Johnson Zariel I, Gogate Shilpa S, Day Rebecca, Binch Abbie, Markova Dessislava Z, Chiverton Neil, Cole Ashley, Conner Matt, Shapiro Irving M, Le Maitre Christine L, Risbud Makarand V
Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, USA.
Biomedical Research Centre, Sheffield Hallam University, Sheffield, UK.
Oncotarget. 2015 May 20;6(14):11945-58. doi: 10.18632/oncotarget.3631.
Objectives of this study were to investigate whether AQP1 and AQP5 expression is altered during intervertebral disc degeneration and if hypoxia and HIF-1 regulate their expression in NP cells. AQP expression was measured in human tissues from different degenerative grades; regulation by hypoxia and HIF-1 was studied using promoter analysis and gain- and loss-of-function experiments. We show that both AQPs are expressed in the disc and that mRNA and protein levels decline with human disease severity. Bioinformatic analyses of AQP promoters showed multiple evolutionarily conserved HREs. Surprisingly, hypoxia failed to induce promoter activity or expression of either AQP. While genomic chromatin immunoprecipitation showed limited binding of HIF-1α to conserved HREs, their mutation did not suppress promoter activities. Stable HIF-1α suppression significantly decreased mRNA and protein levels of both AQPs, but HIF-1α failed to induce AQP levels following accumulation. Together, our results demonstrate that AQP1 and AQP5 expression is sensitive to human disc degeneration and that HIF-1α uniquely maintains basal expression of both AQPs in NP cells, independent of oxemic tension and HIF-1 binding to promoter HREs. Diminished HIF-1 activity during degeneration may suppress AQP levels in NP cells, compromising their ability to respond to extracellular osmolarity changes.
本研究的目的是调查水通道蛋白1（AQP1）和水通道蛋白5（AQP5）的表达在椎间盘退变过程中是否发生改变，以及低氧和缺氧诱导因子-1（HIF-1）是否调节其在髓核细胞中的表达。在不同退变程度的人体组织中检测AQP的表达；通过启动子分析以及功能获得和功能缺失实验研究低氧和HIF-1的调节作用。我们发现，两种水通道蛋白均在椎间盘中表达，且mRNA和蛋白质水平随人类疾病严重程度而下降。对AQP启动子的生物信息学分析显示存在多个进化上保守的缺氧反应元件（HRE）。令人惊讶的是，低氧未能诱导任何一种AQP的启动子活性或表达。虽然基因组染色质免疫沉淀显示HIF-1α与保守的HRE结合有限，但对其进行突变并未抑制启动子活性。稳定抑制HIF-1α可显著降低两种AQP的mRNA和蛋白质水平，但HIF-1α在积累后未能诱导AQP水平升高。总之，我们的结果表明，AQP1和AQP5的表达对人类椎间盘退变敏感，并且HIF-1α独特地维持髓核细胞中两种AQP的基础表达，这与血氧张力以及HIF-1与启动子HRE的结合无关。退变过程中HIF-1活性降低可能会抑制髓核细胞中AQP的水平，损害其对细胞外渗透压变化作出反应的能力。
