Mitochondria and TFAM in Osteoblast Biology

摘要 曾经被认为只是细胞状态的结果，现在已知新陈代谢发挥着关键作用 决定细胞分化。非氧化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，例如缺氧 诱导因子 1a (HIF1) 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氧化磷酸。然而， OxPhos 在体内成骨细胞生物学中的作用很大程度上尚未被探索。为了填补这一知识空白，我们生成了 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 调节编码线粒体基因的转录 电子传递链的 13 个亚基，从而控制 OxPhos。 3 周龄 PRX 分析；TFAMf/f 骨骼显示突变体存在严重的低骨量表型，并伴有自发性骨折。 因此，我们的数据表明间充质 TFAM 对于骨量增长是必要的。此外，我们还提供了 初步证据表明TFAM的缺失抑制骨髓基质细胞的体外分化 (BMSC) 进入成骨细胞并显着降低其细胞内 ATP 水平。 OxPhos 的损伤是 TFAM 缺失带来的最强大、最一致和最具特征的生物学后果 许多细胞类型。” “然而，TFAM 也调节线粒体 DNA 的复制，并且线粒体具有 其功能超出了 OxPhos 和 ATP 生产的范围。因此，要确定 OxPhos 和 因此，细胞内 ATP 减少是 PRX;TFAMf/f 骨表型的主要原因，我们询问 通过强制上调非氧化糖酵解来纠正 ATP 水平是否可以防止 PRX；TFAMf/f 小鼠骨量低。为此，我们将 PRX;TFAMf/f 突变体与小鼠杂交 在相同细胞中过表达组成型稳定的 HIF1 (PRX;HIF1dPAf/f)。 HIF1 已知可促进 非氧化糖酵解并损害 OxPhos.. PRX;TFAMf/f;HIF1dPAf/f 双突变体的初步分析 小鼠研究表明，HIF1 活性的增加纠正了 PRX;TFAMf/f 突变体的骨表型。建立在 根据这些发现，我们现在将检验成骨细胞谱系细胞中的 TFAM 对骨骼至关重要的假设 通过促进 OxPhos 来增加和维持质量，从而确保细胞内 ATP 的适当水平。 我们将在三个目标中检验我们的假设。线粒体活性逐渐受损 与许多与衰老相关的疾病有关，但尚不确定这种关联是否合理，至少在 部分，功能失调的 OxPhos。我们在此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， 成骨细胞分化以及骨量增加和维持的调节。 ”

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. "