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.
