Toll-Like Receptor-2 Ligand Peptidoglycan Upregulates Expression and Ubiquitin Ligase Activity of CHIP through JNK Pathway

Abstract

Background: Peptidoglycan (PGN) is a component of cell wall in Gram-positive bacteria that stimulates inflammatory responses through Toll-like receptor 2 (TLR2). The carboxyl terminus of constitutive heat shock cognate 70 (HSC70)-interacting protein (CHIP, also known as Stub1) is a U-box-type E3 ubiquitin ligase, which plays an important role in protein quality control and inflammation through ubquitin-mediated proteasomal degradation. However, it is unclear whether TLR2 agonist PGN regulates the expression and activation of CHIP. Methods/Results: In this study, we showed that PGN significantly up-regulated the expression of CHIP in both mRNA and protein levels in RAW264.7 cells in a time-dependant manner, and the expression of CHIP induced by PGN was abolished in TLR2 knockout macrophages. No significant change in CHIP was observed after lipopolysaccharide (LPS, TLR4 agonist) and cytosine-phosphorous-guanine oligonucleotide (CpG ODN, TLR9 agonist) treatment. Moreover, PGN markedly induced the expression and activity of CHIP in macrophages, whereas this effect was attenuated by SP600125, a selective inhibitor of JNK. Conclusion: Our study for the first time demonstrates that TLR2 activation enhances the expression and activity of CHIP through JNK signaling pathway.

The effects of an ActRIIb receptor Fc fusion protein ligand trap in juvenile simian immunodeficiency virus‐infected rhesus macaques

“…Myostatin has been reported to activate JNK1 in muscle of aging mice (42). Interestingly, JNK activation is involved in up‐regulation of proteasomal activity (43) and increased proteasomal protein degradation (44). This is in line with our findings of the reduced proteasomal activity in skeletal muscle of ActRIIB.Fc‐treated animals.…”

Ubiquitin‐activating enzyme E1 inhibitor PYR41 attenuates angiotensin II‐induced activation of dendritic cells via the IκBa/NF‐κB and MKP1/ERK/STAT1 pathways

“…For immunoprecipitation assays, cell lysates were pre‐cleared with protein A/G plus agarose for 1 hr at 4° and then 2 μg of anti‐TRAF6 or NEMO and 30 μl protein A/G agarose was added and incubated overnight at 4° to capture primary immune complexes. The beads were washed and fractionated by 10% SDS–PAGE followed by immunoblotting as previously described …”

“…The beads were washed and fractionated by 10% SDS-PAGE followed by immunoblotting as previously described. 17,18 RNA extraction and quantitative real-time PCR Total RNA was isolated from DCs using Trizol reagent according to the manufacturer's instructions. First-strand cDNA synthesis was performed with 1 lg of total RNA in a reaction volume of 20 ll using PrimeScript â RT reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan).…”

The E3 Ligase CHIP: Insights into Its Structure and Regulation

“…Treatment of RAW264.7 cells by peptidoglycan activates TLR2 receptors and through JNK pathway upregulates expression of CHIP. Interestingly, although CHIP is crucial for proper TLR2/4/7/9 signaling, its expression seems to be controlled by only TLR2 signaling as LPS (TLR4 ligand) or CpG ODN (TLR9 ligand) does not stimulate endogenous CHIP overexpression [37, 38] (Figure 2(c)). …”