Matsumura Hiroyoshi, Nakayama Yohei, Takai Hideki, Ogata Yorimasa
Department of Periodontology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, 271-8587, Japan.
J Bone Miner Metab. 2015 Mar;33(2):142-53. doi: 10.1007/s00774-014-0576-8. Epub 2014 Mar 15.
Interleukin-11 (IL-11) is a bone marrow stromal fibroblast-derived cytokine with a wide spectrum of activities in different biological systems. IL-11 and IL-6 are two cytokines known to rely on osteoblast-osteoclast communication for their effects on osteoclast differentiation. Bone sialoprotein (BSP) is a mineralized connective tissue-specific protein expressed in differentiated osteoblasts, odontoblasts, and cementoblasts. To determine the molecular basis of the transcriptional regulation of the human BSP gene by IL-11, we conducted real-time polymerase chain reactions (PCR), transient transfection analyses with chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene, gel mobility shift assays, and a chromatin immunoprecipitation assay using human osteoblast-like Saos2 cells. IL-11 (20 ng/ml) increased BSP, Runx2, and Osterix mRNA levels at 6 h and the alkaline phosphatase (ALP) mRNA level at 12 h in osteoblast-like Saos2 cells. In a transient transfection assay, IL-11 (20 ng/ml, 12 h) increased luciferase activities of constructs between -60LUC and -868LUC including the human BSP gene promoter. Transcriptional stimulations by IL-11 were partially inhibited in the constructs that included 2-bp mutations in the cAMP response element 1 (CRE1, -72 to -79) and CRE2 (-667 to -674). When mutations were made in pairs of CRE1 and CRE2 in -868LUC, the effect of IL-11 on luciferase activity was almost totally abrogated. Transcriptional activities induced by IL-11 were inhibited by protein kinase A, tyrosine kinase, ERK1/2, and PI3-kinase inhibitors. Gel mobility shift analyses showed that IL-11 increased nuclear proteins binding to CRE1 and CRE2. CREB1, phospho-CREB1, c-Fos, and c-Jun antibodies disrupted the formation of CRE1 and CRE2 protein complexes. These data demonstrate that IL-11 stimulates BSP gene transcription via CRE1 and CRE2 elements in the human BSP gene promoter.
白细胞介素-11（IL-11）是一种由骨髓基质成纤维细胞产生的细胞因子，在不同生物系统中具有广泛的活性。IL-11和IL-6是已知依赖成骨细胞-破骨细胞通讯来影响破骨细胞分化的两种细胞因子。骨唾液蛋白（BSP）是一种在分化的成骨细胞、成牙本质细胞和成牙骨质细胞中表达的矿化结缔组织特异性蛋白。为了确定IL-11对人BSP基因转录调控的分子基础，我们使用人成骨样Saos2细胞进行了实时聚合酶链反应（PCR）、将人BSP基因启动子与荧光素酶报告基因连接的嵌合构建体的瞬时转染分析、凝胶迁移率变动分析以及染色质免疫沉淀分析。IL-11（20 ng/ml）在6小时时增加了成骨样Saos2细胞中BSP、Runx2和Osterix mRNA水平，在12小时时增加了碱性磷酸酶（ALP）mRNA水平。在瞬时转染实验中，IL-11（20 ng/ml，12小时）增加了包含人BSP基因启动子的-60LUC至-868LUC构建体的荧光素酶活性。在cAMP反应元件1（CRE1，-72至-79）和CRE2（-667至-674）中包含2个碱基对突变的构建体中，IL-11的转录刺激作用被部分抑制。当在-868LUC中对CRE1和CRE2进行成对突变时，IL-11对荧光素酶活性的影响几乎完全消除。蛋白激酶A、酪氨酸激酶、ERK1/2和PI3激酶抑制剂抑制了IL-11诱导的转录活性。凝胶迁移率变动分析表明，IL-11增加了与CRE1和CRE2结合的核蛋白。CREB1、磷酸化CREB1、c-Fos和c-Jun抗体破坏了CRE1和CRE2蛋白复合物的形成。这些数据表明，IL-11通过人BSP基因启动子中的CRE1和CRE2元件刺激BSP基因转录。
