细胞周期蛋白D1作为甲状旁腺激素（PTH）和甲状旁腺激素相关蛋白（PTHrP）对早期成骨细胞增殖作用的靶点。

Cyclin D1 as a target for the proliferative effects of PTH and PTHrP in early osteoblastic cells.

作者信息

Datta Nabanita S, Pettway Glenda J, Chen Chen, Koh Amy J, McCauley Laurie K

机构信息

Department of Periodontics and Oral Medicine, University of Michigan, Ann Arbor, Michigan 48109-1078,, USA.

出版信息

J Bone Miner Res. 2007 Jul;22(7):951-64. doi: 10.1359/jbmr.070328.

DOI:10.1359/jbmr.070328

PMID:17501623

Abstract

UNLABELLED

PTHrP induced a proliferative cyclin D1 activation in low-density osteoblastic cells. The process was PKA and MAPK dependent and involved both AP-1 and CRE sites. In ectopic ossicles generated from implanted bone marrow stromal cells, PTH upregulated cyclin D1 after acute or intermittent anabolic treatment. These data suggest a positive role of PTH and PTHrP in the cell cycle of early osteoblasts.

INTRODUCTION

The mechanisms underlying the actions of PTH and its related protein (PTHrP) in osteoblast proliferation, differentiation, and bone remodeling remain unclear. The action of PTH or PTHrP on the cell cycle during osteoblast proliferation was studied.

MATERIALS AND METHODS

Mouse calvarial MC3T3-E1 clone 4 cells were synchronized by serum starvation and induced with 100 nM PTHrP for 2-24 h under defined low serum conditions. Western blot, real-time PCR, EMSAs, and promoter/luciferase assays were performed to evaluate cyclin D1 expression. Pharmacological inhibitors were used to determine the relevant signaling pathways. Ectopic ossicles generated from implanted bone marrow stromal cells were treated with acute (a single 8- or 12-h injection) or intermittent anabolic PTH treatment for 7 days, and RNA and histologic analysis were performed.

RESULTS

PTHrP upregulated cyclin D1 and CDK1 and decreased p27 expression. Cyclin D1 promoter/luciferase assays showed that the PTHrP regulation involved both activator protein-1 (AP-1) and cyclic AMP response element binding protein (CRE) sites. AP-1 and CRE double mutants completely abolished the PTHrP effect of cyclin D1 transcription. Upregulation of cyclin D1 was found to be protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) dependent in proliferating MC3T3-E1 cells. In vivo expression of cyclin D1 in ectopic ossicles was upregulated after a single 12-h PTH injection or intermittent anabolic PTH treatment for 7 days in early developing ossicles.

CONCLUSIONS

These data indicate that PTH and PTHrP induce cyclin D1 expression in early osteoblastic cells and their action is developmental stage specific.

摘要

未标记

甲状旁腺激素相关蛋白（PTHrP）在低密度成骨细胞中诱导增殖性细胞周期蛋白D1激活。该过程依赖蛋白激酶A（PKA）和丝裂原活化蛋白激酶（MAPK），并涉及活化蛋白-1（AP-1）和环磷腺苷反应元件（CRE）位点。在植入骨髓基质细胞生成的异位骨中，甲状旁腺激素（PTH）在急性或间歇性促合成治疗后上调细胞周期蛋白D1。这些数据表明PTH和PTHrP在早期成骨细胞的细胞周期中起积极作用。

引言

PTH及其相关蛋白（PTHrP）在成骨细胞增殖、分化和骨重塑中的作用机制尚不清楚。研究了PTH或PTHrP在成骨细胞增殖过程中对细胞周期的作用。

材料与方法

通过血清饥饿使小鼠颅骨MC3T3-E1克隆4细胞同步化，并在确定的低血清条件下用100 nM PTHrP诱导2 - 24小时。进行蛋白质印迹、实时聚合酶链反应（PCR）、电泳迁移率变动分析（EMSA）和启动子/荧光素酶测定以评估细胞周期蛋白D1的表达。使用药理抑制剂确定相关信号通路。对植入骨髓基质细胞生成的异位骨用急性（单次8或12小时注射）或间歇性促合成PTH治疗7天，并进行RNA和组织学分析。

结果

PTHrP上调细胞周期蛋白D1和细胞周期蛋白依赖性激酶1（CDK1）并降低p27表达。细胞周期蛋白D1启动子/荧光素酶测定表明，PTHrP调节涉及活化蛋白-1（AP-1）和环磷腺苷反应元件结合蛋白（CRE）位点。AP-1和CRE双突变体完全消除了PTHrP对细胞周期蛋白D1转录的影响。发现在增殖的MC3T3-E1细胞中，细胞周期蛋白D1的上调是蛋白激酶A（PKA）和丝裂原活化蛋白激酶（MAPK）依赖性的。在早期发育的异位骨中，单次12小时PTH注射或间歇性促合成PTH治疗7天后，细胞周期蛋白D1的体内表达上调。