Resveratrol: Proteasome Inhibitor and Immunomodulator

Abstract

IntroductionProteasomes (P) are large protein complexes which controls a variety of physiological and cellular processes by regulating selective degradation of proteins. The catalytic activities of P mainly reside in three subunits: X (β5), Y (β1) and Z (β2) which corresponds to chymotrypsin‐like (CT), post‐acidic (PA) and trypsin‐like (T) activities respectively. After priming with IFNγ or LPS; X (β5), Y (β1) and Z (β2) subunits can be induced to LMP7 (β5i), LMP2 (β1i) and LMP10 (β2i) immunoproteasome (IP) subunits. Human monocytes mainly possess IP subunits. We have previously reported that naturally‐occurring compounds such as trans‐resveratrol, trans‐pterostilbene, morin hydrate, and nicotinic acid inhibit chymotrypsin‐like, trypsin‐like, and post‐acidic activities, as well as TNF‐α and nitric oxide (NO). Recently, proteasome inhibitors (PIs) are being used for therapeutic interventions in chronic inflammatory diseases such as arthritis.ObjectivesIn the current study, we explored the immunomodulatory effects of non‐toxic, and naturally occurring trans‐resveratrol (Res) on human blood monocytes.MethodsMononuclear CD14+ cells were separated from blood by mixing monocyte enrichment cocktail and granulocyte depletion cocktail. After dilution, blood sample was layered on top of the Ficoll‐Paque PLUS and centrifuged to separate enriched CD14+ cells. Western blot, ELISA and RT‐PCR was performed to investigate the effect of resveratrol on expression of different proteins. Purified human erythrocyte 20S P and peripheral blood monocyte 20S IP (R&D Systems) were assayed for different peptidase activities using fluorogenic tri‐and tetra‐peptide substrates.ResultsRes (0–100μM) inhibited all three protease activities of purified human blood P and IP, although predominantly IP. Moreover, Res was more specific for CT‐like activity as compared to PA and T‐like. Western blot analysis revealed the accumulation of ubiquitinated proteins after 4h stimulation of CD14+ monocytes with resveratrol. In addition, Res inhibited LPS‐induced expression of ICAM1, resistin and TNF‐α. LPS‐induced activation of TNF‐α which is primarily controlled by nuclear factor NF‐κB, largely depends on phosphorylation dependent ubiquitination and degradation of inhibitor of kappa B (IκB) proteins. Therefore, we performed immunoblotting of phosphorylated IkBα and IkBα proteins after lysis of CD14+ monocytes treated with Res and LPS. We detected that 30 min incubation with LPS alone, increased cytoplasmic pIκBα protein and decreased IκBα protein levels. However, preincubation with Res for 60 min inhibited the degradation of pIκBα and decreased IκBα protein levels.ConclusionCollectively, our data demonstrates that Res inhibits proteasome activity, thereby, affecting inflammatory responses in human monocytes partly via degradation of Ik‐B and reduced NF‐kB activation hence can effectively be used in various inflammatory diseases like arthritis, atherosclerosis, sepsis, and neurodegeneration.Support or Funding InformationThis study was supported in part by grants R01‐GM 102631 (NQ), and R01‐GM102631S1 (NQ) from NIH, NIGMS