Beum Paul V, Bastola Dhundy R, Cheng Pi-Wan
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198-4525, USA.
Am J Respir Cell Mol Biol. 2003 Jul;29(1):48-56. doi: 10.1165/rcmb.2002-0147OC. Epub 2003 Jan 10.
Enzymes which exhibit core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) activity play important roles in physiologic processes including the inflammatory response and immune system function, and C2GnT activity is regulated during processes, such as T cell activation and cellular differentiation. In this study, we have examined the regulation of C2GnT activity in the H292 airway epithelial cell line by epidermal growth factor (EGF), which has been previously shown to upregulate expression of the airway mucin MUC5AC in this cell line. We found that EGF suppressed C2GnT activity in a time- and dose-dependent fashion, and also suppressed core 4 beta1,6 N-acetylglucosaminyltransferase (C4GnT) activity. Consistent with the suppression of C4GnT activity, Northern blotting results showed that EGF preferentially inhibited the M isoform of C2GnT, which forms core 2, core 4, and blood group I beta1,6 branched carbohydrate structures, while the L isoform, which forms only the core 2 structure, was only modestly affected. Furthermore, EGF treatment resulted in a shift in the carbohydrate structure of FLAG-tagged MUC1 expressed in the cells from core 2-based toward core 1-based structures, consistent with the inhibitory effects of EGF on C2GnT. Transforming growth factor alpha mimicked the effect of EGF on C2GnT, implicating the EGF receptor (EGF-R) in C2GnT suppression, and the EGF-R tyrosine kinase inhibitor AG1478 blocked C2GnT suppression, confirming the role of EGF-R in the inhibition of C2GnT expression. Also, PD98059, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1/2 in the Ras-mitogen-activated protein kinase pathway, completely blocked the EGF suppressive effect, suggesting possible involvement of the Ras-mitogen-activated protein kinase pathway in EGF-mediated downregulation of C2GnT. The results of this study suggest that exposure of airway cells to EGF may result in remodeling of mucin carbohydrate structure, potentially altering the biological properties of the cells.
具有核心2β1,6 - N - 乙酰葡糖胺基转移酶（C2GnT）活性的酶在包括炎症反应和免疫系统功能在内的生理过程中发挥重要作用，并且C2GnT活性在诸如T细胞活化和细胞分化等过程中受到调控。在本研究中，我们检测了表皮生长因子（EGF）对H292气道上皮细胞系中C2GnT活性的调控作用，此前已表明EGF可上调该细胞系中气道粘蛋白MUC5AC的表达。我们发现EGF以时间和剂量依赖性方式抑制C2GnT活性，并且还抑制核心4β1,6 - N - 乙酰葡糖胺基转移酶（C4GnT）活性。与C4GnT活性的抑制一致，Northern印迹结果显示EGF优先抑制形成核心2、核心4和血型Iβ1,6分支碳水化合物结构的C2GnT的M同工型，而仅形成核心2结构的L同工型仅受到适度影响。此外，EGF处理导致细胞中表达的FLAG标记的MUC1的碳水化合物结构从基于核心2的结构向基于核心1的结构转变，这与EGF对C2GnT的抑制作用一致。转化生长因子α模拟了EGF对C2GnT的作用，表明表皮生长因子受体（EGF - R）参与C2GnT的抑制，并且EGF - R酪氨酸激酶抑制剂AG1478阻断了C2GnT的抑制，证实了EGF - R在抑制C2GnT表达中的作用。此外，PD98059是Ras - 丝裂原活化蛋白激酶途径中丝裂原活化蛋白激酶/细胞外信号调节激酶激酶（MEK）1/2的特异性抑制剂，它完全阻断了EGF的抑制作用，提示Ras - 丝裂原活化蛋白激酶途径可能参与EGF介导的C2GnT下调。本研究结果表明，气道细胞暴露于EGF可能导致粘蛋白碳水化合物结构重塑，潜在地改变细胞的生物学特性。
