Civitelli R, Hruska K A, Jeffrey J J, Kahn A J, Avioli L V, Partridge N C
Division of Bone and Mineral Metabolism, Jewish Hospital of St. Louis, Washington University Medical Center, Missouri.
Endocrinology. 1989 Jun;124(6):2928-34. doi: 10.1210/endo-124-6-2928.
Recent work indicates that PTH can stimulate osteoblastic cells to secrete neutral collagenase, an enzyme thought to be linked to bone matrix turnover. Since recent studies suggest that the calcium/protein kinase-C (PKC) message system is involved in signal transduction stimulated by PTH, we examined the role of these putative second messengers of PTH in the regulation of collagenase production by the osteoblastic tumor cell line UMR 106-01. Immunohistochemical staining of cells exposed to PTH (10(-7) M) revealed that about 20% of the entire population was positive for collagenase, compared to less than 3% staining positively in control untreated cells. Incubation with the cAMP analog 8-bromo-cAMP (8BrcAMP) increased the number of collagenase-staining cells in a dose-dependent manner (ED0.5 = 2.5 x 10(-4) M), but to a lower level than PTH, with the maximal effect producing about 15% positive cells. The calcium ionophore ionomycin (10(-7) M) was ineffective, whereas phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased collagenase-specific staining to about 5%, but only at high concentrations (10(-5) M). Incubation of UMR 106-01 cells with ionomycin and PMA did not change the effect of the latter. When the three agents were used in combination, an additive effect was observed, which fully reproduced that of PTH. Similarly, the amount of collagenase released into the medium by cells stimulated with maximal concentrations of 8BrcAMP (10(-3) M) was only 80% of that induced by maximal doses of PTH (10(-7) M). PMA (10(-5) M) was slightly stimulatory, and ionomycin was ineffective alone, but they were synergistic with submaximal doses of 8BrcAMP (10(-4) M). In agreement with the immunohistochemical results, the full hormonal effect was reproduced when the three second messenger analogs were used in combination. In conclusion, signal transduction from PTH receptor to collagenase production is mediated mainly by cAMP; the Ca2+/PKC system appears to have a contributory role necessary for the full expression of hormonal response. These results support the hypothesis of a dual pathway of target cell activation by PTH.
近期研究表明，甲状旁腺激素（PTH）可刺激成骨细胞分泌中性胶原酶，该酶被认为与骨基质更新有关。由于近期研究提示钙/蛋白激酶-C（PKC）信号系统参与了PTH刺激的信号转导过程，我们研究了这些假定的PTH第二信使在成骨细胞瘤细胞系UMR 106-01的胶原酶产生调节中的作用。对暴露于PTH（10⁻⁷ M）的细胞进行免疫组织化学染色显示，在整个细胞群体中约20%的细胞胶原酶呈阳性，而未处理的对照细胞中阳性染色的细胞不到3%。用环磷酸腺苷（cAMP）类似物8-溴-cAMP（8BrcAMP）孵育细胞，胶原酶染色阳性的细胞数量呈剂量依赖性增加（半数有效剂量=2.5×10⁻⁴ M），但低于PTH的作用，最大效应时约15%的细胞呈阳性。钙离子载体离子霉素（10⁻⁷ M）无效，而PKC激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）仅在高浓度（10⁻⁵ M）时可使胶原酶特异性染色增加至约5%。用离子霉素和PMA孵育UMR 106-01细胞不会改变PMA的作用效果。当将这三种试剂联合使用时，观察到相加效应，完全重现了PTH的作用效果。同样，用最大浓度的8BrcAMP（10⁻³ M）刺激细胞释放到培养基中的胶原酶量仅为最大剂量PTH（10⁻⁷ M）诱导量的80%。PMA（10⁻⁵ M）有轻微刺激作用，离子霉素单独使用无效，但它们与亚最大剂量的8BrcAMP（10⁻⁴ M）具有协同作用。与免疫组织化学结果一致，当联合使用这三种第二信使类似物时，可重现完整的激素效应。总之，从PTH受体到胶原酶产生的信号转导主要由cAMP介导；Ca²⁺/PKC系统似乎在激素反应的完全表达中起必要的辅助作用。这些结果支持了PTH激活靶细胞的双途径假说。
