Function of transient receptor potential channels in bone-forming osteoblasts

瞬时受体电位通道在成骨成骨细胞中的功能

• 批准号: RGPIN-2014-05594
• 负责人: Moreau, Robert
• 金额: $ 1.92万
• 依托单位: Université du Québec à Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=588499
• 关键词: Function; transient; receptor; potential; channels

Bones fulfill three important functions: mechanical (protection and locomotion), metabolic (reservoir of calcium) and hematopoietic (niches for stem cells). Bone integrity is preserved by the balance between bone degradation mediated by osteoclasts and bone formation achieved by osteoblasts (OBs). The role for OBs in the maintenance of bones requires tuned regulation of their proliferation, migration, differentiation, secretory functions for matrix deposition and mineralization. Given the high incidence of bone diseases in the elderly population, there is a need for improved understanding of the regulation of bone metabolism. The long-term goal of my research program is to establish the contribution for ion channels of the transient receptor potential (TRP) family in OB physiology, through modulation of calcium influx and signaling as second messenger. Over the present funding term we established an expression inventory of TRP channels in OBs, focussing on the major subfamilies TRP Canonical (TRPC), TRP Melastatin (TRPM) and TRP Vanilloid (TRPV). We demonstrated that TRPM7 is critical for proliferation, migration and differentiation of OBs. We also evidenced that TRPM7 gene expression is enhanced during OB differentiation and others reported upregulation of TRPV4 expression with OB differentiation. We recently found that bone anabolic factors promote TRPV4-mediated calcium influx in OBs. SHORT-TERM OBJECTIVES: For the current application, we intend to focus on TRPM7 and TRPV4, and address 3 specific objectives to unravel the roles of these channels in OB function. 1) Study in vitro the mechanism responsible for TRPV4 activation by bone anabolic factors: We propose to detail the mechanisms by which the anabolic factors PTH and Wnt5a support TRPV4 activation, by focusing on the cAMP-dependent and tyrosine-dependent channel phosphorylation. Activation mechanisms will be investigated with selective activators/blockers, by detection of TRPV4 phosphorylation and molecular biology through targeted mutation. These approaches will be combined with measurements of intracellular calcium and functional assays, all techniques that are well established in the laboratory. 2) Determine the mechanism responsible for enhanced expression of TRPM7 and TRPV4 in OB differentiation: The enhanced TRPM7 and TRPV4 gene expressions during OB differentiation suggest sustained contribution for these channels in OB functions. We will investigate how up-regulation of TRPM7 and TRPV4 expression occurs, by evaluating the transcription rate of TRPM7 and TRPV4 genes during OB differentiation, and by determining the contribution of miRNA for the regulation of TRPV4 and TRPM7 expression. 3) Determine the roles of TRPM7 in bone metabolism in vivo: Since our in vitro data suggest that TRPM7 regulates OB proliferation, migration and differentiation, we will document in bone tissue the impact of Trpm7 gene inactivation in OBs. For this, tamoxifen-inducible transgenic mice (Cre-Col1a1/ERT2) will be crossed with the Trpm7tm1clph mouse line, which contains loxP sites flanking the Trpm7 exon 17. Deletion of TRPM7 specifically in OBs will be induced in the mutant mouse line with administration of tamoxifen at different time of bone development. Analysis of the bone status of tamoxifen-treated mice will allow identifying in vivo the input for TRPM7 in OB functions. EXPECTED IMPACT: Given the gradual loss of bone mass with aging, a main challenge in the bone field is to increase knowledge on the bone-forming process. Intracellular calcium levels constitute an underestimated limiting step in various signaling pathways and this research will allow identifying regulatory roles for transient receptor potential channels in osteoblast physiology.

骨骼有三个重要的功能：机械（保护和运动）、代谢（钙的储存）和造血（干细胞的小生境）。通过破骨细胞介导的骨降解与成骨细胞（OB）实现的骨形成之间的平衡来保持骨完整性。OBs在维持骨骼中的作用需要调谐调节其增殖、迁移、分化、分泌功能以进行基质沉积和矿化。鉴于老年人群中骨疾病的高发病率，有必要提高对骨代谢调节的认识。我的研究计划的长期目标是建立在OB生理学的瞬时受体电位（TRP）家族的离子通道的贡献，通过调节钙内流和信号作为第二信使。在目前的资助期限内，我们建立了一个表达库存的TRP通道在OB，集中在主要的亚家族TRP经典（TRPC），TRP Melastatin（TRPM）和TRP香草（TRPV）。我们证明了TRPM 7对OB的增殖、迁移和分化至关重要。我们还证明了TRPM 7基因表达在OB分化过程中增强，其他人报道了TRPV 4表达随OB分化而上调。我们最近发现，骨合成代谢因子促进TRPV 4介导的骨钙内流。 短期替代品：对于当前的应用，我们打算专注于TRPM 7和TRPV 4，并解决3个具体目标，以阐明这些通道在OB功能中的作用。 1)体外研究骨合成代谢因子激活TRPV 4的机制：我们建议通过关注cAMP依赖性和酪氨酸依赖性通道磷酸化来详细说明合成代谢因子PTH和Wnt 5a支持TRPV 4激活的机制。将使用选择性激活剂/阻断剂，通过检测TRPV 4磷酸化和靶向突变的分子生物学研究激活机制。这些方法将与细胞内钙和功能测定的测量相结合，所有这些技术都在实验室中得到了很好的建立。 2)确定在OB分化中TRPM 7和TRPV 4表达增强的机制：在OB分化期间TRPM 7和TRPV 4基因表达增强表明这些通道在OB功能中的持续贡献。我们将研究TRPM 7和TRPV 4表达的上调是如何发生的，通过评估TRPM 7和TRPV 4基因在OB分化过程中的转录速率，以及通过确定miRNA对TRPV 4和TRPM 7表达调控的贡献。 3)确定TRPM 7在体内骨代谢中的作用：由于我们的体外数据表明，TRPM 7调节OB增殖，迁移和分化，我们将在骨组织中记录OB中Trpm 7基因失活的影响。为此，他莫昔芬诱导的转基因小鼠（Cre-Col 1a 1/ERT 2）将与Trpm 7 tm 1clph小鼠系杂交，该小鼠系包含Trpm 7外显子17侧翼的loxP位点。在骨发育的不同时间给予他莫昔芬，将在突变小鼠系中诱导特异性OBs中TRPM 7的缺失。对他莫昔芬处理的小鼠的骨状态的分析将允许在体内鉴定OB功能中TRPM 7的输入。 预期影响：鉴于随着年龄的增长，骨量逐渐丢失，骨领域的主要挑战是增加对骨形成过程的了解。细胞内钙离子水平构成了一个被低估的限制步骤，在各种信号通路，这项研究将允许确定在成骨细胞生理学的瞬时受体电位通道的调节作用。