The long-term goal is to determine the mechanisms by which pluripotent stem cells of the vascular stroma of adipose tissue undergo commitment to the adipocyte lineage.
The SPECIFIC AIMS are: 1. (A) To identify BMP4-induced genes and proteins, and (B) to identify molecules that undergo phosphorylation on serine, threonine or tyrosine residues upon BMP4 treatment in C3H10TI/2 pluripotent stem cells. 2. To determine whether expression of genes """"""""identified"""""""" above causes commitment to the adipocyte lineage in cell culture or in vivo contexts and to determine the mechanistic basis for such commitment. 3. To identify, using a proteomic approach, secreted proteins that initiate, or are involved in, the commitment process. Changes in gene expression induced by BMP4 will be examined at both the mRNA transcript and protein levels. At the transcript level, changes in expression will be monitored with oligonucleotide arrays. Since mRNA and protein levels do not always reflect regulation by post-transcriptional mechanisms, differences in protein levels will also be directly assessed by mass spectrometry-based proteomic methods. This combined approach should not only provide verification of the transcript data, but also allow identification of molecules whose mRNA levels do not change and would otherwise be missed by microarray-based methods. Other advantages of mass spectrometry-based methods include identification of proteins that undergo post-translational modifications such as BMP4-induced phosphorylation and identification of novel molecules by de novo sequencing. Our studies should result in a detailed map of the adipocyte commitment process, as we should not only be able to identify the extracellular factors that influence this process but also elucidate the intracellular signaling pathways that are involved.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK066627-04
Application #
7098681
Study Section
Special Emphasis Panel (ZRG1-NMS (50))
Program Officer
Haft, Carol R
Project Start
2003-09-15
Project End
2008-08-31
Budget Start
2006-09-01
Budget End
2008-08-31
Support Year
4
Fiscal Year
2006
Total Cost
$399,144
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Bowers, Robert R; Lane, M Daniel (2008) Wnt signaling and adipocyte lineage commitment. Cell Cycle 7:1191-6
Bowers, Robert R; Lane, M Daniel (2007) A role for bone morphogenetic protein-4 in adipocyte development. Cell Cycle 6:385-9
Kim, Jae-woo; Monila, Henrik; Pandey, Akhilesh et al. (2007) Upstream stimulatory factors regulate the C/EBP alpha gene during differentiation of 3T3-L1 preadipocytes. Biochem Biophys Res Commun 354:517-21
Kim, Jae-Woo; Tang, Qi-Qun; Li, Xi et al. (2007) Effect of phosphorylation and S-S bond-induced dimerization on DNA binding and transcriptional activation by C/EBPbeta. Proc Natl Acad Sci U S A 104:1800-4
Otto, Tamara C; Bowers, Robert R; Lane, M Daniel (2007) BMP-4 treatment of C3H10T1/2 stem cells blocks expression of MMP-3 and MMP-13. Biochem Biophys Res Commun 353:1097-104
Bowers, Robert R; Kim, Jae Woo; Otto, Tamara C et al. (2006) Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: role of the BMP-4 gene. Proc Natl Acad Sci U S A 103:13022-7