Transcription-independent turnover of I kappa B alpha during monocyte adherence: implications for a translational component regulating I kappa B alpha/MAD-3 mRNA levels.
We identified I kappa B alpha/MAD-3 as an immediate-early gene in human monocytes that is expressed in response to a variety of signals, including adhesion, lipopolysaccharide, and phorbol myristate acetate. Within 5 min of monocyte adhesion, the level of the I kappa B alpha protein is markedly diminished but is rapidly replaced in a cycloheximide-sensitive manner within 20 min. Accompanying the rapid turnover of the I kappa B alpha protein is simultaneous translocation of NF-kappa B-related transcription factors to nuclei of adhered monocytes. The demonstration that NF-kappa B can regulate I kappa B alpha/MAD-3 gene transcription in other cell types suggested that the rapid increase in steady-state I kappa B alpha/MAD-3 mRNA levels we observed within 30 min of monocyte adherence would result from NF-kappa B-dependent transcriptional stimulation of the I kappa B alpha/MAD-3 gene. Nuclear run-on analyses indicated that, instead, while several immediate-early cytokine genes, such as the interleukin 1 beta (IL-1 beta) gene, were transcriptionally activated during monocyte adhesion, the rate of I kappa B alpha/MAD-3 gene transcription remained constant. The adherence-dependent increase in I kappa B alpha/MAD-3 mRNA levels was also not a consequence of mRNA stabilization events. Interestingly, while increases in both IL-1 beta and I kappa B alpha/MAD-3 mRNA levels were detected in nuclei of adherent monocytes, cytoplasmic levels of IL-1 beta mRNA increased during adherence whereas those of I kappa B alpha/MAD-3 mRNA did not. Taken together, our data suggest that two interactive mechanisms regulate monocytic I kappa B alpha/MAD-3 mRNA levels. We propose that adherent monocytes regulate nuclear processing (or decay) of I kappa B alpha/MAD-3 mRNA, thereby increasing mRNA levels without stimulating I kappa B alpha/MAD-3 gene transcription. Moreover, since inhibition of protein synthesis leads to accumulation of I kappa B alpha/MAD-3 mRNA without stimulating I kappa B alpha/MAD-3 gene transcription, we suggest that low cytoplasmic levels of I kappa B alpha/MAD-3 mRNA are maintained by a translation-dependent degradation mechanism.
Lofquist
A K
AK
UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599-7295.
Mondal
K
K
Morris
J S
JS
Haskill
J S
JS
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015153:Blotting, Western; D002448:Cell Adhesion; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D020672:I-kappa B Proteins; D019012:Integrin beta1; D016023:Integrins / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D007700:Kinetics; D009000:Monocytes / Q000378:metabolism / Q000502:physiology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D019012:Integrin beta1; D016023:Integrins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D012333:RNA, Messenger; D000072000:NF-KappaB Inhibitor alpha