Fukayama S, Kearns A K, Skurat R M, Tashjian A H, Bringhurst F R
Endocrine Unit, Massachusetts General Hospital, Boston.
Cell Regul. 1991 Nov;2(11):889-96. doi: 10.1091/mbc.2.11.889.
We have established mutant SaOS-2 cell lines that express a cyclic AMP (cAMP)-resistant phenotype to investigate the regulation and functional importance of orthophosphoric-monoester phosphohydrolase alkaline optimum (ALPase) in the action of parathyroid hormone (PTH). Cells were stably transfected with a plasmid that directs the synthesis of a mutant form of the type I regulatory subunit of protein kinase A (PKA) under the control of the metallothionein promotor. There was no significant difference between parental SaOS-2 cells and the mutant lines in the affinity or number of receptors for 125I-Nle8,18Tyr34bPTH1-34NH2, either in the absence or presence of Zn2+. When cAMP-dependent gene transcription was examined using transient transfection with a somatostatin promoter-chloramphenicol acetyl transferase (CAT) reporter plasmid, CAT activity stimulated by human PTH and dibutyryl cAMP (DBcAMP) was inhibited by greater than 90% in the presence of Zn2+ in the mutant cell lines. In contrast, activation by a phorbol ester of a pentameric collagenase promoter/CAT construct containing five tandem copies of the AP-1 response element (5x-TRE-CAT) was unaffected in Zn(2+)-treated mutant cells. The inhibitory actions of PTH and DBcAMP on ALPase release were blunted by up to 80-90% in the mutant cell lines in the presence of Zn2+; there were no significant differences in the magnitude of inhibitory effects between these agonists. We conclude that the inhibitory action of PTH on ALPase release in SaOS-2 cells is mediated via activation of PKA. These cAMP-resistant cell lines will be especially useful in elucidating signal transduction mechanism(s) for PTH in human osteoblastic cells.
我们建立了表达抗环磷酸腺苷（cAMP）表型的突变型SaOS-2细胞系，以研究正磷酸单酯磷酸水解酶碱性最佳值（ALPase）在甲状旁腺激素（PTH）作用中的调节及其功能重要性。用一种质粒稳定转染细胞，该质粒在金属硫蛋白启动子的控制下指导合成蛋白激酶A（PKA）I型调节亚基的突变形式。在不存在或存在Zn2+的情况下，亲本SaOS-2细胞与突变细胞系在125I-Nle8,18Tyr34bPTH1-34NH2受体的亲和力或数量上没有显著差异。当使用生长抑素启动子-氯霉素乙酰转移酶（CAT）报告质粒进行瞬时转染来检测cAMP依赖性基因转录时，在突变细胞系中存在Zn2+的情况下，人PTH和二丁酰cAMP（DBcAMP）刺激的CAT活性被抑制了90%以上。相比之下，在Zn(2+)处理的突变细胞中，含五个串联AP-1反应元件拷贝（5x-TRE-CAT）的五聚体胶原酶启动子/CAT构建体被佛波酯激活的情况不受影响。在存在Zn2+的情况下，突变细胞系中PTH和DBcAMP对ALPase释放的抑制作用减弱了80%-90%；这些激动剂之间的抑制作用大小没有显著差异。我们得出结论，PTH对SaOS-2细胞中ALPase释放的抑制作用是通过PKA的激活介导的。这些抗cAMP细胞系在阐明人成骨细胞中PTH的信号转导机制方面将特别有用。
