Koh A J, Beecher C A, Rosol T J, McCauley L K
The University of Michigan, Department of Periodontics/Prevention/Geriatrics, Ann Arbor 48109, USA.
Endocrinology. 1999 Jul;140(7):3154-62. doi: 10.1210/endo.140.7.6872.
PTH has anabolic and catabolic effects in bone through activation of the PTH-1 (PTH/PTHrP) receptor and the cAMP/protein kinase A pathway. The effects of agents that regulate cAMP in nontransformed osteoblasts in relation to cell differentiation have not been described. The purpose of this study was to determine the effects of PTH fragments with differing cAMP-stimulating activity, and nonPTH cAMP regulators on PTH-1 receptor expression and activity, and osteoblast differentiation in vitro using MC3T3-E1 and primary rat calvarial cells. PTH (1-34), but not PTH (53-84), (7-34), or PTHrP (107-139) treatment (24 h) resulted in down-regulation of steady-state messenger RNA for the PTH-1 receptor. Forskolin (a stimulator of cAMP accumulation) also down regulated the PTH-1 receptor, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (an inhibitor of adenylyl cyclase) had no effect. Similarly, PTH (1-34) treatment for 48 h abolished PTHrP binding to cell surface receptors; however, neither the PTH analogs nor the cAMP regulating agents altered PTH binding or numbers of binding sites on osteoblastic cells. Basal levels of cAMP were reduced in cultured cells treated for 6 days with PTH (7-34) or THFA compared with controls. In contrast, PTH-stimulated cAMP levels were significantly increased in cultures treated with PTH (7-34) and THFA for 6 days during osteoblast differentiation and were decreased in cultures treated with PTH (1-34) and forskolin compared with controls. To evaluate effects of the cAMP pathway on osteoblast differentiation, cultures were treated continuously with PTH analogs and cAMP regulators during an 18-day differentiation regime, total RNA was isolated at multiple time points, and Northern blot analysis for osteocalcin (OCN) was performed. THFA and PTH (7-34)-treated cultures had increased OCN expression; whereas, PTH (1-34) and forskolin reduced OCN expression. Interestingly, PTH (7-34) and THFA-treated cultures had increased mineralized nodule formation, in contrast to PTH (1-34) and forskolin treatment, which reduced nodule formation. Similarly, calcium accumulation in cultures was significantly increased in the PTH (7-34) and THFA-treated cultures and reduced in the PTH (1-34) and forskolin-treated cultures. These data demonstrate that agents that increase cAMP down regulate PTH-1 receptor messenger RNA and inhibit osteoblast differentiation in vitro. Agents that reduce or block adenylyl cyclase or cAMP activity do not alter PTH-1 receptor expression or binding, but have striking effects on promoting osteoblast differentiation. We conclude that many effects of PTH on osteoblasts may be mimicked or antagonized by agents that alter cAMP activity and bypass the PTH-1 receptor.
甲状旁腺激素（PTH）通过激活甲状旁腺激素-1（PTH/PTHrP）受体和环磷酸腺苷（cAMP）/蛋白激酶A途径，在骨骼中发挥合成代谢和分解代谢作用。关于非转化成骨细胞中调节cAMP的药物对细胞分化的影响尚未见报道。本研究的目的是使用MC3T3-E1细胞和原代大鼠颅骨细胞，在体外确定具有不同cAMP刺激活性的PTH片段以及非PTH cAMP调节剂对PTH-1受体表达和活性以及成骨细胞分化的影响。PTH（1-34）处理（24小时）可导致PTH-1受体的稳态信使核糖核酸（mRNA）下调，但PTH（53-84）、（7-34）或PTHrP（107-139）处理则无此作用。福斯可林（一种cAMP积累刺激剂）也下调PTH-1受体，而9-（四氢-2-呋喃基）腺嘌呤（THFA）（一种腺苷酸环化酶抑制剂）则无影响。同样，PTH（1-34）处理48小时可消除PTHrP与细胞表面受体的结合；然而，PTH类似物和cAMP调节药物均未改变PTH与成骨细胞的结合或结合位点数量。与对照组相比，用PTH（7-34）或THFA处理6天的培养细胞中，基础cAMP水平降低。相反，在成骨细胞分化过程中，用PTH（7-34）和THFA处理6天的培养物中，PTH刺激的cAMP水平显著升高，而用PTH（1-34）和福斯可林处理的培养物中该水平则降低。为了评估cAMP途径对成骨细胞分化的影响，在18天的分化过程中，用PTH类似物和cAMP调节剂连续处理培养物，在多个时间点分离总RNA，并进行骨钙素（OCN）的Northern印迹分析。THFA和PTH（7-34）处理的培养物中OCN表达增加；而PTH（1-34）和福斯可林则降低OCN表达。有趣的是，与PTH（1-34）和福斯可林处理减少结节形成相反，PTH（7-34）和THFA处理的培养物中矿化结节形成增加。同样，PTH（7-34）和THFA处理的培养物中钙积累显著增加，而PTH（1-34）和福斯可林处理的培养物中钙积累减少。这些数据表明，增加cAMP的药物在体外下调PTH-1受体信使核糖核酸并抑制成骨细胞分化。降低或阻断腺苷酸环化酶或cAMP活性的药物不会改变PTH-1受体的表达或结合，但对促进成骨细胞分化有显著作用。我们得出结论，PTH对成骨细胞的许多作用可能被改变cAMP活性并绕过PTH-1受体的药物模拟或拮抗。
