Biochemistry Faculty

Philip H. Pekala
Professor and Interim Chair of Biochemistry & Molecular Biology
B.S., St. Vincent College, 1971
Ph.D., Virginia Tech, 1978
Fellowship, The Johns Hopkins Univ. School of Medicine

Molecular and Cellular Biology of Adipocyte Differentiation

Tissue of Interest: Adipose

Genes of Interest: The transcription factor C/EBPβ and the RNA binding protein HuR

Focus of Study: RNA binding protein involvement in cellular differentiation: mRNA transport, control of translation and mRNA stability.

Selected Publications

*LATEST PUBLICATION FROM THE PEKALA LAB!!

Cherry, J., Jones, H., Karschner, V., and Pekala, Phillip H. (2008) The involvement of HuR in control of C/EBPβ expression: Translocation to the cytosol and mRNA stability. J. Biol. Chem. 283: 30812-30820.

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Curriculum vitæ (CV)

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Tissue of Interest:

Adipose tissue plays a critical role with respect to both diabetes ad obesity and has become recognized as a dynamic endocrine-like tissue, synthesizing 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. (top)

Genes of Interest – HuR and C/EBPβ

Adipocyte differentiation is a complex process regulated in large part by the temporarily controlled expression and activation of numerous transcription factors. Among these proteins, the C/EBP and PPAR families of transcriptional activators have been identified as critical to initiation of the differentiation process as well as maintenance of the adipocyte phenotype. Functional roles for these factors have been established at least in part through use of the 3T3-L1 preadipocyte model system. When growth arrested 3T3-L1 preadipocytes are included to differentiate, the cells reenter the cell cycle and undergo mitotic clonal expansion followed by growth arrest and expression of the adipocyte phenotype. During this process C/EBPβ is expressed on induction of differentiation and is essential not only for mitotic clonal expansion but the transcriptional activation of PPARTy and C/EBPα. The indispensable nature of carefully timed C/EBPβ expression was demonstrated in studies with C/EBPβ-/- MEFs which when treated with the differentiation inducers could neither reneter the cell cycle and undergo mitotic clonal expansion nor express the adipocyte phenotype (4). Similar results were observed in 3T3-L1 cells expressing a dominant negative C/EBP.

Messenger RNA export from the nucleus, mRNA turnover as well as translation initiation are important control points in the post-transcriptional regulation of gene expression. At least in part, control of these processes is exerted through recognition of cis elements in the mRNA by specific binding proteins. One of these binding proteins is HuR, which was first reported to bind to AU-rich elements of sequence AU₃A within AU-rich backgrounds in the 3’UTRs of early response genes such as cytokines and protoncogenes. The binding of HuR to the ARE of a ligand message results in a significant stabilization of that mRNA. HuR is a 32-kDa protein that belongs to the Hu/ELAV family (embryonic lethal, abnormal vision) of RNA binding proteins is ubiquitously expressed and localized predominately to the nucleus but has been demonstrated to shuttle between the nucleus and cytoplasm. The shuttling activity has suggested that HuR functions by binding to nascent mRNAs in the nucleus and protecting them from degradation by actively participating in their nucleo-cytoplasmic transport as well as controlling their stability. Thus, HuR appears to be an important regulatory protein that is involved in the posttranscriptional processing of certain mRNAs. Studies characterizing the translocation process in HeLa cells have demonstrated that HuR contains a nucleo-cytoplasmic shuttling sequence and functions as an adapter protein in the nuclear export of mRNAs that contain an ARE in their 3’ untranslated regions. Very recent evidence has suggested that additional non-AU-rich HuR binding sites may exist within particular mRNAs that are recognized by the protein based on structural features and while interaction with the ARE may be necessary for stability, these structural sites may function in nuclear export. Thus, suggesting that at least in some mRNAs, functional HuR binding sites for control of translocation and stability may be separable.

In the 3T3-L1 cells HuR is constitutively expressed and localized predominately to the nucleus in the preadipocytes. However within 30 min of exposure to the differentiation stimulus, the HuR content in the cytosol increased, consistent with HuR regulating the availability of relevant mRNAs for translation. The C/EBPβ message, one of the earliest mRNAs expressed after induction of differentiation, is a ligand for HuR. Within 30 min of initiation of the differentiation process, HuR complexes containing C/EBPβ mRNA selectively formed in the nucleus, the complex then appeared to translocate to the cytosol. The proximity of this event to the induction of differentiation and the importance of C/EBPβ to the differentiation process led us to hypothesize a role for HuR in the regulation of the onset of adipogenesis. Further support for this hypothesis was obtained in our study when siRNA suppression of HuR protein content resulted in an inhibition of C/EBPβ protein expression and an attenuation of the differentiation process. (top)

Focus of Studies:

In our current work we propose to define key components and the mechanism of a post-transcriptional regulatory network associated with the differentiation of preadipocytes leading to adipocytes. Our hypothesis is that HuR participates in the post-transcriptional control of expression of gene products necessary to initiate and maintain the adipocyte phenotype. While C/EBPβ may be a primary target, other critical mRNAs i.e. cell cycle related, may also play a role. Thus, it can be argued that HuR by selecting specific mRNA ligands and controlling their expression is capable of controlling critical events in the cell cycle as well as the differentiation process. In addition, once the master switch is flipped, we believe that HuR participates in regulating the expression of genes necessary for maintenance of the adipocyte phenotype.

Through characterization of the interaction of RNA binding proteins with specific mRNAs, we will identify and define a critical regulatory process that encompass the selection of the mRNA in the nucleus, transport of the message to the cytosol where the binding protein controls both stability and translational efficiency of the mRNA. Understanding the mechanism of HuR function will provide detail on an RNA binding protein involved in a very focused regulation impacting on gene expression, RNA trafficking and adipocyte biology. These studies are expected to yield novel information on an understudied target for control of adipocyte differentiation.

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

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Selected Publications

Cherry, J., Jones, H., Karschner, V.A., and Pekala, P.H. (2007) Expression of C/EBPβ and the control of adipocyte differentiation: Influence of the C/EBPβ 3’UTR and the binding of HuR. Submitted.

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.

Blasts from the past – some old favorites that still have impact on our work

Jain, R.G., Andrews, L.G., McGowan, K.M., Gao, F., Keene, J., and Pekala, P.H. (1997) Messenger RNA binding protein, Hel-N1 accelerates differentiation of adipocytes and increases levels of the glucose transporter (GLUT1) Mol. Cell. Bio. 17, 954-962.

Stephens, J. M. and Pekala, P. H. (1992) Transcriptional repression of the C/EBP and GLUT-4 genes in 3T3-L1 : regulation is coordinate and independenta-adipocytes by tumor necrosis Factor of protein synthesis. J. Biol. Chem. 267, 13580-13584.

Kaestner, K. H., Christy, R. J., McLenithan, J. C., Braiterman, L. T., Cornelius, P., Pekala, P. H., and Lane, M. D. (1989) Sequence, tissue distribution and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3-L1 adipocytes. Proc. Natl. Acad. Sci. USA86, 3150-3154.

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Contact Information

Professor & Interim Chair of Biochemistry & Molecular Biology

Brody 5S-17

The Brody School of Medicine at East Carolina University

Greenville, NC 27834

phone: 252.744.2684

email: pekalap@ecu.edu

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