title: Role of UBC9 in the regulation of the adipogenic program in 3T3-L1 adipocytes.
abstract: The small ubiquitin-like modifier-conjugating enzyme UBC9, involved in protein modification through covalent attachment of small ubiquitin-like modifier and other less defined mechanisms, has emerged as a key regulator of cell proliferation and differentiation. To explore the role of UBC9 in adipocyte differentiation, the UBC9 protein levels were examined in differentiating 3T3-L1 cells. UBC9 mRNA and protein levels were increased 2.5-fold at d 2 and then gradually declined to basal levels at d 8 of differentiation. In addition, UBC9 was expressed predominantly in the nucleus of preadipocytes but shifted to cytoplasmic compartments after d 4, after induction of differentiation. UBC9 knockdown was then achieved in differentiating 3T3-L1 preadipocytes using a specific small interfering RNA. Oil-Red-O staining demonstrated accumulation of large triglyceride droplets in approximately 90% of control cells, whereas lipid droplets were smaller and evident in only 30% of cells treated with the UBC9-specific small interfering RNA. CCAAT/enhancer-binding protein (C/EBP)-δ, peroxisome proliferator-activated receptor-γ, and C/EBPα mRNA levels were increased severalfold 2-6 d after induction of differentiation in control cells, whereas the expression of these transcription factors was significantly lower in the presence of UBC9 gene silencing. Adenovirus-mediated overexpression of a catalytically inactive mutant UBC9 protein in 3T3-L1 cells resulted in no changes in expression of adipogenic transcription factors and conversion to mature adipocytes as compared with control. In conclusion, UBC9 appears to play an important role in adipogenesis. The temporal profile of UBC9 induction and its ability to affect C/EBPδ mRNA induction support a role for this protein during early adipogenesis.
authors.affiliation: Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, Andrology, and Metabolic Diseases, Università degli Studi di Bari Aldo Moro, I-70124 Bari, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Cignarelli
authors.forename: Mariangela
authors.initials: M
authors.lastname: Melchiorre
authors.forename: Alessandro
authors.initials: A
authors.lastname: Peschechera
authors.forename: Antonella
authors.initials: A
authors.lastname: Conserva
authors.forename: Lucia Adelaide
authors.initials: LA
authors.lastname: Renna
authors.forename: Sara
authors.initials: S
authors.lastname: Miccoli
authors.forename: Annalisa
authors.initials: A
authors.lastname: Natalicchio
authors.forename: Sebastio
authors.initials: S
authors.lastname: Perrini
authors.forename: Luigi
authors.initials: L
authors.lastname: Laviola
authors.forename: Francesco
authors.initials: F
authors.lastname: Giorgino
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D050156:Adipogenesis / Q000502:physiology*; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D051379:Mice; D047495:PPAR gamma / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D047495:PPAR gamma; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9