Stimulation of T and B cells by various agents is followed by expression of the GM-CSF gene in these cells. In T cells, we have shown that the expression of the GM-CSF gene is post-transcriptionally controlled mainly through stabilization of the mRNA. The mechanism of mRNA degradation and stabilization is being examined using murine EL-4 thymoma cells as a model system for T cells. Induction of GM-CSF mRNA in these cells by phorbol esters (TPA) and lectins has been shown to occur by stabilization of the mature transcript in the cytoplasm. Using CAT constructs in which portions of the GM-CSF 3' UTR have been substituted for the SV40 poly A adenilation signals and transfections of EL-4 and NIH3T3 cells, we have determined which sequences in the GM-CSF mRNA regulate degradation and stabilization. The AU-rich elements in the 3' UTR are principally responsible for mRNA degradation and do not function in a cell-specific manner. mRNA stabilization, on the other hand, is not mediated by the AU- rich elements, and is highly cell-specific. Using PCR, we have produced linker-substitution mutations of the GM-CSF 3' UTR and have mapped the TPA-responsive elements of the 3' UTR to a 60 bp region which is 160 bases upstream from the AU rich area. The mechanism of GM-CSF gene expression in B cells was investigated in 2 murine B cell lines. IL1 stimulated GM-CSF production in the M12.4.1 lymphoma and Th2.2 hybridoma lines in a dose-and-time-dependent fashion. Nuclear run-on transcription assays revealed constitutive transcription of the GM-CSF gene in unstimulated M12.4.1 cells that did not change after IL1 treatment. By using PCR combined with actinomycin D chase experiments we showed that IL1 increased GM-CSF mRNA stability in the cells, indicating that GM-CSF gene expression in murine B cell lines can be induced by IL1 and that this event is post-transcriptionally controlled mainly at the level of mRNA stabilization.
