Zeng Yan, Danielson Keith G, Albert Todd J, Shapiro Irving M, Risbud Makarand V
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA, USA.
J Bone Miner Res. 2007 Dec;22(12):1851-61. doi: 10.1359/jbmr.070620.
The regulation of galectin-3 expression in skeletal tissues is not completely understood. Our studies indicate that HIF-1 alpha regulates galectin-3 expression by interacting with hypoxia regulatory elements in the promoter region. Finally, we show that galectin-3 serves a prosurvival role in the intervertebral disc.
Earlier reports indicated that galectin-3 (gal-3) is highly expressed in the epiphyseal growth plate cartilage and the intervertebral disc. Because these skeletal tissues have a limited vascular supply and the cells reside in a low O2 environment, we determined if the oxemic status modulates gal-3 expression.
Cells were cultured in normoxia (21% O2) or hypoxia (2% O2), and gal-3 expression and promoter activity were evaluated. Interaction of hypoxia inducible factor (HIF)-1 alpha with the gal-3 promoter was confirmed by gel shift and site-directed mutagenesis.
There was minimal oxygen-dependent change in HIF-1 alpha levels and no change in gal-3 expression and promoter activity in nucleus pulposus cells. In contrast, hypoxia induced gal-3 mRNA, protein, and promoter activity in HeLa cells and mouse embryonic fibroblasts (MEFs) from HIF-1 alpha wildtype but not HIF-1-null mice. To evaluate the importance of HIF-1 in regulation of gal-3 expression, we overexpressed HIF-1 alpha or constitutively active-HIF-1 alpha in null MEF. An increase in gal-3 promoter activity was observed in both normoxia and hypoxia. Similarly, suppression of HIF-1 alpha in nucleus pulposus cells, and wildtype MEF, using siRNA and pharmacological inhibitors resulted in suppression of gal-3 promoter activity and mRNA levels. Analysis of the gal-3 promoter indicated that it contained two hypoxia response elements (HREs). Gel-shift and chromatin immunoprecipitation analysis confirmed that there was binding of HIF-1 alpha to the gal-3 HRE. Furthermore, site-directed mutagenesis of HRE completely blocked hypoxic induction of gal-3 promoter activity. In nucleus pulposus cells, suppression of gal-3 expression promoted FasL-mediated apoptosis.
Together, these studies showed that gal-3 is a HIF-1-regulated lectin that plays an important role in nucleus pulposus cell survival.
骨骼组织中半乳糖凝集素-3表达的调控尚未完全明确。我们的研究表明，缺氧诱导因子-1α（HIF-1α）通过与启动子区域的缺氧调控元件相互作用来调节半乳糖凝集素-3的表达。最后，我们发现半乳糖凝集素-3在椎间盘中发挥促生存作用。
早期报道指出，半乳糖凝集素-3（gal-3）在骨骺生长板软骨和椎间盘中高表达。由于这些骨骼组织血管供应有限，细胞处于低氧环境中，我们确定血氧状态是否调节gal-3的表达。
细胞在常氧（21% O₂）或缺氧（2% O₂）条件下培养，评估gal-3的表达和启动子活性。通过凝胶迁移和定点诱变证实缺氧诱导因子（HIF）-1α与gal-3启动子的相互作用。
髓核细胞中HIF-1α水平的氧依赖性变化极小，gal-3的表达和启动子活性无变化。相反，缺氧诱导HIF-1α野生型而非HIF-1基因敲除小鼠的HeLa细胞和小鼠胚胎成纤维细胞（MEF）中gal-3的mRNA、蛋白质和启动子活性。为评估HIF-1在gal-3表达调控中的重要性，我们在基因敲除的MEF中过表达HIF-1α或组成型活性HIF-1α。在常氧和缺氧条件下均观察到gal-3启动子活性增加。同样，使用小干扰RNA（siRNA）和药物抑制剂抑制髓核细胞和野生型MEF中的HIF-1α，导致gal-3启动子活性和mRNA水平受到抑制。对gal-3启动子的分析表明，它包含两个缺氧反应元件（HRE）。凝胶迁移和染色质免疫沉淀分析证实HIF-1α与gal-3的HRE存在结合。此外，HRE的定点诱变完全阻断了gal-3启动子活性的缺氧诱导。在髓核细胞中，抑制gal-3表达促进FasL介导的细胞凋亡。
总之，这些研究表明gal-3是一种受HIF-1调控的凝集素，在髓核细胞存活中起重要作用。
