TRPM8 is a novel regulator of bone homeostasis through neural and cell-autonomous mechanisms

TRPM8 是一种通过神经和细胞自主机制调节骨稳态的新型调节剂

• 批准号: 9108599
• 负责人: Katherine Jean Motyl
• 金额: $ 12.68万
• 依托单位: MAINEHEALTH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-10 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9108599
• 关键词: Absence of pain sensation; Adipocytes; Adipose tissue; Adult; Afferent Neurons; Alleles; Body Temperature; Bone Density; Bone Growth; Bone Marrow; Bone Resorption; Bone remodeling; Breeding; CRISPR/Cas technology; Calvaria; Cations; Cell Communication; Cells; Coculture Techniques; Collaborations; Conditioned Culture Media; Cyclic AMP-Dependent Protein Kinases; Data; Defect; Dendrites; Development; Ear; Employee Strikes; Esthesia; Femur; Figs - dietary; Foundations; Future; Genes; Homeostasis; In Vitro; Injection of therapeutic agent; Length; Marrow; Mediating; Menthol; Mesenchymal; Microfluidics; Mus; Nerve Fibers; Neurons; Obesity; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; Pain; Pathway interactions; Phenotype; Role; Signal Transduction; Site; Stromal Cells; Sympathetic Nervous System; TRPV1 gene; Techniques; Temperature Sense; Testing; Thermogenesis; Thick; Transgenic Mice; Work; adipocyte differentiation; bone; bone cell; bone loss; bone mass; cell type; in vitro Model; in vivo; knock-down; lipid metabolism; long bone; novel; osteoblast differentiation; osteogenic; paracrine; public health relevance; receptor; relating to nervous system; release of sequestered calcium ion into cytoplasm; research study; skeletal; small hairpin RNA; spine bone structure; substantia spongiosa; voltage

 DESCRIPTION (provided by applicant): The discovery of modulators of bone remodeling is crucial to developing new treatments for osteoporosis. The transient receptor potential melastatin 8 (TRPM8, a.k.a the cold and menthol receptor) is a voltage-gated cation channel that is open below 26°C. Neural TRPM8 is important for cold sensation, cold pain and cold analgesia, and thermogenesis. However, a non-neuronal role for TRPM8 is beginning to emerge. Adult Trpm8- /- mice have reduced trabecular bone volume fraction in the vertebrae and to a lesser extent in the long bones. This could be due to suppressed bone formation and/or increased bone resorption. In vitro, we found no difference in Trpm8-/- osteoclast differentiation compared to wildtype. Alternately, Trpm8-/- bone marrow stromal cells (BMSCs) and calvarial osteoblasts (OB) have reduced differentiation. Consistent with the in vivo phenotype of reduced bone marrow adiposity, BMSCs and ear MSCs (eMSCs) fail to fully differentiate into adipocytes in vitro. This novel finding demonstrates there is a significant defect in differentiation into eiter OBs or adipocytes. However, the majority of TRPM8 expression in vivo is on sensory neurons and we have evidence that neurons expressing TRPM8 are present in the bone marrow cavity. These could influence osteoblasts through paracrine pathways. My overarching hypotheses are that TRPM8 supports osteoblast and adipocyte differentiation through its direct expression in MSC-derived precursors (Aim 1) and that neural TRPM8 supports osteoblast differentiation through paracrine mechanisms (Aim 2). Finally, I will utilize novel Trpm8fl/fl mice to selectively delete Trpm8 in mesenchymal cells versus sensory neurons (Aim 3), where I will be able to delineate the extent to which each cell type contributes to the reduced bone mass phenotype. SPECIFIC AIM 1: In this aim, I will test the hypothesis that TRPM8 signaling in mesenchymal cells activates PKA to promote adipocyte and osteoblast differentiation utilizing shRNA and calcium flux analyses. SPECIFIC AIM 2: Despite striking in vitro differences in MSC differentiation in the absence of TRPM8, the majority of TRPM8 expression in vivo is on sensory neurons. Aim 2a: Test the hypothesis that sensory neurons expressing TRPM8 are present in bone. Aim 2b: Test the hypothesis that TRPM8 activation in sensory neurons supports osteoblast differentiation through dendrite:osteoblast contact. SPECIFIC AIM 3: Trpm8fl/fl mice will be crossed with SynapsinCre/+ and Prrx1Cre/+ mice to test whether effects of in vivo Trpm8 deletion are mediated through neural or osteoblast TRPM8 expression, respectively. The proposed experiments will identify mechanisms though which TRPM8 regulates osteoblast and adipocyte lineage, generate novel data on neural- mesenchymal cell interactions, and set forth a foundation for future work examining sensory neuron control of bone remodeling.

 描述（由申请人提供）：骨重塑调节剂的发现对于开发骨质疏松症新疗法至关重要。瞬态受体电位 melastatin 8（TRPM8，又名冷薄荷醇受体）是电压门控阳离子通道，在 26°C 以下开放。神经TRPM8对于冷感觉、冷痛和冷镇痛以及生热作用很重要。然而，TRPM8 的非神经元作用开始显现。成年Trpm8-/-小鼠椎骨中的骨小梁体积分数减少，长骨中的骨小梁体积分数减少。这可能是由于骨形成受到抑制和/或骨吸收增加所致。在体外，我们发现与野生型相比，Trpm8-/-破骨细胞分化没有差异。另外，Trpm8-/- 骨髓基质细胞 (BMSC) 和颅骨成骨细胞 (OB) 的分化程度降低。与骨髓脂肪减少的体内表型一致，BMSC 和耳 MSC (eMSC) 在体外无法完全分化为脂肪细胞。这一新发现表明，分化为 OB 或脂肪细胞时存在显着缺陷。然而，体内 TRPM8 的大部分表达是在感觉神经元上，我们有证据表明表达 TRPM8 的神经元存在于骨髓腔中。这些可能通过旁分泌途径影响成骨细胞。我的总体假设是 TRPM8 通过在 MSC 衍生的前体中直接表达来支持成骨细胞和脂肪细胞分化（目标 1），而神经 TRPM8 通过旁分泌机制支持成骨细胞分化（目标 2）。最后，我将利用新型 Trpm8fl/fl 小鼠选择性删除间充质细胞与感觉神经元中的 Trpm8（目标 3），这样我将能够描绘出每种细胞类型对骨量减少表型的贡献程度。具体目标 1：在此目标中，我将利用 shRNA 和钙流分析来测试间充质细胞中的 TRPM8 信号传导激活 PKA 以促进脂肪细胞和成骨细胞分化的假设。具体目标 2：尽管在缺乏 TRPM8 的情况下 MSC 分化在体外存在显着差异，但体内大部分 TRPM8 表达位于感觉神经元上。目标 2a：检验骨骼中存在表达 TRPM8 的感觉神经元的假设。目标 2b：检验感觉神经元中 TRPM8 激活通过树突：成骨细胞接触支持成骨细胞分化的假设。具体目标 3：将 Trpm8fl/fl 小鼠与 SynapsinCre/+ 和 Prrx1Cre/+ 小鼠杂交，以测试体内 Trpm8 缺失的影响是否分别通过神经或成骨细胞 TRPM8 表达介导。拟议的实验将确定 TRPM8 调节成骨细胞和脂肪细胞谱系的机制，生成有关神经间充质细胞相互作用的新数据，并为未来检查感觉神经元对骨重塑的控制的工作奠定基础。