Li Zhongyong, Zhou Zhiyi, Saunders Marnie M, Donahue Henry J
Division of Musculoskeletal Sciences, Department of Orthopaedics & Rehabilitation, Pennsylvania State University College of Medicine 500 University Dr., Hershey, 17033, USA.
Am J Physiol Cell Physiol. 2006 Apr;290(4):C1248-55. doi: 10.1152/ajpcell.00428.2005. Epub 2005 Nov 30.
Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43(-)). hFOB/Cx43(-) cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43(-) cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43(-) cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor alpha1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43(-). Osteopontin mRNA levels were increased in hFOB/Cx43(-) relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43(-) and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells.
细胞间的间隙连接通道为调节性离子和小分子的交换提供了一条途径。我们之前证明，连接蛋白的表达和细胞间通讯与成骨细胞分化平行，并且间隙连接通讯的非特异性药理抑制剂会抑制碱性磷酸酶活性。在本研究中，我们将连接蛋白（Cx）43反义cDNA稳定转染至永生化的人胎儿成骨细胞系hFOB 1.19（hFOB/Cx43(-)）中。相对于对照 [hFOB/质粒载体对照（pvc）]，hFOB/Cx43(-)细胞表达较低水平的Cx43蛋白和mRNA，并且间隙连接细胞间通讯减少50%。这表明，其他连接蛋白，如在hFOB/Cx43(-)细胞和hFOB/pvc细胞中表达程度相似的Cx45，对hFOB 1.19细胞中的细胞间通讯有贡献。尽管细胞间通讯仅减少了50%，但我们观察到hFOB/Cx43(-)细胞中的碱性磷酸酶活性几乎完全受到抑制。这表明了一种有趣的可能性，即Cx43的表达本身，独立于细胞间通讯，会影响碱性磷酸酶活性，也许还会影响骨细胞分化。定量实时RT-PCR显示，骨钙素和核心结合因子α1（Cbfa1）的mRNA水平在hFOB/pvc中随时间增加，但在hFOB/Cx43(-)中受到抑制。相对于hFOB/pvc，hFOB/Cx43(-)中的骨桥蛋白mRNA水平升高，并且在hFOB/Cx43(-)和hFOB/pvc中均随时间降低。用Cx43反义转染不影响hFOB 1.19细胞中I型胶原蛋白的表达。这些结果表明，间隙连接细胞间通讯和Cx43的表达有助于成骨细胞中的碱性磷酸酶活性以及骨钙素、骨桥蛋白和Cbfa1的表达。
