Function of transient receptor potential channels in bone-forming osteoblasts

骨骼有三个重要的功能：机械功能(保护和移动)、代谢功能(钙库)和造血细胞功能(干细胞的利基)。通过破骨细胞介导的骨降解和成骨细胞(OBS)实现的骨形成之间的平衡来保持骨的完整性。OBS在维持骨骼中的作用需要对其增殖、迁移、分化、基质沉积和矿化的分泌功能进行调节。鉴于骨病在老年人群中的高发病率，有必要提高对骨代谢调节的了解。我的研究计划的长期目标是通过调节钙内流和作为第二信使的信号来确定瞬时受体潜力(Trp)家族的离子通道在OB生理学中的贡献。在目前的资金期限内，我们建立了OB中TRP通道的表达清单，重点关注主要亚家族TRP典范(TRPC)、Trp Melastatin(TRPM)和Trp Vanilloid(TRPV)。我们证明TRPM7在OBS的增殖、迁移和分化过程中起关键作用。我们还证实了TRPM7基因在OB分化过程中的表达增强，其他人报道了随着OB分化，TRPV4的表达上调。我们最近发现骨合成因子促进TRPV4介导的成骨细胞内钙内流。短期目标：对于当前的应用，我们打算专注于TRPM7和TRPV4，并解决3个具体目标，以揭示这些渠道在OB功能中的作用。1)体外研究骨合成因子激活TRPV4的机制：我们建议通过cAMP依赖和酪氨酸依赖的通道磷酸化来详细研究合成因子PTH和WNT5a支持TRPV4激活的机制。将通过检测TRPV4的磷酸化和通过靶向突变的分子生物学来研究选择性激活剂/阻断剂的激活机制。这些方法将与细胞内钙测量和功能分析相结合，所有这些技术都在实验室得到了很好的确立。2)探讨成骨细胞分化过程中TRPM7和TRPV4基因表达增强的机制：成骨细胞分化过程中TRPM7和TRPV4基因表达增强，提示这些通道在成骨细胞功能中持续发挥作用。我们将通过评估TRPM7和TRPV4基因在OB分化过程中的转录速率，以及通过确定miRNA对TRPV4和TRPM7表达的调节作用，来研究TRPM7和TRPV4表达上调是如何发生的。3)确定TRPM7在体内骨代谢中的作用：由于我们的体外数据表明TRPM7调控OB的增殖、迁移和分化，我们将在骨组织中记录Trpm7基因失活对OB的影响。为此，将他莫昔芬诱导的转基因小鼠(CRE-Col1a1/ERT2)与Trpm7tm1clph小鼠品系杂交，该品系含有Trpm7外显子17两侧的loxP位点。在突变的小鼠系中，在不同的骨骼发育时间给予他莫昔芬将诱导OBS特异性的TRPM7缺失。对他莫昔芬治疗的小鼠骨骼状态的分析将允许在体内确定TRPM7在OB功能中的输入。预期影响：鉴于随着年龄的增长，骨量逐渐减少，骨领域的一个主要挑战是增加对骨形成过程的了解。细胞内钙水平在各种信号通路中是一个被低估的限制步骤，这项研究将有助于确定瞬时受体电位通道在成骨细胞生理学中的调节作用。

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.