Tristetraprolin Regulates CXCL1 (KC) mRNA Stability

Abstract

mRNAs encoding proinflammatory chemokines are regulated posttranscriptionally via adenine-uridine-rich sequences (AREs) located in the 3′ untranslated region of the message, which are recognized by sequence-specific RNA-binding proteins. One ARE binding protein, tristetraprolin (TTP), has been implicated in regulating the stability of several ARE-containing mRNAs, including those encoding TNF-α and GM-CSF. In the present report we examined the role of TTP in regulating the decay of the mouse chemokine KC (CXCL1) mRNA. Using tetR-regulated control of transcription in TTP-deficient HEK293 cells, KC mRNA half-life was markedly decreased in the presence of TTP. Deletion and site-specific mutagenesis were used to identify multiple AUUUA sequence determinants responsible for TTP sensitivity. Although a number of studies suggest that the destabilizing activity of TTP is subject to modulation in response to ligands of Toll/IL-1 family receptors, decay mediated by TTP in 293 cells was not sensitive to stimulation with IL-1α. Using primary macrophages from wild-type and TTP-deficient mice, KC mRNA instability was found to be highly dependent on TTP. Furthermore, LPS-mediated stabilization of KC mRNA is blocked by inhibition of the p38 MAPK in macrophages from wild-type but not TTP-deficient mice. These findings demonstrate that TTP is the predominant regulator of KC mRNA decay in mononuclear phagocytes acting via multiple 3′-untranslated region-localized AREs. Nevertheless, KC mRNA remains highly unstable in cells that do not express TTP, suggesting that additional determinants of instability and stimulus sensitivity may operate in cell populations where TTP is not expressed.

Genetic Regulation ofZfp30, CXCL1, and Neutrophilic Inflammation in Murine Lung

“…ZFP30 is a C2H2-type zinc finger that contains a Krupple-associated box (KRAB) domain, which are known to repress gene expression (Witzgall et al 1994), perhaps through the induction of heterochromatin (Groner et al 2010). Zfp30 expression was negatively associated with CXCL1 protein (and PMN) but was not associated with lung Cxcl1 RNA in preCC mice (not shown), suggesting that Cxcl1 RNA is not a direct target of ZFP30 (in contrast to ZFP36, tristetraprolin, which is well-known regulator of Cxcl1 RNA levels; Datta et al 2008). The targets and sequence specificity of ZFP30 are clearly important questions to address in the future and will be necessary to understanding how variation in this gene affects immune response.…”

Suppression of Lipopolysaccharide-stimulated Tumor Necrosis Factor-α Production by Adiponectin Is Mediated by Transcriptional and Post-transcriptional Mechanisms

“…TTP was immunoprecipitated from the cell lysates by overnight incubation with 10 g/ml anti-TTP antibody followed by the addition of 50 l of protein G-agarose beads. The beads were then washed, and RNA was purified (35,36). The total RNA sample was reverse transcribed, and 5% of the total reverse transcribed product was used for PCR.…”

“…Cells were stimulated or not with 100 ng/ml LPS during the last 2 h of gAcrp exposure. Cells were removed from the cell culture dish, washed twice with cold phosphate-buffered saline, and fixed in 0.1% formaldehyde, and lysates were prepared as described previously (35,36). TTP was immunoprecipitated from the cell lysates by overnight incubation with 10 g/ml anti-TTP antibody followed by the addition of 50 l of protein G-agarose beads.…”

MAPKAP Kinase 2 Blocks Tristetraprolin-directed mRNA Decay by Inhibiting CAF1 Deadenylase Recruitment

“…p38 MAPK also regulates mRNA stability by direct phosphorylation and inactivation of another ARE-binding protein, KH domain-splicing regulatory protein (10,11). p38 MAPK inhibitors fail to destabilize mRNAs of the inflammatory response in macrophages from TTP Ϫ/Ϫ mice (8,12). Thus, it appears that in these cells, at least, TTP is entirely responsible for the effects on mRNA stability mediated by the p38 MAPK pathway.…”