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Y-box (YB) protein-1 serves as a master regulator in gene transcription and mRNA translation. YB-1 itself is regulated at various levels, e.g. through post-translational modifications. In our previous work, we identified RANTES/CCL5 as a transcriptional target of YB-1. We previously demonstrated that YB-1 protein is transiently up-regulated during monocyte/macrophage differentiation evidenced in monocytic cells (THP-1 cells) that were differentiated using phorbol myristate acetate (PMA). Here we provide evidence that YB-1 phosphorylation, specifically at its serine residue 102 (Ser-102), increases early on in THP-1 cells following PMA treatment as well as in differentiated primary human monocytes. This process is mediated through the Akt signaling pathway. Ser-102-phosphorylated YB-1 displays stronger binding affinity and trans-activating capacity at the CCL5 gene promoter. Notably, Ser-102-phosphorylated YB-1 disappears at later stages of the monocyte/macrophage differentiation process. We demonstrate that serine-threonine phosphatase calcineurin (CN) dephosphorylates YB-1 preventing it from binding to and trans-activating the CCL5 promoter. Co-immunoprecipitation assays prove a direct YB-1/CN interaction. Furthermore, analyses in kidney tissues from mice that were treated with the CN inhibitor cyclosporine A revealed an in vivo effect of CN on the YB-1 phosphorylation status. We conclude that YB-1 phosphorylation at Ser-102 is an important prerequisite for CCL5 promoter activation during macrophage differentiation. Our findings point to a critical role of YB-1 in the resolution of inflammatory processes which may largely be due to CN-mediated dephosphorylation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

TCR-αβ(+)CD3(+)CD4(-)CD8(-) "double negative" T cells are expanded in the peripheral blood of patients with systemic lupus erythematosus (SLE) and lupus-prone mice. Double negative T cells have been claimed to derive from CD8(+) cells that down-regulate CD8 co-receptors and acquire a distinct effector phenotype that includes the expression of proinflammatory cytokines. This, along with the fact that double negative T cells have been documented in inflamed organs, suggests that they may contribute to disease expression and tissue damage. We recently linked the transcription factor cAMP responsive element modulator (CREM) α, which is expressed at increased levels in T cells from SLE patients and lupus prone MRL/lpr mice, with trans-repression of a region syntenic to the murine CD8b promoter. However, the exact molecular mechanisms that result in a stable silencing of both CD8A and CD8B genes remain elusive. Here, we demonstrate that CREMα orchestrates epigenetic remodeling of the CD8 cluster through the recruitment of DNA methyltransferase (DNMT) 3a and histone methyltransferase G9a. Thus, we propose that CREMα is essential for the expansion of double negative T cells in SLE. CREMα blockade may have therapeutic value in autoimmune disorders with DN T cell expansion.

In the mammalian retina, glutamate uptake is mediated by members of a family of glutamate transporters known as "excitatory amino acid transporters (EAATs)." Here we cloned and functionally characterized two retinal EAATs from mouse, the GLT-1/EAAT2 splice variant GLT-1c, and EAAT5. EAATs are glutamate transporters and anion-selective ion channels, and we used heterologous expression in mammalian cells, patch-clamp recordings and noise analysis to study and compare glutamate transport and anion channel properties of both EAAT isoforms. We found GLT-1c to be an effective glutamate transporter with high affinity for Na(+) and glutamate that resembles original GLT-1/EAAT2 in all tested functional aspects. EAAT5 exhibits glutamate transport rates too low to be accurately measured in our experimental system, with significantly lower affinities for Na(+) and glutamate than GLT-1c. Non-stationary noise analysis demonstrated that GLT-1c and EAAT5 also differ in single-channel current amplitudes of associated anion channels. Unitary current amplitudes of EAAT5 anion channels turned out to be approximately twice as high as single-channel amplitudes of GLT-1c. Moreover, at negative potentials open probabilities of EAAT5 anion channels were much larger than for GLT-1c. Our data illustrate unique functional properties of EAAT5, being a low-affinity and low-capacity glutamate transport system, with an anion channel optimized for anion conduction in the negative voltage range.

T cell receptor-αβ(+) CD3(+)CD4(-)CD8(-) "double-negative" T cells are expanded in the peripheral blood of patients with systemic lupus erythematosus and autoimmune lymphoproliferative syndrome. In both disorders, double-negative T cells infiltrate tissues, induce immunoglobulin production, and secrete proinflammatory cytokines. Double-negative T cells derive from CD8(+) T cells through down-regulation of CD8 surface co-receptors. However, the molecular mechanisms orchestrating this process remain unclear. Here, we demonstrate that the transcription factor cAMP-responsive element modulator α (CREMα), which is expressed at increased levels in T cells from systemic lupus erythematosus patients, contributes to transcriptional silencing of CD8A and CD8B. We provide the first evidence that CREMα trans-represses a regulatory element 5' of the CD8B gene. Therefore, CREMα represents a promising candidate in the search for biomarkers and treatment options in diseases in which double-negative T cells contribute to the pathogenesis.

Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase involved in essential cellular functions. T cells from patients with systemic lupus erythematosus (SLE) express high levels of the catalytic subunit of PP2A (PP2Ac). A mouse overexpressing PP2Ac in T cells develops glomerulonephritis in an IL-17-dependent manner. Here, using microarray analyses, we demonstrate that increased expression of PP2Ac grants T cells the capacity to produce an array of proinflammatory effector molecules. Because IL-17 is important in the expression of glomerulonephritis, we studied the mechanism through which PP2Ac dysregulation facilitates its production. We report that PP2Ac is involved in the regulation of the Il17 locus by enhancing histone 3 acetylation through a mechanism that involves activation of interferon regulatory factor 4. Increased histone 3 acetylation of the Il17 locus is shared between T cells of PP2Ac transgenic mice and patients with SLE. We propose that, by promoting the inflammatory capacity of T cells, PP2Ac dysregulation contributes to the pathogenesis of SLE.

Notch signaling constitutes an evolutionarily conserved pathway that transduces signals between neighboring cells and determines major decisions in cell proliferation, survival, and differentiation. Notch signaling has been shown to play a pivotal role during T cell lineage determination. T lymphocytes from patients with systemic lupus erythematosus (SLE) display a severely altered phenotype with several molecular and functional aberrations, including defective capacities to up-regulate Notch-1 receptor expression upon T cell receptor activation. Here, we demonstrate that basal Notch-1 expression is decreased in T cells from active SLE patients at the mRNA and protein levels in various T cell subpopulations. Notch-1 transcript numbers inversely correlate with disease activity in SLE patients. We provide evidence that both enhanced histone H3 methylation and CpG DNA methylation of the human Notch-1 promoter contribute to decreased Notch-1 expression in SLE T cells. Previous data from our group identified cAMP-responsive element modulator α (CREMα), which is up-regulated in SLE T cells, as a key regulator of epigenetic patterns and gene transcription, e.g. that of IL2 and IL17 genes. In this study, we observed increased CREMα binding to the Notch-1 promoter, which eventually resulted in significantly reduced Notch-1 promoter activity and gene transcription. Notably, decreased Notch-1 levels were associated with elevated IL-17A levels. Our data suggest a role for Notch-1 in SLE immunopathogenesis, and for the first time, we present molecular mechanisms that mediate dysregulated Notch-1 expression in SLE T cells.

Th17 lymphocytes play a key role during immune responses against bacteria and fungi and are involved in the pathophysiology of multiple autoimmune disorders. The co-stimulatory molecules SLAMF3 and SLAMF6 have been implicated in the formation of Th17 phenotypes and IL-17A expression. Increased surface expression of SLAMF3 and SLAMF6 has been linked with disease activity in systemic lupus erythematosus. Here we demonstrate that in human total T lymphocytes the canonical CD28 and the non-canonical SLAMF3/SLAMF6 co-stimulatory pathways cooperate in the recruitment of the transcription factor NFAT1 to the IL17A promoter. Furthermore, the dominance of the SLAMF3/SLAMF6 pathway in inducing IL-17A production can be attributed to an increased nuclear abundance and recruitment of RORγt to the IL17A promoter. Thus, we have identified an additional mechanism that may be central for the specific control of IL17A gene regulation in systemic lupus erythematosus T lymphocytes.

The proinflammatory cytokines IL-17A and IL-17F are primarily produced by Th17 lymphocytes. Both are involved in host defense mechanisms against bacterial and fungal pathogens and contribute to the development of various autoimmune diseases. T lymphocytes from patients with systemic lupus erythematosus (SLE) display increased expression of transcription factor cAMP-responsive element modulator α (CREMα), which has been documented to account for aberrant T cell function and contributes to the pathogenesis of SLE. Here, we provide evidence that IL-17F expression is reduced in SLE T cells. We demonstrate that CREMα binds to a yet unidentified CRE site within the proximal promoter. This results in reduced IL-17F expression in SLE T lymphocytes and is independent of activating epigenetic patterns (increased histone H3 Lys-18 acetylation, reduced histone H3 Lys-27 trimethylation, and CpG-DNA demethylation). Forced CREMα expression in human T lymphocytes results in reduced IL-17F expression. Our findings demonstrate extended involvement of CREMα in cytokine dysregulation in SLE by contributing to a disrupted balance between IL-17A and IL-17F. An increased IL-17A/IL-17F ratio may aggravate the proinflammatory phenotype of SLE.

IL-17A is a proinflammatory cytokine that is produced by specialized T helper cells and contributes to the development of several autoimmune diseases such as systemic lupus erythematosus (SLE). Transcription factor cAMP-responsive element modulator (CREM)α displays increased expression levels in T cells from SLE patients and has been described to account for aberrant T cell function in SLE pathogenesis. In this report, we provide evidence that CREMα physically binds to a cAMP-responsive element, CRE (-111/-104), within the proximal human IL17A promoter and increases its activity. Chromatin immunoprecipitation assays reveal that activated naïve CD4(+) T cells as well as T cells from SLE patients display increased CREMα binding to this site compared with T cells from healthy controls. The histone H3 modification pattern at the CRE site (-111/-104) and neighboring conserved noncoding sequences within the human IL17A gene locus suggests an accessible chromatin structure (H3K27 hypomethylation/H3K18 hyperacetylation) in activated naïve CD4(+) T cells and SLE T cells. H3K27 hypomethylation is accompanied by decreased cytosine phosphate guanosine (CpG)-DNA methylation in these regions in SLE T cells. Decreased recruitment of histone deacetylase (HDAC)1 and DNA methyltransferase (DNMT)3a to the CRE site (-111/-104) probably accounts for the observed epigenetic alterations. Reporter studies confirmed that DNA methylation of the IL17A promoter indeed abrogates its inducibility. Our findings demonstrate an extended role for CREMα in the immunopathogenesis of SLE because it contributes to increased expression of IL-17A.

IL-2 is a key cytokine during proliferation and activation of T lymphocytes and functions as an auto- and paracrine growth factor. Regardless of activating effects on T lymphocytes, the absence of IL-2 has been linked to the development of autoimmune pathology in mice and humans. Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease and characterized by dysregulation of lymphocyte function, transcription factor and cytokine expression, and antigen presentation. Reduced IL-2 expression is a hallmark of SLE T lymphocytes and results in decreased numbers of regulatory T lymphocytes which play an important role in preventing autoimmunity. Reduced IL-2 expression was linked to overproduction of the transcription regulatory factor cAMP-responsive element modulator (CREM)α in SLE T lymphocytes and subsequent CREMα binding to a CRE site within the IL2 promoter (-180 CRE). In this study, we demonstrate the involvement of CREMα-mediated IL2 silencing in T lymphocytes from SLE patients through a gene-wide histone deacetylase 1-directed deacetylation of histone H3K18 and DNA methyltransferase 3a-directed cytosine phosphate guanosine (CpG)-DNA hypermethylation. For the first time, we provide direct evidence that CREMα mediates silencing of the IL2 gene in SLE T cells though histone deacetylation and CpG-DNA methylation.

The transcriptional repressor cAMP response element modulator (CREM) α has important roles in normal T cell physiology and contributes to aberrant T cell function in patients with systemic lupus erythematosus (SLE). Recently, we characterized a specificity protein-1-dependent promoter located upstream of the CREM gene that accounts for increased basal CREM expression in SLE T cells and reflects disease activity. Here, we identify a novel intronic CREM promoter (denoted P2) in front of the second exon of the CREM gene that harbors putative binding sites for TATA-binding proteins and the transcriptional activator AP-1. DNA binding studies, chromatin immunoprecipitation, and reporter assays confirmed the functional relevance of these sites, and T cell activation through CD3/CD28 stimulation or phorbol 12-myristate 13-acetate/ionomycin treatment enhances P2 promoter activity. Although the basal CREM levels are increased in T cells from SLE patients compared with healthy controls, there are remarkable differences in the regulation of CREM expression in response to T cell activation. Whereas T cells from healthy individuals display increased CREM expression after T cell activation, most likely through AP-1-dependent up-regulation of the P2 promoter, SLE T cells fail to further increase their basal CREM levels upon T cell activation due to a decreased content of the AP-1 family member c-Fos. Because CREM trans-represses c-fos transcription in SLE T cells, we propose an autoregulatory feedback mechanism between CREM and AP-1. Our findings extend the understanding of CREM gene regulation in the context of T cell activation and disclose another difference in the transcriptional machinery in SLE T cells.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with numerous abnormalities recorded at the cellular, molecular, and genetic level. Expression of the basic leucine zipper transcription factor cAMP-responsive element modulator (CREM)α was reported to be abnormally increased in T cells from SLE patients. CREMα suppresses IL-2 and T cell receptor ζ chain gene transcription by direct binding to the respective promoters. Here, we show that increased CREM expression is the result of enhanced promoter activity. DNA binding analyses reveal direct binding of transcription factor specificity protein-1 (SP-1) to the CREM promoter resulting in enhanced transcriptional activity and increased CREM expression. Protein phosphatase 2A is known to activate SP-1 through dephosphorylation at its serine residue 59. Our results show that nuclei from SLE T cells contain lower levels of Ser(59)-phosphorylated SP-1 protein and a stronger SP-1 binding to the CREM promoter. We conclude that protein phosphatase 2A accounts for enhanced SP-1 dephosphorylation at Ser(59) in SLE T cells. More importantly, CREM promoter activity mirrors reliably disease activity in SLE patients, underscoring its potential role as a biomarker for the prediction of flares in SLE patients.

Y-box (YB) protein-1 is secreted by mesangial and immune cells after cytokine challenge, but extracellular functions are unknown. Here, we demonstrate that extracellular YB-1 associates with outer cell membrane components and interacts with extracellular Notch-3 receptor domains. The interaction appears to be specific for Notch-3, as YB-1-green fluorescent protein binds to the extracellular domains and full-length forms of Notch-3 but not to Notch-1. YB-1-green fluorescent protein and Notch-3 proteins co-localize at cell membranes, and extracellular YB-1 activates Notch-3 signaling, resulting in nuclear translocation of the Notch-3 intracellular domain and up-regulation of Notch target genes. The YB-1/Notch-3 interaction may be of particular relevance for inflammatory mesangioproliferative disease, as both proteins co-localize in an experimental nephritis model and receptor activation temporally and spatially correlates with YB-1 expression.

The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5' splicing site selection and formation of the 12 S isoform.

Glutamate transport by the neuronal excitatory amino acid carrier (EAAC1) is accompanied by the coupled movement of one proton across the membrane. We have demonstrated previously that the cotransported proton binds to the carrier in the absence of glutamate and, thus, modulates the EAAC1 affinity for glutamate. Here, we used site-directed mutagenesis together with a rapid kinetic technique that allows one to generate sub-millisecond glutamate concentration jumps to locate possible binding sites of the glutamate transporter for the cotransported proton. One candidate for this binding site, the highly conserved glutamic acid residue Glu-373 of EAAC1, was mutated to glutamine. Our results demonstrate that the mutant transporter does not catalyze net transport of glutamate, whereas Na(+)/glutamate homoexchange is unimpaired. Furthermore, the voltage dependence of the rates of Na(+) binding and glutamate translocation are unchanged compared with the wild-type. In contrast to the wild-type, however, homoexchange of the E373Q transporter is completely pH-independent. In line with these findings the transport kinetics of the mutant EAAC1 show no deuterium isotope effect. Thus, we suggest a new transport mechanism, in which Glu-373 forms part of the binding site of EAAC1 for the cotransported proton. In this model, protonation of Glu-373 is required for Na(+)/glutamate translocation, whereas the relocation of the carrier is only possible when Glu-373 is negatively charged. Interestingly, the Glu-373-homologous amino acid residue is glutamine in the related neutral amino acid transporter alanine-serine-cysteine transporter. The function of alanine-serine-cysteine transporter is neither potassium- nor proton-dependent. Consequently, our results emphasize the general importance of glutamate and aspartate residues for proton transport across membranes.