Effect of proteasome inhibitors on monocytic IκB-α and -β depletion, NF-κB activation, and cytokine production

Abstract

Abstract:We investigated the effect of proteasome inhibitors on the lipopolysaccharide (LPS)-induced expression of several monocytic cytokines, which may be dependent on the transcription factor, nuclear factor-B (NF-B). Exposure of human monocytic THP-1 cells to ALLN and Mu873 prevented the LPS-induced degradation of I B-␣ and -␤, as did the more potent proteasome inhibitor, PSI, whereas several calpain inhibitors were ineffective. This was accompanied by the inhibition of nuclear NF-B binding activity and NF-B transcriptional activation. At the mRNA level, the inhibitors blocked the expression of tumor necrosis factor (TNF) and interleukin-1␤ (IL-1␤), whereas IL-8 remained unaffected by ALLN and was only partially reduced by the highest dose of PSI. The latter effect appears to be due to an increase in IL-8 mRNA stability in the presence of proteasome inhibitors. Furthermore, the production of TNF was efficiently suppressed by ALLN and PSI, less by Mu873, and not at all by calpain inhibitors. In primary human blood monocytes ALLN also prevented the LPS-induced degradation of I B-␣ and -␤, efficiently blocked the production of TNF and, to a lesser extent, IL-1␤, whereas that of IL-8 was not inhibited. The expression of NF-B-dependent monocytic cytokines may be selectively controlled by the proteasome, offering a potential therapeutic target in inflammatory disease.

Antifibroblast antibodies in systemic sclerosis induce fibroblasts to produce profibrotic chemokines, with partial exploitation of toll‐like receptor 4

“…Nuclear translocation of NF-B is dependent on the degradation by proteasome of its inhibitor, IB (46), and we have observed that AFAs induce NF-B nuclear accumulation. Thus, in agreement with the literature, our data are consistent with the requirement of the role of NF-B in CXCL8 and CCL2 gene transcription (47). In addition, since JNK inhibition resulted in the selective abrogation of CCL2 production, our data indicate that in dermal fibroblasts, CCL2 production also requires the transcription factor activator protein 1 (AP-1).…”

Differential Effects of Lipopolysaccharide and Tumor Necrosis Factor on Monocytic IκB Kinase Signalsome Activation and IκB Proteolysis

“…Polyacrylamide Gel Electrophoresis and Western Blot Analysis-Cytosolic extracts were isolated as described earlier (42). Electrophoresis was performed with 12.5% polyacrylamide gels (0.1% SDS) as described previously (40).…”

“…Transfection of THP-1 Cells-The following reporter plasmids were utilized in transfection studies: 3xB.luci, a firefly luciferase reporter plasmid containing three copies of a prototypic (5Ј-GGGACTTTCC-3Ј) B site (42); TNFkop.luci, comprising 1108 base pairs of the TNF promoter region (42); and pGL2-IL-8, containing 420 base pairs of the IL-8 promoter region (44). Overexpression plasmids used in our studies included IKK-␣, IKK-␤, NIK (wild type and mutated forms), IKAP (all gifts from Tularik Inc., South San Francisco, CA), NEMO, IB-⑀ (wild type and mutated), IB-␤ (gifts from Prof. Alain Israël and co-workers, Institut Pasteur), and antisense IKK-␣ (a gift from Prof. Michael Karin, University of California-San Diego, La Jolla, CA).…”

Inhibition of Lipopolysaccharide-Induced Signal Transduction in Endotoxin-Tolerized Mouse Macrophages: Dysregulation of Cytokine, Chemokine, and Toll-Like Receptor 2 and 4 Gene Expression

“…min with constant shaking. Luc and ␤-gal activities were measured as described (57), and data were presented as a ratio of Luc relative light units divided by ␤-gal relative light units, according to Haas et al (58).…”