O-glycosylation of Sp1 and transcriptional regulation of the calmodulin gene by insulin and glucagon.
Both insulin and glucagon stimulate steady-state levels of Sp1 transcription factor, but only insulin stimulates transcription of the calmodulin (CaM) gene in liver. Because O-glycosylation of Sp1 by O-linked N-acetylglucosamine (O-GlcNAc) is thought to regulate its ability to activate transcription, we assayed the levels of Sp1 with anti-Sp1 and anti-O-GlcNAc antibodies in Western blots by use of extracts of H-411E liver cells treated with insulin (10,000 microU/ml) or glucagon (1.5 x 10(-5) M). We also assessed subcellular localization of the native and glycosylated Sp1 in H411E cells treated with either hormone in the presence of deoxynorleucine (DON, an indirect inhibitor of O-glycosylation) or streptozotocin (STZ, an indirect stimulator of O-glycosylation). Insulin stimulated both total and O-GlcNAc-modified Sp1 primarily in the nucleus and induced CaM gene transcription (P < 0.0001). In contrast, glucagon promoted accumulation of Sp1 in the cytoplasm but not the nucleus, without significantly stimulating (P = not significant) either its O-glycosylation or transcription of the CaM gene. DON inhibited O-glycosylation of Sp1 and its ability to migrate to the nucleus and transactivate CaM gene transcription. In contrast, cotreatment of cells with STZ and glucagon enhanced O-glycosylation of Sp1, promoting its migration to the nucleus and resulting in increased CaM gene transcription. Thus O-glycosylation of Sp1 by insulin, but not glucagon, apparently enhances its (Sp1) nuclear recruitment and results in activation of CaM gene transcription.
Majumdar
Gipsy
G
Research Services, Veterans Affairs Medical Center, Memphis, TN 38104, USA.
Harmon
Ashley
A
Candelaria
Rosalind
R
Martinez-Hernandez
Antonio
A
Raghow
Rajendra
R
Solomon
Solomon S
SS
D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D002147:Calmodulin / Q000235:genetics*; D006528:Carcinoma, Hepatocellular; D005765:Gastrointestinal Agents / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005934:Glucagon / Q000378:metabolism / Q000494:pharmacology*; D006031:Glycosylation; D007004:Hypoglycemic Agents / Q000378:metabolism / Q000494:pharmacology*; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D009646:Norleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013311:Streptozocin / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D002147:Calmodulin; D005765:Gastrointestinal Agents; D007004:Hypoglycemic Agents; D007328:Insulin; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D013311:Streptozocin; D009646:Norleucine; D005934:Glucagon; D000117:Acetylglucosamine