甲状旁腺素（1-34）对破骨细胞抑制性凝集素表达的调节及信号转导机制

Parathyroid hormone （1-34） regulates the expression of osteoclast inhibitory lectin via multiple signaling pathways in UMR106 osteoblast-like cells

目的探讨甲状旁腺素（parathyroidhormone，PTH）（1_34）对UMRl06成骨细胞表达破骨细胞抑制性凝集素（osteoclastinhibitorylectin，OCIL）基因mRNA的调节及其信号转导机制。方法培养大鼠UMRl06成骨细胞，采用胛H（1—34）及蛋白激酶A（PKA）、蛋白激酶C（PKC）、钙离子、MAPK通路的激动剂或阻断剂干预细胞不同时间后，收集细胞提取总RNA，实时荧光定量PCR检测OCILmRNA的表达水平。结果PTH（1-34）以剂量和时间依赖方式促进OCILmRNA表达。10nmol／LPTH（1-34）作用6h后促进OCILmRNA表达，24h上调效应最显著，约为对照组[未加入PTH（1-34）]的2．8倍。PTH（1_34）对OCILmRNA表达促进作用的起始时间及高峰时间均晚于其对RANKL的诱导和对OPG的抑制。蛋白激酶A通路激动剂福司可林（FSK）、双丁酰基环腺苷磷酸（db．cAMP）及钙离子载体A23187均不同程度促进OCILmRNA表达，最大上调效应分别约为对照组的4．2、4．5和5．1倍。蛋白激酶C激动剂佛波酯（PMA）作用早期（2～6h）抑制OCILmRNA表达，作用6h后最大抑制率达50％；PMA作用后期（24h）对OCILmRNA的抑制作用逆转为促进效应。PKA阻断剂KT5720、钙调蛋白（CaMK）阻断剂W-7、钙调蛋白激酶Ⅱ阻断剂KN-62及丝裂原激活的蛋白激酶（MAPK）阻断剂PD98059均下调胛H（1-34）诱导的OCILmRNA表达，最大抑制率分别为56％、61％、63％和50％。各信号通路间存在交互作用。MAPK通路阻断剂PD98059减少PKA激动剂FSK或db—cAMP诱导的OCILmRNA表达，抑制率分别为98％和63％；但不影响A23187诱导的OCILmRNA水平增高。结论PKA通路、钙／钙调蛋白通路和MAPK通路可介导PTH（1-34）诱导的OCILmRNA表达。

Objective To investigate the regulation of parathyroid hormone（I-34） on mRNA expression of osteoelast in- hibitory lectin （OCIL） gene in UMR106 osteoblastie-like cells and involved signaling pathway. Methods Rat UMR106 osteoblas- tic-like cells were cultured and treated with various concentration of PTH（1-34） and specific agonists or inhibitors of PKA, PKC, Ca2~/calmodulin- dependent protein kinase （CaMK） and mitogen-activated protein kinase （MAPK） signal pathways for indicated time intervals. Then the cells were gathered at indicated time points and total RNA were extracted. OCIL mRNA expression was analyzed using real-time PCR technique. Results PTH（1-34） stimulated OCIL mRNA expression in a time- and dose-depen- dent manner. A dose of 10 nmol/L PTH（1-34） started to induce OCIL mRNA from 6 h,with a highest increase of about 2.8-fold vs. control group （without PTH treatment） at 24 h. The up-regulation of OCIL mRNA began and reached maximum later than RANKL induction and OPG suppression effected by PTH（1-34）. Protein Kinase A （PKA） signaling activators forskolin（FSK） and dibutyryl cAMP （rib-cAMP）, as well as calcium ionophore A23187 all up-regulated OCIL mRNA with the maximal induction of about 4.2- fold, 4.5-fold and 5.1-fold. Protein Kinase C （PKC） activator phorbol-12-myristate-13-aeetate（PMA） reduced OCIL mRNA expres- sion at the early stage（2-6 h）, with the highest down-regulation of 50% at 6 h. However, the inhibitory effect on OCIL mRNA turned into slightly stimulatory effect later （24 h）. PKA inhibitor KT5720, calmodulin antagonist W-7, CaMK II inhibitor KN-62 and mitogen-aetivated protein kinase （MAPK） inhibitor PD98059 all blocked PTH（1-34）-indueed OCIL mRNA expression by the maximal reduction of 56%, 61%, 63% and 50% respectively. There also exist cross-talks between different signal pathways. MAPK inhibitor PD98059 blocked the expression of OCIL mRNA which was stimulated by PKA activators FSK or db-cAMP, with the reduction of 98% and 63% respectively, while the OCIL mRNA expression stimulated by A23187 remained unaffected. Con- clusion PTH（1-34） increased OCIL mRNA expression in vitro through cAMP/PKA, CaZ＋/CaMK and MAPK signaling pathways.