CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor
CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor
复制标题
DOI：
10.1074/jbc.m113.454652
复制
发表时间：
2013-07-05
影响因子：
4.8
通讯作者：
Yamaguchi, Akira
中科院分区：
文献类型：
--
作者：
Matsushita, Yuki;Sakamoto, Kei;Yamaguchi, Akira
CCN蛋白家族成员CCN 3在体外抑制成骨细胞分化。然而，CCN 3在骨再生中的作用尚未得到很好的阐明。在这项研究中，我们研究了CCN 3在骨再生中的作用。我们通过微阵列分析在小鼠骨再生模型中将Ccn 3基因鉴定为骨再生早期高表达的基因。我们通过RT-PCR、Western blot和免疫荧光分析证实了Ccn 3在骨再生早期的上调。Ccn 3转基因小鼠，其中Ccn 3的表达是由2.3 kb的Col 1a 1启动子驱动，表现出骨质减少与野生型小鼠相比，但Ccn 3敲除小鼠与野生型小鼠相比，没有表现出骨骼变化。我们通过显微计算机断层扫描和组织学分析，分析了Ccn 3转基因小鼠和Ccn 3基因敲除小鼠的骨再生过程。与野生型小鼠相比，Ccn 3基因敲除小鼠的骨再生加速。RT-PCR结果显示，Ccn 3基因敲除小鼠成骨细胞相关基因（Runx 2、Sp 7、Col 1a 1、Alpl和Bglap）的mRNA表达水平比野生型小鼠早上调。Ccn 3转基因小鼠的骨再生与野生型小鼠相比无明显变化。与野生型小鼠相比，在Ccn 3 KO小鼠的骨再生位点，Smad 1/5的磷酸化高度上调。这些结果表明，CCN 3在骨再生的早期阶段被上调，并且充当骨再生的负调节剂。这项研究可能有助于骨再生治疗新策略的发展。
CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wildtype mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.