Expression of the osteoblast differentiation factor RUNX2 (Cbfa1/AML3/Pebp2αA) is inhibited by tumor necrosis factor-α
Expression of the osteoblast differentiation factor RUNX2 (Cbfa1/AML3/Pebp2αA) is inhibited by tumor necrosis factor-α
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DOI：
10.1074/jbc.m106339200
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发表时间：
2002-01-25
影响因子：
4.8
通讯作者：
Nanes, MS
中科院分区：
文献类型：
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作者：
Gilbert, L;He, XF;Nanes, MS
转录因子RUNX2（Cbfal/AML 3/Pebp-2alphaA）是成骨细胞分化的关键调节因子。我们研究了炎性细胞因子肿瘤坏死因子α（TNF）对RUNX2表达的影响，因为已知TNF抑制成骨细胞从多能祖细胞分化。通过逆转录-PCR测量，胎儿颅骨前体细胞或MC3T3-E1克隆前成骨细胞的TNF处理引起RUNX2稳态mRNA的剂量依赖性抑制。TNF抑制的IC 50为0.6 ng/ml。使用北方分析证实了TNF对RUNX2 mRNA的抑制。使用位于两种主要RUNX2同种型独特区域侧翼的同种型特异性引物研究TNF的作用。TNF抑制编码较短MRIPV同种型的mRNA表达> 90%，而抑制较长MASNS同种型的mRNA表达50%。RUNX2核含量通过使用大鼠骨钙素启动子结合序列作为探针的电泳迁移率变动分析和Western分析进行评价。TNF减少核RUNX2蛋白。放线菌酮抑制新的蛋白质合成未能阻止TNF抑制RUNX2 mRNA，这表明新翻译的蛋白质不介导TNF的作用。RUNX2 mRNA半衰期为1.8 h，TNF降低至0.9 h。在MC3T3-E1细胞中使用0.6kb RUNX2启动子-荧光素酶报告基因评估TNF对RUNX2基因转录的影响。TNF引起剂量依赖性的转录抑制至对照值的50%。翻译起始位点上游-108位核苷酸的缺失保留了TNF的抑制作用;然而，-108位核苷酸下游的定位由于基础活性的丧失而变得模糊。我们的研究结果表明，TNF调节RUNX2的表达在多个水平，包括mRNA的不稳定和转录抑制。RUNX2核蛋白的不成比例抑制表明可能发生了其他转录后机制。TNF对RUNX2的抑制可能会降低成骨细胞的分化，并抑制TNF过量状态下的骨形成。
The transcription factor RUNX2 (Cbfal/AML3/Pebp-2alphaA) is a critical regulator of osteoblast differentiation. We investigated the effect of the inflammatory cytokine tumor necrosis factor alpha (TNF) on the expression of RUNX2 because TNF is known to inhibit differentiation of ostcoblasts from pluripotent progenitor cells. TNF treatment of fetal calvaria precursor cells or MC3T3-E1 clonal pre-osteoblastic cells caused a dose-dependent suppression of RUNX2 steady state mRNA as measured by reverse transcription-PCR. The IC50 for TNF inhibition was 0.6 ng/ml. TNF suppression of RUNX2 mRNA was confirmed using Northern analysis. The effect of TNF was studied using isoform-specific primers that flanked unique regions of two major RUNX2 isoforms. TNF suppressed expression of the mRNA coding for the shorter MRIPV isoform by >90% while inhibiting expression of the mRNA for the longer MASNS isoform by 50%. RUNX2 nuclear content was evaluated by electrophoretic mobility shift assay using a rat osteocalcin promoter binding sequence as probe and by Western analysis. TNF reduced nuclear RUNX2 protein. Inhibition of new protein synthesis with cycloheximide failed to prevent TNF inhibition of RUNX2 mRNA, suggesting that a newly translated protein did not mediate the TNF effect. RUNX2 mRNA half-life was 1.8 h and reduced to 0.9 h by TNF. The effect of TNF on RUNX2 gene transcription was evaluated using a 0.6.kb RUNX2 promoter-luciferase reporter in MC3T3-E1 cells. TNF caused a dose-dependent inhibition of transcription to 50% of control values. The inhibitory effect of TNF was preserved with deletions to nucleotide -108 upstream of the translational start site; however, localization downstream of nucleotide -108 was obscured by loss of basal activity. Our results indicate that TNF regulates RUNX2 expression at multiple levels including destabilization of mRNA and suppression of transcription. The disproportionate inhibition of RUNX2 nuclear protein suggests that additional post-transcriptional mechanisms may be occurring. Suppression of RUNX2 by TNF may decrease ostcoblast differentiation and inhibit bone formation in TNF excess states.