H2O2 oxidation of cysteine residues in c-Jun N-terminal kinase 2 (JNK2) contributes to redox regulation in human articular chondrocytes

Abstract

Reactive oxygen species (ROS), in particular HO, regulate intracellular signaling through reversible oxidation of reactive protein thiols present in a number of kinases and phosphatases. HO has been shown to regulate mitogen-activated protein kinase (MAPK) signaling depending on the cellular context. We report here that in human articular chondrocytes, the MAPK family member c-Jun N-terminal kinase 2 (JNK2) is activated by fibronectin fragments and low physiological levels of HO and inhibited by oxidation due to elevated levels of HO The kinase activity of affinity-purified, phosphorylated JNK2 from cultured chondrocytes was reversibly inhibited by 5-20 μm HO Using dimedone-based chemical probes that react specifically with sulfenylated cysteines (RSOH), we identified Cys-222 in JNK2, a residue not conserved in JNK1 or JNK3, as a redox-reactive site. MS analysis of human recombinant JNK2 also detected further oxidation at Cys-222 and other cysteines to sulfinic (RSOH) or sulfonic (RSOH) acid. HO treatment of JNK2 resulted in detectable levels of peptides containing intramolecular disulfides between Cys-222 and either Cys-213 or Cys-177, without evidence of dimer formation. Substitution of Cys-222 to alanine rendered JNK2 insensitive to HO inhibition, unlike C177A and C213A variants. Two other JNK2 variants, C116A and C163A, were also resistant to oxidative inhibition. Cumulatively, these findings indicate differential regulation of JNK2 signaling dependent on HO levels and point to key cysteine residues regulating JNK2 activity. As levels of intracellular HO rise, a switch occurs from activation to inhibition of JNK2 activity, linking JNK2 regulation to the redox status of the cell.

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“…Depending on the cell type and experimental conditions, JNK signaling has been found to either promote cell survival or cell death . We previously showed that, under conditions of oxidative stress in cultured human chondrocytes, JNK is inactivated while the p38 MAP kinase remains active . In further studies, we demonstrated that prolonged oxidative stress induced chondrocyte death, which was blocked when cells were treated with a p38 inhibitor .…”

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“…6). The less effective treatment in inducing apoptosis (PAM + Pyr) produces signals related to sublethal levels of oxidative stress and proliferation such as c-JUN 65 and AKT 64 . It should be underlined that treatment with PAM (+/− Pyr) as well as with H 2 O 2 , affects proteins related to mitochondrial stress and apoptosis like HSP60 66 , or STAT3 pathway 67 .…”

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“…For instance, in human chondrocytes, while sublethal hydrogen peroxide concentrations (0.5 and 1 µM) activated JNK2, on the contrary, lethal concentrations (higher than 10 µM) decreased its activity. 52 Conversely, in hepatocytes, low hydrogen peroxide concentrations (5–10 μM) stimulated cell proliferation through Akt, whereas high concentrations (20 μM) decreased Akt activation and instead activated JNK. 119 In human uterosacral ligament fibroblasts, sublethal doses (200 and 400 µM) induced collagen synthesis through TGF-β, whereas high doses reduced TGF-β signaling and led to tissue degeneration.…”

“…While the first one is mainly involved in cell growth, survival, and differentiation, depending on target tissue, the other two also trigger inflammatory and apoptotic processes. 49 Depending on cell type, low oxidative stress activated p38, 50,51 JNK, 4,50,52,53 canonical MAPK Erk, 51,54–56 Erk5 (also known as big MAPK1 [BMK1]), 57,58 or even JNK2α2, a rare member of the noncanonical JNK family, which in turn stimulated Erk1/2 signaling. 59 ROS-mediated MAPK activation generally resulted in increased cell migration 5,58,60 and proliferation, 51,58–62 as it upregulated cyclin D 1 , thereby facilitating G1/S transition.…”

“…While several ROS mechanisms of action have been widely investigated, only few studies have investigated the mechanisms underlying the switch toward a cytotoxic effect, and their results only emphasized a high intercellular variability. For instance, while in chondrocytes, low—but not high—oxidative stress activated JNK kinase; 52 in hepatocytes, JNK activation was instead associated with the response to lethal doses of ROS. 119 Furthermore, while Erk1/2 was generally associated with the triggering of cell growth and proliferation, it mediated ROS induction of an apoptotic cascade in epithelial cells.…”