The expression of Clcn7 and Ostm1 in osteoclasts is coregulated by microphthalmia transcription factor
The expression of Clcn7 and Ostm1 in osteoclasts is coregulated by microphthalmia transcription factor
复制标题
DOI：
10.1074/jbc.m608572200
复制
发表时间：
2007-01-19
影响因子：
4.8
通讯作者：
Cassady, Alan I.
中科院分区：
文献类型：
--
作者：
Meadows, Nicholas A.;Sharma, Sudarshana M.;Cassady, Alan I.
小眼炎转录因子(MITF)通过调节抗酒石酸酸性磷酸酶(TRAP)和组织蛋白酶K等基因的表达来调节破骨细胞的功能，以响应核因子-kappa B受体激活剂(RANKL)诱导的信号传导。为了确定新的MITF靶基因，我们在小鼠巨噬细胞系RAW264.7亚克隆4(RAW/C4)中过表达了MITF，并检测了sRANKL刺激破骨细胞生成后的基因表达谱。基因芯片分析发现了一组MITF过表达诱导的基因，包括Clcn7(氯离子通道7)和Ostm1(骨化相关跨膜蛋白1)。利用凝胶迁移率改变分析，我们在Clcn7启动子中发现了两个MITF结合位点(M-box)，在Ostm1启动子中发现了一个单独的M-box。抗MITF抗体使两个基因中的启动子位置的DNA-蛋白质复合体发生超位，而MITF结合因这些位置的突变而取消。在报告基因检测中，共表达MITF可反式激活Clcn7启动子。一个Clcn7 M盒的突变阻止了MITF的反式激活，但第二个MITF结合位点的突变只降低了基础活性。染色质免疫沉淀实验证实，两个Clcn7 MITF体外结合区和反应区与基因组DNA中的MITF结合。Clcn7在显性负性突变MITF小鼠mi/mi中的表达受到抑制，这表明在这些小鼠中看到的骨吸收失调可以部分归因于Clcn7的转录抑制。MITF对TRAP、Cathepsin K、Clcn7和Ostm1基因的调节对破骨细胞的吸收至关重要，这表明MITF的作用比人们之前认为的更重要，MITF可能是破骨细胞功能和骨吸收的主要调节因子。
Microphthalmia transcription factor (MITF) regulates osteoclast function by controling the expression of genes, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K in response to receptor activator of nuclear factor-kappa B ligand (RANKL)-induced signaling. To identify novel MITF target genes, we have overexpressed MITF in the murine macrophage cell line RAW264.7 subclone 4 (RAW/C4) and examined the gene expression profile after sRANKL-stimulated osteoclastogenesis. Microarray analysis identified a set of genes superinduced by MITF overexpression, including Clcn7 (chloride channel 7) and Ostm1 (osteopetrosis-associated transmembrane protein 1). Using electrophoretic mobility shift assays, we identified two MITF-binding sites (M-boxes) in the Clcn7 promoter and a single M-box in the Ostm1 promoter. An anti-MITF antibody supershifted DNA-protein complexes for promoter sites in both genes, whereas MITF binding was abolished by mutation of these sites. The Clcn7 promoter was transactivated by coexpression of MITF in reporter gene assays. Mutation of one Clcn7 M-box prevented MITF transactivation, but mutation of the second MITF-binding site only reduced basal activity. Chromatin immunoprecipitation assays confirmed that the two Clcn7 MITF binding and responsive regions in vitro bind MITF in genomic DNA. The expression of Clcn7 is repressed in the dominant negative mutant Mitf mouse, mi/mi, indicating that the dysregulated bone resorption seen in these mice can be attributed in part to transcriptional repression of Clcn7. MITF regulation of the TRAP, cathepsin K, Clcn7, and Ostm1 genes, which are critical for osteoclast resorption, suggests that the role of MITF is more significant than previously perceived and that MITF may be a master regulator of osteoclast function and bone resorption.