Retinoic acid modulates the retinoblastoma protein during adipocyte terminal differentiation (original) (raw)
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Genes & Development, 1996
To define a mechanism by which retinoblastoma protein (Rb) functions in cellular differentiation, we studied primary fibroblasts from the lung buds of wild-type (RB + / +) and null-mutant (RB-/-) mouse embryos. In culture, the RB +/+ fibroblasts differentiated into fat-storing cells, either spontaneously or in response to hormonal induction; otherwise syngenic RB-/-fibroblasts cultured in identical conditions did not. Ectopic expression of normal Rb, but not Rb with a single point mutation, enabled RB-/-fibroblasts to differentiate into adipocytes. Rb appears in murine fibroblasts to activate CCAAT/enhancer-binding proteins (C/EBPs), a family of transcription factors crucial for adipocyte differentiation. Physical interaction between Rb and C/EBPs was demonstrated by reciprocal coimmunoprecipitation, but occurred only in differentiating cells. Wild-type Rb also enhanced the binding of C/EBP to cognate DNA sequences in vitro and the transact[vat[on of a C/EBPl$-responsive promoter in cells. Taken together, these observations establish a direct and positive role for Rb in terminal differentiation. Such a role contrasts with the function of Rb in arresting cell cycle progression in G1 by negative regulation of other transcription factors like E2F-1.
Proceedings of the National Academy of Sciences, 2007
The role of the tumor suppressor retinoblastoma protein (pRb) has been firmly established in the control of cell cycle, apoptosis, and differentiation. Recently, it was demonstrated that lack of pRb promotes a switch from white to brown adipocyte differentiation in vitro . We used the Cre-Lox system to specifically inactivate pRb in adult adipose tissue. Under a high-fat diet, pRb-deficient (pRb ad−/− ) mice failed to gain weight because of increased energy expenditure. This protection against weight gain was caused by the activation of mitochondrial activity in white and brown fat as evidenced by histologic, electron microscopic, and gene expression studies. Moreover, pRb −/− mouse embryonic fibroblasts displayed higher proliferation and apoptosis rates than pRb +/+ mouse embryonic fibroblasts, which could contribute to the altered white adipose tissue morphology. Taken together, our data support a direct role of pRb in adipocyte cell fate determination in vivo and suggest that pRb...
Differentiation-dependent expression of retinoid-proteins in BFC-1β adipocytes
Journal of Biological Chemistry
Recently, we demonstrated that adipose tissue plays an important role in retinol storage and retinol-binding protein (RBP) synthesis. Our data suggested that RBP expression in adipose tissue is dependent on the state of adipocyte differentiation. To examine this possibility, we explored the differentiation-dependent expression of RBP using BFC-1 j 3 preadipocytes, which can be stimulated to undergo adipose differentiation. Total RNA was isolated from undifferentiated (preadipocytes) and differentiated (adipocytes) BFC-1j3 cells and analyzed by Northern blotting. RBP mRNA was not detected in the preadipocytes, but considerable RBP mRNA was present in differentiated BFC-lj3 cells. In BFC-1j3 cells, induced to differentiate with insulin and thyroid hormone, RBP mRNA was first detected after 4 days, reached a maximum level by day 10, and remained at this maximum level for at least 2 more days. Cellular retinol-binding protein was expressed at low levels in the BFC-1B preadipocytes and the level of expression increased for 6 days after induction to differentiate and slowly declined on later days. Neither the maximum level of RBP expression nor the day on which this level was reached was influenced by the level of retinol provided in the BFC-1j3 culture medium. BFC-lj3 cells secreted newly synthesized RBP into the culture medium at a rate of 43 2 14 ng RBP/24 h/106 adipocytes. When the BFC-lj3 adipocytes were provided 1.0 PM retinol in the medium, they accumulated the retinol and synthesized retinyl esters. These studies with BFC-lj3 cells confirm that RBP synthesis and secretion and retinol accumulation are intrinsic properties of differentiated adipocytes. Furthermore, they suggest that RBP and cellular retinolbinding protein gene expression are regulated as part of a package of genes which are modulated during adipocyte differentiation.
Proceedings of the National Academy of Sciences, 2004
Adipocyte precursor cells give raise to two major cell populations with different physiological roles: white and brown adipocytes. Here we demonstrate that the retinoblastoma protein (pRB) regulates white vs. brown adipocyte differentiation. Functional inactivation of pRB in wild-type mouse embryo fibroblasts (MEFs) and white preadipocytes by expression of simian virus 40 large T antigen results in the expression of the brown fat-specific uncoupling protein 1 (UCP-1) in the adipose state. Retinoblastoma genedeficient (Rb ؊/؊ ) MEFs and stem cells, but not the corresponding wild-type cells, differentiate into adipocytes with a gene expression pattern and mitochondria content resembling brown adipose tissue. pRB-deficient MEFs exhibit an increased expression of the Forkhead transcription factor Foxc2 and its target gene cAMPdependent protein kinase regulatory subunit RI␣, resulting in increased cAMP sensitivity. Suppression of cAMP-dependent protein kinase activity in Rb ؊/؊ MEFs blocked the brown adipocyte-like gene expression pattern without affecting differentiation per se. Immunohistochemical studies revealed that pRB is present in the nuclei of white but not brown adipocyte precursor cells at a developmental stage where both cell types begin to accumulate lipid and brown adipocytes express UCP-1. Furthermore, pRB rapidly undergoes phosphorylation upon cold-induced neodifferentiation and up-regulation of UCP-1 expression in brown adipose tissue. Finally, down-regulation of pRB expression accompanies transdifferentiation of white into brown adipocytes in response to 3-adrenergic receptor agonist treatment. We propose that pRB acts as a molecular switch determining white vs. brown adipogenesis, suggesting a previously uncharacterized function of this key cell cycle regulator in adipocyte lineage commitment and differentiation.
Retinoids are positive effectors of adipose cell differentiation
Molecular and Cellular Endocrinology, 1994
Retinoids, especially all-truns retinoic acid (t-RA), have been reported in the last decade to inhibit the differentiation of preadipose cells. In those studies, however, the concentrations of t-RA were supraphysiological (O.l-10,~M range). In contrast we show that, when present at concentrations below or close to the Kd values of retinoic acid receptors, retinoids behave as potent adipogenic hormones (1 pM to 10 nM range). As shown by the use of specific ligands for each RAR subtype, these positive effects on adipose differentiation involve in particular the RARa subtype, and have been observed in 0b17 cells exposed to serum-supplemented or serum-free medium, and in rat preadipocytes exposed to serum-free medium. Among the two classes of retinoid acid receptors (RARs) and retinoid X receptors (RXRs), RARa, RARy, RXRa and RXRg mRNAs could be detected in growing adipoblasts and were found to be increased in committed preadipocytes and differentiated cells upon retinoid treatment. Like other adipogenic hormones, retinoids were only effective in the terminal differentiation process leading from preadipocytes to adipocytes.
BMC Research Notes, 2011
Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs (Rb-/- MEFs). Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1.To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like) adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment. In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte differentiation of Rb-/- MEFs could be identified. These data and the analysis provide a starting point for further experimental studies to identify target genes for pharmacological intervention and ultimately remodeling of white adipose tissue into brown adipose tissue.
European Journal of Cell Biology, 1998
pRB-ClEBPaadipocytescell growthdifferentiation We investigated the expression of the retinoblastoma protein (pRB) in adipocytes and its possible interaction with the adipogenic transcription factor CCAAT/enhancer-binding protein a (C/EBPa) in controUing the acquisition of the terminally differentiated adipocyte phenotype. The pRB was expressed (as measured by immunoblotting and/or immuno-Duorescence) 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 eDNA probe) and in the case of brown adipocytes DCP•1 (measured using specific antibodies), but was undetectable in proliferative undifferentiated preadipocytes. lransient transfection experiments revealed a functional interaction between pRB and CIEBPa affecting transcription from the ucp-] gene promoter. Thus, in immortalized brown adipocytes, co-transfection of both a CIEBPa and a pRB expression vectors maximally enhanced the expression of reporter chloramphenicol acetyltransferase driven by the ucp-] promoter. Interestingly, C/EBPa 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-] promoter could be detected in C/EBPa-lfibroblasts only after forced to overexpress CIEBPa, suggesting that the effect of pRB is dependent on its interaction with CIEBPa. We also found evidence that pRB and CIEBPa 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 func• tional interaction between pRB and C/EBPa that affects the transcriptional activity of the later on a brown adipocyte-specific gene.
Differentiation, 1994
Adipocyte differentiation of 3T3-Ll cells is a complex process which is inhibited by retinoic acid (RA). Since RA acts by nuclear receptors which directly regulate gene expression, we postulate that the primary targets of RA action in this system are genes which are regulated early in adipose conversion. In this study, we demonstrate the use of the differential display technique to search for early events in adipose commitment which are sensitive to RA. A mRNA was identified on the basis of its RA-dependent gene expression 24 h after initiation of a standard differentiation protocol. Molecular cloning of the cDNA revealed it to be identical to the rus recision gene (rrg), for lysyl oxidase. Indeed, two mRNAs identical to those recognized by lysyl oxidase probes were expressed in preadipocytes and tandemly repressed with 24 h of exposure to differentiation conditions. Lysyl oxidase activity was similarly reduced in the media of differentiated cells. RA completely blocked the differentiation related reduction in rrgllysyl oxidase gene expression, although RA had no independent stimulatory effect on rrgllysyl oxidase expression in cells not exposed to differentiating conditions. Thus, differential display has been successfully used to identify rrgllysyl oxidase as an early marker for adipose conversion that is responsive to RA.