Conditional Deletion of Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Gene Reveals Its Essential Role in Chondrocyte Function and Endochondral Bone Formation
Conditional Deletion of Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Gene Reveals Its Essential Role in Chondrocyte Function and Endochondral Bone Formation
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DOI：
10.1210/en.2015-1473
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发表时间：
2016-01-01
期刊：
影响因子：
4.8
通讯作者：
Mohan, Subburaman
中科院分区：
文献类型：
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作者：
Cheng, Shaohong;Xing, Weirong;Mohan, Subburaman
缺氧的生长板软骨需要缺氧诱导因子（HIF）介导的通路来维持软骨细胞的存活和分化。HIF蛋白受到含脯氨酰羟化酶结构域蛋白2（Phd2）介导的蛋白酶体降解的严格调控。我们通过将Phd2基因敲除小鼠与Ⅱ型胶原蛋白α1 - Cre转基因小鼠杂交，有条件地破坏了软骨细胞中的Phd2基因，发现Phd2条件性敲除（cKO）小鼠的长骨和腰椎骨小梁骨量大量增加（>50%），这是由于骨小梁数量和厚度显著增加以及骨小梁间距减小所致。cKO小鼠股骨中段皮质厚度和组织矿物质密度也显著增加。动态组织形态计量学分析显示，cKO小鼠初级松质骨的纵向长度和每骨表面的类骨质表面增加，这表明从肥大软骨细胞到矿化骨基质的转化率提高，以及初级松质骨的骨形成增加。在次级松质骨中，以每骨表面矿化表面和矿物质沉积率衡量的骨形成没有改变，但吸收略有减少。通过定量RT - PCR检测到cKO小鼠生长板中SRY（性别决定区Y）盒9、成骨相关转录因子（Osx）、Ⅱ型胶原蛋白、聚集蛋白聚糖、碱性磷酸酶、骨涎蛋白、血管内皮生长因子、促红细胞生成素和糖酵解酶的mRNA水平升高。免疫组织化学显示cKO小鼠肥大软骨细胞中HIF - 1α蛋白水平升高。用腺病毒Cre感染从Phd2基因敲除小鼠分离的软骨细胞，导致了与在cKO生长板软骨细胞中观察到的相似的基因表达模式。我们的研究结果表明，在小鼠中，Phd2部分通过HIF依赖机制抑制软骨内骨形成。
The hypoxic growth plate cartilage requires hypoxia-inducible factor (HIF)-mediated pathways to maintain chondrocyte survival and differentiation. HIF proteins are tightly regulated by prolyl hydroxylase domain-containing protein 2 (Phd2)-mediated proteosomal degradation. We conditionally disrupted the Phd2 gene in chondrocytes by crossing Phd2 floxed mice with type 2 collagen-alpha 1-Cre transgenic mice and found massive increases (>50%) in the trabecular bone mass of long bones and lumbar vertebra of the Phd2 conditional knockout (cKO) mice caused by significant increases in trabecular number and thickness and reductions in trabecular separation. Cortical thickness and tissue mineral density at the femoral middiaphysis of the cKO mice were also significantly increased. Dynamic histomorphometric analyses revealed increased longitudinal length and osteoid surface per bone surface in the primary spongiosa of the cKO mice, suggesting elevated conversion rate from hypertrophic chondrocytes to mineralized bone matrix as well as increased bone formation in the primary spongiosa. In the secondary spongiosa, bone formation measured by mineralizing surface per bone surface and mineral apposition rate were not changed, but resorption was slightly reduced. Increases in the mRNA levels of SRY (sex determining region Y)-box 9, osterix (Osx), type 2 collagen, aggrecan, alkaline phosphatase, bone sialoprotein, vascular endothelial growth factor, erythropoietin, and glycolytic enzymes in the growth plate of cKO mice were detected by quantitative RT-PCR. Immunohistochemistry revealed an increased HIF-1 alpha protein level in the hypertrophic chondrocytes of cKO mice. Infection of chondrocytes isolated from Phd2 floxed mice with adenoviral Cre resulted in similar gene expression patterns as observed in the cKO growth plate chondrocytes. Our findings indicate that Phd2 suppresses endochondral bone formation, in part, via HIF-dependent mechanisms in mice.