Molecular and Cellular Biology of Adipocyte Differentiation
Genes of Interest: C/EBPβ, HuR and Zfp206
Current Focus: The regulation of adipogenesis by transcription factors and RNA binding proteins.
Tissue of Interest: Adipose
Obesity develops when energy intake exceeds energy expenditure. The obese phenotype has reached epidemic proportions and is a major factor in the development of a number of common medical conditions including type 2 diabetes, hyperlipidemias, fatty liver, cardiovascular disease, Alzheimer's disease and even some cancers. Additionally, obesity has been shown to have substantial negative effect on longevity, reducing the lifespan of severely obese people by an estimated five to 20 years. In fact, despite a 1000-year trend of increasing life expectancy, studies predict that obesity might actually reduce life expectancy in the US in the coming years. In short, obesity is rapidly becoming perhaps the major health concern in our society today and thus there continues to be a substantial need for new diagnostic biomarkers and treatment options. Our studies will address this need by detailing novel regulatory mechanisms in the early stages of adipogenesis.
Adipose tissue plays a critical role with respect to the pathology of obesity as well as diabetes and has become recognized as a dynamic endocrine-like tissue, synthesizing and secreting proteins responsible for regulation of the balance between energy storage and energy expenditure. If we are to identify new diagnostic biomarkers and discover new treatment options, we need to understand the regulation of the differentiation process as well as the maintenance of the differentiated phenotype in this complex tissue. Fundamental to obesity is adipocyte hyperplasia that occurs through recruitment and proliferation of preadipose cells present in the vascular stroma of adipose tissue. This hyperplasia is mimicked ex vivo by postconfluent 3T3-L1 preadipocytes which when induced to differentiate, synchronously re-enter the cell cycle and undergo several rounds of mitotic clonal expansion prior to growth arrest and expression of the adipocyte phenotype.
Genes of Interest – HuR and C/EBPβ
The differentiation program in the 3T3-L1 adipocytes is controlled by a cascade of transcription factors, the CCAAT/enhancer binding proteins (C/EBPs) and a nuclear hormone receptor, peroxisome proliferator activated receptor γ (PPARγ). C/EBPβ and C/EBPδ are rapidly induced in the early stage of adipogenesis and subsequently drive C/EBPα and PPARγ expression. C/EBPα and PPARγ can induce each other in a positive feedback loop and directly activate many of the genes for terminal adipocyte differentiation. PPARγ is considered the master regulator of adipogenesis as it alone can induce differentiation in C/EBPα deficient murine embryonic fibroblasts (MEFs), while C/EBPα is incapable of driving differentiation in the absence of PPARγ.
Superimposed on this framework of C/EBPs and PPARγ we have recently identified the transient expression of the embryonic stem cell transcription factor Zfp206 and its splice variant Zscan. We propose that they play a critical role in the control of not only the adipogenic differentiation program of the 3T3-L1 cells but of human preadipocytes also.
Zfp206 is a SCAN-ZFP gene located in a gene cluster on Chr. 17 and has been implicated in the maintenance of embryonic stem cell (ESC) pluripotency. Support for the function of the Zfp206 gene as a pluripotency factor is based on it being highly expressed in undifferentiated ESC and the inner cell mass of the preimplantation embryo, but not in differentiated ESC or trophectoderm. Knockdown of Zfp206 expression induces ESC differentiation, whereas it's sustained over expression impedes differentiation, thus establishing that Zfp306 is a regulator of pluripotency.
Based on the limited information available on Zfp206 function, our hypothesis is that its expression is part of a mechanism for maintenance of the differentiation potential of preadipocytes and as the adipocyte phenotype is established, i.e. as PPARγ expression locks in the adipocyte phenotype, Zfp206 expression is down regulated. Our first approach to understanding the mechanism of Zfp206 action will be through knock outs, which we propose will result in an enhanced initiation of the differentiation program. Complementary to those experiments will be over expression studies, with the expectation that forced expression of Zfp206 will attenuate the differentiation program. Both knock outs and Zfp206 over expressing cells will be induced to differentiate, characterization of the alterations in the acquisition of the adipocyte phenotype will be diagnostic of Zfp206 function. (top)
Control of Zfp206 mRNA metabolism. Expression of the RNA binding protein HuR is essential for the 3T3-L1 differentiation program. We have demonstrated that within 30 min of induction of differentiation the Zfp206 mRNA becomes a ligand for the RNA binding protein HuR, consistent with HuR playing a role in the metabolism of this message. As it was through formation of this mRNP complex that we discovered Zfp206, we will determine the mechanism by which HuR controls Zfp206 expression by investigation of its effects on polyadenylation, export from the nucleus, stabilization and/or translational efficiency of the Zfp206 mRNA. Understanding the regulation of Zfp206 mRNA metabolism, much like understanding control of transcription, provides information on mechanisms that control the levels of Zfp206 protein, and thus mechanisms that can be targeted for intervention.
The role of Zfp206 and Zscan in the 3T3-L1 differentiation program. Based on the limited information available on Zfp206 function, our hypothesis is that its expression is part of a mechanism for maintenance of the differentiation potential of the preadipocyte, as the adipocyte phenotype is established, its expression is diminished. It may be that as PPARγ expression is established, locking in the adipocyte phenotype, maintenance of the differentiation potential is no longer needed and Zfp206 expression is down regulated.To that end we will approach identification of the Zfp206 mechanism of action first through shRNA knock out experiments, which we propose will result in an enhanced initiation of the differentiation program. Complementary to these studies will be Zfp206 over expression studies, expecting that forced expression of Zfp206 will suppress the initiation of the differentiation program. In both approaches, characterization of the alterations in the acquisition of the adipocyte phenotype as well as identification of the genes activated or suppressed as Zfp206 expression is altered will be diagnostic of function.
Transcriptional control of Zfp206 expression. Limited information exists on control of Zfp206 transcription. The embryonic stem (ES) cell transcription factors Oct4 and Sox2 are reported to mediate activation of transcription of the Zfp206 gene in ES cells. Our preliminary data demonstrates that neither Oct4 nor Sox2 are expressed in the 3T3-L1 cells consistent with a novel regulation of Zfp206 gene expression. Genomatix MatInspector software analysis identified 1150 bases of the 5’ flanking region to the purported transcription start site as the promoter region and detailed 211 transcription factor binding sites. Notable was the presence of three sites for C/EBPβ in the first 300 bases. In this portion of the study, the control elements of the Zfp206 gene will be identified to gain further insight into its role in the differentiation program.
Karschner VA, Pekala PH. (2009) HuR involvement in mitotic clonal expansion during acquisition of the adipocyte phenotype. Biochem Biophys Res Commun. (2009) 383:203-5
Cherry, J., Jones, H., Karschner, V., and Pekala, Phillip H. (2008) Post Transcriptional Control of C/EBPVβ Expression: Formation of a Nuclear HuR-C/EBPβ mRNA Complex Determines the Amount of Message Reaching the Cytosol. J. Biol. Chem. 283: 30812-30820
Karschner VA, Pekala PH. (2009) HuR involvement in mitotic clonal expansion during acquisition of the adipocyte phenotype. Biochem Biophys Res Commun. (2009) 383:203-5.
Cherry, J., Jones, H., Karschner, V.A., and Pekala, P.H. (2008) Post Transcriptional Control of C/EBPβ Expression: Formation of a Nuclear HuR-C/EBPβ mRNA Complex Determines the Amount of Message Reaching the Cytosol. J. Biol. Chem. 283: 30812-30820
Gantt, K., Cherry, J., Atasoy, U., Karschner, V., Richardson, M., and Pekala, Phillip H. (2006) Maintenance of the adipocyte phenotype; HuR is a ligand for GLUT1 and leptin mRNAs. J. Cellular Biochemistry 99: 565-574.
Pessler-Cohen, D., Pekala, P.H., Kovsan, J., Bloch-Damti, A., Rudich, A., and Bashan, N. (2006) Glut4 repression in response to oxidative stress is associated with reciprocal alterations in C/EBPα anddisoforms in 3T3-L1 adipocytes. Arch. Physiol. Biochem. 112: 3-12.
Cherry, J., Karschner, V., Jones, H., and Pekala, P.H. (2006) HuR, an RNA binding protein involved in cellular differentiation. In Vivo 20: 17-24.
Gantt, K., Cherry, J., Tenney, T., Karschner, V., and Pekala, Phillip H. (2005) An early event in adipogenesis, the nuclear selection of C/EBPbmRNA by HuR and it’s translocation to the cytosol. J. Biol. Chem. 280: 24768-24774.
Tenney, R.E. and Pekala, P. H. (2004) Interleukin 11 treatment alters the protein content of Gai2 and adipogenic transcription factors in 3T3-L1 adipocytes. Cytokine 27: 1-6.
Tenney, R.E., Stansfield, K.A., and Pekala, Phillip H. (2004) Interleukin 11 signaling in 3T3-L1 adipocytes. J. Cell. Physiol. 202: 160-166.
Former Students of the Pekala Lab:
Russ Price, Ph.D. Professor, Departments of Medicine and Physiology, Emory University School of Medicine.
Peter Cornelius, Ph.D. Senior Research Scientist, Department of Cardiovascular and Metabolic Diseases, Pfizer Central Research.
Jackie Stephens, Ph.D. Professor, Department of Physiology and Zoology, LSU, Baton Rouge, LA.
Kevin McGowan, Ph.D. Assistant Professor, Department of Medicine, UNC-Chapel Hill School of Medicine.
Sheree Long, Ph.D. Application Specialist, Biacore, Inc.
Renu Jain, Ph.D. 1997, Director, Clinical Trials, Pediatric Aids Drug Therapy Program Glaxo-Welcome