Osteoarthritis is a degenerative joint disease characterized by a progressive and irreversible loss of the articular cartilage, due in main part to the cleavage of type II collagen within the matrix by the enzyme matrix metalloproteinase (MMP)13. Here, we examined the methylation status of MMP13 promoter and report the demethylation of specific CpG dinucleotides within its promoter in osteoarthritic compared to normal cartilage, which correlates with increased MMP13 expression. Of the promoter CpG sites examined, the -104 CpG was consistently demethylated following treatment of human articular chondrocytes with 10 μM DNA-methyltransferase inhibitor 5-aza-2'-deoxycytidine, again correlating with increased MMP13 expression. Methylation of the -104 CpG site resulted in reduced promoter activity in the chondrosarcoma cell line SW1353 as shown by CpG-free luciferase reporter. Using electrophoretic mobility shift assays, we identified CREB as the regulating factor able to only bind to the MMP13 promoter when the -104 CpG is demethylated, and confirmed this binding by chromatin immunoprecipitation. Finally, we demonstrated that CREB induces MMP13 expression only following treatment of SW1353 with 0.5 μM Ca(2+) ionophore A23187. In summary, the -104 CpG is demethylated in osteoarthritic cartilage, correlating with the elevated MMP13 expression and cartilage destruction, providing a highly novel link between epigenetic status and arthritic disease.

Reactive oxygen species (ROS) are physiological mediators of cellular signaling and play potentially damaging roles in human diseases. In this study, we found that the catalytic activity of the Ser/Thr kinase Aurora A was inhibited by the oxidation of a conserved cysteine residue (Cys290) that lies adjacent to Thr288, a critical phosphorylation site in the activation segment. Cys is present at the equivalent position in ~100 human Ser/Thr kinases, a residue that we found was important not only for the activity of human Aurora A but also for that of fission yeast MAPK-activated kinase (Srk1) and PKA (Pka1). Moreover, the presence of this conserved Cys predicted biochemical redox sensitivity among a cohort of human CAMK, AGC, and AGC-like kinases. Thus, we predict that redox modulation of the conserved Cys290 of Aurora A may be an underappreciated regulatory mechanism that is widespread in eukaryotic Ser/Thr kinases. Given the key biological roles of these enzymes, these findings have implications for understanding physiological and pathological responses to ROS and highlight the importance of protein kinase regulation through multivalent modification of the activation segment.

Poly(adenosine diphosphate ribose) polymerases (PARPs) are multifunctional proteins which play a role in many cellular processes. Namely, PARP1 and PARP2 have been shown to be involved in DNA repair, and therefore are valid targets in cancer treatment with PARP inhibitors, such as rucaparib, currently in clinical trials. Proton magnetic resonance spectroscopy ( H-MRS) was used to study the impact of rucaparib in vitro and ex vivo in liver tissue from mice, via quantitative analysis of nicotinamide adenosine diphosphate (NAD ) spectra, to assess the potential of MRS as a biomarker of the PARP inhibitor response. SW620 (colorectal) and A2780 (ovarian) cancer cell lines, and PARP1 wild-type (WT) and PARP1 knock-out (KO) mice, were treated with rucaparib, temozolomide (methylating agent) or a combination of both drugs. H-MRS spectra were obtained from perchloric acid extracts of tumour cells and mouse liver. Both cell lines showed an increase in NAD levels following PARP inhibitor treatment in comparison with temozolomide treatment. Liver extracts from PARP1 WT mice showed a significant increase in NAD levels after rucaparib treatment compared with untreated mouse liver, and a significant decrease in NAD levels in the temozolomide-treated group. The combination of rucaparib and temozolomide did not prevent the NAD depletion caused by temozolomide treatment. The H-MRS results show that NAD levels can be used as a biomarker of PARP inhibitor and methylating agent treatments, and suggest that in vivo measurement of NAD would be valuable.

Peroxiredoxins (Prdx) utilize reversibly oxidized cysteine residues to reduce peroxides but also to promote H2O2 signal transduction, including H2O2-induced activation of P38 MAPK. Prdx form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here we show that a genetically-encoded fusion between Prdx and the P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. In yeast, we demonstrate that a P38-Prdx fusion protein compensates for the loss of a scaffold protein and upstream MAP3K kinase activity, driving entry into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides a scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx can be sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.

Elevated pro-inflammatory signalling coupled with catabolic metalloproteinase expression is a common feature of arthritis, leading to cartilage damage, deterioration of the joint architecture and the associated pain and immobility. Countering these processes, histone deacetylase inhibitors (HDACi) have been shown to suppress matrix metalloproteinase (MMP) expression, block cytokine-induced signalling and reduce the cartilage degradation in animal models of the arthritis. In order to establish which specific HDACs account for these chondro-protective effects an HDAC1-11 RNAi screen was performed. HDAC6 was required for both the interleukin (IL)-1 induction of MMP expression and pro-inflammatory interleukin expression in chondrocytes, implicating an effect on NF-κB signalling. Depletion of HDAC6 post-transcriptionally up-regulated inhibitor of κB (IκB), prevented the nuclear translocation of NF-κB subunits and down-regulated NF-κB reporter activation. The pharmacological inhibition of HDAC6 reduced MMP expression in chondrocytes and cartilage collagen release. This work highlights the important role of HDAC6 in pro-inflammatory signalling and metalloproteinase gene expression, and identifies a part for HDAC6 in the NF-κB signalling pathway. By confirming the protection of cartilage this work supports the inhibition of HDAC6 as a possible therapeutic strategy in arthritis.