Commitment is the process of restriction of cell fate from multiple differentiation potentials to a single differentiation pathway. Little is known about the genes which are involved in the commitment process, or the signaling pathways that regulate them. In the bone marrow, multipotential mesechymal cells can undergo commitment to the adipocyte or the osteoblast lineage. In conditions of reduced bone strength, such as osteoporosis, immobilization, or in aging, fat in the bone marrow increases as the number of osteoblasts decreases, suggesting a reciprocal relationship in the numbers of fat and bone cells in this system. Thus, elucidation of the molecular basis of the commitment decision process between adipocyte and osteoblast may provide a basis for development of therapeutics targeted at improving bone health. Previous research in our lab has demonstrated that it is possible to derive fully differentiated osteoblasts from committed preadipocytes by a combination of bone morphogenetic protein (BMP) and retinoic acid (RA) signaling. This application builds upon these findings and proposes to characterize the global patterns of gene expression that accompany adipocyte commitment, as well as the switch in commitment mediated by BMP/RA.
Aim 1 is to examine differential gene expression patterns in the committed preadipocyte cell line 3T3-F442A vs. the matched, uncommitted fibroblast line 3T3-C2, as well as their immediate/early responses to BMP and/or RA treatment, by microarray analysis. Genes will be clustered by similar expression patterns and subjected to bio-informatics analysis/phylogenetic footprinting to determine possible common promoter sequence elements. Genes clustered by functions will allow assessment of the kinds changes in biological processes accompanying adipocyte commitment.
Aim 2 is to functionally dissect the BMP and RA signaling pathways to determine the basis for cooperation between them that results in commitment switching. Individual components of each signaling pathway will be inactivated by expression of dominant negative proteins, RNAi, or pharmacological inhibitors. The corresponding effect on the ability to respond to BMP/RA by undergoing commitment switching will be tested, and compared to the corresponding gene expression profile assessed by microarray. Together, these experiments should identify candidate adipocyte commitment genes suitable for continued investigation of their functions and role in commitment regulation.
