Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation.

骨稳态（bone homeostasis）似乎由神经系统与调控骨重塑的“骨神经介质（osteo-neuromediators）”之间精细微妙的“交叉对话”所调控。本研究旨在评估神经肽（neuropeptides）与人类骨形态发生蛋白2（human bone morphogenetic protein 2, hBMP2）之间的相互作用对人类成骨细胞（human osteoblasts）的影响。此外，本研究还探讨了神经肽与hBMP2对间隙连接细胞间通讯（gap junction intercellular communication, GJIC）的作用。实验中，我们以三种浓度的神经肽Y（neuropeptide Y, NPY）、P物质（substance P, SP）或hBMP2处理成骨细胞。在处理后的不同时间点，采用MTT法（MTT assay）检测细胞活力。此外，分别通过比色法（colorimetric assay）与放射免疫分析法（radioimmunoassay）测定细胞碱性磷酸酶（cellular alkaline phosphatase, ALP）活性与骨钙素（osteocalcin）水平。采用激光扫描共聚焦显微镜术（laser scanning confocal microscopy）检测NPY、SP及hBMP对GJIC的影响。结果显示，经神经肽与hBMP2处理的细胞活力呈时间依赖性显著升高，但与处理剂浓度呈负相关。暴露于神经肽与hBMP2联合处理的成骨细胞中，ALP活性与骨钙素水平均有所降低。神经肽与hBMP2可显著增强成骨细胞的GJIC。上述结果表明，神经肽与hBMP2可通过增强GJIC提升成骨细胞活性。鉴定能够调控骨细胞细胞活动的GJIC介导信号转导（signal transduction）通路，为研究骨骼神经支配的生物学特性提供了全新的研究思路。