Regulation of chemokine mRNA stability by lipopolysaccharide and IL-10.
IL-10 has been reported to inhibit the expression of LPS-induced proinflammatory cytokines and chemokines by altering the rate of specific mRNA decay although the molecular target(s) for its action remain unknown. In the present study, using primary peritoneal exudate macrophages and a cell culture model in which a tetracycline-responsive promoter controls transcription of CXC ligand 1 (KC) mRNA, we demonstrate that LPS promotes a time-dependent increase in KC mRNA stability. Although IL-10 had no direct effect on mRNA decay, this treatment antagonized the stabilizing action of LPS. The mechanisms involved were further explored using a cell-free mRNA degradation system. A 5'-capped, polyadenylated in vitro transcript derived from the 3'-untranslated region of KC mRNA exhibited time-dependent decay in the presence of protein extracts prepared from untreated RAW264.7 macrophages. Extracts prepared from LPS-treated RAW264.7 cells had reduced decay activity and this change was antagonized if the cells were costimulated with IL-10. A substrate in which the AU-rich element motifs were mutated exhibited minimal decay that did not vary using extracts prepared from cells treated with LPS or LPS and IL-10. A nonadenylated RNA substrate was also degraded and that activity was diminished by LPS. In concert, these findings demonstrate that KC mRNA stability is regulated by LPS-induced alterations in activities that govern both deadenylation and degradation of the mRNA body. The effects of IL-10 on KC mRNA stability reflect antagonism of the response to LPS.
Biswas
Roopa
R
Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Datta
Shyamasree
S
Gupta
Jaydip Das
JD
Novotny
Michael
M
Tebo
Julie
J
Hamilton
Thomas A
TA
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D002478:Cells, Cultured; D054360:Chemokine CXCL1; D018925:Chemokines / Q000235:genetics* / Q000378:metabolism*; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004306:Dose-Response Relationship, Immunologic; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D016753:Interleukin-10 / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000276:immunology / Q000378:metabolism; D051379:Mice; D020871:RNA Stability / Q000276:immunology*; D014162:Transfection
D054360:Chemokine CXCL1; D018925:Chemokines; D019743:Chemokines, CXC; D002630:Chemotactic Factors; C475895:Cxcl1 protein, mouse; D016207:Cytokines; D036341:Intercellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D016753:Interleukin-10; C074302:keratinocyte-derived chemokines