Involvement of the retinoblastoma protein in brown and white adipocyte cell differentiation: functional and physical association with the adipogenic transcription factor C/EBPalpha.
We investigated the expression of the retinoblastoma protein (pRB) in adipocytes and its possible interaction with the adipogenic transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) in controlling the acquisition of the terminally differentiated adipocyte phenotype. The pRB was expressed (as measured by immunoblotting and/or immunofluorescence) in mice brown and white adipose tissue and in cultured adipocytes that showed lipid accumulation and expressed specific differentiation markers such as aP2 (measured using a specific cDNA probe) and in the case of brown adipocytes UCP-1 (measured using specific antibodies), but was undetectable in proliferative undifferentiated preadipocytes. Transient transfection experiments revealed a functional interaction between pRB and C/EBPalpha affecting transcription from the ucp-1 gene promoter. Thus, in immortalized brown adipocytes, co-transfection of both a C/EBPalpha and a pRB expression vectors maximally enhanced the expression of reporter chloramphenicol acetyltransferase driven by the ucp-1 promoter. Interestingly, C/EBPalpha inhibited reporter gene expression in CHO cells in an effect that was also potentiated in the presence of pRB. A positive effect of pRB on transcription from the ucp-1 promoter could be detected in C/EBPalpha-/-fibroblasts only after forced to overexpress C/EBPalpha, suggesting that the effect of pRB is dependent on its interaction with C/EBPalpha. We also found evidence that pRB and C/EBPalpha can directly bind to each other in vitro. Our results show that the expression of pRB is restricted to differentiated adipocytes, and provide evidence of a physical and functional interaction between pRB and C/EBPalpha that affects the transcriptional activity of the later on a brown adipocyte-specific gene.
Puigserver
P
P
Departament de Biologia Fonamental i Ciències de la Salut, Laboratori de Biologia Molecular, Nutrició i Biotecnologia, Universitat de les Illes Balears, Palma de Mallorca, Spain.
Ribot
J
J
Serra
F
F
Gianotti
M
M
Bonet
M L
ML
Nadal-Ginard
B
B
Palou
A
A
D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D002001:Adipose Tissue, Brown / Q000166:cytology / Q000378:metabolism; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D002454:Cell Differentiation; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors