Protein kinase Cδ oxidation contributes to ERK inactivation in lupus T cells.

OBJECTIVE: CD4+ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes, and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T(505) in the protein kinase Cδ (PKCδ) activation loop. However, the mechanisms that prevent PKCδ T(505) phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We undertook this study to test our hypothesis that nitration inactivates PKCδ, contributing to impaired ERK pathway signaling in lupus T cells. METHODS: CD4+ T cells were purified from lupus patients and controls and then stimulated with phorbol myristate acetate (PMA). Signaling protein levels, nitration, and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation. RESULTS: Treating CD4+ T cells with peroxynitrite nitrated PKCδ, preventing PKCδ T(505) phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKCδ levels than did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKCδ, but not unmodified PKCδ, was refractory to PMA-stimulated T(505) phosphorylation, similar to PKCδ in peroxynitrite-treated cells. CONCLUSION: Oxidative stress causes PKCδ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKCδ as a link between oxidative stress and the T cell epigenetic modifications in lupus.

Stimulatory and inhibitory killer Ig-like receptor molecules are expressed and functional on lupus T cells.

T cells from lupus patients have hypomethylated DNA and overexpress genes normally suppressed by DNA methylation that contribute to disease pathogenesis. We found that stimulatory and inhibitory killer cell Ig-like receptor (KIR) genes are aberrantly overexpressed on experimentally demethylated T cells. We therefore asked if lupus T cells also overexpress KIR, and if the proteins are functional. T cells from lupus patients were found to overexpress KIR genes, and expression was proportional to disease activity. Abs to the stimulatory molecule KIR2DL4 triggered IFN-gamma release by lupus T cells, and production was proportional to disease activity. Similarly, cross-linking the inhibitory molecule KIR3DL1 prevented the autoreactive macrophage killing that characterizes lupus T cells. These results indicate that aberrant T cell KIR expression may contribute to IFN overproduction and macrophage killing in human lupus, and they suggest that Abs to inhibitory KIR may be a treatment for this disease.

Decreased ERK and JNK signaling contribute to gene overexpression in "senescent" CD4+CD28- T cells through epigenetic mechanisms.

An inflammatory and cytotoxic CD4+CD28- T cell subset infiltrates atherosclerotic plaques and is implicated in plaque rupture and myocardial infarctions. This pathologic subset develops with replicative stress and is found in patients with chronic inflammatory diseases such as RA as well as with aging. CD4+CD28- cells overexpress genes normally suppressed by DNA methylation in CD4+CD28+ T cells, such as KIR, perforin, and CD70. How this subset over expresses methylation-sensitive genes is unknown. DNA methylation patterns are maintained in proliferating cells by Dnmts, which are up-regulated during mitosis by the ERK and JNK signaling pathways. We hypothesized that defects in these signaling pathways contribute to altered gene expression in human CD4+CD28- cells through effects on DNA methylation. We report that signaling through the ERK and JNK pathways is decreased in CD4+CD28- relative to CD4+CD28+ cells from the same individuals and that ERK and JNK pathway inhibition decreases Dnmt1 and -3a levels, which in turn, causes demethylation and overexpression of the TNFSF7 (CD70) gene. We also report that CD4+CD28- T cells overexpress PP5, a stress-induced inhibitor of the ERK and JNK signaling pathways that may contribute to the signaling defects. We conclude that decreased ERK and JNK signaling in the CD4+CD28- subset, arising with replicative stress, can lead to the overexpression of normally suppressed genes through effects on Dnmts and consequently, chromatin structure.