Cited2 Modulates Hypoxia-Inducible Factor-Dependent Expression of Vascular Endothelial Growth Factor in Nucleus Pulposus Cells of the Rat Intervertebral Disc
Cited2 Modulates Hypoxia-Inducible Factor-Dependent Expression of Vascular Endothelial Growth Factor in Nucleus Pulposus Cells of the Rat Intervertebral Disc
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DOI：
10.1002/art.24073
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发表时间：
2008-12-01
影响因子：
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通讯作者：
Risbud, Makarand V.
中科院分区：
文献类型：
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作者：
Agrawal, Amit;Gajghate, Sachin;Risbud, Makarand V.
目标。目的：检测椎间盘髓核细胞是否表达缺氧诱导因子2 α (HIF-2 α)，并探讨HIF-1和HIF-2在控制cited2和血管内皮生长因子（VEGF）表达中的作用。在常氧（21% O-2）或缺氧（2% O-2）条件下培养大鼠细胞，评估HIF-2靶基因的表达和启动子活性。通过获得或丧失功能的实验来研究HIF异构体对cited2活性的贡献以及cited2在调节VEGF表达中的作用。我们发现HIF-2 α蛋白在体内均有表达，在常氧和缺氧条件下，HIF-2 α蛋白和信使RNA的表达相似。然而，在缺氧条件下HIF-2 α的活化显著增加。在功能活性方面，与大多数其他组织的情况不同，HIF-2未能增加超氧化物歧化酶2和fraataxin的转录活性，这是两个参与自由基突变的常见靶基因。然而，在缺氧条件下，HIF-2优先调节p300结合蛋白cited2的表达和启动子活性。当HIF-2 α或HIF-1 α被抑制时，cited2启动子活性被抑制。最后，我们发现强迫表达或抑制cited2会导致髓核细胞中HIF-1和HIF-2的共同靶基因VEGF的表达发生相应的变化。本研究结果表明，在髓核细胞中，HIF-2和HIF-1通过cited2调节各自的转录活性。我们认为调节回路的两条臂膀在维持健康椎间盘的生存活动和抑制血管生成方面起作用。
Objective. To determine whether nucleus pulposus cells of the intervertebral disc express hypoxia-inducible factor 2 alpha (HIF-2 alpha), and to assess the role of HIF-1 and HIF-2 in controlling cited2 and vascular endothelial growth factor (VEGF) expression.Methods. Rat cells were cultured under normoxic (21% O-2) or hypoxic (2% O-2) conditions, and expression and promoter activity of HIF-2 target genes were evaluated. Gain- or loss-of-function experiments were performed to investigate the contribution of HIF isoforms to cited2 activity as well as the role of cited2 in regulating VEGF expression.Results. We found that HIF-2 alpha protein was expressed in vivo and that protein and messenger RNA expression were similar under both normoxic and hypoxic conditions. However, there was a significant increase in HIF-2 alpha transactivation under hypoxic conditions. With respect to functional activity, unlike the case in most other tissues, HIF-2 failed to increase the transcriptional activities of superoxide dismutase 2 and frataxin, 2 common target genes involved in radical dismutation. However, under hypoxic conditions, HIF-2 preferentially regulated the expression and promoter activity of cited2, a p300 binding protein. When HIF-2 alpha or HIF-1 alpha was suppressed, cited2 promoter activity was inhibited. Finally, we showed that forced expression or suppression of cited2 resulted in corresponding changes in expression of VEGF, a common target gene for HIF-1 and HIF-2 in the nucleus pulposus cells.Conclusion. Results of this study indicate that in nucleus pulposus cells, HIF-2 and HIF-1 modulate their own transcriptional activity through cited2. We suggest that the 2 arms of the regulatory circuit serve to maintain survival activities and inhibit angiogenesis in the healthy disc.