Published ahead of print on January 10, 2003, doi:10.1165/rcmb.2002-0147OC
© 2003 American Thoracic Society DOI: 10.1165/rcmb.2002-0147OC Mucin BiosynthesisEpidermal Growth Factor Downregulates Core 2 Enzymes in a Human Airway Adenocarcinoma Cell LineDepartment of Biochemistry and Molecular Biology, College of Medicine; and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska Address correspondence to: Pi-Wan Cheng, Ph.D., Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525. E-mail: pcheng{at}unmc.edu
Enzymes which exhibit core 2 ß1,6 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 ß1,6 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 ß1,6 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
Abbreviations: bovine serum albumin, BSA • UDP-GlcNAc:Galß1-3 GalNAc (GlcNAc to GalNAc) ß1-6 GlcNAc transferase, C2GnT • UDP-GlcNAc:GlcNAcß1-3 GalNAc (GlcNAc to GalNAc) ß1-6 GlcNAc transferase, C4GnT • complimentary DNA, cDNA • cytidine monophosphate, CMP • UDP-Gal:GalNAc ß1-3 Gal transferase, Core 1 Gal TF • epidermal growth factor, EGF • EGF receptor, EGF-R • extracellular signal-regulated kinase, ERK • DYKDDDD epitope, FLAG • galactose, Gal • glyceraldehyde 3-phosphate dehydrogenase, GAPDH • N-acetylgalactosamine, GalNAc • N-acetylglucosamine, GlcNAc • insulin-like growth factor-1, IGF-1 • mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, MEK • messenger RNA, mRNA • platelet-derived growth factor, PDGF • UDP-GalNAc:polypeptide GalNAc transferase, pGalNAc TF • Raf oncogenes-encoded protein-serving/threonine kinases, Raf • Ras oncogenes-encoded small GTP-binding proteins, Ras • transferase, TF • transforming growth factor
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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.