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LPS-Induced Formation of Immunoproteasomes: TNF-α and Nitric Oxide Production are Regulated by Altered Composition of Proteasome-Active Sites

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Abstract

Stimulation of mouse macrophages with LPS leads to tumor necrosis factor (TNF-α) secretion and nitric oxide (NO) release at different times through independent signaling pathways. While the precise regulatory mechanisms responsible for these distinct phenotypic responses have not been fully delineated, results of our recent studies strongly implicate the cellular cytoplasmic ubiquitin–proteasome pathway as a key regulator of LPS-induced macrophage inflammatory responses. Our objective in this study was to define the relative contribution of specific proteasomal active-sites in induction of TNF-α and NO after LPS treatment of RAW 264.7 macrophages using selective inhibitors of these active sites. Our data provide evidence that LPS stimulation of mouse macrophages triggers a selective increase in the levels of gene and protein expression of the immunoproteasomes, resulting in a modulation of specific functional activities of the proteasome and a corresponding increase in NO production as compared to untreated controls. These findings suggest the LPS-dependent induction of immunoproteasome. In contrast, we also demonstrate that TNF-α expression is primarily dependent on both the chymotrypsin- and the trypsin-like activities of X, Y, Z subunits of the proteasome. Proteasome-associated post-acidic activity alone also contributes to LPS-induced expression of TNF-α. Taken together; our results indicate that LPS-induced TNF-α in macrophages is differentially regulated by each of the three proteasome activities. Since addition of proteasome inhibitors to mouse macrophages profoundly affects the degradation of proteins involved in signal transduction, we conclude that proteasome-specific degradation of several signaling proteins is likely involved in differential regulation of LPS-dependent secretion of proinflammatory mediators.

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Acknowledgments

These studies were supported in part by NIH Grants GM-50870 (NQ), AI-18797 (SNV), and CA-124634 (AFK). We thank Drs. Alfred L. Goldberg and John J. Monaco for helpful suggestions. We thank Dr. Peter Silverstein for help with the real-time RT-PCR of proteasome subunits in macrophages.

Conflict of interest

The authors have no financial conflict of interest.

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Authors and Affiliations

  1. Department of Basic Medical Science, Shock/Trauma Research Center, School of Medicine, University of Missouri, 2411 Holmes Street, Kansas City, MO, 64108, USA

    Julia Reis, Xiu Qin Guan, Christopher J. Papasian, Asaf A. Qureshi, David C. Morrison & Nilofer Qureshi

  2. Department of Surgery, Shock/Trauma Research Center, University of Missouri, Kansas City, MO, 64108, USA

    Charles W. Van Way III

  3. Departments of Pharmacology and Toxicology, Pharmacy, University of Missouri, Kansas City, MO, 64108, USA

    Julia Reis & Nilofer Qureshi

  4. Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, NH, 03756, USA

    Alexei F. Kisselev

  5. Department of Microbiology and Immunology, University of Maryland, Baltimore, MD, 21201, USA

    Stefanie N. Vogel

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  1. Julia Reis
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Correspondence to Nilofer Qureshi.

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Reis, J., Guan, X.Q., Kisselev, A.F. et al. LPS-Induced Formation of Immunoproteasomes: TNF-α and Nitric Oxide Production are Regulated by Altered Composition of Proteasome-Active Sites. Cell Biochem Biophys 60, 77–88 (2011). https://doi.org/10.1007/s12013-011-9182-8

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