Deletion of mouse embryo fibroblast N-acetylglucosaminyltransferase V stimulates alpha5beta1 integrin expression mediated by the protein kinase C signaling pathway.
An N-linked glycan often increased during oncogenic transformation contains beta(1,6)-linked GlcNAc, synthesized by the N-acetylglucosaminyltransferase V (GnT-V). The progression of polyoma middle T-antigen oncoprotein-induced mammary carcinomas in GnT-V null mice was significantly retarded compared with that observed in wild-type mice. The matrix adhesion of mouse embryonic fibroblasts (MEF) from GnT-V null and wild-type mice was investigated to understand the mechanism by which deletion of GnT-V could retard tumor progression. GnT-V null MEF displayed enhanced adhesion to and spreading on fibronectin-coated plates with concomitant inhibition of cell migration. GnT-V null MEF also showed increased focal adhesion kinase tyrosine phosphorylation, consistent with decreased cell motility on fibronectin-coated plates. Expression of GnT-V cDNA in the null MEF reversed these abnormal characteristics, indicating the direct involvement of N-glycosylation events in these phenotypic changes. The alpha5beta1 fibronectin receptors exhibited increased clustering on the null MEF cell surfaces, consistent with previous studies that observed less integrin clustering in cells overexpressing GnT-V. Most surprisingly, GnT-V null MEF displayed increased expression levels of both alpha5 and beta1 subunits in lysates and on the cell surface. Increased alpha5beta1 expression in the null MEF was because of increased alpha5beta1 transcript levels that declined after re-expression of GnT-V cDNA, confirming that increased alpha5beta1 expression in null MEF was because of changes in GnT-V expression. The increased null MEF transcripts were shown to be caused at least in part by increased integrin promoter activity. Moreover, increased alpha5beta1 integrin transcripts in GnT-V null MEF were not due to a differential response to fibronectin; rather, they appeared to be mediated by activation of a protein kinase C signaling pathway. These results demonstrate that deletion of MEF GnT-V resulted in enhanced integrin clustering and activation of alpha5beta1 transcription by protein kinase C signaling, which in turn up-regulated levels of cell surface alpha5beta1 fibronectin receptors that resulted in increased matrix adhesion and inhibition of migration.
Guo
Hua-Bei
HB
Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA.
Lee
Intaek
I
Bryan
Beau T
BT
Pierce
Michael
M
D000818:Animals; D016466:CHO Cells; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002469:Cell Separation; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000201:enzymology* / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D017353:Gene Deletion; D006031:Glycosylation; D015151:Immunoblotting; D047468:Immunoprecipitation; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000502:physiology*; D010641:Phenotype; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017469:Receptors, Fibronectin / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D015854:Up-Regulation
D015820:Cadherins; D016895:Culture Media, Serum-Free; D005353:Fibronectins; D039081:Integrin alpha5beta1; D016023:Integrins; D037102:Lectins; D012333:RNA, Messenger; D017469:Receptors, Fibronectin; D014443:Tyrosine; D008156:Luciferases; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase; D011493:Protein Kinase C