The proposed studies focus on one aspect of the complex mechanism by which retinoic acid (RA) regulates cell growth and differentiation. RA is the active metabolite of a dietary factor, vitamin A, necessary for proper development in juveniles; a morphogen during embryogenesis; and a chemotherapeutic agent regulating growth and differentiation of neoplastic cells. The studies utilize HL-60 human leukemia cells, an uncommitted hematopoietic precursor cell that can undergo myeloid or monocytic differentiation and GO arrest. RA causes MAPK signaling dependent myeloid differentiation and GO arrest of these cells. The studies will determine the contribution of RA-induced expression of selected membrane receptors and adapter signaling molecules to the unanticipated activation of MAPK signaling and to the diversity of gene expression changes caused by RA. They thus focus on two basic biological questions: 1) what is the significance of MAPK signaling in Ra-induced growth arrest and differentiation when such signaling is usually considered the prototypical signal for mitogenesis; 2) how are the diverse sequential gene expression changes needed to evoke growth arrest and differentiation generated by RA. Searches by our laboratory, as well as others, using a combination of differential display, a proteomics based differential search, or querying candidate molecules revealed that RA induces the expression of four receptors, c-FMS, BLR1, CD32, and CD38, which differ significantly in character. The downstream signaling consequences of these receptors are thus likely to differ and regulate the expression of different ensembles of genes. However, a common feature of these receptors is that all have been implicated with utilizing MAPK signaling. RA also induces the expression of several adapter molecules, in particular paxillin, SLP-76, and dok. These, too, have been connected with MAPK signaling and in some cases explicitly to some of the aforementioned receptors. The working hypothesis is that RA induces the expression of cell surface receptors, whose signaling is regulated by adapter molecules, resulting in MAPK signal activation and consequential changes in protein expression ultimately leading to GO arrest and differentiation. Different combinations of receptors and adapters then direct changes in the expression of different ensembles of proteins, each combination contributing to the total changes attributed to RA. The experimental aims are to ectopically express these receptors and adapters, singly and in combination, to determine their ability to stimulate MAPK signaling and cause gene expression changes that contribute to the total RA-induced changes, and to test the functional significance of their induced expression to RA-induced differentiation or GO arrest.
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