Inhibition of protein kinase C beta II downregulates tristetraprolin expression in activated macrophages.

OBJECTIVE AND DESIGN: Tristetraprolin (TTP) is a 3'-UTR-binding protein known to destabilize mRNAs of TNFalpha and some other cytokines and to act as an anti-inflammatory factor. The aim of this study was to investigate the role of classical protein kinase C isoenzymes (cPKC) in the regulation of TTP expression in activated macrophages. MATERIALS AND METHODS: The expression of TTP in J774 macrophages was induced by a combination of LPS and phorbol myristate acetate (PMA). The effects of cPKC inhibitors and the effects of cPKC activation and downregulation by PMA on TTP protein and mRNA expression were determined by Western blotting and quantitative RT-PCR, respectively. Also, the effect of PKC beta II inhibitor CGP53353 on the activation of transcription factors AP-2, NF-kappaB, EGR1 and Sp1 was assessed. RESULTS: cPKC inhibitors RO318220, GO6976, LY333531 and CGP53353 inhibited LPS and PMA-induced expression of TTP protein and mRNA. Similar effects were obtained when cPKC isoenzymes were downregulated by PMA. In addition, CGP53353 decreased the activation of transcription factor AP-2. CONCLUSIONS: The results suggest that cPKCs, most likely PKC beta II, upregulate TTP expression in activated macrophages. This regulation is possibly mediated through the activation of transcription factor AP-2, and serves as an additional mechanism how PKC beta regulates the inflammatory process.

TGFbeta inhibits IL-1 -induced iNOS expression and NO production in immortalized chondrocytes.

OBJECTIVE: The balance between anti-inflammatory (e.g. TGFbeta) and proinflammatory cytokines (e.g. IL-1 and TNFalpha), regulates destructive processes in OA cartilage. IL-1 and TNFalpha enhance nitric oxide (NO) production in OA cartilage through the inducible nitric oxide synthase (iNOS) pathway and NO mediates many of the destructive effects of these cytokines. The aim of the present study was to investigate the effects of TGFbeta on NO production in immortalized H4 chondrocytes exposed to IL-1. RESULTS: IL-1 induced NO production in chondrocytes through nuclear factor kappa B (NF-kappaB) sensitive and dexamethasone insensitive expression of iNOS. TGFbeta inhibited IL-1 -induced iNOS expression and NO production in chondrocytes, but it did not have any effect on iNOS mRNA levels. iNOS protein levels were similar in cells treated with IL-1 or IL-1+TGFbeta when measured after 8 h incubation, whereas when measured after 12 h and 24 h incubations, iNOS protein levels were 50% and 80% lower in cells treated with IL-1+TGFbeta than in cells treated with IL-1 alone. CONCLUSION: TGFbeta suppressed IL-1-induced iNOS expression and NO production in chondrocytes, probably by enhancing iNOS protein degradation. This finding suggests an additional mechanism for TGFbeta to counteract the destructive effects of IL-1 in OA.