Egan J J, Gronowicz G, Rodan G A
Laboratory of Cellular and Developmental Biology, National Institutes of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20891.
J Cell Biochem. 1991 Jan;45(1):101-11. doi: 10.1002/jcb.240450117.
Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X-100 (1%) nonextractable proteins. After 2-5 minutes, PTH caused a rapid and transient decrease of 50-60% in polymerized actin and myosin associated with the Triton X-100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose-dependent with an IC50 of about 1 nM, and was partially inhibited by the (3-34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin-2/AM (10 microM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (PI 4.9) myosin light chain in cells preincubated with [32P]-orthophosphate. The phosphorylation of this protein decreased within 2-3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A-23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine-conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP.
甲状旁腺激素（PTH）在体内和体外均可改变成骨细胞的形态。在本研究中，我们通过对经1% Triton X-100处理后不可提取蛋白质进行聚丙烯酰胺凝胶电泳，检测了PTH对细胞骨架肌动蛋白和肌球蛋白的影响。2 - 5分钟后，PTH导致与Triton X-100不可提取细胞骨架相关的聚合肌动蛋白和肌球蛋白迅速且短暂地减少50 - 60%。聚合肌动蛋白在30分钟时恢复到对照水平。PTH的作用呈剂量依赖性，IC50约为1 nM，且被（3 - 34）PTH拮抗剂部分抑制。PTH使这些细胞中的环磷酸腺苷（cAMP）迅速短暂升高，在4分钟时达到峰值，而细胞骨架肌动蛋白和肌球蛋白的最低点出现在约5分钟时。细胞内钙螯合剂Quin-2/AM（10 μM）也使细胞骨架肌动蛋白和肌球蛋白减少，减少程度与100 nM的PTH相同。为了区分cAMP升高和Ca++减少作为PTH作用的介导因素，我们测量了用[32P] - 正磷酸盐预孵育的细胞中20 kD（PI 4.9）肌球蛋白轻链的磷酸化情况。添加PTH后2 - 3分钟内该蛋白的磷酸化减少，5分钟后恢复到对照水平。钙离子载体A-23187并未拮抗PTH的这种作用。用罗丹明标记的鬼笔环肽对微丝进行可视化显示，PTH通过减少应力纤维的数量改变细胞骨架。细胞骨架的这些变化与细胞形态从伸展状态变为具有回缩突起的星状形态的变化相平行。上述发现表明，PTH引起的成骨细胞形态改变涉及细胞骨架组织中相对快速且短暂的变化，这些变化似乎由cAMP介导。
