The RCAN1.4-calcineurin/NFAT signaling pathway is essential for hypoxic adaption of intervertebral discs
The RCAN1.4-calcineurin/NFAT signaling pathway is essential for hypoxic adaption of intervertebral discs
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RCAN1.4-钙调神经磷酸酶/NFAT信号通路对于椎间盘的缺氧适应至关重要
DOI：
10.1038/s12276-020-0441-x
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发表时间：
2020-05-29
影响因子：
12.8
通讯作者：
Zhao, Fengdong
中科院分区：
文献类型：
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作者：
Huang, Bao;He, Yongqing;Zhao, Fengdong
Calcipressin-1，也称为钙调磷酸酶调节剂 1 (RCAN1)，可以在钙调磷酸酶 A 催化结构域或其附近特异性结合钙调磷酸酶，并下调钙调磷酸酶活性。然而，RCAN1是否通过钙调神经磷酸酶/NFAT信号通路影响缺氧椎间盘（IVD）表型仍不清楚。首先，我们通过HE、番红O/固绿和阿尔新蓝染色证实了退变髓核（NP）的特征，并通过免疫组织化学检测了退变NP中RCAN1水平的增加。然后，我们证明RCAN1.4的蛋白水平高于RCAN1.1，并且从对照组到Pfirrmann V级组逐渐升高。在体外，缺氧（1% O2）和 HIF-1α 过表达均以剂量和时间依赖性方式降低大鼠 NP 细胞中 RCAN1.4 的蛋白水平。我们进一步发现miRNA-124通过非降解途径（没有蛋白酶体或溶酶体）抑制RCAN1.4的表达。正如预期的那样，在缺氧或RCAN1.4 siRNA转染下，NP细胞中的钙调神经磷酸酶被激活，并主要促进NFATc1的核转位。此外，SOX9、II型胶原和MMP13在缺氧、RCAN1.4 siRNA转染或NFATc1过表达下升高。使用染色质免疫沉淀 (ChIP) 和荧光素酶报告基因测定（带突变），我们阐明 NFATc1 越来越多地结合 SOX9 启动子区域 (bp -367~-357)。 HIF-1α 和 NFATc1 的相互作用促进 MMP13 转录。最后，我们发现 FK506 逆转了 NP 细胞和离体模型中缺氧诱导的钙调神经磷酸酶/NFAT 信号通路的激活。总之，这些发现表明 RCAN1.4-钙调神经磷酸酶/NFAT 信号通路在 NP 细胞的缺氧表型中起着至关重要的作用。 RCAN1.4 可能是退行性椎间盘疾病的治疗靶点。
Calcipressin-1, also known as regulator of calcineurin 1 (RCAN1), can specifically bind calcineurin at or near the calcineurin A catalytic domain and downregulate calcineurin activity. However, whether RCAN1 affects the hypoxic intervertebral disc (IVD) phenotype through the calcineurin/NFAT signaling pathway remains unclear. First, we confirmed the characteristics of the degenerative nucleus pulposus (NP) by HE, safranin O/fast green and Alcian blue staining, and detected increased RCAN1 levels in the degenerative NP by immunohistochemistry. Then, we demonstrated that the protein level of RCAN1.4 was higher than that of RCAN1.1 and progressively elevated from the control group to the Pfirrmann grade V group. In vitro, both hypoxia (1% O2) and overexpression of HIF-1α reduced the protein level of RCAN1.4 in rat NP cells in a dose- and time-dependent manner. We further found that miRNA-124, through a nondegradative pathway (without the proteasome or lysosome), suppressed the expression of RCAN1.4. As expected, calcineurin in NP cells was activated and primarily promoted nuclear translocation of NFATc1 under hypoxia or RCAN1.4 siRNA transfection. Furthermore, SOX9, type II collagen and MMP13 were elevated under hypoxia, RCAN1.4 siRNA transfection or NFATc1 overexpression. Using chromatin immunoprecipitation (ChIP) and a luciferase reporter assay (with mutation), we clarified that NFATc1 increasingly bound the SOX9 promotor region (bp −367~−357). Interaction of HIF-1α and NFATc1 promoted MMP13 transcription. Finally, we found that FK506 reversed hypoxia-induced activation of the calcineurin/NFAT signaling pathway in NP cells and an ex vivo model. Together, these findings show that the RCAN1.4-calcineurin/NFAT signaling pathway has a vital role in the hypoxic phenotype of NP cells. RCAN1.4 might be a therapeutic target for degenerative disc diseases.