EphrinB2 regulation by PTH and PTHrP revealed by molecular profiling in differentiating osteoblasts
EphrinB2 regulation by PTH and PTHrP revealed by molecular profiling in differentiating osteoblasts
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DOI：
10.1359/jbmr.080324
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发表时间：
2008-08-01
影响因子：
6.2
通讯作者：
Martin, T. John
中科院分区：
文献类型：
--
作者：
Allan, Elizabeth H.;Hausler, Karl D.;Martin, T. John
为了鉴定成骨细胞中PTH（1-34）和PTH相关蛋白1-141 [PTHrP（1-141）]调控的新途径和基因，本研究使用小鼠骨髓基质细胞系Kusa 4 b10进行，该细胞系在长期培养条件下获得成骨细胞表型的特征。在Kusa 4 b10细胞中出现功能性PTH受体1（PTHR 1）后，用PTH（134）或PTHrP（1-141）处理它们，并对RNA进行Affyssin全小鼠基因组阵列。使用定量实时RT-PCR对来自分化的Kusa 4 b10、UMR 106骨肉瘤细胞和原代小鼠颅骨成骨细胞的独立制备的RNA样品进行验证，以及在体内使用来自干骺端骨的RNA对3周龄和6月龄的卵巢切除大鼠进行单次PTH注射后验证微阵列数据。在微阵列上使用的45，101个探针中，4675个差异表达>= 1.5倍，错误发现率< 0.1。在受调节的基因中，ephrinB 2 mRNA在PTH和PTHrP的反应中上调。这通过体外和体内定量实时PCR证实。通过蛋白质印迹法也显示了体外ephrinB 2蛋白的增加，股骨切片的免疫染色显示ephrinB 2在成骨细胞和成骨细胞中。在Kusa 4 b10细胞的分化过程中，ephrinB 2以及其他ephrins或Eph家族成员的产生没有变化。阻断ephrinB 2/EphB 4相互作用导致Kusa 4 b10细胞矿化抑制。连同所显示的ephrinB 2通过作用于EphB 4促进成骨细胞分化和骨形成的作用，数据提高了PTH或PTHrP可能调节ephrinB 2以旁分泌或自分泌方式作用于成骨细胞中的EphB 4或EphB 2的可能性，作为局部事件对PTH或PTHrP的合成代谢作用做出贡献。
With the aim of identifying new pathways and genes regulated by PTH(1-34) and PTH-related protein 1-141 [PTHrP(1-141)] in osteoblasts, this study was carried out using a mouse marrow stromal cell line, Kusa 4b10, that acquires features of the osteoblastic phenotype in long-term culture conditions. After the appearance of functional PTH receptor 1 (PTHR1) in Kusa 4b10 cells, they were treated with either PTH(134) or PTHrP(1-141), and RNA was subjected to Affymetrix whole mouse genome array. The microarray data were validated using quantitative real-time RT-PCR on independently prepared RNA samples from differentiated Kusa 4b10, UMR106 osteosarcoma cells, and primary mouse calvarial osteoblasts, as well as in vivo using RNA from metaphyseal bone after a single PTH injection to 3-wk-old and 6-mo-old ovariectomized rats. Of the 45,101 probes used on the microarray, 4675 were differentially expressed by >= 1.5 fold, with a false discovery rate < 0.1. Among the regulated genes, ephrinB2 mRNA was upregulated in response to both PTH and PTHrP. This was confirmed by quantitative real-time PCR in vitro and in vivo. Increased ephrinB2 protein was also shown in vitro by Western blotting, and immunostaining of femur sections showed ephrinB2 in both osteoblasts and osteoblasts. Production of ephrinB2, as well as other ephrins or Eph family members, did not change during differentiation of Kusa 4b10 cells. Blockade of ephrinB2/EphB4 interaction resulted in inhibition of mineralization of Kusa 4b10 cells. Together with the shown effect of ephrinB2 promoting osteoblast differentiation and bone formation through action on EphB4, the data raise the possibility that PTH or PTHrP might regulate ephrinB2 to act in a paracrine or autocrine manner on EphB4 or EphB2 in the osteoblast, contributing as a local event to the anabolic action of PTH or PTHrP.