Systemic lupus erythematosus (SLE) is an incompletely understood chronic, relapsing autoimmune disease. Persuasive evidence indicates that genetic factors contribute to disease development. However, a lack of complete disease concordance in identical twins and the relapsing nature of the disease indicate that environmental factors are important as well (1). The full repertoire of the environmental factors, and the mechanism(s) by which the environmental agents contribute to the onset of lupus and lupus flares, remain unknown. Our group has reported that T cell DNA demethylation contributes to the pathogenesis of human lupus by causing overexpression of immune genes, leading to T cell autoreactivity and the development of lupus in genetically predisposed hosts (2). This suggests that the environment may trigger lupus flares by inhibiting T cell DNA methylation. The mechanisms by which environmental agents cause T cell DNA demethylation in lupus are also unknown.
DNA methylation patterns are replicated each time a cell divides by DNA methyltransferase 1 (DNMT-1) (3), whose levels are regulated in part by the ERK signaling pathway (4–6). We reported that treating proliferating T cells with ERK pathway inhibitors, such as hydralazine and U0126, or with DNMT inhibitors, such as procainamide, is sufficient to demethylate the DNA and that the demethylated T cells are sufficient to cause a lupus-like disease in murine models (7, 8). Further, transgenic mice with an inducible T cell ERK pathway signaling defect develop a lupus-like disease (9). We also reported that T cells from patients with active lupus have impaired ERK pathway signaling, low DNMT-1 levels, and overexpress methylation-sensitive genes (10), suggesting that impaired ERK pathway signaling contributes to DNA demethylation in human lupus. Interestingly, CD4+ lupus T cells demethylate the same genetic regulatory elements demethylated by ERK pathway inhibitors and DNMT inhibitors (11), and the level of lupus disease activity is directly related to the degree of signaling impairment and DNA demethylation (10). These studies thus suggest an important role of impaired ERK pathway signaling in triggering lupus flares.
More recent studies traced the lupus ERK pathway signaling defect to impaired protein kinase Cδ (PKCδ) phosphorylation (12). PKCδ is a key molecule that is phosphorylated in response to a variety of signals, and thus activates other signaling pathways including the ERK pathway (13–15). PKCδ in T cells from patients with active lupus or in hydralazine-treated T cells is not properly phosphorylated in response to phorbol myristate acetate (PMA) or other stimuli, and the defect correlates with the lupus ERK pathway defect. Transfecting human T cells with a kinase-inactive PKCδ causes decreased ERK phosphorylation, indicating that PKCδ is critically required for ERK pathway activation. Additionally, the transfected cells exhibited demethylation and overexpression of the TNFSF7 (CD70) gene, similar to findings in lupus patients (12). However, the mechanisms inhibiting PKCδ phosphorylation in lupus T cells are unknown.
Inflammation is associated with the generation of reactive oxygen species (ROS), and patients with active lupus have increased levels of ROS and reactive nitrogen intermediates (RNIs), as well as decreased levels of oxidant scavengers (16–18). This imbalance causes increased levels of superoxide (O2−), hydrogen peroxide (H2O2), and peroxynitrite (ONOO−), all highly reactive metabolites that cause direct toxicity by inducing chemical modifications in lipids, proteins, and DNA (19). ONOO− nitrates Tyr residues to prevent phosphorylation (20) and may thus affect signaling pathways. However, the consequences of oxidative stress on T cell signaling are also poorly understood.
Since T cell PKCδ kinase activity is decreased in patients with active lupus and active lupus is characterized by the generation of ROS/RNIs and oxidative protein damage, we hypothesized that PKCδ may be covalently modified by ROS/RNIs in lupus T cells, preventing its activation. The goal of this study was to determine if oxidative modifications produced by ONOO− contribute to impaired T cell PKCδ phosphorylation, causing the decreased ERK pathway signaling observed in patients with active lupus.
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The goal of this study was to investigate mechanisms causing the PKCδ phosphorylation defect responsible for decreased ERK pathway signaling in lupus T cells. PKCδ belongs to the PKC family of related serine/threonine kinases with active roles in growth regulation and apoptosis. PKC isoforms contain a highly conserved C-terminal catalytic domain. However, they are subdivided into 3 subfamilies according to their N-terminal regulatory domains. Conventional isoforms comprise PKCα, PKCβ, and PKCγ, bind diacylglycerol (DAG)/PMA in their C1 domain, and bind anionic phospholipids in a calcium-dependent manner in their C2 domain. Novel isoforms include PKCδ, PKCε, PKCη, PKCμ, and PKCθ and are activated by DAG/PMA without a calcium requirement. Atypical isoforms, PKCζ and PKCλ/ι, are DAG/PMA and calcium independent (37).
PKCδ is ubiquitously expressed among cells and tissues and is the only isoform that can be activated by 3 different mechanisms: through Ser/Thr phosphorylation, through tyrosine phosphorylation, and by caspase 3–dependent proteolytic cleavage. These are independent mechanisms that regulate PKCδ activity, substrates, and cellular localization and play critical roles during cell growth, differentiation, and programmed cell death as well as the cellular response to oxidative stress (38).
Our previous work identified decreased PKCδ phospho-T505 levels in stimulated lupus T cells, which correlated with the decreased ERK pathway signaling previously reported in lupus T cells. We also demonstrated that PKCδ is upstream of ERK and that impaired PKCδ/ERK pathway signaling in T cells causes demethylation and overexpression of methylation-sensitive genes (12). Importantly, transgenic mice lacking T cell PKCδ activity develop a lupus-like disease with decreased ERK signaling, overexpression of methylation-sensitive genes, and production of anti–double-stranded DNA antibodies similar to those observed in lupus patients (ref.39 and Gorelik GJ, et al: unpublished observations), strongly supporting the hypothesis that defective PKCδ signaling is sufficient to cause lupus.
PDK-1 phosphorylates PKCδ at T505 in the activation loop, promoting alignment of these residues with the catalytic pocket and controlling catalytic activity of the enzyme (24). We demonstrated that phosphorylation of Ser241, which is required for PDK-1 kinase activity, is not appreciably affected in lupus T cells relative to T cells from healthy donors and under the same assay conditions in which PKCδ phospho-T505 levels were decreased. This implies that another mechanism inhibits phosphorylation at T505 in PKCδ in lupus T cells.
Chronic inflammation is associated with oxidative stress, and a prooxidative state has been described in patients with lupus (27, 34). In this prooxidative state, higher levels of ROS/RNIs are present and can directly cause toxicity through posttranslational modifications of proteins and alterations of lipids and DNA. Nitric oxide (NO) is overproduced in lupus (40) and can combine with O2− to form ONOO−, a highly reactive and potentially pathogenic molecule. Multiple markers of protein oxidation have been found in SLE patient sera, including protein carbonyls, protein-bound methionine sulfoxide, decreased levels of protein thiols (27), and increases in protein 3-nitrotyrosines that correlate with worsening disease status (16, 33, 41), indicating that protein oxidation and, in particular, nitrating pathways may play an important role in the pathogenesis of SLE (33, 34, 36). However, while it is recognized that overproduction of ROS/RNIs alters and modifies T cell signaling, their target molecules and the intracellular mechanisms that they affect are not well understood.
Based on these observations, we investigated whether the defective PKCδ activation in lupus T cells was caused by oxidative damage. ONOO− was used as the oxidizing agent and caused PKCδ nitration that resulted in decreased phosphorylation of T505. The fact that PKCδ was the only PKC isoform catalytically affected indicates that the ONOO− inhibitory effect is selective and specific to PMA-stimulated PKCδ phospho-T505.
PKCδ is not phosphorylated at the activation loop (T505) in resting T cells, but phosphorylation increases following PMA stimulation and translocation to the cytoplasmic membrane. In our studies, oxidation modified the phosphorylation pattern, increasing tyrosine phosphorylation while decreasing threonine phosphorylation, indicating specific effects on PKCδ phosphorylation regulation. This differential pattern of PKCδ phosphorylation also modifies its intracellular translocation, resulting in changes to the interaction of PKCδ with downstream targets (42). Recent studies indicate that ROS can also modulate PKCδ activity in other cell types. In keratinocytes, oxidation increases PKCδ tyrosine phosphorylation, resulting in reduced enzymatic activity (43), whereas in other cells, oxidation enhances the enzymatic activity (44) or even modifies the enzymatic specificity (45), indicating that oxidative modifications of PKCδ activity may be cell type dependent and/or phosphorylated residue dependent (28). The precise effects of oxidation on T cell PKCδ have not been explored, but it is possible that oxidation of certain residues in the molecule causes distortions in the secondary and tertiary structure, resulting in decreased accessibility of T505 to phosphorylation. It is also possible that the rate of dephosphorylation increases with oxidation.
The phospho-serine/threonine phosphatases PP-1c, PP-2Ac, and PP-2Cα dephosphorylate PKCδ, with PP-2Ac demonstrating the highest specific activity toward PKCδ (31). However, our results show that T cells transfected with catalytically inactive PP-2Ac mutants or with the corresponding siRNA did not restore PKCδ phosphorylation following ONOO− treatment, suggesting that modifications of PP-2Ac activity are unlikely to be responsible for the decreased T505 phosphorylation observed in oxidized PKCδ. This observation is consistent with a study demonstrating that even though the messenger RNA, protein, and enzymatic activity of the PP-2A catalytic subunit (PP-2Ac) is increased in patients with lupus, the defect is independent of disease activity (23), while the PKCδ phosphorylation impairment is directly related to disease activity (12). The present work shows that oxidation of PKCδ results in a selective decrease in PKCδ phospho-T505 levels and directly correlates with decreased phospho-ERK levels in T cells, consistent with our previous results.
The consequences of an oxidative environment on ERK activity are not clearly defined and vary depending on the cell system and stimulus. Decreased phosphorylation of ERK induced by ONOO− in kidneys of βs sickle cell mice has been reported (46), and similarly, an alteration of the cellular redox state leads to increased MAPK phosphatase 1 expression in fibroblasts, resulting in ERK inactivation (47). This suggests that additional mechanisms may contribute to the decreased ERK phosphorylation caused by oxidative stress in lupus T cells.
ONOO− may alter protein structure and function by interacting with different amino acids. By oxidating cysteine residues (S-nitrosylation), ONOO− inactivates many enzymes involved in energetic processes in addition to protein tyrosine phosphatases that may enhance tyrosine phosphorylation–dependent signaling (20). ONOO− also oxidizes tyrosine residues (O-nitration) to form 3-nitrotyrosine derivatives (20). Tyrosine nitration is considered to be a major cause of ONOO−-mediated cytotoxicity because it affects protein structure and function that may result in the generation of antigenic epitopes (48), changes in the catalytic activity of enzymes, and impaired cell signal transduction (49). Nitration is a highly selective process limited to specific tyrosine residues on a limited number of proteins. In general, protein nitration is associated with loss of function (46). Others have reported an increased amount of NO-mediated oxidation products, such as 3-nitrotyrosine proteins, due to elevated levels of ONOO− in the serum of lupus patients (41).
We found higher levels of nitrotyrosine-containing proteins in T cells from patients with active lupus than in T cells from patients with inactive lupus or in T cells from healthy controls. The levels of nitrated PKCδ were also greater in T cells from patients with active lupus, and PMA-stimulated T505 phosphorylation of the nitrated proteins was impaired. Similar results were observed in control T cells pretreated with ONOO−. Therefore, the presence of significant levels of nitrated PKCδ in T cells from patients with active lupus may explain the decrease in PKCδ phospho-T505 levels observed in T cells from lupus patients, the consequent decrease in ERK signaling pathway, and their correlation with disease severity. Protein nitration is often associated with pathologic states related to inflammation, but is also recognized as a normal physiologic process (20), suggesting an explanation for the presence of nitrated proteins in T cells from healthy donors and patients with inactive lupus. The relatively lower level of PKCδ phosphorylation that we found in normal T cells in the nitrated fraction could be due to a lesser degree of oxidation that normally takes place during physiologic metabolism. However, our studies also demonstrate that human T cell PKCδ is directly modified by oxidation. The oxidation selectively decreases PMA-stimulated PKCδ T505 phosphorylation and is directly related to a decrease in ERK signaling, suggesting that higher levels of nitration cause structural changes in PKCδ that make it catalytically inactive.
Our study reveals that the impaired T cell PKCδ kinase activation observed in patients with active lupus is likely due to oxidative damage and causes the impaired ERK pathway signaling in T cells from lupus patients. These studies point to PKCδ as a link between oxidative stress, caused by environmental agents, and the epigenetic changes observed in lupus T cells. Other poorly understood autoimmune diseases also result from gene–environment interactions and involve epigenetic mechanisms that include not only DNA methylation, but also histone modifications and microRNA (50). Oxidative stress may also contribute to these disorders through mechanisms such as altered signaling.