| The Macrophage-Specific Transcription Factor Can Be Modified Posttranslationally by Ubiquitination in the Lipopolysaccharide-Treated Macrophages. |
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Jae Woo Jung, Jae Chol Choi, Jae Yeol Kim, In Won Park, Byoung Whui Choi, Jong Wook Shin, John William Christman |
1Divisioin of Allergy, Respiratory and Critical Care Medicine, Department of Internal Medicine, Chung-Ang University College of Medicine, Seoul, Korea. basthma@cau.ac.kr
2Department of Pulmonary, Critical Care and Sleep Medicine, University of Illinois College of Medicine, Chicago, Illinois, USA. |
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| Abstract |
BACKGROUND
Macrophages are one of the most important inflammatory cells in innate immunity. PU.1 is a macrophage-specific transcription factor. Ubiquitins are the ultimate regulator of eukaryotic transcription. The ubiquitination process for PU.1 is unknown. This study investigated the lipopolysaccharide (LPS)-induced activation of PU.1 and its relation to ubiquitins in the macrophages. METHODS: Raw264.7 cells, the primary cultured alveolar, pulmonary, and bone marrow derived macrophages were used. The Raw264.7 cells were treated with MG-132, NH4Cl, lactacytin and LPS. Nitric oxide and prostaglandin D2 and E2 were measured. Immunoprecipitation and Western blots were used to check ubiquitination of PU.1. RESULTS: The PU.1 ubiquitination increased after LPS (1 microg/mL) treatment for 4 hours on Raw264.7 cells. The ubiquitination of PU.1 by LPS was increased by MG-132 or NH4Cl pretreatment. Two hours of LPS treatment on macrophages, PU.1 activation was not induced nor increased with the inhibition of proteasomes and/or lysosomes. The ubiquitination of PU.1 was increased in LPS-treated Raw264.7 cells at 12- and at 24 hours. LPS-treated cells increased nitric oxide production, which was diminished by MG-132 or NH4Cl. LPS increased the production of PGE2 in the alveolar and peritoneal macrophages of wild type mice; however, PGE2 was blocked or diminished in Rac2 null mice. Pretreatment of lactacystin increased PGE2, however it decreased the PGD2 level in the macrophages derived from the bone marrow of B57/BL6 mice. CONCLUSION: LPS treatment in the macrophages ubiquitinates PU.1. Ubiquitination of PU.1 may be involved in synthesis of nitric oxide and prostaglandins. |
| Key Words:
Macrophages, PU.1, ubiquitin, Proteasome, Lysosomes |
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