Nanes M S, Kuno H, Demay M B, Kurian M, Hendy G N, DeLuca H F, Titus L, Rubin J
Division of Endocrinology and Metabolism, Emory University School of Medicine, Veterans Affairs Medical Center, Atlanta, Georgia 30033.
Endocrinology. 1994 Mar;134(3):1113-20. doi: 10.1210/endo.134.3.8119149.
Tumor necrosis factor-alpha (TNF alpha) is one of several autocrine/paracrine factors known to exert potent inhibitory effects on bone. We have shown that TNF alpha inhibition of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-stimulated synthesis of the bone-specific protein osteocalcin (OC) occurs by decreasing steady state levels of OC mRNA, suggesting a pretranslational mechanism. In many genes, TNF alpha action is mediated by the transcription factor NF kappa B. Analysis of OC 5'-flanking DNA revealed a sequence structurally homologous to the previously described NF kappa B-binding site and, thus, a potential TNF alpha response element. Deletion analysis was performed to identify the sequences mediating the response to TNF alpha in osteoblastic ROS 17/2.8 cells by transient transfection with reporter constructs containing rat OC 5'-flanking DNA [chloramphenicol acetyltransferase (CAT)] that retained or deleted homologous NF kappa B sites or a previously defined 1,25-(OH)2D3 response element (VDRE). Transfection with all reporter constructs resulted in low basal CAT activity, measured 72 h after transfection. 1,25-(OH)2D3 stimulated CAT activity 2.8- to 4.5-fold in cells transfected with constructs that included the VDRE. TNF alpha inhibited 1,25-(OH)2D3-stimulated, but not basal, CAT activity. Deletion analysis localized the effect of TNF alpha to a sequence between -522 and -306 relative to the OC transcription start site, an area that included the VDRE but deleted a homologous NF kappa B element. Transfection of cells with a heterologous reporter containing one copy of the OC VDRE inserted in correct orientation or two copies in inverse orientation was sufficient to confer a response to TNF alpha. Gel mobility shift analysis of DNA-nuclear protein interaction revealed that 1,25-(OH)2D3 stimulated an increase in binding of nuclear proteins to an OC 32P-VDRE probe. Preincubation of nuclear extract with specific monoclonal antibodies confirmed that the proteins binding the VDRE included the vitamin D receptor and retinoid-X receptor. TNF alpha treatment of cells inhibited the 1,25-(OH)2D3-stimulated increase in nuclear protein binding to the VDRE. These results suggest 1) the VDRE is sufficient to confer a response to the inhibitory effect of TNF alpha on 1,25-(OH)2D3-stimulated rat OC gene transcription; 2) the action of TNF alpha does not require homologous NF kappa B response elements; and 3) the mechanism of TNF alpha inhibition of 1,25-(OH)2D3-stimulated OC gene expression includes modulation of binding of the vitamin D receptor/retinoid-X receptor heterodimer to the VDRE.
肿瘤坏死因子-α（TNFα）是已知对骨骼具有强大抑制作用的几种自分泌/旁分泌因子之一。我们已经表明，TNFα对1,25-二羟基维生素D3[1,25-(OH)2D3]刺激的骨特异性蛋白骨钙素（OC）合成的抑制作用是通过降低OC mRNA的稳态水平来实现的，这提示了一种转录前机制。在许多基因中，TNFα的作用是由转录因子NF-κB介导的。对OC 5'-侧翼DNA的分析揭示了一个与先前描述的NF-κB结合位点结构同源性的序列，因此是一个潜在的TNFα反应元件。通过用含有大鼠OC 5'-侧翼DNA[氯霉素乙酰转移酶（CAT）]的报告构建体进行瞬时转染，进行缺失分析以鉴定在成骨细胞ROS 17/2.8细胞中介导对TNFα反应的序列，该报告构建体保留或缺失同源NF-κB位点或先前定义的1,25-(OH)2D3反应元件（VDRE）。用所有报告构建体转染后，在转染后72小时测量基础CAT活性较低。1,25-(OH)2D3在转染了包含VDRE的构建体的细胞中刺激CAT活性增加2.8至4.5倍。TNFα抑制1,25-(OH)2D3刺激的但非基础的CAT活性。缺失分析将TNFα的作用定位到相对于OC转录起始位点-522至-306之间的序列，该区域包括VDRE但缺失了同源NF-κB元件。用含有一个以正确方向插入的OC VDRE拷贝或两个以反向插入的拷贝的异源报告基因转染细胞足以赋予对TNFα的反应。DNA-核蛋白相互作用的凝胶迁移率变动分析表明，1,25-(OH)2D3刺激核蛋白与OC 32P-VDRE探针的结合增加。用特异性单克隆抗体对核提取物进行预孵育证实，与VDRE结合的蛋白质包括维生素D受体和视黄酸X受体。用TNFα处理细胞抑制了1,25-(OH)2D3刺激的核蛋白与VDRE结合的增加。这些结果提示：1）VDRE足以赋予对TNFα对1,25-(OH)2D3刺激的大鼠OC基因转录抑制作用的反应；2）TNFα的作用不需要同源NF-κB反应元件；3）TNFα抑制1,25-(OH)2D3刺激的OC基因表达的机制包括调节维生素D受体/视黄酸X受体异二聚体与VDRE的结合。
