PHD/HIF-1 upregulates CA12 to protect against degenerative disc disease: a human sample, in vitro and ex vivo study
PHD/HIF-1 upregulates CA12 to protect against degenerative disc disease: a human sample, in vitro and ex vivo study
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PHD/HIF-1 上调 CA12 以预防退行性椎间盘疾病：人体样本、体外和离体研究。
DOI：
10.1038/labinvest.2016.32
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发表时间：
2016-05-01
影响因子：
5
通讯作者：
Fan, Shun-Wu
中科院分区：
文献类型：
--
作者：
Chen, Shuai;Fang, Xiang-Qian;Fan, Shun-Wu
腰椎间盘退变是引起腰痛的主要原因。髓核是椎间盘的重要组成部分。最近的研究表明，碳酸酐酶12（CA 12）是一种新的NP标记。然而，CA12的调节机制及其生理功能尚不清楚。在我们的研究中，CA 12，缺氧诱导因子1 α（HIF-1 α）和HIF-2 α的表达水平在81人变性NP样品中使用实时RT-PCR，免疫组化和western blot检测。在低氧环境中培养大鼠NP细胞，并检测低氧诱导的CA 12表达。用HIF-1 α siRNA或脯氨酰羟化酶（PHD）抑制剂二甲基草酰甘氨酸（DMOG）处理大鼠NP细胞以评估PHD/HIF-1在调节CA 12表达中的作用。用CA12 siRNA处理大鼠NP细胞以确定CA12的功能。建立大鼠离体模型以证实PHD、HIF-1和CA 12在椎间盘退变中具有重要作用。我们发现，CA12显着下调，在退化的人NP样品在mRNA和蛋白质水平。CA12表达在缺氧时急剧增加约30倍。HIF-1 α的表达，而不是HIF-2 α，也在退化的人NP样品中降低，并与CA12表达呈正相关。缺氧条件下HIF-1 α的敲低降低了CA12 mRNA和蛋白的表达水平。DMOG处理增加HIF-1 α和CA 12表达。CA12敲低显著抑制合成代谢蛋白的表达，而分解代谢酶保持不变。离体实验支持了我们对PHD/HIF-1/CA 12作用的体外研究。总之，CA12在变性的NP中下调，其表达可能受PHD/HIF-1轴调节。CA12表达减少可能导致细胞外基质合成减少，这有助于退行性椎间盘疾病的进展。
Intervertebral disc degeneration is a major cause of low back pain. The nucleus pulposus (NP) is an important intervertebral disc component. Recent studies have shown that carbonic anhydrase 12 (CA12) is a novel NP marker. However, the mechanism by which CA12 is regulated and its physiological function are unclear. In our study, CA12, hypoxia-inducible factor 1 alpha (HIF-1 alpha) and HIF-2 alpha expression levels were examined in 81 human degenerated NP samples using real-time RT-PCR, immunohistochemistry and western blot. Rat NP cells were cultured in a hypoxic environment, and hypoxia-induced CA12 expression was examined. Rat NP cells were treated with HIF-1 alpha siRNA or the prolyl hydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG) to evaluate the role of PHD/HIF-1 in regulating CA12 expression. Rat NP cells were treated with CA12 siRNA to determine the function of CA12. A rat ex vivo model was established to confirm that PHD, HIF-1, and CA12 have important roles in disc degeneration. We found that CA12 was significantly downregulated in degenerated human NP samples at the mRNA and protein levels. CA12 expression sharply increased by similar to 30-fold in response to hypoxia. The expression of HIF-1 alpha, but not HIF-2 alpha, also decreased in degenerated human NP samples and was positively correlated with CA12 expression. HIF-1 alpha knockdown under hypoxia reduced the CA12 mRNA and protein expression levels. DMOG treatment increased HIF-1 alpha and CA12 expression. CA12 knockdown significantly inhibited anabolic protein expression, whereas catabolic enzymes remained unchanged. The ex vivo experiments supported our in vitro studies of the role of PHD/HIF-1/CA12. In conclusion, CA12 is downregulated in degenerated NPs, and its expression may be regulated by the PHD/HIF-1 axis. Decreased CA12 expression may lead to decreased extracellular matrix synthesis, which contributes to degenerative disc disease progression.