Effect of TNFR1 and TNFR2 on the stability of GM-CSF mRNA.

BMSCs were prepared as described in “Materials and methods” and were stimulated with 1 μg/mL LPS for 2 hours. After that, actinomycin D (5 μg/mL) was added, and the cells were harvested for RNA at the indicated times. Northern blots from these experiments are shown at the top, with the 2 species of GM-CSF mRNA and the single species of cyclophilin indicated. On the bottom is the PhosphorImager quantitation of the GM-CSF mRNA levels (total hybridizable mRNA, ie, both species together) after normalization to cyclophilin mRNA levels. The data are expressed as a percentage of the value at time 0, which was assigned the value of 100%. In the presence of TTP (TNFR1KO, TNFR2KO), GM-CSF mRNA appeared as a typical doublet of 0.8 and 1 kb. However, when TTP was absent (TTP/TNFR1KO, TTP/TNFR2KO), the larger species appeared to be predominant. Note also that in the absence of TTP, the stability of the mRNA was increased, particularly when both TTP and TNFR2 were absent. The experiment shown here is representative of 2 independent experiments with cells derived from different groups of animals.