Henriksen Zanne, Hiken Jeffrey F, Steinberg Thomas H, Jørgensen Niklas R
Osteoporosis and Bone Metabolic Unit, Dept. 545, Departments of Endocrinology and Clinical Biochemistry, Copenhagen University Hospital Hvidovre, Kettegaard Allé 30, DK-2650 Hvidovre, Denmark.
Cell Calcium. 2006 May;39(5):435-44. doi: 10.1016/j.ceca.2006.01.012. Epub 2006 Mar 20.
Intercellular calcium waves (ICW) are calcium transients that spread from cell to cell in response to different stimuli. We previously demonstrated that human osteoblast-like cells in culture propagate ICW in response to mechanical stimulation by two mechanisms. One mechanism involves autocrine activation of P2Y receptors, and the other requires gap junctional communication. In the current work we ask whether long-term culture of osteoblast-like cells affects the propagation of ICW by these two mechanisms. Human osteoblast-like cells were isolated from bone marrow. Mechanically induced ICW were assessed by video imaging of Fura-2 loaded cells after 1, 2 and 4 months culture. The P2Y2 receptor and the gap junction protein Cx43 were assessed by Western blot and real-time PCR. In resting conditions, P2Y mediated ICW prevailed and spread rapidly to about 13 cells. P2Y receptor desensitization by ATP disclosed gap junction-mediated ICW which diffused more slowly and involved not more than five to six cells. After 2 months in culture, ICW appeared slower and wave propagation was much less inhibited by P2Y desensitization, suggesting an increase in gap junction-mediated ICW. After 4 months in culture cells still responded to addition of ATP, but P2Y desensitization did not inhibit ICW propagation. Our data indicate that the relative role of P2Y-mediated and gap junction-mediated ICW changes during osteoblast differentiation in vitro. In less differentiated cells, P2Y-mediated ICW predominate, but as cells differentiate in culture, gap-junction-mediated ICW become more prominent. These results suggest that P2Y receptor-mediated and gap junction-mediated mechanisms of intercellular calcium signaling may play different roles during differentiation of bone-forming cells.
细胞间钙波（ICW）是一种钙瞬变现象，可响应不同刺激在细胞间传播。我们之前证明，培养的人成骨样细胞可通过两种机制对机械刺激产生细胞间钙波。一种机制涉及P2Y受体的自分泌激活，另一种机制则需要缝隙连接通讯。在当前研究中，我们探究成骨样细胞的长期培养是否会影响这两种机制介导的细胞间钙波传播。从骨髓中分离出人成骨样细胞。在培养1、2和4个月后，通过对负载Fura-2的细胞进行视频成像来评估机械诱导的细胞间钙波。通过蛋白质免疫印迹和实时定量PCR评估P2Y2受体和缝隙连接蛋白Cx43。在静息状态下，P2Y介导的细胞间钙波占主导，且能迅速传播至约13个细胞。ATP使P2Y受体脱敏后，可揭示缝隙连接介导的细胞间钙波，其扩散速度较慢，涉及的细胞不超过五六个。培养2个月后，细胞间钙波出现得较慢，且P2Y脱敏对波传播的抑制作用明显减弱，这表明缝隙连接介导的细胞间钙波有所增加。培养4个月后，细胞仍对添加ATP有反应，但P2Y脱敏并未抑制细胞间钙波传播。我们的数据表明，在体外成骨细胞分化过程中，P2Y介导和缝隙连接介导的细胞间钙波的相对作用发生了变化。在分化程度较低的细胞中，P2Y介导的细胞间钙波占主导，但随着细胞在培养中分化，缝隙连接介导的细胞间钙波变得更加突出。这些结果表明，P2Y受体介导和缝隙连接介导的细胞间钙信号传导机制在成骨细胞分化过程中可能发挥不同作用。
