大电导钙激活钾通道在骨重建中的作用和机制

Bone remodeling 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, osteopetrosis, rickets, etc. Although our understanding of bone-remodeling diseases and targeted therapies is rapidly evolving, it is remain necessary for the development of even more effective therapies from a better understanding the pathogenesis of bone-remodeling diseases. It is well-established that calcium activated large conductance potassium channel (big K channel, BK) plays important roles in excitatory tissues. Recent study showed the BK knockout mice exhibited osteopenic phenotype, suggesting that BK was also involved in the functional regulation of non-excitatory tissues. However, the molecular mechanism of BK regulating bone biology remains elusive. To address this scientific question, we hypothesize that BK plays unique roles in bone remodeling and participates in the development of bone disorders such as osteoporosis,through the bi-directional signaling of integrin. Integrin outside-in signaling to BK is mediated by non-receptor tyrosine kinases, whereas the inside-out signaling is mediated by small GTPase rap1 pathway. In this study, molecular biological skills and electrophysiology will be used to investigate the expression profile, electrophysiological and pharmacological property of BK alpha sub-units and beta-subunits, in bone tissue. In addition, the role of BK in regulating proliferation, differentiation, migration, secretion, mineralization and bone resorption of osteoblast and osteoclast, will be investigated. Our prelimnary data suggested that the underlying mechanism is related to the cross-talk between BK and integrin receptors. Finally, osteoporosis animal model and BK modulator treatment will be used to explore the contribution of BK to the pathogenesis of bone-remodeling diseases. This study will be helpful for understanding the pathophysiological role of BK in bone tissue, and developing new therapeutic strategy for bone-remodeling diseases.

骨质疏松、肾性骨营养不良等骨重建性疾病严重危害人类健康，但病理机制尚不完全清楚，更缺乏有效治疗手段。最近研究发现大电导钙激活钾通道（BK）基因敲除小鼠有骨重建异常表型，此外我们前期工作显示BK通道多种亚型在成骨细胞表达，但其在骨重建生理及病理过程中的生物学作用尚不清楚。针对该科学问题，我们假设：骨组织中不同亚型BK通道通过与整合素的双向信号转导机制调节骨重建过程并参与骨重建性疾病的发生发展。本课题深入研究BK通道在骨组织细胞中存在的亚型基础和对其功能的影响、以及在骨重建异常动物模型中的变化，可能通过整合素-非受体酪氨酸激酶参与的内向转导和小分子GTP酶通路参与的外向转导机制。本课题以期阐明BK通道在骨重建生理及病理过程中的重要生物学作用。研究结果将不仅丰富人们对骨重建调控机制的认识，而且为防治骨质疏松、肾性骨营养不良等骨重建性疾病提供治疗基础。

大电导钙激活钾通道(large conductance calcium activated potassium channel, BK, maxi K, KCa1.1，KCNMA1，Slo1)在体内分布广泛，其在可兴奋性细胞如平滑肌细胞，神经细胞，分泌细胞中的作用已有深入研究，但其在非兴奋性细胞如骨细胞中的研究近年来才引起重视。骨质疏松、肾性骨营养不良等骨重建性疾病严重危害人类健康，但病理机制尚不完全清楚，更缺乏有效治疗手段。最近研究发现大电导钙激活钾通道（BK）基因敲除小鼠有骨重建异常表型，此外我们前期工作显示BK通道多种亚型在成骨细胞表达，但其在骨重建生理及病理过程中的生物学作用尚不清楚。本项目对去卵巢大鼠骨质疏松模型中BK通道的表达变化及其作用机制进行了研究。发现在骨质疏松病理状态下，骨组织的 BK通道mRNA和蛋白的表达量都明显降低，同时整合素受体的表达量也明显降低，并且，我们通过免疫荧光共定位实验发现BK通道与整合素受体的表达位点具有高度的一致性，这些结果提示BK通道及整合素受体在骨质疏松动物体内的变化具有相关性，两者之间可能存在着重要的相互协调作用。因此，我们进一步在成骨细胞上探究了两者之间的关系。通过在成骨细胞上外源性转入BK质粒使其过表达，或利用shRNA使其沉默的方法，调节BK通道的表达量，发现整合素受体的表达也随着BK的变化而变化；为探究二者相互作用的机制，我们检测了整合素受体信号通路下游磷酸化过程中的磷酸化相关蛋白，如p-erk1/2，β-catenin蛋白等，发现当沉默成骨细胞中BK基因时，整合素受体信号通路下游的磷酸化相关蛋白的表达也明显降低。我们的研究表明BK通道在骨质疏松疾病中有调节作用，其机制与整合素受体通路相关。本研究丰富了人们对骨重建调控机制的认识，可能为防治骨质疏松、肾性骨营养不良等骨重建性疾病提供治疗基础。