Genetic determination of the role of PU.1 in macrophage gene expression.
PU.1, an Ets family transcription factor, mediates macrophage effector function in inflammation by regulating gene expression. But, the extent and nature of PU.1 function in gene expression has not been genetically determined because ablation of PU.1 gene abolishes macrophage development. Here, we epigenetically suppressed PU.1 by stably expressing PU.1 specific siRNA in macrophages, and determined the effect of PU.1 deficiency on expressions of key inflammatory genes: Toll-like receptor 4 (TLR4), cyclooxygenase-2 (COX-2), and macrophage inflammatory protein-1alpha (MIP-1alpha). PU.1-silenced cell lines expressed lower TLR4 mRNA and COX-2 protein, but higher MIP-1alpha protein, than controls. Over-expression of PU.1 suppressed lipopolysaccharide-induced MIP-1alpha production. PU.1 occupied proximal and distal cognate sites in the endogenous MIP-1alpha promoter, but dissociated only from the distal sites in response to lipopolysaccharide, suggesting a novel negative regulatory mechanism by PU.1. Together, our results defined PU.1 function in differentially regulating expressions of TLR4, COX-2, and MIP-1alpha.
Joo
Myungsoo
M
Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, B1222 MCN, Nashville, TN 37232-2650, USA. Myungsoo.Joo@vanderbilt.edu
Kwon
Minjae
M
Azim
Anser C
AC
Sadikot
Ruxana T
RT
Blackwell
Timothy S
TS
Christman
John W
JW
D000818:Animals; D002460:Cell Line; D054405:Chemokine CCL3 / Q000235:genetics*; D051546:Cyclooxygenase 2 / Q000235:genetics*; D005786:Gene Expression Regulation; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051197:Toll-Like Receptor 4 / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D015854:Up-Regulation; D000099232:Proto-Oncogene Protein Spi-1
D054405:Chemokine CCL3; D051546:Cyclooxygenase 2; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D051197:Toll-Like Receptor 4; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C495347:Tlr4 protein, rat; C077623:Ptgs2 protein, mouse