Osteoblastic Respiration and IRS Signaling

成骨细胞呼吸和 IRS 信号传导

• 批准号: 8823029
• 负责人: CLIFFORD JAMES ROSEN
• 金额: $ 23.42万
• 依托单位: MAINEHEALTH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8823029
• 关键词: AKT Signaling Pathway; ATP Synthesis Pathway; Abbreviations; Affect; Attenuated; Binding; Bioenergetics; Biogenesis; Biology; Bone Density; Bone Diseases; C3H/HeJ Mouse; Calvaria; Cell Respiration; Cells; Collagen; Complex; Data; Defect; Energy Metabolism; Enzymes; Exhibits; Gene Expression; Generations; Genes; Glycolysis; Glycolysis Induction; Goals; Hormones; IRS1 gene; Image; Impairment; In Situ; In Vitro; Inbred Strain; Inbred Strains Mice; Individual; Insulin-Like Growth Factor I; Lead; Longevity; Measures; Mediating; Membrane; Membrane Potentials; Membrane Proteins; Microscopy; Mitochondria; Mus; Nature; Neonatal; Nuclear; Nutrient; Osteoblasts; Osteocalcin; Osteogenesis; Osteoid; Osteoporosis; Oxidative Phosphorylation; Oxygen Consumption; Parathyroid gland; Pathway interactions; Peptides; Proto-Oncogene Proteins c-akt; Raptors; Relative (related person); Respiration; Role; Signal Pathway; Signal Transduction; Stress; Sum; Techniques; Testing; Therapeutic Agents; Time; Work; bone; bone cell; bone mass; cellular imaging; density; energy balance; high risk; human FRAP1 protein; in vivo; insight; loss of function mutation; mineralization; mitochondrial membrane; mouse model; mutant; novel; osteoblast differentiation; public health relevance; response; skeletal; skeletal disorder; transcriptome sequencing; two-photon

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to delineate how energy utilization in bone cells is regulated and in turn, how substrate availability affects osteoblast differentiation and bone formation. Optimal bone formation is required for peak bone acquisition and skeletal remodeling across the lifespan. We previously showed IGF-I is necessary for terminal OB differentiation through the mTORC1 pathway. Moreover, PTH induces skeletal IGF-I and this peptide is required for the anabolic actions of PTH on bone. Previously we noted that in C57BL/6J (B6) and C3H/HeJ (C3H) mice, the latter exhibits higher bone density, greater bone formation, and more skeletal Igf1 expression. Importantly, C3H COBs have higher rates of oxidative phosphorylation (OxPhos) and glycolysis (Glyc) than B6. We then established that during terminal OB differentiation glycolysis (Glyc) is favored over OxPhos in both strains and that PTH, which stimulates IGF-I, increases Glyc in both COBs and calvariae ex vivo. To understand the role of the IGF pathway in the bioenergetics of OB differentiation, we have been studying the sml/sml mouse that has a defect in mTOR signaling due to a 'loss of function' mutation in the Irs1 gene. These mice have very low bone mass, reduced bone formation but normal OB number and osteoid volume. Remarkably, sml/sml COBs have significantly reduced OxPhos and Glyc during OB differentiation in vitro and in vivo. Taken together these data point to the importance of energy metabolism during bone formation, and the central role of IGF-I in cellular respiration. As such, in this proposal our over- arching hypothesis is that PTH stimulates bone formation by enhancing Glyc through the induction of IGF-I, which in turn activates IRS1/ mTorc/AKT signaling. Thus we propose 2 specific aims in a high risk, high impact strategy to delineate the importance of substrate availability during PTH induced bone formation using novel single cell imaging and bioenergetic studies of OBs and calvariae: 1-Determine the context-specific nature of OB respiration and Glyc as well as its relationship to bone formation in response to PTH in +/+ and sml/sml mice in vitro and ex vivo: We propose there is a time dependent switch during COB differentiation under the influence of PTH when Glyc is activated, leading to enhanced synthesis and mineralization of matrix. Novel in situ imaging and cellular respiration studies will be employed to determine PTH actions on single cell bioenergetics and will be compared with in vitro studies. 2-Define the importance of the IRS/ mTORC1 mTORC2/AKT signaling pathway for PTH-stimulated changes in energy balance in differentiated OBs. We postulate that mTORC2 activation via the IRS/AKT pathway is critical for up regulating Glyc during the OB differentiation following PTH. We will delineate te relationship of individual signaling components of the mTOR pathway to OB bioenergetics. Findings from these studies could lead to novel insights into the fundamental biology of OBs and enhance the possibility of newer anabolic approaches for skeletal disorders.

描述（由申请人提供）：该提案的长期目标是描述骨细胞中的能量利用是如何调节的，以及底物可用性如何影响成骨细胞分化和骨形成。在整个生命周期中，骨获取和骨骼重塑需要最佳的骨形成。我们之前表明 IGF-I 对于通过 mTORC1 途径的终末 OB 分化是必需的。此外，PTH 诱导骨骼 IGF-I，这种肽是 PTH 对骨的合成代谢作用所必需的。之前我们注意到，在 C57BL/6J (B6) 和 C3H/HeJ (C3H) 小鼠中，后者表现出更高的骨密度、更多的骨形成和更多的骨骼 Igf1 表达。重要的是，C3H COB 比 B6 具有更高的氧化磷酸化 (OxPhos) 和糖酵解 (Glyc) 率。然后我们确定，在终末 OB 分化期间，两种菌株中的糖酵解 (Glyc) 均优于 OxPhos，并且刺激 IGF-I 的 PTH 会在体外增加 COB 和颅盖中的 Glyc。为了了解 IGF 通路在 OB 分化的生物能学中的作用，我们一直在研究由于 Irs1 基因“功能丧失”突变而导致 mTOR 信号传导缺陷的 sml/sml 小鼠。这些小鼠的骨量非常低，骨形成减少，但 OB 数量和类骨质体积正常。值得注意的是，sml/sml COB 在体外和体内 OB 分化过程中显着降低了 OxPhos 和 Glyc。综上所述，这些数据表明能量代谢在骨形成过程中的重要性，以及 IGF-I 在细胞呼吸中的核心作用。因此，在本提案中，我们的首要假设是 PTH 通过诱导 IGF-I 增强糖基来刺激骨形成，IGF-I 反过来又激活 IRS1/mTorc/AKT 信号传导。因此，我们在高风险、高影响策略中提出了 2 个具体目标，以利用新型单细胞成像和 OB 和颅骨的生物能研究来描述 PTH 诱导骨形成过程中底物可用性的重要性： 1-确定 OB 呼吸的特定背景性质和 Glyc 及其与体外和离体 +/+ 和 sml/sml 小鼠中 PTH 响应的骨形成的关系：我们提出，在 COB 分化过程中，在当 Glyc 被激活时，PTH 会增强基质的合成和矿化。将采用新颖的原位成像和细胞呼吸研究来确定 PTH 对单细胞生物能的作用，并将与体外研究进行比较。 2-定义 IRS/mTORC1 mTORC2/AKT 信号通路对于分化 OB 中 PTH 刺激的能量平衡变化的重要性。我们假设通过 IRS/AKT 途径激活 mTORC2 对于 PTH 后 OB 分化过程中的 Glyc 上调至关重要。我们将描述 mTOR 通路的各个信号成分与 OB 生物能学的关系。这些研究的结果可能会对 OB 的基础生物学产生新的见解，并增强针对骨骼疾病采用新的合成代谢方法的可能性。