Novel Regulators of Fgf23 Expression and Mineralization in Hyp Bone

We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts regulating Fgf23 production and mineralization in Hyp mice. We discovered that activation of Fgf receptor- and Wnt-pathways contribute to increased Ffg23 gene transcription in Hyp bone. We found evidence in Hyp bone for increased expression of Fgf1, Fgf7, and Egr2 in the Fgf-signaling pathway and decrements in Sost and Cpz and increments in Sfrp1 and 4 in the Wnt-signaling pathway. Moreover, activation of Fgf and Wnt-signaling stimulated, whereas Tgf β inhibited Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the Fgf23 regulatory extracellular matrix protein Dmp1. These findings suggest that elevation of Fgf23 expression in osteocytes is regulated by interactions between cell surface expression of Phex, extracellular matrix proteins and paracrine effects of Fgf and Wnt. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced post-translational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regards to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone. We isolated total RNAs from long bones of both WT and Hyp mice at 12 days of age. Since the RNA yields from the long bones are very low, we combined 2 bone samples with same genotype (WT or Hyp) for one RNA extraction. We will compare the difference of the gene expressions between Hyp and WT. We will use 4 samples in each animal condition.我们通过对皮质骨开展基因芯片分析，鉴定出了Phex依赖的基因转录本，这些转录本可调控Hyp小鼠体内成纤维细胞生长因子23（Fgf23）的生成与矿化过程。本研究发现，成纤维细胞生长因子受体（Fgf receptor）通路与Wnt信号通路（Wnt-pathways）的激活，可促进Hyp小鼠骨骼中Fgf23基因的转录上调。在Hyp小鼠的骨骼组织中，我们观察到成纤维细胞生长因子信号通路内的Fgf1、Fgf7及Egr2表达水平升高，而Wnt信号通路中的Sost、Cpz表达量降低，Sfrp1与Sfrp4表达量升高。此外，成纤维细胞生长因子与Wnt信号通路的激活可增强成骨细胞中Fgf23启动子的活性，而转化生长因子β（TGF-β）则会抑制该启动子活性。 我们还发现，金属蛋白酶Bmp1的表达出现下调——该酶可代谢调控Fgf23的细胞外基质蛋白Dmp1。上述研究结果表明，骨细胞中Fgf23表达的升高，受到Phex的细胞表面表达、细胞外基质蛋白的相互作用，以及成纤维细胞生长因子与Wnt的旁分泌效应的共同调控。 此外，我们在调控Fgf23翻译后加工与稳定性的酶类中也发现了表达异常：糖基转移酶（glycosyltransferase）Galnt3与前蛋白转化酶（proprotein convertase）Pcsk5的表达均出现下调。我们进一步证实，Hyp小鼠骨骼中Pcsk5与Galnt3的表达水平降低，这提示Fgf23的翻译后加工受损，可能同样参与了Hyp小鼠体内Fgf23水平升高的病理过程。 关于矿化缺陷相关研究，我们鉴定出了更多可解释Hyp小鼠成骨细胞内在矿化异常的候选基因：包括矿化抑制剂基质Gla蛋白（Mgp）与血小板反应蛋白4（Thbs4）的表达上调，以及调节局部pH的因子——碳酸酐酶12（Car12）、碳酸酐酶3（Car3），与钠依赖性柠檬酸转运体（Slc13a5）的表达上调。 本研究揭示了失活性Phex突变所伴随的骨骼基因表达改变的复杂性，并明确了可协同调控Hyp小鼠骨骼中Fgf23表达与细胞外基质矿化的全新通路。 实验部分：我们于小鼠出生后12天时，从野生型（Wild Type, WT）与Hyp小鼠的长骨中分离总RNA。由于长骨的RNA提取产量极低，我们将同基因型（野生型或Hyp）的2份骨组织样本合并，用于一次RNA提取实验。本研究将比较Hyp小鼠与野生型小鼠的基因表达差异，每组动物条件下将使用4份样本。