（〜50％）。除p-p53以外，Mn还激活了许多途径，包括AKT和mTOR途径。因此，我们探索了Mn在STHdh细胞中对这些途径的激活以及其他途径抑制剂的作用。加入NU7441（5μM）或LY294002（7μM）后，Mn对p-AKT和p-S6的激活几乎被完全阻断，从而支持PI3K在AKT / mTOR途径中的上游作用。我们还研究了PI3K抑制是否阻止其他细胞系中的Mn吸收。LY294002暴露并未降低ST14A，Neuro2A，HEK293，MEF或hiPSC衍生的神经祖细胞中的Mn摄取。接下来，我们试图确定是否通过直接阻断未知的PI3K / p53相互作用或间接降低细胞内Mn来抑制PI3K从而阻断p53磷酸化，降低p-p53表达。使用多种浓度的Mn暴露进行的细胞内Western和CFMEA实验表明，添加或不添加LY294002，细胞内Mn水平与p-p53表达直接相关。最后，我们检查了PI3K抑制是否能够阻断hiPSC衍生的纹状体神经祖细胞中Mn诱导的p-p53活性。正如预期的那样，LY294002不会阻断Mn诱导的p-p53，因为PI3K抑制不能降低该细胞系中Mn的净摄取，这表明LY294002对Mn摄取的作用相对于STHdh小鼠纹状体细胞系是相对特异性的。我们检查了PI3K抑制作用是否能够阻断hiPSC衍生的纹状体神经祖细胞中Mn诱导的p-p53活性。正如预期的那样，LY294002不会阻断Mn诱导的p-p53，因为PI3K抑制不能降低该细胞系中Mn的净摄取，这表明LY294002对Mn摄取的作用相对于STHdh小鼠纹状体细胞系是相对特异性的。我们检查了PI3K抑制作用是否能够阻断hiPSC衍生的纹状体神经祖细胞中Mn诱导的p-p53活性。正如预期的那样，LY294002不会阻断Mn诱导的p-p53，因为PI3K抑制不能降低该细胞系中Mn的净摄取，这表明LY294002对Mn摄取的作用相对于STHdh小鼠纹状体细胞系是相对特异性的。