Mitochondria and TFAM in Osteoblast Biology

摘要 新陈代谢曾经被认为仅仅是细胞状态的结果，现在人们知道它起着关键作用 在决定细胞分化方面。非氧化性糖酵解和氧化磷酸化（OxPhos）是两种 细胞内ATP的来源。增加成骨细胞活性和骨量的因素，如缺氧， 诱导因子1a（HIF 1）已被证明可以激活非氧化糖酵解。体外成骨 间充质祖细胞的分化增加了非氧化糖酵解和OxPhos。但 OxPhos在体内成骨细胞生物学中的作用在很大程度上未被探索。为了填补这一知识空白，我们生成了一个 未定型间充质祖细胞中缺乏线粒体转录因子A（TFAM）的突变小鼠 及其后代（PRX;TFAMf/f）。TFAM调节线粒体基因的转录， 电子传递链的13个亚基，从而控制OxPhos。3周龄PRX的分析;TFAMf/f 骨骼显示突变体中存在严重的低骨量表型，伴有自发性骨折。 因此，我们的数据表明，间充质TFAM是必要的骨量积累。此外，我们还提供 TFAM缺失抑制骨髓基质细胞体外分化的初步证据 （BMSCs）进入成骨细胞并显著降低其细胞内ATP水平。OxPhos的损害是 TFAM丢失的最强大，一致和最好表征的生物学后果， 多种细胞类型。”“然而，TFAM也调节线粒体DNA的复制，线粒体具有 功能超越OxPhos和ATP生产。因此，为了确定OxPhos和 因此，细胞内ATP减少是PRX;TFAMf/f骨表型的主要原因， 通过强制上调非氧化性糖酵解来纠正ATP水平是否会阻止 PRX;TFAMf/f小鼠的低骨量。为此，我们将PRX;TFAMf/f突变体与小鼠杂交， 在相同的细胞中过表达组成型稳定的HIF 1（PRX; HIF 1dPAf/f）。已知HIF 1促进 非氧化糖酵解和损害OxPhos。PRX;TFAMf/f; HIF 1dPAf/f双突变体的初步分析 小鼠显示，增加的HIF 1活性校正了PRX;TFAMf/f突变体的骨表型。基础上 这些发现，我们现在将测试的假设，TFAM在细胞的成骨细胞系是至关重要的骨 通过促进OxPhos并因此确保细胞内ATP的适当水平来实现质量积累和维持。 我们将在三个目标中检验我们的假设。线粒体活性的进行性损害已经被 与许多与衰老有关的疾病有关，但目前还不确定这种联系是否是由于，至少在 一部分，一个功能失调的OxPhos。我们在这方面提出的实验的成功完成， 应用将扩大和加深我们对能量代谢作用的认识，特别是OxPhos， 成骨细胞分化的调控及骨量的增加和维持。 "

ABSTRACT Once thought to be a mere consequence of the state of the cell, metabolism is now known to play a critical role in dictating cell differentiation. Non-oxidative glycolysis and oxidative phosphorylation (OxPhos) are the two sources of intracellular ATP. Factors that increase osteoblast activity and bone mass such as the Hypoxia- Inducible Factor 1a (HIF1) have been shown to activate non-oxidative glycolysis. In vitro osteogenic differentiation of mesenchymal progenitors increases both non-oxidative glycolysis and OxPhos. However, the role of OxPhos in osteoblast biology in vivo is largely unexplored. To fill this gap in knowledge, we generated a mutant mouse lacking Mitochondrial Transcription Factor A (TFAM) in uncommitted mesenchymal progenitors and their descendants (PRX;TFAMf/f). TFAM regulates transcription of the mitochondrial genes that encode thirteen subunits of the electron transport chain and thus controls OxPhos. Analysis of 3-week-old PRX;TFAMf/f bones revealed the presence of a severe low bone mass phenotype with spontaneous fractures in mutants. Our data thus indicate that mesenchymal TFAM is necessary for bone mass accrual. In addition, we provided preliminary evidence that loss of TFAM inhibits the in vitro differentiation of bone marrow stromal cells (BMSCs) into osteoblasts and significantly reduces their intracellular levels of ATP. Impairment of OxPhos is the most powerful, consistent and best characterized biological consequence of loss of TFAM across numerous cell types." " However, TFAM also regulates duplication of mitochondrial DNA, and mitochondria have functions that go beyond OxPhos and ATP production. Therefore, to establish if the impairment of OxPhos and thus the decreased intracellular ATP is the primary cause of the PRX;TFAMf/f bone phenotype, we asked whether correcting the ATP levels through forced upregulation of non-oxidative glycolysis would prevent the low bone mass of PRX;TFAMf/f mice. For this purpose, we crossed PRX;TFAMf/f mutants with mice overexpressing a constitutively stabilized HIF1 in the same cells (PRX;HIF1dPAf/f). HIF1 is known to promote non-oxidative glycolysis and to impair OxPhos.. Preliminary analysis of PRX;TFAMf/f;HIF1dPAf/f double mutant mice revealed that increased HIF1 activity corrected the bone phenotype of PRX;TFAMf/f mutants. Building on these findings, we will now test the hypothesis that TFAM in cells of the osteoblast lineage is crucial for bone mass accrual and maintenance by promoting OxPhos and thus ensuring the proper levels of intracellular ATP. We will test our hypothesis in three Aims. Progressive impairment of mitochondrial activity has been associated with numerous aging-related diseases, but it is uncertain whether this association is due, at least in part, to a dysfunctional OxPhos. The successful accomplishment of the experiments we propose in this application will expand and deepen our knowledge of the role of energy metabolism, particularly OxPhos, in the regulation of osteoblast differentiation and bone mass accrual and maintenance. "