The Transcription Factor Paired Box-5 Promotes Osteoblastogenesis Through Direct Induction of Osterix and Osteocalcin
The Transcription Factor Paired Box-5 Promotes Osteoblastogenesis Through Direct Induction of Osterix and Osteocalcin
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DOI：
10.1002/jbmr.1708
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发表时间：
2012-12-01
影响因子：
6.2
通讯作者：
Yoneda, Yukio
中科院分区：
文献类型：
--
作者：
Hinoi, Eiichi;Nakatani, Eri;Yoneda, Yukio
虽然骨骼异常被认为是在小鼠缺乏特定的配对盒（Pax）家族蛋白，很少有人注意到他们在成骨细胞的作用。在这里，我们研究了可能参与的几个Pax家族成员的机制，潜在的调节成骨细胞的分化和成熟。在测试的不同Pax家族成员中，Pax 5在培养前的小鼠颅骨成骨细胞中不明显表达，但在分化成熟的成骨细胞中进行性表达。免疫反应性Pax 5是高度可检测到的原代培养的成熟成骨细胞免疫印迹和成骨细胞附着在松质骨的小鼠胫骨切片的免疫组化，分别。通过内源性Pax 5的小干扰RNA（siRNA）敲低，除了α 1（I）胶原表达和碱性磷酸酶（ALP）染色之外，还通过基因反式激活的恶化导致骨钙素和Osterix的表达的显著抑制，而不影响成骨细胞MC 3 T3-E1细胞中的runt相关转录因子-2（Runx 2）表达和细胞活力。在MC 3 T3-E1细胞中，Pax 5的引入以依赖于配对结构域的方式增强了骨钙素和Osterix的启动子活性。在小鼠骨钙素和Osterix的5 '侧翼区域鉴定了推定的Pax 5结合位点，而染色质免疫沉淀测定揭示了Pax 5与MC 3 T3-E1细胞中骨钙素和Osterix启动子的特定区域的直接结合。在MC 3 T3-E1细胞中，Pax 5的过表达显著增加骨钙素、Osterix和a1（I）胶原蛋白的表达、ALP活性和Ca 2+积累，而不影响Runx 2的表达。在成骨细胞中主要表达Pax 5的转基因小鼠的椎骨中，骨体积与组织体积的比率和骨形成率显著增加。这些研究结果表明，Pax 5可以积极调节成骨细胞的分化走向成熟，在体外，除了促进骨形成和重建在体内，作为一个转录因子控制成骨细胞独立的Runx 2。(C)2012年美国骨与矿物质研究学会。
Although skeletal abnormalities are seen in mice deficient of particular paired box (Pax) family proteins, little attention has been paid to their role in osteoblastogenesis so far. Here, we investigated the possible involvement of several Pax family members in mechanisms underlying the regulation of differentiation and maturation of osteoblasts. Among different Pax family members tested, Pax5 was not markedly expressed in murine calvarial osteoblasts before culture, but progressively expressed by osteoblasts under differentiation toward maturation. Immunoreactive Pax5 was highly detectable in primary cultured mature osteoblasts on immunoblotting and in osteoblastic cells attached to cancellous bone in mouse tibial sections on immunohistochemistry, respectively. Knockdown by small interfering RNA (siRNA) of endogenous Pax5 led to significant inhibition of the expression of Osteocalcin, and Osterix through deterioration of gene transactivation, in addition to a1(I)Collagen expression and alkaline phosphatase (ALP) staining, without affecting runt-related transcription factor-2 (Runx2) expression and cell viability in osteoblastic MC3T3-E1 cells. The introduction of Pax5 enhanced promoter activities of Osteocalcin and Osterix in a manner dependent on the paired domain in MC3T3-E1 cells. Putative Pax5 binding sites were identified in the 5'-flanking regions of mouse Osteocalcin and Osterix, whereas chromatin immunoprecipitation assay revealed the direct binding of Pax5 to particular regions of Osteocalcin and Osterix promoters in MC3T3-E1 cells. Overexpression of Pax5 significantly increased Osteocalcin, Osterix, and a1(I)Collagen expression, ALP activity, and Ca2+ accumulation, without affecting Runx2 expression, in MC3T3-E1 cells. In vertebrae of transgenic mice predominantly expressing Pax5 in osteoblasts, a significant increase was seen in the ratio of bone volume over tissue volume and the bone formation rate. These findings suggest that Pax5 could positively regulate osteoblastic differentiation toward maturation in vitro, in addition to promoting bone formation and remodeling in vivo, as one of the transcription factors essential for controlling osteoblastogenesis independently of Runx2. (C) 2012 American Society for Bone and Mineral Research.