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

 描述（由申请人提供）：骨重建调节剂的发现对于开发骨质疏松症的新疗法至关重要。瞬时受体电位melastatin 8（TRPM 8，又称冷和薄荷醇受体）是一种电压门控阳离子通道，在26°C以下开放。神经TRPM 8对于冷感觉、冷痛和冷镇痛以及产热是重要的。然而，TRPM 8的非神经元作用开始出现。成年Trpm 8- /-小鼠在椎骨中具有降低的骨小梁体积分数，并且在长骨中具有较小程度的降低。这可能是由于骨形成抑制和/或骨吸收增加。在体外，我们发现Trpm 8-/-破骨细胞分化与野生型相比没有差异。或者，Trpm 8-/-骨髓基质细胞（BMSC）和颅骨成骨细胞（OB）具有降低的分化。与骨髓肥胖减少的体内表型一致，BMSC和耳MSC（eMSC）在体外不能完全分化成脂肪细胞。这一新的发现表明，在分化成脂肪细胞或脂肪细胞方面存在明显缺陷。然而，TRPM 8在体内的大部分表达是在感觉神经元上，并且我们有证据表明表达TRPM 8的神经元存在于骨髓腔中。这些可以通过旁分泌途径影响成骨细胞。我的首要假设是，TRPM 8支持成骨细胞和脂肪细胞分化，通过其直接表达在MSC衍生的前体（目的1）和神经TRPM 8支持成骨细胞分化，通过旁分泌机制（目的2）。最后，我将利用新的Trpm 8 fl/fl小鼠选择性地删除间充质细胞与感觉神经元中的Trpm 8（目标3），在那里我将能够描绘每种细胞类型对骨量减少表型的贡献程度。具体目标1：在这个目标中，我将测试的假设，TRPM 8信号在间充质细胞激活PKA，以促进脂肪细胞和成骨细胞分化利用shRNA和钙流分析。具体目标2：尽管在不存在TRPM 8的情况下MSC分化的体外差异显著，但体内大多数TRPM 8表达在感觉神经元上。目的2a：检验表达TRPM 8的感觉神经元存在于骨中的假设。目的2b：测试感觉神经元中TRPM 8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Trpm 8 fl/fl小鼠与SynapsinCre/+和PrrxlCre/+小鼠杂交，以测试体内Trpm 8缺失的作用是否分别通过神经或成骨细胞TRPM 8表达介导。所提出的实验将确定TRPM 8调节成骨细胞和脂肪细胞谱系的机制，产生关于神经-间充质细胞相互作用的新数据，并为检查感觉神经元控制骨重建的未来工作奠定基础。

 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.