Yamanaka I, Kuraoka A, Inai T, Ishibashi T, Shibata Y
Department of Anatomy, Faculty of Medicine, Kyushu University, Fukuoka/Japan.
Eur J Cell Biol. 1997 Feb;72(2):166-73.
Using specific antibodies and cDNA probe, we examined the expression pattern of a major gap junction protein, connexin43 (Cx43), in rat mammary glands during pregnancy and lactation. Double immuno-fluorescence revealed that the labeling of Cx43 was superimposed in the alpha-smooth muscle actin-positive cells, suggesting that myoepithelial cell were interconnected by gap junctions formed of Cx43. Just after delivery, the Cx43-labeled plaques were enlarged and increased in intensity. Northern and Western blot analyses confirmed the dramatic induction of Cx43 at both mRNA and protein levels on the day of parturition. Cx43 mRNA transcript immediately declined, while the increase of Cx43 protein continued for a few days. During pregnancy, immunoblots showed two bands of almost equal amounts at 43 and 45 kDa. Following delivery, the 45-kDa band gradually increased in intensity with a concomitant decrease of the 43-kDa band. From the sixth day of lactation, Cx43 was always detected as a single band at 45 kDa. Alkaline phosphatase treatment of immunoprecipitated Cx43 revealed that both bands represented phosphorylated forms, thus indicating that Cx43 was naturally phosphorylated and that it altered its phosphorylation states during lactation stages. These results suggest that the induction of Cx43 with the changes in the phosphorylation states plays an important role in the lactating function of myoepithelial cells in rat mammary glands. This is the first report on the changes of Cx43 phosphorylation states during physiological stages in vivo.
我们使用特异性抗体和cDNA探针，检测了大鼠乳腺在妊娠和哺乳期主要缝隙连接蛋白连接蛋白43（Cx43）的表达模式。双重免疫荧光显示，Cx43的标记与α-平滑肌肌动蛋白阳性细胞重叠，表明肌上皮细胞通过由Cx43形成的缝隙连接相互连接。分娩后，Cx43标记的斑块立即增大且强度增加。Northern和Western印迹分析证实，分娩当天Cx43在mRNA和蛋白质水平均有显著诱导。Cx43 mRNA转录本立即下降，而Cx43蛋白的增加持续了几天。在怀孕期间，免疫印迹显示在43 kDa和45 kDa处有两条几乎等量的条带。分娩后，45 kDa条带的强度逐渐增加，同时43 kDa条带的强度下降。从哺乳期第6天开始，Cx43始终检测为45 kDa的单一条带。对免疫沉淀的Cx43进行碱性磷酸酶处理后发现，两条条带均代表磷酸化形式，因此表明Cx43天然磷酸化，且在哺乳期各阶段其磷酸化状态发生改变。这些结果表明，Cx43的诱导及其磷酸化状态的变化在大鼠乳腺肌上皮细胞的泌乳功能中起重要作用。这是关于体内生理阶段Cx43磷酸化状态变化的首次报道。
