Olf1/EBF associated zinc finger protein interfered with antinuclear antibody production in patients with systemic lupus erythematosus
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The aim of the study was to determine whether Olf1/EBF associated zinc finger protein (OAZ), a transcription factor encoded by a positional systemic lupus erythematosus (SLE) candidate gene, plays a functional role in the pathogenesis in SLE.
Gene expression levels in peripheral blood cells (PBLs) measured using quantitative real-time polymerase chain reaction (qPCR) were assessed for association with disease activity and the presence of specific autoantibodies. Peripheral blood mononuclear cells (PBMCs) were incubated with specific siRNAs for three days, then cells were harvested for measuring mRNA levels using qPCR, and supernatants for levels of total immunoglobulin (Ig)G and IgM as well as secreted cytokines, chemokine and antinuclear antibodies (ANA) using ELISA. Indirect immunofluorescence was also applied for ANA detection.
OAZ gene expressions in PBLs from 40 ANA-positive SLE patients were significantly increased than those from 30 normal controls (P < 0.0001) and 18 patients with rheumatoid arthritis (P < 0.01). In SLE patients, OAZ transcripts were positively correlated with SLE disease activity index (SLEDAI) score (r = 0.72, P < 0.0001) and higher in those positive for anti-dsDNA or anti-Sm antibodies (both P < 0.05). Co-culturing with OAZ siRNAs reduced mRNA levels of OAZ by 74.6 ± 6.4% as compared to those co-cultured with non-targeting siRNA and OAZ silencing resulted in reduced total IgG, ANA, interferon (IFN)-γ, interleukin (IL)-10, IL-12 and IL-21, but elevated CCL2 levels in culture supernatants (P < 0.05). The declined ANA levels correlated with inhibited OAZ expression (r = 0.88, P = 0.05), reduced IL-21 levels (r = 0.99, P < 0.01), and elevated chemokine (C-C motif) ligand 2 levels (r = -0.98, P < 0.01). Expressions of ID1-3 were significantly down-regulated by 68.7%, 70.2% and 67.7% respectively after OAZ silence, while ID3 was also highly expressed in SLE PBLs (P < 0.0001) and associated with disease activity (r = 0.76, P < 0.0001) as well as anti-dsDNA or anti-Sm antibodies (both P < 0.05).
Elevated expression of OAZ transcripts in SLE PBLs were strongly correlated with disease activity. Suppression of OAZ expression inhibited downstream ID levels, and secretion of ANA and IL-21, implicating a role of OAZ pathway in the pathogenesis of SLE.
KeywordsSystemic Lupus Erythematosus Rheumatoid Arthritis Patient Systemic Lupus Erythematosus Patient Negative Control Group Peripheral Blood Cell
anti-RNA binding protein
chemokine (C-C motif) ligand 2
early B cell factor
inhibitor of differentiation/inhibitor of DNA binding
lymphocyte antigen 6 complex, locus E
olf1/EBF associated zinc finger protein
peripheral blood cells
peripheral blood mononuclear cells
quantitative real-time PCR
human ribosomal protein, large, P0
systemic lupus erythematosus
SLE disease activity index.
Systemic lupus erythematosus (SLE) is a prototype autoimmune disease with relatively strong genetic components that genome wide linkage scans performed in the past decade have identified chromosome 16p12.3 to q12.2 to be the second-strongest locus linked to SLE after HLA (6p22.3 to 6p21.1) [1, 2, 3, 4, 5, 6, 7, 8]. A positional candidate gene, ITGAM located at 16p11.2, has been recently identified and validated as a lupus susceptibility gene by different groups [9, 10, 11]. However, fine mapping experiments of the chromosome 16p locus have shown multiple association signals clustering in several regions [8, 12], suggesting more than one gene may be involved in the increased risk of SLE.
We have previously shown overall skewing of the transmission of D16S517 (a microsatellite marker) alleles and preferential transmission of one of its alleles from heterozygous parents to offspring affected with SLE of Chinese descent [13, 14]. D16S517 is located within intron 5 of a gene named Olf1/EBF associated zinc finger protein (OAZ, also known as zinc finger protein 423; ZNF423) in 16q12. Genotyping of five SNPs within intron 4 and intron 5 of OAZ reveals preferential transmission of haplotypes containing SNPs and/or the SLE-associated D16S517 allele , suggesting OAZ may be a positional candidate gene within the 16q interval.
Currently there is no evidence indicating OAZ in the pathogenesis of SLE. OAZ is a transcriptional factor that binds to DNA through its zinc fingers, which acts as a bone morphogenetic protein (BMP) induced co-regulator of the Smad1 to Smad4 complex . Through model biology study, OAZ is found to be necessary in BMP-4 induced gene activation . Among the various genes activated via BMP signaling, ID (inhibitor of differentiation or inhibitor of DNA binding) proteins involved in the regulation of cell differentiation and proliferation may be the most important targets . Four ID proteins (ID1 to 4) have been identified in mammals, in which ID3 is required for normal B cell functions . Thus, it is plausible that OAZ may act through these IDs to regulate immune responses.
The presence of antinuclear antibodies (ANA), found in 95% of the patients, is the most prevalent feature of SLE that may play a pivotal role in the disease pathogenesis. These antibodies bind to a variety of macromolecules, including DNA, RNA and proteins, and some of them, notably anti-dsDNA and anti-Sm, are specific for the disease. Both the B-cell and T-cell compartments exhibit functional abnormalities that could lead to the autoantibody production in SLE . Here we showed that OAZ expression levels were elevated in patients affected with SLE but not RA, correlated with disease activity and associated with positivity for anti-dsDNA and anti-RNA binding protein (anti-RBP) antibodies. In addition, silencing OAZ expression correlated with decreased production of ANA, IL-21, but elevated CCL2. These findings support a role of OAZ in the pathogenesis of SLE.
Materials and methods
Patients and controls
Demographics of SLE patients, normal controls and disease controls*
SLE patients (n = 40)
Normal controls (n = 30)
Disease controls (n = 18)+
33.48 ± 2.11 (14 to 70)
29.23 ± 1.13 (19 to 42)
38.39 ± 2.02 (18 to 54)
Disease duration, years
5.91 ± 0.94 (0.08 to 20)
7.70 ± 0.90 (1 to 27)
At the beginning of the study, each SLE patient was assessed for the presence of ANA and IgG antibodies to anti-dsDNA and anti-extractable nuclear antigen using reagents from Euroimmun AG (Lübeck, Germany) and disease activity at the time of blood drawn was assessed by their rheumatologists and verified by two of the authors (RL and BH) using the SLE disease activity index (SLEDAI) . Among the 40 patients having their gene expressions measured in peripheral blood, 38 were treated with steroids (an average dose of 34.6 ± 2.6 mg per day of methylprednisolone (from 6 mg to 80 mg according to their disease activity)), 18 were taking hydrochloroquine, 5 were on cellcept or cyclosporin and 8 were on non-steroidal anti-inflammatory drug at the time of blood draw.
Sample collection and RNA processing
A 2 to 3 ml sample of blood was collected in BD Vacutainer spray-coated K2EDTA Tubes (BD Biosciences, San Jose, CA, USA) and total RNA from whole cells was extracted immediately using Trizol (Invitrogen, Carlsbad, CA, USA). A 1 to 2 μg aliquot of total RNA was reverse transcribed into complementary DNA (cDNA) using Superscript II reverse transcriptase (Invitrogen, USA). All RNA and cDNA samples were stored at -70°C before use.
Gene expression measuring
Primers for real-time PCR
CTG CTC ACA GTG CCC TCA GAA G
ACT GTG CGT GCT GGC TCA TC
GTA AAC GTG CTG CTC TAC GAC ATG A
AGC TCC AAC TGA AGG TCC CTG A
TGT CAG CCT GCA TCA CCA GA
CCA CAC AGT GCT TTG CTG TCA
TCA GCT TAG CCA GGT GGA AAT C
GGC TGT CTG GAT GGG AAG GT
GAT CCT GCA GCA CGT TAT CGA CT
AAT GCT GTC GCC CTG CTT G
GTT TCA TTG TGG GAG CAG ACA
CAT GGT GTT CTT GCC CAT CA
Experiments were performed in triplicate for each sample in 96-well plates using SYBR Green I Dye in the Applied Biosystems 7500 real-time PCR system. Reactions were performed in a 20 μl reaction volume and cycling times and temperatures were as follows: initial denaturation was carried out for 10 seconds at 95°C, followed by 45 cycles of denaturation at 95°C for 5 seconds and combined primer annealing/extension at 60°C for 34 seconds. Data were displayed using SDS software, version 2.0 (Applied Biosystems, USA). The ΔCT value was determined by subtracting the RPLP0 (the house keeping gene) CT value from the target gene CT value. Relative gene expressions were calculated as 2-(ΔCT each-ΔCT mean) where ΔCT each = ΔCT value of each sample, ΔCT mean = mean ΔCT values of normal controls.
Accell siRNA sequences
GUA AUG AAU AUA AUC GGU U
AAC CGA UUA UAU UCA UUA C
GCA UCA ACC ACG AGU GUA A
UUA CAC UCG UGG UUG AUG C
GGA GGA UGA AUC AAU UUA C
GUA AAU UGA UUC AUC CUC C
CCU UCA UGU AUU AUA UUG A
UCA AUA UAA UAC AUG AAG G
UUC UCC GAA CGU GUC ACG U
ACG UGA CAC GUU CGG AGA A
GUA UGA CAA CAG CCU CAA G
CUU GAG GCU GUU GUC AUA C
Blood was drawn from five randomly selected female SLE patients (40.40 ± 1.69 yrs old) and OAZ gene silencing was performed according to the manufacturer's instructions (Dharmacon). Briefly, mononuclear cells were collected by Ficoll-Hypaque discontinuous gradient, resuspended in siRNA buffer, distributed to 96-well plate at 2 × 105 per well, and then divided into OAZ silence group (OAZ sil, added with OAZ siRNA mixture using SMARTpool technique), positive control group (added with GAPDH siRNA), negative control group (Neg ctl, added with non-targeting siRNA) and mock group (only culture medium added). For each sample, 4 to 10 replications in each group were applied. Cells were incubated at 37°C with 5% CO2 for 72 hour and trypan blue exclusion test of cell viability was done by counting 200 cells before and after cell culture. After culturing, supernatants were collected and cells were harvested, RNA was extracted and then reverse transcribed to cDNA. Specific inhibition efficacy of OAZ, ID1-4 and GAPDH was confirmed by qPCR.
Immunoglobulin and antinuclear antibody detection
Total IgG and IgM levels were measured by commercial ELISA kits (Westang Bio-Tech Co., Shanghai, China). Cultured supernatants were diluted in 1:10 for IgG test and undiluted for IgM test. ANA levels were detected by ELISA and also indirect immunofluorescence according to manufacturer's instruction (Euroimmun, Lübeck, Germany). An ANA value for each sample was calculated by OD sample/OD standard obtained from the ELISA test. As for the immunofluorescence test, undiluted samples were layered on HEp-2 cell spots and incubated for 30 minutes. After washing, fluorescein-labeled anti-human globulin was added to each spot and incubated for another 30 minutes. Slides were then embedded and read under the fluorescent microscope (Olympus; Tokyo, Japan). The criterion for assigning intensity of fluorescence was as follows: 4+ (very bright green), 3+ (bright green), 2 + (green), 1 + (faint green).
Cytokine and chemokine measurements
Interferon γ (IFN-γ), interleukin 4 (IL-4), IL-10, IL-12, IL-21 and chemokine (C-C motif) ligand 2 (CCL2) levels in supernatants obtained from the previous step were measured by EIA kits (R & D Systems, Minneapolis, MN, USA). For each cytokine, samples and diluted standards were added to pre-embedded plate and incubated for two hours at 37°C. Then biotinylated anti-human detection antibody was added to each well. The plate was incubated for one hour at 37°C and developed with the addition of Streptavidin-HRP and tetramethylbenzidine as a substrate. The optical density for each well was documented with a microplate reader (Tecan Sunrise, Männedorf, Switzerland) set to 450 nm and levels of cytokines and chemokine (pg/ml) were calculated according to their standard curves.
Results are presented as means ± SEM. Because gene expressions were extremely altered in certain patients, those levels were transformed to log10 values for analysis. To observe the changes of gene expressions, cytokine/chemokine levels and ANA values due to OAZ silence, an excitation-inhibition ratio (EIR) for each sample was calculated according to the formula: Open image in new window . For comparing the results between two groups, Student's t-test was conducted if the variance was normally distributed, whereas the Mann-Whitney U test was used if the variance was not normally distributed. Correlation between groups was evaluated using the Pearson rank test. Data were analyzed using the Prism 3.0 program (GraphPad, La Jolla, CA, USA), and P < 0.05 was considered significant.
OAZ highly expressed in SLE patients and correlated with disease activity
OAZ associated with autoantibody production in SLE patients
Since the production of antinuclear autoantibodies is the most ubiquitous feature in SLE, SLE predisposing factors should impact on ANA production . Within our 40 patients, all had a positive ANA (titers >1:100) at the time of blood draw, while 15 had concurrent IgG anti-dsDNA, 19 had anti-Sm, 18 had anti-SSA, 14 had anti-SSB and 9 had anti-U1 RNP. We found OAZ expressions were higher in patients with positive anti-dsDNA antibodies as well as in those with positive anti-Sm antibodies (both P < 0.05, Figure 1c). However, there were no associations between OAZ expression and anti-SSA, SSB or U1RNP antibodies (data not shown).
Diminished ANA production after OAZ silence
ANA were present in all studied SLE patients, making ANA the most useful test to measure the change of autoantibodies after OAZ knockdown. Because most SLE patients did not have positive anti-dsDNA antibody and anti-Sm antibody in their serum, we measured ANA in culture supernatants as an alternative. As shown in Figure 2c, ANA value was 2.30 ± 0.20 in the negative control group and 0.13 ± 0.03 in the OAZ knockdown group measured by ELISA (P < 0.01). There was no difference in ANA value between negative control group and GAPDH knockdown group (P > 0.05). Consistent with ELISA results, intensity of ANA fluorescence was decreased from 2 to approximately 3+ in the control group to 1+ in OAZ silenced group assayed using indirect immunofluorescence (Figure 2d). There was a positive correlation between the EIR of OAZ expression and ANA value (r = 0.88, P = 0.05) (Figure 2e), suggesting that OAZ might play a role in ANA production.
To further confirm a relationship between OAZ expression and antibody production, total IgG and IgM in culture supernatants were measured. IgG levels were significantly decreased in the OAZ knockdown group as compared to those in the negative control group (26.8 ± 3.7 ug/ml vs 44.3 ± 6.4 ug/ml, P < 0.05), while there was no difference in IgM levels between the two groups (4.13 ± 0.56 ug/ml vs 2.82 ± 0.36 ug/ml, P = 0.09).
Alteration of cytokine and chemokine levels after OAZ silence
To identify the relationship between OAZ expression and specific cytokines and/or chemokine, EIR was calculated as described before. There was a positive trend of correlation between the EIR of OAZ expression and the EIR of IL-21 level (r = 0.83, P = 0.08, Figure 3b), suggesting the more decreased levels of OAZ, the lower levels of IL-21. The EIR of OAZ expression was negatively correlated with the EIR of CCL2 level (r = -0.89, P < 0.05, Figure 3c), indicating a strong negative correlation between OAZ transcripts and the secreted levels of CCL2 in cultures of peripheral blood cells from SLE patients.
Strikingly, there was a perfect positive correlation between EIR of IL-21 levels and EIR of ANA value, and a negative correlation between EIR of CCL2 levels and EIR of ANA value (r = 0.99 and r = -0.98, both P < 0.01) (Figure 3d, e), providing evidence that these two factors might be important in the production of ANA.
ID3 gene participated in OAZ pathway in SLE
Among the four IDs, ID3 that had putative immunofunctions  was selected as a representative for in vivo studies. To assess whether ID3 expressions were related to the disease status, qPCR was applied using peripheral blood cells from 40 SLE patients and 20 normal controls. Our data showed that ID3 was highly expressed in SLE patients compared to that in normal controls (0.75 ± 0.09 vs 0 ± 0.11, P < 0.0001) and there was a positive correlation between ID3 expression level and SLEDAI score (r = 0.76, P < 0.0001) (Figure 4b, c). Meanwhile, a correlation between OAZ and ID3 expression levels was observed (r = 0.55, P = 0.0002). ID3 expressions were also elevated in those with positive anti-dsDNA or anti-Sm antibodies (Figure 4d), indicating that ID3 could play a role in the regulation of OAZ driven ANA production.
Abnormal activation of the type I interferon pathway presents in most SLE patients particularly in those expressing anti-RBP antibodies . To address whether siRNA for OAZ might non-specifically inactivate the type I interferon pathway, the expression level of LY6E, a type I interferon inducible gene , was measured after OAZ knockdown. No significant alteration of LY6E expression was observed among groups treated with non-targeting GAPDH- or OAZ- targeting SiRNA (data not shown).
To our knowledge, this is the first report to implicate a functional role of OAZ, a novel positional lupus candidate gene, in the pathogenesis of SLE. Dysregulation of OAZ expression modulated the ANA production of SLE patients. Our studies showed that OAZ mRNA levels in peripheral blood cells were elevated in SLE patients as compared to those in either normal or disease controls (RA patients). Transcript levels of OAZ were highly correlated with disease activity (r = 0.72, P < 0.0001) as well as associated with the presence of autoantibodies to dsDNA or Sm (Figure 1). OAZ expressions were negatively correlated with methylprednisolone doses (data not shown), implying that there should be an even greater elevation of OAZ expression in those patients with high SLEDAI scores because they were treated with high dose of steroids. When OAZ expression in peripheral blood mononuclear cells was silenced in vitro, mRNA levels of OAZ and ID1-3 were concomitantly decreased, together with a reduction of IFN-γ, IL-10, IL-12 and IL-21 levels, an elevation of CCL2 level, and decreased levels of total IgG and ANA (Figures 2 and 3). The extent of decreased ANA level was correlated with the inhibition of OAZ expression, the reduction of IL-21 level and the elevation of CCL2 level. Interestingly, ID3, a gene that was down-regulated after OAZ silence, was also highly expressed in SLE patients and associated with disease activity as well as the presence of anti-dsDNA or anti-Sm autoantibodies (Figure 4).
OAZ is present in cell nucleus and acts as a DNA-binding partner of BMP-Activated Smads . According to our results, mRNA expression of OAZ is very low in peripheral blood cells (approximately 0.002% of the expression level of the house-keeping gene), making it difficult to be detected in the protein level. To ensure a specific amplification of this gene, primers have been blasted after the designation . Besides, only OAZ but not EHZF, an OAZ homology gene that highly expressed in primitive human hematopoietic cells , showed differential expression in bone marrow cells between SLE patients and healthy donors in our preliminary study. The Accell siRNA delivery method, which has recently been widely used to silence genes in many cell types including those difficult-to-transfect immune cells [30, 31, 32], provides us a highly efficient way to study genes with low expressions but important functions.
In this paper, OAZ was found to be associated with autoantibody production in vivo and in vitro, suggesting that highly expressed OAZ in SLE patients might promote differentiation of B lymphocytes to antibody-secreting cells. Support evidence for this hypothesis includes that 1) overexpression of OAZ in the neuroblastoma cells leads to enhanced differentiation , and 2) suppression of OAZ expression reduces cell growth and contributes to the blast crisis stage in chronic myelogenous leukemia mouse model as the result of the differentiation block . However, it is not clear how OAZ mediates its role in cell differentiation at present. As one of the largest members of the Kruppel-like zinc finger protein family, OAZ provides specificity in BMP signaling while ID genes are thought to be important targets of BMP signaling [15, 35]. Based on the study of osteoblast differentiation of mesenchymal stem cells, both RNA interference-mediated knockdown and constitutive overexpression of Id genes are associated with diminished cell differentiation, implicating that Id proteins could play a dual role in BMP-mediated differentiation .
Among the four ID proteins, ID3 is known to have important functions in the immune system, although its specific role is yet to be delineated. Young ID3-deficient mice (6 to 12 weeks old) have defective immune responses displaying as a B-cell defect in the proliferation and attenuated antibody production . Meanwhile, the production of IgG2a (a Th1 response immunoglobulin) is impaired along with a decreased expression of IFN-γ when stimulated with anti-CD3 in these mice. When E2A, an ID3 antagonist gene, was knocked down, mice frequently developed ANA and proteinuria at age eight months or older . Thus a sustained high expression of ID3 might trigger abnormal immunity in SLE patients, facilitating the production of autoantibodies. To support this notion, our current data have shown a good correlation between ID3 and OAZ expressions and an association of ID3 expression with both anti-dsDNA and anti-Sm antibodies. Besides ID pathway, OAZ has been shown to be a component of retinoic acid signaling, and plays a key role in retinoic acid-induced differentiation through the regulation of retinoic acid receptors . In addition, OAZ may act as a corepressor of EBF (early B cell factor) , and thus inhibit the transcription of a lot B lineage genes including Pax-5  and consequently affect plasma cell differentiation [40, 41]. It is reasonable that these pathways could also be helpful in OAZ induced B lymphocyte differentiation and antibody production in SLE patients.
Studies have implicated that cytokines and chemokines are involved in the differentiation of B lymphocytes into plasma cells [42, 43]. The decreased level of Th1cytokines (IFN-γ and IL-12) in our case after OAZ silence is similar to that found in ID3-deficient mice , supporting a role of OAZ-ID3 pathway in this process. IL-10 is a well-known mediator of human B cell differentiation that has a powerful effect on the stimulation of immunoglobulin secretion by B cells in SLE . IL-21 has a critical role in terminal B cell differentiation to plasma cells and has recently been identified as an important component in the development of autoimmune disease [45, 46, 47]. Our in vitro results suggested that OAZ could regulate IL-10 and IL-21 level in SLE. The near perfect correlation between the extent of IL-21 and ANA suppression by OAZ knockdown suggested that this cytokine could be a key mediator for OAZ induced B cell differentiation and ANA production. In addition to these cytokines, a chemokine, CCL2, has been shown to suppress plasma cell immunoglobulin production via STAT3 inactivation and PAX5 induction . Of note, the expression of CCL2 is effectively regulated by BMP members . In our experiment, CCL2 might work as a negative regulator of plasma cell differentiation when upregulated via OAZ silence.
OAZ has been previously considered as a positional candidate gene for susceptibility to SLE. Our data provide evidence for functional relevance of OAZ in SLE in the production of antinuclear antibody, a hallmark of SLE, along with the alterations of several cytokines and chemokine especially those involved in the regulation of plasma cell function. As a complex network, BMP-Smad-IDs, retinoic acid receptor and Olf-1/EBF pathways could participate in OAZ-driven ANA production. Further studies are required for elucidating the molecular mechanisms of the OAZ gene in the development of autoimmune diseases.
We thank all the patients and healthy volunteers participating in this study. This work was supported by Jiangsu Natural Science Foundation Grant (BK2004009), Chinese National Natural Science Fund (30972735) and Jiangsu Province 135 Talented Grant (2007002). BPT was supported by NIH RO1 43824.
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