Pyruvate dehydrogenase kinase 1 is essential for transplantable mouse bone marrow hematopoietic stem cell and progenitor function

Background
Accumulating evidence suggests that hypoxic areas in the bone marrow are crucial for maintenance of hematopoietic stem cells (HSCs) by supporting a quiescent state of cell cycle and regulating the transplantation capacity of long-term (LT)-HSCs. In addition, HSCs seem to express a metabolic profile of energy production away from mitochondrial oxidative phosphorylation in favor of glycolysis. At oxygen deprivation, hypoxia inducible factor 1α (HIF-1α) is known to induce glycolytic enzymes as well as suppressing mitochondrial energy production by inducing pyruvate dehydrogenase kinase 1 (Pdk1) in most cell types. It has not been established whether PDK1 is essential for HSC function and mediates hypoxia-adapting functions in HSCs. While the Pdk gene family contains four members (Pdk1-4), it was recently shown that Pdk2 and Pdk4 have an important role in regulating LT-HSCs.
Principle findings
Here we demonstrate that PDK1 activity is crucial for transplantable HSC function. Whereas Pdkl, Pdk2, and Pdk3 transcripts were expressed at higher levels in different subtypes of HSCs compared to differentiated cells, we could not detect any major differences in expression between LT-HSCs and more short-term HSCs and multipotent progenitors. When studying HIF-1α-mediated regulation of Pdk activity in vitro, Pdk1 was the most robust target regulated by hypoxia, whereas Pdk2, Pdk3, and Pdk4 were not affected. Contrary, genetic ablation in a cre-inducible Hif-1α knockout mouse did not support a link between HIF-1α and Pdk1. Silencing of Pdk1 by shRNA lentiviral gene transfer partially impaired progenitor colony formation in vitro and had a strong negative effect on both long-term and short-term engraftment in mice.
Conclusions
Our study demonstrates that PDK1 has broad effects in hematopoiesis and is a critical factor for engraftment of both HSCs and multipotent progenitors upon transplantation to recipient mice. While Pdk1 was a robust hypoxia-inducible gene mediated by HIF-1α in vitro, we could not find evidence of any in vivo links between Pdk1 and HIF-1α.

研究背景
越来越多的证据表明，骨髓内的缺氧区域通过维持造血干细胞（hematopoietic stem cells, HSCs）的细胞周期静息状态，并调控长期造血干细胞（long-term (LT)-HSCs）的移植能力，在造血干细胞维持过程中发挥关键作用。此外，造血干细胞似乎呈现出一种远离线粒体氧化磷酸化、偏向糖酵解的能量代谢特征。已知在多数细胞类型中，缺氧条件下缺氧诱导因子1α（hypoxia inducible factor 1α, HIF-1α）可诱导糖酵解相关酶的表达，并通过诱导丙酮酸脱氢酶激酶1（pyruvate dehydrogenase kinase 1, Pdk1）抑制线粒体能量产生。目前尚不清楚PDK1是否对造血干细胞功能至关重要，以及其是否介导造血干细胞的缺氧适应功能。尽管Pdk基因家族包含4个成员（Pdk1-4），但近期研究表明Pdk2和Pdk4在调控长期造血干细胞中具有重要作用。

主要研究结果
本研究证实PDK1活性对可移植造血干细胞的功能至关重要。与分化细胞相比，Pdk1、Pdk2和Pdk3的转录本在不同亚型的造血干细胞中表达水平更高，但我们未检测到长期造血干细胞与短期造血干细胞及多能祖细胞之间存在显著表达差异。体外研究HIF-1α介导的Pdk活性调控时发现，Pdk1是受缺氧调控最显著的靶点，而Pdk2、Pdk3和Pdk4的表达未受影响。与之相反，在可诱导敲除Hif-1α的Cre重组酶小鼠中进行基因敲除实验，并未证实HIF-1α与Pdk1之间存在关联。通过短发夹RNA（shRNA）慢病毒基因转导沉默Pdk1，可在体外部分削弱祖细胞集落形成能力，并对小鼠体内的长期及短期造血干细胞植入均产生显著抑制作用。

研究结论
本研究证实PDK1在造血过程中具有广泛调控作用，是造血干细胞及多能祖细胞移植至受体小鼠后成功植入的关键因子。尽管体外实验中Pdk1是受HIF-1α介导的显著缺氧诱导基因，但我们未发现Pdk1与HIF-1α在体内存在关联的证据。