Universidade Católica de Brasília, Pós-Graduação em Ciências Genômicas e Biotecnologia, SGAN Quadra 916, Av. W5 Norte, 70790-160 Brasília, DF, Brazil.
Biochem Biophys Res Commun. 2011 Jan 28;404(4):997-1003. doi: 10.1016/j.bbrc.2010.12.098. Epub 2010 Dec 25.
Hypoxia has emerged as a key determinant of osteogenesis. HIF-1α is the transcription factor mediating hypoxia responses that include induction of VEGF and related bone induction. Inflammatory signals antagonize bone repair via the NF-κB pathway. The present investigation explored the functional relationship of hypoxia (HIF-1α function) and inflammatory signaling (NF-κB) in stem like and osteoprogenitor cell lines. The potential interaction between HIF-1α and NF-κB signaling was explored by co-transfection studies in hFOB with p65, HIF-1α and 9x-HRE-luc or HIF-1α target genes reporter plasmids. Nuclear cross-talk was directly tested using the mammalian Gal4/VP16 two-hybrid, and confirmed by co-immunoprecipitation/western blotting assays. The results show that inflammatory stimulation (TNF-α treatment) causes a marked inhibition of HIF-1α function at the HRE in all cell lines studied. Also, co-transfection with p65 expression vector leads to reduced hVEGFp transcription after DFO-induced hypoxia. However, TNF-α treatment had little effect on HIF-1α mRNA levels. The functional interaction of Gal4-HIF-1α and VP16-p300 fusion proteins is effectively blocked by expression of p65 in a dose dependent manner. It was concluded that NF-κB-mediated inflammatory signaling is able to block HIF-1α transactivation at HRE-encoding genes by direct competition for p300 binding at the promoter. Inflammation may influence the stem cell niche and tissue regeneration by influencing cellular responses to hypoxia.
缺氧已成为成骨的关键决定因素。HIF-1α 是介导缺氧反应的转录因子，包括诱导 VEGF 和相关的骨诱导。炎症信号通过 NF-κB 途径拮抗骨修复。本研究探讨了类似干细胞和成骨前体细胞系中缺氧（HIF-1α 功能）和炎症信号（NF-κB）的功能关系。通过共转染研究在 hFOB 中用 p65、HIF-1α 和 9x-HRE-luc 或 HIF-1α 靶基因报告质粒探索了 HIF-1α 和 NF-κB 信号之间的潜在相互作用。使用哺乳动物 Gal4/VP16 双杂交直接测试核串扰，并通过共免疫沉淀/ Western blot 测定进行验证。结果表明，炎症刺激（TNF-α 处理）导致所有研究细胞系中 HRE 处的 HIF-1α 功能明显抑制。此外，DFO 诱导缺氧后，共转染 p65 表达载体导致 hVEGFp 转录减少。然而，TNF-α 处理对 HIF-1α mRNA 水平影响不大。Gal4-HIF-1α 和 VP16-p300 融合蛋白的功能相互作用可通过表达 p65 以剂量依赖的方式有效阻断。结论是，NF-κB 介导的炎症信号能够通过在启动子处直接竞争与 p300 的结合来阻断 HRE 编码基因的 HIF-1α 反式激活。炎症可能通过影响细胞对缺氧的反应来影响干细胞龛和组织再生。
