Kawane T, Mimura J, Yanagawa T, Fujii-Kuriyama Y, Horiuchi N
Department of Biochemistry, Ohu University School of Dentistry, Koriyama 963-8611, Japan.
J Endocrinol. 2003 Aug;178(2):247-56. doi: 10.1677/joe.0.1780247.
Parathyroid hormone (PTH) regulates osteoblast function via a G protein-linked PTH/PTH-related protein (PTHrP) receptor. We have studied the mechanisms of PTH/PTHrP receptor gene repression by PTH in UMR-106 osteoblast-like cells. Inhibition of PTH/PTHrP receptor mRNA expression by rat (r) PTH(1-34) and Insulin-like growth factor-I (IGF-I) at 10(-7)M was significant at 1 h and 3 h, and maximal at 2 h and 6 h. A maximal decrease in receptor mRNA abundance by rPTH(1-34) and IGF-I was maintained for 24 h. Inhibition of receptor gene expression by rPTH(1-34) was mimicked in UMR-106 cells by the addition of forskolin (an adenylyl cyclase activator), or 8-(4-chlorophenylthio)-adenine 3',5'-cyclic monophosphate (8-pCPTcAMP; a cAMP analogue). Although H89, a selective protein kinase A (PKA) inhibitor, completely inhibited PKA activity stimulated by rPTH(1-34), forskolin or 8-pCPTcAMP, suppression of PTH/PTHrP receptor mRNA synthesis induced by these substances in UMR-106 cells was not affected by H89. In primary osteoblast cultures, rPTH(1-34) inhibited synthesis of PTH/PTHrP receptor mRNA irrespective of H89. The down-regulation effect of rPTH(1-34) was also unaltered by PD98059 (an extracellularly regulated kinase 1/2 mitogen-activated protein kinase pathway inhibitor). Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter the inhibition of PTH/PTHrP receptor mRNA expression by rPTH(1-34), indicating that receptor mRNA suppression does not require new protein synthesis. Transcriptional activation of PTH/PTHrP receptor gene promoter (U3P or U4P)-luciferase constructs was decreased by rPTH(1-34), forskolin and 8-pCPTcAMP irrespective of H89. Thus, PTH transcriptionally down-regulates PTH/PTHrP receptor gene expression in osteoblast-like cells via a cAMP-dependent, PKA-independent pathway.
甲状旁腺激素（PTH）通过G蛋白偶联的PTH/甲状旁腺激素相关蛋白（PTHrP）受体调节成骨细胞功能。我们研究了PTH在UMR-106成骨细胞样细胞中抑制PTH/PTHrP受体基因的机制。10⁻⁷M的大鼠（r）PTH（1-34）和胰岛素样生长因子-I（IGF-I）在1小时和3小时时对PTH/PTHrP受体mRNA表达的抑制作用显著，在2小时和6小时时达到最大，rPTH（1-34）和IGF-I对受体mRNA丰度的最大降低持续24小时。在UMR-106细胞中，加入福斯可林（一种腺苷酸环化酶激活剂）或8-（4-氯苯硫基）-腺嘌呤3'，5'-环磷酸（8-pCPTcAMP；一种cAMP类似物）可模拟rPTH（1-34）对受体基因表达的抑制作用。尽管H89（一种选择性蛋白激酶A（PKA）抑制剂）完全抑制了rPTH（1-34）、福斯可林或8-pCPTcAMP刺激的PKA活性，但这些物质在UMR-106细胞中诱导的PTH/PTHrP受体mRNA合成的抑制不受H89影响。在原代成骨细胞培养物中，无论有无H89，rPTH（1-34）均抑制PTH/PTHrP受体mRNA的合成。rPTH（1-34）的下调作用也不受PD98059（一种细胞外调节激酶1/2丝裂原活化蛋白激酶途径抑制剂）的影响。用蛋白质合成抑制剂环己酰亚胺预处理不会改变rPTH（1-34）对PTH/PTHrP受体mRNA表达的抑制作用，这表明受体mRNA的抑制不需要新的蛋白质合成。无论有无H89，rPTH（1-34）、福斯可林和8-pCPTcAMP均可降低PTH/PTHrP受体基因启动子（U3P或U4P）-荧光素酶构建体的转录激活。因此，PTH通过cAMP依赖性、PKA非依赖性途径在成骨细胞样细胞中转录下调PTH/PTHrP受体基因表达。
