Yanaka N, Akatsuka H, Kawai E, Omori K
Discovery Research Laboratory, Tanabe Seiyaku, Osaka 532-8505, Japan.
Am J Physiol. 1998 Dec;275(6):E965-73. doi: 10.1152/ajpendo.1998.275.6.E965.
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], a key regulator of mineral metabolism, regulates expression of several genes related to bone formation. The present study examined the 1,25(OH)2D3-mediated regulation of natriuretic peptide receptor-C (NPR-C) expression in osteoblasts. 1,25(OH)2D3 treatment significantly increased NPR-C-dependent atrial natriuretic peptide-binding activity and synthesis of the NPR-C protein in mouse osteoblastic cells in a cell-specific manner. Western blot analysis also demonstrated that 1, 25(OH)2D3 upregulated expression of NPR-C protein in slow kinetics. Next, Northern blot analysis revealed a significant increase in the steady-state NPR-C mRNA level by 1,25(OH)2D3. Sequence analysis of the 9 kb of the 5'-flanking region of the mouse NPR-C gene revealed an absence of consensus vitamin D-response elements, and promoter analysis using osteoblastic cells stably transfected with mouse NPR-C promoter-reporter constructs showed a slight increase of promoter activity with 1,25(OH)2D3 treatment. In addition, a nuclear run-on assay exhibited that the transcriptional rate of the NPR-C gene was unchanged by 1,25(OH)2D3, whereas that of the osteopontin gene was increased. Evaluation of NPR-C mRNA half-life demonstrated that 1,25(OH)2D3 significantly increased the NPR-C mRNA stability in osteoblastic cells. 1,25(OH)2D3 attenuated intracellular cGMP production in osteoblastic cells stimulated by C-type natriuretic peptide (CNP) without a significant change of the natriuretic peptide receptor-B mRNA level, suggesting enhancement of the clearance of exogenously added CNP via NPR-C. Furthermore, NPR-C and osteopontin mRNAs in mouse calvariae were significantly increased by administration of 1,25(OH)2D3, and immunohistological analysis demonstrated that NPR-C is actually and strongly expressed in mouse periosteal fibroblasts. These findings suggest that 1,25(OH)2D3 can play a critical role for determination of the natriuretic peptide availability in bones by regulation of NPR-C expression through stabilizing its mRNA.
1,25 - 二羟基维生素D3[1,25(OH)2D3]是矿物质代谢的关键调节因子，可调节多个与骨形成相关基因的表达。本研究检测了1,25(OH)2D3对成骨细胞中利钠肽受体 - C（NPR - C）表达的调节作用。1,25(OH)2D3处理以细胞特异性方式显著增加了小鼠成骨细胞中依赖NPR - C的心房利钠肽结合活性及NPR - C蛋白的合成。蛋白质印迹分析还表明，1,25(OH)2D3以慢动力学上调NPR - C蛋白的表达。接下来，Northern印迹分析显示1,25(OH)2D3使NPR - C mRNA的稳态水平显著增加。对小鼠NPR - C基因5' - 侧翼区域9 kb的序列分析显示不存在共有的维生素D反应元件，使用稳定转染小鼠NPR - C启动子 - 报告基因构建体的成骨细胞进行的启动子分析表明，1,25(OH)2D3处理使启动子活性略有增加。此外，核转录分析显示1,25(OH)2D3对NPR - C基因的转录速率无影响，而骨桥蛋白基因的转录速率增加。对NPR - C mRNA半衰期的评估表明，1,25(OH)2D3显著增加了成骨细胞中NPR - C mRNA的稳定性。1,25(OH)2D3减弱了C型利钠肽（CNP）刺激的成骨细胞内cGMP的产生，而利钠肽受体 - B mRNA水平无显著变化，提示通过NPR - C增强了外源性添加的CNP的清除。此外，给予1,25(OH)2D3后，小鼠颅骨中的NPR - C和骨桥蛋白mRNA显著增加，免疫组织学分析表明NPR - C在小鼠骨膜成纤维细胞中实际且强烈表达。这些发现提示，1,25(OH)2D3可通过稳定其mRNA来调节NPR - C的表达，从而在决定骨骼中利钠肽的可用性方面发挥关键作用。
