成骨细胞BK通道对整合素信号介导骨代谢的分子调控机制

Bone metabolism is a physiological process in which old or damaged bone is removed by osteoclasts, and then replaced by new bone formed by osteoblasts. Nonetheless, the imbalance between bone resorption and bone formation may occur under certain pathological conditions, which leads to abnormal bone remodeling and the development of bone disorders, such as osteoporosis, renal osteodystrophy etc. Although our understanding of bone-remodeling diseases and targeted therapies is rapidly evolving, it remains necessary for the development of even more effective therapies from a better understanding the pathogenesis of bone-remodeling diseases. .Integrin is a family of transmembrane alpha beta heterodimer adhesion molecules that conveys signals to (outside-in signaling) and from (inside-out signaling) the cytosol across the plasma membrane. Recently, several lines of evidence have shown that integrin can be an important target for regulation of bone metabolism, whereas little is known about how to regulate its expression and activity on bone tissues. Our preliminary data showed that large conductance calcium activated potassium (BK) channels were expressed in osteoblast cells and had protein-protein interaction with integrin. Silencing BK channels on osteoblasts led to decrease of integrin expressions. Meanwhile, we found that the expression of BK and integrin were both reduced in metabolic bone disease models. Based on those results, we speculate that BK channel plays a pivotal role in bone metabolism while integrin is the key downstream molecules that participant in the pathophysiological mechanism. In this study, molecular biological and electrophysiological techniques such as patch clamp, FRET, flow cytometry, micro-CT and immunohistochemistry will be performed to investigate the expression profile, electrophysiological and pharmacological property of BK in osteoblasts. The aim of the study: (1) Whether BK channel regulates integrin signaling pathway and the function of osteoblasts; (2) In osteoblast conditional BK knock-out mice, whether the characteristics of bone phenotype and bone metabolism is related to integrin signaling pathway; (3) How BK channel and integrin signaling pathway are altered in osteoporosis animal and its osteoblast precursor--mesenchymal stem cells (MSC), and whether BK gene therapy is able to rescue the MSC function and BK opener is able to benefit bone building in animal model. This study will provide deep insight into understanding the pathogenesis of metabolic bone disease and explore the new therapeutic target.

整合素是调控骨代谢的关键分子，但其作为治疗骨代谢疾病的直接靶标有较大副作用。因此，寻找其上下游信号分子作为治疗疾病的药靶至关重要。我们前期研究发现，大电导钙激活钾通道（BK）参与成骨细胞功能，并对整合素信号通路有调节作用。鉴此我们推测，BK是影响整合素调控骨代谢的上游信号分子，但具体机制尚不明确。本项目拟通过膜片钳、免疫共沉淀、荧光能量共振转移、流式细胞术、micro-CT和免疫组化等方法，从不同角度阐明（1）成骨细胞BK调节整合素受体及其信号通路的分子机制，以及对成骨细胞功能的影响；（2）在成骨细胞BK条件性敲除小鼠模型中，骨表型及骨代谢特点与整合素受体及其信号通路分子之间的联系；（3）在骨代谢异常（骨质疏松）的动物和细胞模型中，BK与整合素受体及其信号通路的关系，并评价BK通道开放剂的治疗作用。本研究丰富了对骨代谢调控机制的认识，为阐明骨代谢疾病的发病机制及探索治疗靶点提供理论依据。

整合素是调控骨代谢的关键分子，但其作为治疗骨代谢疾病的直接靶标有较大副作用如可能增加癌症发生风险。因此，寻找其上下游信号分子作为调控骨代谢的靶标至关重要。BK通道在兴奋性细胞中的作用已有较为广泛且深入的研究，而在非兴奋细胞如骨细胞等中的作用近年来才引起重视。我们前期研究发现BK通道在成骨细胞中的转导途径与骨功能密切相关，但机制尚不清楚。本课题研究BK通道在成骨细胞上的作用及整合素介导的调节机制。我们通过检测15周龄的BK基因敲除（BKO）和成骨细胞条件性敲除小鼠的胫骨和腰椎骨，发现其骨密度和骨小梁体积下降，与骨形成和成骨细胞活性相关的指标异常。上调或下调成骨细胞BK通道对成骨细胞增殖、分化有显著影响。ALP染色、茜素红染色及CCK-8结果显示，BK通道被抑制后成骨细胞分化能力下降，增殖能力下调。Western blot 及免疫荧光结果显示BK上调或下调后，integrin β1也随之变化，免疫共沉淀及免疫荧光共定位证实成骨细胞中BK与integrin β1之间有蛋白相互作用，且相互作用部位在BK通道的α亚基C-末端与整合素β1蛋白；下调成骨细胞中的整合素并不影响BK的表达量，提示我们BK可能位于integrin β1的上游；调节成骨细胞中BK的表达量，integrin β1下游的FAK, ERK的表达量均随之改变，并会影响转录因子RUNX2的表达；ERK通路被抑制后，BK引起的RUNX2的上升也随之消失。此外，骨质疏松模型动物也显示BK蛋白及integrin β1蛋白表达量减少。综上所述，我们发现BK通道对成骨细胞分化及增殖的调节作用是通过整合素介导的，BK通道与整合素受体β 1之间存在蛋白相互作用，当调节BK的活性时，会引起整合素受体下游FAK，ERK的磷酸化，进而影响ERK对成骨转录因子RUNX2的磷酸化，最终对成骨细胞相关分化基因的表达产生影响，并且在骨质疏松病理状态下BK及整合素表达均表现异常。本项目为离子通道蛋白与细胞外基质在成骨细胞调节中的相互作用提供了新的见解，并揭示了一种新的纠正骨形成缺陷的潜在策略，为骨质疏松等疾病的治疗提供潜在靶点。

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