. The molecular events which control the differentiation of pluripotent stem cells into differentiated cells are not yet well understood. One approach has utilized multipotent stem cells grown in culture that can be induced to differentiate. The human promyelocytic leukemia cell line, HL-60, can be induced to differentiate into one of four general cell types by a variety of agents. Treatment of HL60 cells with phorbol esters causes them to differentiate into macrophage-like cells. Phorbol esters like TPA, activate the protein kinase C (PKC) pathway leading to changes in gene expression. Amongst the genes transcriptionally activated are members of the fos and jun gene families, leading to an increase in the transcription factor, AP-1, which is the product of these genes. The working hypothesis of this proposal is that AP-1 induction is important in macrophage differentiation. The principal investigator will determine the level of expression of jun and fos gene family members during HL-60 differentiation and identify the specific components of the induced AP-1 activity. In addition to analyzing the levels of mRNA and protein, phosphorylation states of the proteins will be analyzed by radiolabelling cultured cells followed by immunoprecipitation using Jun and Fos specific antibodies. Correlation of the levels and phosphorylation state of fos and jun to the appearance of AP-1 activity will be achieved by gel retardation experiments and supershift experiments using fos- and jun-specific antibodies. The investigator will also identify the phorbol ester response elements in the jun-B promoter that mediate its transcriptional activation. Characterization of the nuclear factor(s) which bind to this element is a long-term goal of the research. The proposed research will also determine whether phorbol ester-induced HL-60 differentiation strictly correlates with AP-1 production by carefully examining cells treated with a PKC activator which does not induce differentiation and also examining cells which have previously been shown to differentiate in the absence of c-fos induction. Finally, to determine whether AP-1 plays a direct role in macrophage differentiation, cells will be transfected with jun and fos expression clones to see if expression of these genes is sufficient to induce differentiation.
