Microphthalmia transcription factor is a target of the p38 MAPK pathway in response to receptor activator of NF-κB ligand signaling
Microphthalmia transcription factor is a target of the p38 MAPK pathway in response to receptor activator of NF-κB ligand signaling
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DOI：
10.1074/jbc.m111696200
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发表时间：
2002-03-29
影响因子：
4.8
通讯作者：
Ostrowski, MC
中科院分区：
文献类型：
--
作者：
Mansky, KC;Sankar, U;Ostrowski, MC
NF-kappaB 配体受体激活剂 (RANKL) 激活调节破骨细胞分化、功能和存活的信号通路。小眼转录因子（MITF）是破骨细胞终末分化所必需的。为了确定 MITF 是否可以成为 RANKL 信号传导的靶标，我们生产了针对保守残基 Ser(307)（一个潜在的丝裂原激活蛋白激酶 (MAPK) 位点）的磷酸特异性 MITF 抗体。使用该抗体，我们可以证明 MITF 在用 RANKL 刺激原代破骨细胞后快速且持续地磷酸化，并且 Ser(307) 的磷酸化与靶基因抗酒石酸酸性磷酸酶的表达相关。 MITF Ser(307) 磷酸化也与 p38 MAPK 的持续激活相关，并且 p38 MAPK 可以利用 MITF Ser(307) 作为体外底物。 p38 MAPK 抑制剂 SB203580 阻断 MITF 的磷酸化和破骨细胞中靶基因表达的激活。在瞬时转染中，组成型活性的 Rac1 或 MKK6 基因可以与 MITF 协同激活依赖于 Ser(307) 的抗酒石酸酸性磷酸酶基因启动子。在瞬时检测中，显性阴性 p38 a 和 0 可以抑制上游信号成分与 MITF 之间的协作。这些结果表明 MITF 是破骨细胞中 RANKL 信号通路的靶标，并且 MITF 磷酸化导致破骨细胞特异性基因表达增加。
Receptor activator of NF-kappaB ligand (RANKL) activates signaling pathways that regulate osteoclast differentiation, function, and survival. The microphthalmia transcription factor (MITF) is required for terminal differentiation of osteoclasts. To determine whether MITF could be a target of RANKL signaling, a phosphospecific MITF antibody directed against conserved residue Ser(307), a potential mitogen-activated protein kinase (MAPK) site, was produced. Using this antibody, we could demonstrate that MITF was rapidly and persistently phosphorylated upon stimulation of primary osteoclasts with RANKL and that phosphorylation of Ser(307) correlated with expression of the target gene tartrate-resistant acid phosphatase. MITF phosphorylation at Ser(307) also correlated with persistent activation of p38 MAPK, and p38 MAPK could utilize MITF Ser(307) as a substrate in vitro. The phosphorylation of MITF and activation of target gene expression in osteoclasts were blocked by p38 MAPK inhibitor SB203580. In transient transfections, a constitutively active Rac1 or MKK6 gene could collaborate with MITF to activate the tartrate-resistant acid phosphatase gene promoter dependent on Ser(307). Dominant negative p38 a and 0 could inhibit the collaboration between upstream signaling components and MITF in the transient assays. These results indicate that MITF is a target for the RANKL signaling pathway in osteoclasts and that phosphorylation of MITF leads to an increase in osteoclast-specific gene expression.