Abou-Samra A B, Jueppner H, Westerberg D, Potts J T, Segre G V
Endocrine Unit, Massachusetts General Hospital, Boston 02114.
Endocrinology. 1989 Mar;124(3):1107-13. doi: 10.1210/endo-124-3-1107.
PTH binds to specific receptors that are coupled to adenylate cyclase and activate cAMP-dependent protein kinase. Since it has been shown that PTH activates phospholipid inositol metabolism, we investigated whether PTH influences protein kinase-C (PKC) activity in rat osteosarcoma (ROS) cells 17/2.8 that contain a large number of PTH receptor. Incubation of ROS cells with PTH or phorbol 12-myristate 13-acetate (PMA) for 1-30 min caused a rapid and transient decrease in PKC activity in the cytosol, which was associated with a transient increase in PKC activity in the membrane fraction. After 1, 5, 15, and 30 min of incubation with PTH, cytosolic PKC activity decreased to 57%, 74%, 84%, and 93% of the control value, whereas membrane PKC activity increased to 156%, 122%, 111%, and 106% of the control value, respectively. After PMA treatment for 1, 5, 15, and 30 min, cytosolic PKC activity decreased by 81%, 74%, 63%, and 44%, whereas membrane-bound PKC activity increased by 83%, 44%, 28%, and 17%, respectively. The effects of PTH and PMA on PKC were dose dependent, with ED50 values of 0.3 nM PTH and 4 nM PMA. Chronic treatment of ROS cells for 3 days with PMA caused depletion of total PKC activity in cytosolic and membrane fractions to less than 10% of that in control cells. Conversely, chronic treatment of ROS cells with PTH did not deplete PKC. In addition, chronic treatment of ROS cells with PTH inhibited the responsiveness of PKC activity to subsequent acute PTH challenge, but not to acute PMA challenge, suggesting specific desensitization of this response by PTH. Activation of cytosolic PKC by diolein, phosphatidylserine, and calcium caused phosphorylation of many cytosolic proteins, including those having apparent mol wt of 39K, 35K, 33K, 25K, 19K, and 16K. Pretreatment of ROS cells with PTH resulted in a transient decrease in the phosphorylation of these cytosolic proteins by PKC. This decrease in cytosolic protein phosphorylation by treatment with PTH is temporally associated with PTH-stimulated translocation of PKC activity from the cytosol to the membranes. These data suggest a potential role for PKC in the mechanism of action of PTH in ROS cells.
甲状旁腺激素（PTH）与特定受体结合，这些受体与腺苷酸环化酶偶联并激活依赖于环磷酸腺苷（cAMP）的蛋白激酶。由于已有研究表明PTH可激活磷脂酰肌醇代谢，我们研究了PTH是否会影响大鼠骨肉瘤（ROS）细胞17/2.8中蛋白激酶C（PKC）的活性，该细胞含有大量的PTH受体。用PTH或佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）孵育ROS细胞1至30分钟，可导致细胞质中PKC活性迅速且短暂下降，这与膜组分中PKC活性的短暂增加相关。用PTH孵育1、5、15和30分钟后，细胞质PKC活性分别降至对照值的57%、74%、84%和93%，而膜PKC活性分别升至对照值的156%、122%、111%和106%。用PMA处理1、5、15和30分钟后，细胞质PKC活性分别下降81%、74%、63%和44%，而膜结合PKC活性分别增加83%、44%、28%和17%。PTH和PMA对PKC的作用呈剂量依赖性，PTH的半数有效剂量（ED50）值为0.3 nM，PMA为4 nM。用PMA对ROS细胞进行3天的慢性处理导致细胞质和膜组分中总PKC活性耗竭至对照细胞的不到10%。相反，用PTH对ROS细胞进行慢性处理并未使PKC耗竭。此外，用PTH对ROS细胞进行慢性处理会抑制PKC活性对随后急性PTH刺激的反应性，但不影响对急性PMA刺激的反应性，这表明PTH对该反应具有特异性脱敏作用。二油精、磷脂酰丝氨酸和钙激活细胞质PKC会导致许多细胞质蛋白磷酸化，包括那些表观分子量为39K、35K、33K、25K、19K和16K的蛋白。用PTH预处理ROS细胞会导致PKC对这些细胞质蛋白的磷酸化作用短暂下降。用PTH处理导致的细胞质蛋白磷酸化下降在时间上与PTH刺激PKC活性从细胞质向膜的转位相关。这些数据表明PKC在ROS细胞中PTH的作用机制中可能发挥作用。
