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1.  Association of PPP2CA polymorphisms with SLE susceptibility in multiple ethnic groups 
Arthritis and rheumatism  2011;63(9):2755-2763.
T cells from patients with SLE express increased amounts of PP2Ac which contribute to decreased production of IL-2. Because IL-2 is important in the regulation of several aspects of the immune response, it has been proposed that PP2Ac contributes to the expression of SLE. This study was designed to determine whether genetic variants of PPP2AC are linked to the expression of SLE and specific clinical manifestations and account for the increased expression of PP2Ac.
We conducted a trans-ethnic study consisting of 8,695 SLE cases and 7,308 controls from four different ancestries. Eighteen single-nucleotide polymorphisms (SNPs) across the PPP2CA were genotyped using an Illumina custom array. PPP2CA expression in SLE and control T cells was analyzed by real-time PCR.
A 32-kb haplotype comprised of multiple SNPs of PPP2CA showed significant association with SLE in Hispanic Americans (HA), European Americans (EA) and Asians but not in African-Americans (AA). Conditional analyses revealed that SNP rs7704116 in intron 1 showed consistently strong association with SLE across Asian, EA and HA populations (pmeta=3.8×10−7, OR=1.3[1.14–1.31]). In EA, the largest ethnic dataset, the risk A allele of rs7704116 was associated with the presence of renal disease, anti-dsDNA and anti-RNP antibodies. PPP2CA expression was approximately 2-fold higher in SLE patients carrying the rs7704116 AG genotype than those carrying GG genotype (p = 0.008).
Our data provide the first evidence for an association between PPP2CA polymorphisms and elevated PP2Ac transcript levels in T cells, which implicates a new molecular pathway for SLE susceptibility in EA, HA and Asians.
PMCID: PMC3163110  PMID: 21590681
2.  Promoter Hypomethylation Results in Increased Expression of Protein Phosphatase 2A in T Cells from Patients with Systemic Lupus Erythematosus 
The catalytic subunit α isoform of protein phosphatase 2A (PP2Acα) activity, protein, and mRNA have been found increased in systemic lupus erythematosus (SLE) T cells and to contribute to decreased IL-2 production. The PP2Acα promoter activity is controlled epigenetically through the methylation of a CpG within a cAMP response element (CRE) motif defined by its promoter. We considered that hypomethylation may account for the increased expression of PP2Acα in patients with SLE. Using bisulfite sequencing, we found that SLE T cells displayed decreased DNA methylation in the promoter region compared with normal T cells. More importantly, we found that the CRE-defined CpG, which binds p-CREB, is significantly less methylated in SLE compared with normal T cells, and the levels of methylation correlated with decreased amounts of DNA methyltransferase 1 transcripts. Methylation intensity correlated inversely with levels of PP2Acα mRNA and SLE disease activity. Chromatin immunoprecipitation assays revealed more binding of p-CREB to the CRE site in SLE T cells, resulting in increased expression of PP2Acα. We propose that PP2Acα represents a new methylation-sensitive gene that, like the previously reported CD70 and CD11a, contributes to the pathogenesis of SLE.
PMCID: PMC3148941  PMID: 21346232
3.  Methylation Status of CpG Islands Flanking a CRE Motif on the Protein Phosphatase 2Acα Promoter Determines CREB Binding and Activity 
Protein phosphatase 2A (PP2A) is a major serine-threonine protein phosphatase in eukaryotic cells and is involved in many essential aspects of cell function. The catalytic subunit of the enzyme (PP2Ac), a part of the core enzyme, has two isoforms, α (PP2Acα) and β (PP2Acβ), of which PP2Acα is the major form expressed in vivo. Deregulation of PP2A expression has been linked to several diseases, but the mechanisms which control the expression of this enzyme are still unclear. We conducted experiments to decipher molecular mechanisms involved in the regulation of the PP2Acα promoter in human primary T cells. After constructing serially truncated PP2Acα promoter luciferase constructs, we found that the region stretching around 240 bases upstream from the translation initiation site was of functional significance and included a cAMP response element (CRE) motif flanked by three GC- boxes. Shift assays revealed that CRE-binding protein (CREB)/phosphorylated CREB (pCREB) and stable protein 1 (Sp1) could bind to the region. Furthermore, we demonstrated that methylation of deoxycytosine (dC) in the CpG islands limited binding of pCREB and the activity of the PP2Acα promoter. In contrast, the binding of Sp1 to a GC-box within the core promoter region was not affected by DNA methylation. Primary T cells treated with 5-azacitidine (5-azaC), a DNA methyltransferase (DNMT) inhibitor, showed increased expression of PP2Acα mRNA. We propose that conditions associated with hypomethylation of CpG islands, such as drug- induced lupus and cancer, permit increased PP2Ac expression.
PMCID: PMC2676107  PMID: 19155497
Human; T Cells; Systemic Lupus Erythematosus; Protein Kinases/Phosphatases; Gene Regulation
4.  Induction of tumour necrosis factor receptor-expressing macrophages by interleukin-10 and macrophage colony-stimulating factor in rheumatoid arthritis 
Despite its potent ability to inhibit proinflammatory cytokine synthesis, interleukin (IL)-10 has a marginal clinical effect in rheumatoid arthritis (RA) patients. Recent evidence suggests that IL-10 induces monocyte/macrophage maturation in cooperation with macrophage-colony stimulating factor (M-CSF). In the present study, we found that the inducible subunit of the IL-10 receptor (IL-10R), type 1 IL-10R (IL-10R1), was expressed at higher levels on monocytes in RA than in healthy controls, in association with disease activity, while their expression of both type 1 and 2 tumour necrosis factor receptors (TNFR1/2) was not increased. The expression of IL-10R1 but not IL-10R2 was augmented on monocytes cultured in the presence of RA synovial tissue (ST) cell culture supernatants. Cell surface expression of TNFR1/2 expression on monocytes was induced by IL-10, and more efficiently in combination with M-CSF. Two-color immunofluorescence labeling of RA ST samples showed an intensive coexpression of IL-10R1, TNFR1/2, and M-CSF receptor in CD68+ lining macrophages. Adhered monocytes, after 3-day preincubation with IL-10 and M-CSF, could produce more IL-1β and IL-6 in response to TNF-α in the presence of dibutyryl cAMP, as compared with the cells preincubated with or without IL-10 or M-CSF alone. Microarray analysis of gene expression revealed that IL-10 activated various genes essential for macrophage functions, including other members of the TNFR superfamily, receptors for chemokines and growth factors, Toll-like receptors, and TNFR-associated signaling molecules. These results suggest that IL-10 may contribute to the inflammatory process by facilitating monocyte differentiation into TNF-α-responsive macrophages in the presence of M-CSF in RA.
PMCID: PMC1779421  PMID: 16859503
5.  The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis 
S100A8 and S100A9, two Ca2+-binding proteins of the S100 family, are secreted as a heterodimeric complex (S100A8/A9) from neutrophils and monocytes/macrophages. Serum and synovial fluid levels of S100A8, S100A9, and S100A8/A9 were all higher in patients with rheumatoid arthritis (RA) than in patients with osteoarthritis (OA), with the S100A8/A9 heterodimer being prevalent. By two-color immunofluorescence labeling, S100A8/A9 antigens were found to be expressed mainly by infiltrating CD68+ macrophages in RA synovial tissue (ST). Isolated ST cells from patients with RA spontaneously released larger amounts of S100A8/A9 protein than did the cells from patients with OA. S100A8/A9 complexes, as well as S100A9 homodimers, stimulated the production of proinflammatory cytokines, such as tumor necrosis factor alpha, by purified monocytes and in vitro-differentiated macrophages. S100A8/A9-mediated cytokine production was suppressed significantly by p38 mitogen-activated protein kinase (MAPK) inhibitors and almost completely by nuclear factor kappa B (NF-κB) inhibitors. NF-κB activation was induced in S100A8/A9-stimulated monocytes, but this activity was not inhibited by p38 MAPK inhibitors. These results indicate that the S100A8/A9 heterodimer, secreted extracellularly from activated tissue macrophages, may amplify proinflammatory cytokine responses through activation of NF-κB and p38 MAPK pathways in RA.
PMCID: PMC1526633  PMID: 16613612
6.  Resistance to IL-10 inhibition of interferon gamma production and expression of suppressor of cytokine signaling 1 in CD4+ T cells from patients with rheumatoid arthritis 
Arthritis Research & Therapy  2004;6(6):R567-R577.
IL-10 has been shown to block the antigen-specific T-cell cytokine response by inhibiting the CD28 signaling pathway. We found that peripheral blood CD4+ T cells from patients with active rheumatoid arthritis (RA) were able to produce greater amounts of interferon gamma after CD3 and CD28 costimulation in the presence of 1 ng/ml IL-10 than were normal control CD4+ T cells, although their surface expression of the type 1 IL-10 receptor was increased. The phosphorylation of signal transducer and activator of transcription 3 was sustained in both blood and synovial tissue CD4+ T cells of RA, but it was not augmented by the presence of 1 ng/ml IL-10. Sera from RA patients induced signal transducer and activator of transcription 3 phosphorylation in normal CD4+ T cells, which was mostly abolished by neutralizing anti-IL-6 antibody. Preincubation of normal CD4+ T cells with IL-6 reduced IL-10-mediated inhibition of interferon gamma production. Blood CD4+ T cells from RA patients contained higher levels of suppressor of cytokine signaling 1 but lower levels of suppressor of cytokine signaling 3 mRNA compared with control CD4+ T cells, as determined by real-time PCR. These results indicate that RA CD4+ T cells become resistant to the immunosuppressive effect of IL-10 before migration into synovial tissue, and this impaired IL-10 signaling may be associated with sustained signal transducer and activator of transcription 3 activation and suppressor of cytokine signaling 1 induction.
PMCID: PMC1064873  PMID: 15535835
CD4+ T cells; IL-10; rheumatoid arthritis; signal transducer and activator of transcription 3; suppressor of cytokine signaling 1

Results 1-6 (6)