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

 描述(由申请人提供)：骨重塑调节剂的发现对开发治疗骨质疏松症的新疗法至关重要。瞬时受体电位Melastatin 8(TRPM8，也称为冷和薄荷醇受体)是一种电压门控的阳离子通道，在26°C以下开放。神经TRPM8在冷感、冷痛和冷镇痛以及产热中起重要作用。然而，TRPM8的非神经性作用正开始显现。成年TRPM8-/-小鼠椎骨中的骨小梁体积分数减少，在长骨中的程度较小。这可能是由于骨形成受到抑制和/或骨吸收增加。在体外，我们发现与野生型相比，TRPM8-/-破骨细胞的分化没有差异。另一方面，TRPM8-/-骨髓基质细胞(BMSCs)和颅骨成骨细胞(OB)分化减少。与体内骨髓脂肪减少的表型一致，BMSCs和EAR MSCs在体外未能完全分化为脂肪细胞。这一新的发现表明，在分化为Eiter Ob或脂肪细胞方面存在显著缺陷。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。这些都可能通过旁分泌途径影响成骨细胞。我的主要假设是，TRPM8通过其在MSC来源的前体细胞中的直接表达支持成骨细胞和脂肪细胞的分化(目标1)，神经性TRPM8通过旁分泌机制支持成骨细胞的分化(目标2)。最后，我将利用新的Trpm8fl/fl小鼠选择性地删除间充质细胞和感觉神经元中的TRPM8(目标3)，在那里我将能够描述每种细胞类型对骨量减少表型的贡献程度。具体目的1：在这个目标中，我将利用shRNA和钙通量分析来验证这一假设，即间充质细胞中的TRPM8信号激活PKA促进脂肪细胞和成骨细胞的分化。特异性目的2：尽管在缺乏TRPM8的情况下，MSC的体外分化存在显著差异，但体内TRPM8的表达主要集中在感觉神经元上。目的2a：验证骨中存在表达TRPM8的感觉神经元的假说。目的2b：验证感觉神经元中TRPM8的激活通过树突：成骨细胞接触支持成骨细胞分化的假设。目的：将Trpm8fl/fl小鼠分别与SynapsinCre/+和Prrx1Cre/+小鼠杂交，以检测体内TRPM8缺失的效应是否分别通过神经或成骨细胞的TRPM8表达来介导。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，产生关于神经-间充质细胞相互作用的新数据，并为未来研究感觉神经元对骨重塑的控制奠定基础。

 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.