Novel insights into the cellular mechanisms of the anti-inflammatory effects of NF-kappaB essential modulator binding domain peptides.

The classical nuclear factor kappaB (NF-kappaB) signaling pathway is under the control of the IkappaB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-kappaB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-kappaB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of kappaB alpha (IkappaB alpha). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.

Protein geranylgeranylation controls collagenase expression in osteoarthritic cartilage.

OBJECTIVE: Statins possess anti-inflammatory properties. This study was undertaken to characterize the mechanism of action of statin drugs on collagenase expression in primary human osteoarthritic cartilage tissue. METHOD: Human articular chondrocytes and cartilage explants from osteoarthritic donors were exposed to simvastatin in the presence or absence of interleukin-1 beta (IL-1beta). Collagenase expression was determined by quantifying levels of matrix metalloproteinase 13 (MMP-13) and MMP-1 mRNA and MMP-13 protein. The mechanism of statin action was tested by addition of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) or by using inhibitors of farnesyl transferase (FT) and geranylgeranyl transferase (GGT-1). RESULTS: Treatment of osteoarthritic chondrocytes with simvastatin decreased mRNA levels of MMP-13 and MMP-1 whether under basal conditions or during stimulation with IL-1beta. MMP-13 protein secreted into the culture media was also decreased. Genes involved in cartilage synthesis (type II collagen and aggrecan) were not down-regulated by simvastatin. Exogenous addition of GGPP completely reversed the statin-mediated decrease in MMP-13 mRNA and protein levels whereas FPP partially reversed the statin-mediated effect. An inhibitor of GGT-1 mimicked the simvastatin-mediated reduction in MMP-13 expression by chondrocytes. Finally, consistent with impacts on MMP-13 and MMP-1 expression, simvastatin as well as the GGT-1 inhibitor both blocked type II collagen degradation in primary human articular cartilage explants. CONCLUSION: These results suggest that statins modulate chondrocyte metabolism by reducing prenylation of key signaling molecules that control the expression of collagen-degrading enzymes. Our results strongly support the hypothesis that protein prenyltransferases including geranylgeranyl transferase regulate chondrocyte collagenase expression in osteoarthritis.