Propst Stacie M, Estell Kim, Schwiebert Lisa M
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Biol Chem. 2002 Oct 4;277(40):37054-63. doi: 10.1074/jbc.M205778200. Epub 2002 Jul 16.
We have reported previously that airway epithelial cells (AEC) express CD40 and that activation of this molecule stimulates the expression of inflammatory mediators, including the chemokine RANTES (regulated on activation normal T cell expressed and secreted). Because NF-kappaB regulates the expression of many inflammatory mediators, such as RANTES, we utilized CD40-mediated induction of RANTES expression to investigate the mechanisms that underlie CD40-mediated activation of NF-kappaB in AEC. Results demonstrate that, in AEC, intact NF-kappaB sites were required for CD40-mediated activation of the RANTES promoter. To examine activation of NF-kappaB binding directly, electrophoretic mobility shift analyses were performed. These analyses revealed that CD40 ligation stimulated NF-kappaB binding and that the activated NF-kappaB complexes were composed of p65 subunits. Additional studies focused on the CD40-triggered signaling pathways that facilitate NF-kappaB activation. Findings show that CD40 engagement activated the IkappaB kinases IKK-alpha and IKK-beta and stimulated IkappaBalpha phosphorylation. Analyses also examined the role of tumor necrosis factor-associated factor (TRAF) molecules in CD40-mediated NF-kappaB activation within AEC. Stable transfectants expressing wild-type or mutant forms of the cytoplasmic domain of CD40 suggested that TRAF3, but not TRAF2, binding was essential for CD40-mediated RANTES expression. Further studies indicated that exogenous expression of wild-type TRAF3 enhanced activation of the RANTES promoter, whereas exogenous expression of wild-type TRAF2 inhibited this activation; TRAF3-mediated enhancement was dependent upon NF-kappaB. Together, these findings suggest that, in AEC, ligation of CD40 regulates the expression of inflammatory mediators, such as RANTES, via activation of NF-kappaB. Moreover, these results suggest that CD40-mediated signaling in AEC differs with previously reported findings observed in other cell models, such as B lymphocytes.
我们之前曾报道，气道上皮细胞（AEC）表达CD40，且该分子的激活会刺激炎症介质的表达，包括趋化因子RANTES（正常T细胞激活时表达和分泌的调节因子）。由于核因子-κB（NF-κB）调节许多炎症介质的表达，如RANTES，我们利用CD40介导的RANTES表达诱导来研究AEC中CD40介导的NF-κB激活的潜在机制。结果表明，在AEC中，完整的NF-κB位点是CD40介导的RANTES启动子激活所必需的。为了直接检测NF-κB结合的激活情况，我们进行了电泳迁移率变动分析。这些分析显示，CD40连接刺激了NF-κB结合，且激活的NF-κB复合物由p65亚基组成。进一步的研究聚焦于促进NF-κB激活的CD40触发信号通路。研究结果表明，CD40结合激活了IκB激酶IKK-α和IKK-β，并刺激了IκBα磷酸化。分析还检测了肿瘤坏死因子相关因子（TRAF）分子在AEC中CD40介导的NF-κB激活中的作用。表达野生型或突变型CD40胞质结构域的稳定转染子表明，TRAF3而非TRAF2的结合对于CD40介导的RANTES表达至关重要。进一步的研究表明，野生型TRAF3的外源性表达增强了RANTES启动子的激活，而野生型TRAF2的外源性表达则抑制了这种激活；TRAF3介导的增强依赖于NF-κB。总之，这些发现表明，在AEC中，CD40的连接通过激活NF-κB来调节炎症介质如RANTES的表达。此外，这些结果表明，AEC中CD40介导的信号传导与先前在其他细胞模型如B淋巴细胞中观察到的结果不同。
