Modulation of mouse RANKL gene expression by Runx2 and PKA pathway
Modulation of mouse RANKL gene expression by Runx2 and PKA pathway
复制标题
DOI：
10.1002/jcb.20891
复制
发表时间：
2006-08-15
影响因子：
4
通讯作者：
Kitazawa, Sohei
中科院分区：
文献类型：
--
作者：
Mori, Kiyoshi;Kitazawa, Riko;Kitazawa, Sohei
Runx 2调节成骨细胞表型特征的靶基因，同时根据成骨细胞的分化阶段对成骨细胞产生不同的、有时是有争议的影响。核因子受体活化因子-κ B（RANK）配体（RANKL）是一种对骨（软骨）断裂必不可少的膜结合细胞因子。在软骨内骨化过程中，虽然Runx 2阳性肥大软骨细胞表达RANKL，但成骨细胞中RANKL基因的稳态表达在后期阶段保持在相对较低的水平，以维持已建立的骨。本研究的目的是阐明Runx 2和蛋白激酶A（PKA）通路调节RANKL表达的机制，特别是从它们在RANKL基本启动子活性和成骨细胞/基质细胞染色质结构变化中的功能的角度。使用来自正常和Runx 2缺陷小鼠的成骨细胞/基质细胞系，通过实时逆转录（RT）-PCR分析内源性RANKL基因表达，通过染色质免疫沉淀分析与RANKL基因5 '侧翼区相关的H3和H4组蛋白的乙酰化状态，以及在稳态和PKA激活条件下外源转染的RANKL基因启动子活性。在这里，我们证明Runx 2通过浓缩染色质抑制稳态RANKL基因表达，同时对RANKL基本启动子活性显示出轻微的积极影响。除了通过RANKL基因启动子的CRE样区域（约0.96 kb）起作用外，毛喉素（FK）处理还通过拮抗Runx 2的功能、降低Runx 2 mRNA表达和打开RANKL基因远上游（超过40 kb）的染色质构象来反式激活RANKL基因。
Runx2 regulates the target genes characteristic of osteoblastic phenotypes, while exerting diverse and sometimes controversial effects on osteoblastic cells depending on their differentiation stage. Receptor activator of nuclear factor-KB (RANK) ligand (RANKL) is a membrane bound cytokine essential for osteo(chondro)clastogenesis. During endochondral ossification, while Runx2-positive hypertrophic chondrocytes express RANKL, the steady-state expression of the RANKL gene in osteoblastic cells is, at later stages, kept at a relatively low level to sustain the established bone. The aim of this study was to elucidate the mechanism whereby Runx2 and the protein kinase A (PKA) pathway modulate RANKL expression, especially from the viewpoint of their functions in RANKL basic promoter activity and in chromatin structural changes in osteoblastic/stromal cells. Osteoblastic/stromal cell lines derived from normal and Runx2-deficient mice were used to analyze endogenous RANKL gene expression by real-time reverse transcription (RT)-PCR, the acetylation status of the H3 and H4 histone proteins associated with the 5'-flanking region of the RANKL gene by chromatin immunoprecipitation, and the exogenously transfected RANKL gene promoter activity both in the steady-state and under PKA-activated conditions. Here, we demonstrate that Runx2 suppresses steady-state RANKL gene expression by condensing chromatin, while showing a slightly positive effect on RANKL basic promoter activity. Besides acting through the CRE-like region (-0.96 kb) of the RANKL gene promoter, forskolin (FK) treatment transactivates the RANKL gene by antagonizing the function of Runx2, by reducing Runx2 mRNA expression and by opening the chromatin conformation far upstream (more than 40 kb) of the RANKL gene.