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J. Biol. Chem. 277 (29): 25893-25903

© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Transcription Factor NFIC Undergoes N-Glycosylation during Early Mammary Gland Involution*

Rosemary KaneDagger §, Janice MurtaghDagger §, Darren FinlayDagger §, Andreas Marti||, Rolf Jaggi, David Blatchford**, Colin Wilde**, and Finian MartinDagger §Dagger Dagger

From the Dagger  Conway Institute of Biomolecular and Biomedical Research and § Department of Pharmacology, University College Dublin, Belfield, Dublin 4, Ireland,  Department of Clinical Research University of Bern, Murtenstrasse 35, Bern, CH-3010 Switzerland, and ** Hannah Research Institute, Ayr, Scotland KA6 5HL, United Kingdom

Expression of a 74-kDa nuclear factor I (NFI) protein is triggered in early involution in the mouse mammary gland, and its expression correlates with enhanced occupation of a twin (NFI) binding element in the clusterin promoter, a gene whose transcription is induced at this time (Furlong, E. E., Keon, N. K., Thornton, F. D., Rein, T., and Martin, F. (1996) J. Biol. Chem. 271, 29688-29697). We now identify this 74-kDa NFI as an NFIC isoform based on its interaction in Western analysis with two NFIC-specific antibodies. A transition from the expression of a 49-kDa NFIC in lactation to the expression of the 74-kDa NFIC in early involution is demonstrated. We show that the 74-kDa NFIC binds specifically to concanavalin A (ConA) and that this binding can be reversed by the specific ConA ligands, methyl alpha -D-mannopyranoside and methyl alpha -D-glucopyranoside. In addition, its apparent molecular size was reduced to ~63 kDa by treatment with the peptide N-glycosidase. The 49-kDa lactation-associated NFIC did not bind ConA nor was it affected by peptide N-glycosidase. Tunicamycin, a specific inhibitor of N-glycosylation, blocked formation of the 74-kDa NFI in involuting mouse mammary gland in vivo when delivered from implanted Elvax depot pellets. Finally, the production of the ConA binding activity could be reiterated in "mammospheres" formed from primary mouse mammary epithelial cells associated with a laminin-rich extracellular matrix. Synthesis of the 74-kDa NFIC was also inhibited in this setting by tunicamycin. Thus, involution triggers the production of an NFIC isoform that is post-translationally modified by N-glycosylation. We further show, by using quantitative competitive reverse transcriptase-PCR, that there is increased expression of the major mouse mammary NFIC mRNA transcript, mNFIC2, in early involution, suggesting that the involution-associated change in NFIC expression also has a transcriptional contribution.

* This work was supported by the Health Research Board, Ireland and Enterprise Ireland, the Irish Science and Technology Agency.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF358455-AF358460.

|| Present address: Novartis Pharma AG, Oncology Research, WKL-125.2.42, 4002 Basel, Switzerland.

Dagger Dagger To whom correspondence should be addressed: Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. Tel.: 353-1-716-2808; Fax: 353-1-269-2016; E-mail: finian.martin@ucd.ie.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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