Differential loading of proinflammatory transcripts onto polysomes following stimulation of cells by TNF‐alpha

Inflammatory mediators such as TNF‐alpha activate signaling pathways that lead to the rapid transcriptional activation of a large number of proinflammatory genes. Although transcriptional profiling of total RNA identifies and quantifies specific proinflammatory transcripts, the method does not distinguish between transcripts that are loaded onto polysomes and those that are not. We asked whether translational efficiency, as determined by polysomal loading, plays a part in the early transcriptional responses to inflammatory mediators. We treated Hep3B liver hepatoma cells with TNF‐alpha, which resulted in the rapid activation of stress‐activated protein kinases, JNK and p38, as well as NF‐kappaB. One hr after the addition of TNF‐alpha, cells were harvested and extracts were separated by sucrose gradient centrifugation, and gradients were fractionated into polysomal and non‐polysomal regions. RNA was extracted and subjected to Affymetrix microarray analysis to determine the transcriptional profiles of RNA that was associated, or not associated, with polysomes. As expected, multiple gene products known to be associated with the inflammatory response were identified in cells exposed to TNF‐alpha. Some transcripts were efficiently loaded onto polysomes, whereas others were not. These data suggest the existence of mechanisms that differentially regulate the translational efficiency of proinflammatory transcripts. 1RO1 A1059335‐02 NIH/NIAID 1RO1 DK066439 ‐01A1 NIH/NIDDK