Regulated Gene Expression and Overexpression of Methylation-Sensitive Genes Similar to Lupus Patients

ORIGINAL ARTICLE Defective T-cell ERK signaling induces interferon- regulated gene expression and overexpression of methylation-sensitive genes similar to lupus patients

AH Sawalha1,2,3, M Jeffries3, R Webb3,QLu4, G Gorelik5, D Ray5, J Osban3, N Knowlton3, K Johnson6 and B Richardson5,7 1US Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA; 2Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; 3Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; 4Second Xiangya Hospital, Central South University, Changsha, China; 5Department of Medicine, University of Michigan, Ann Arbor, MI, USA; 6Department of Pathology, University of Michigan, Ann Arbor, MI, USA and 7US Department of Veterans Affairs Medical Center, Ann Arbor, MI, USA

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against a host of nuclear antigens. The pathogenesis of lupus is incompletely understood. Environmental factors may play a role via altering DNA methylation, a mechanism regulating gene expression. In lupus, genes including CD11a and CD70 are overexpressed in T cells as a result of promoter hypomethylation. T-cell DNA methyltransferase expression is regulated in part by the extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we investigate the effects of decreased ERK pathway signaling in T cells using transgenic animals. We generated a transgenic mouse that inducibly expresses a dominant- negative MEK in T cells in the presence of doxycycline. We show that decreased ERK pathway signaling in T cells results in decreased expression of DNA methyltransferase 1 and overexpression of the methylation-sensitive genes CD11a and CD70, similar to T cells in human lupus. Our transgenic animal model also develops anti-dsDNA antibodies. Interestingly, microarray expression assays revealed overexpression of several interferon-regulated genes in the spleen similar to peripheral blood cells of lupus patients. This model supports the contention that ERK pathway signaling defects in T cells contribute to the development of autoimmunity. Genes and Immunity (2008) 9, 368–378; doi:10.1038/gene.2008.29

Keywords: epigenetics; T cell; autoimmunity; interferon; methylation; lupus

Introduction ing in autoreactive T cells capable of promoting autoimmunity. The epigenetic mechanism most strongly Genetic and environmental factors contribute to human linked to lupus-like autoimmunity is DNA methylation.4 lupus. Evidence for a genetic contribution comes from a DNA methylation is the postsynthetic methylation of higher concordance rate among monozygotic twins, cytosine bases occurring in CG pairs. Promoters of active familial aggregation, a number of known lupus-asso- genes are typically hypomethylated, and methylation of ciated genetic polymorphisms and evidence for linkage promoter sequences renders the genes transcriptionally at multiple loci across the human genome.1 Observations inactive.5 Methylation patterns are established during that the majority of lupus is sporadic, concordance in development by the de novo DNA methyltransferases monozygotic twins is incomplete and drugs such as Dnmt3a and 3b, then replicated during mitosis by the procainamide and hydralazine can trigger lupus-like maintenance methyltransferase Dnmt1.6,7 Resting T cells autoimmunity indicate a role for exogenous agents.2,3 express relatively little Dnmt1, but levels are upregulated How environmental agents contribute to the develop- during mitosis by signals through the extracellular ment of autoimmunity is unclear. Recent evidence, signal-regulated kinase (ERK) and Jun N-terminal kinase though, suggests that the environment can modify T-cell pathways.8,9 Inhibiting DNA methyltransferase activity gene expression through epigenetic mechanisms, result- with inhibitors such as 5-azacytidine, or decreasing Dnmt1 levels with ERK pathway inhibitors, inhibits methylation of newly synthesized DNA, causing over- Correspondence: Dr AH Sawalha, Department of Medicine, Uni- expression of genes suppressed by this mechanism.10 versity of Oklahoma Health Sciences Center, Oklahoma Medical CD4 þ T cells treated with DNA methylation inhibi- Research Foundation, 825 NE 13th Street, MS no. 24, Oklahoma City, tors overexpress LFA-1 (CD11a/CD18) and become OK 73104, USA. autoreactive, responding to self-class II MHC without E-mail: [email protected] 11 Received 26 November 2007; revised 15 February 2008; accepted 11 the addition of exogenous antigen. The autoreactivity is March 2008 due to demethylation and overexpression of the ITGAL ERK pathway signaling and epigenetic regulation in lupus AH Sawalha et al 369 (CD11a) gene,12,13 and T cells overexpressing LFA-1 by tively in T cells in the presence of doxycycline. The transfection display identical autoreactivity.14 Hypo- dnMEK mutant, kindly donated by Dr Kun-Liang Guan, methylated, autoreactive CD4 þ cells also spontaneously contains triple mutations in the regulatory and kinase kill autologous macrophages (Mø), causing release of domains of wild-type MEK1 (substitutions of serines 218, antigenic nucleosomes through mechanisms including 222, and lysine 97 by alanine), that block Raf-1-mediated demethylation and subsequent overexpression of the MEK phosphorylation. Expression of this construct leads PRF1 (perforin) gene.15–17 The hypomethylated cells also to B60% reduction in ERK phosphorylation in Swiss 3T3 overstimulate autologous B-cell antibody production, cells and does not impair cell survival.30 This mutant was due to demethylation and overexpression of the genes cloned into pTRE-2, containing a tetracycline response encoding CD70 (TNFSF7), and CD40L (CD40LG).18,19 element, then injected into C57BL/6 Â (C57BL/6 Â SJL) Importantly, procainamide and hydralazine, which cause F1 mouse eggs and implanted into psuedopregnant anti-nuclear antibodies in a majority of people and a females. Mice with the transgene were backcrossed onto lupus-like disease in a subset, are DNA methylation a C57BL/6 background 6–10 times, then bred with a BL/ inhibitors.20 Procainamide is a competitive Dnmt1 6 transgenic strain containing the reverse tetracycline inhibitor,21,22 whereas hydralazine inhibits ERK pathway transactivator under the control of a CD2 promoter signaling, thereby decreasing Dnmt1 expression.23 Adop- (CD2-rtTA). The double-transgenic mice (dnMEK/CD2- tive transfer of CD4 þ T cells treated with these drugs or rtTA) would be predicted to express the dnMEK when 5-azacytidine causes anti-DNA antibodies and a lupus- doxycycline is added to their drinking water (Figure 1a). like disease in mice.23–25 Although all strains studied ‘Leakiness’ and inducibility of dnMEK expression in develop anti-DNA antibodies, the clinical manifestations the dnMEK/CD2-rtTA mice was tested in various tissues are strain dependent,24,25 suggesting a genetic require- including heart, lung, kidney, brain, thymus and spleen ment for full disease development similar to hydrala- by real-time RT-PCR and primers specific for the dnMEK zine-induced autoimmunity in people.26 in mice with and without doxycycline. As expected, Further, CD4 þ T cells from patients with active lupus significant expression of dnMEK mRNA was detected in demethylate the same regulatory sequences, overexpress the lymph nodes, spleen and thymus in the presence of the same genes, and display the same autoreactive Mø doxycycline but not in its absence (Figure 1b). Further- killing and B cell overstimulation as cells demethylated more, the dnMEK was expressed only in CD3 þ T cells in in vitro with 5-azacytidine, procainamide and hydrala- the presence of doxycycline (Figure 1c). To test whether zine.17,27,28 Together, these observations suggest that the expression of dnMEK decreases ERK signaling in T T-cell DNA demethylation may contribute to the devel- cells, CD3 þ cells isolated from spleens of dnMEK/CD2- opment of idiopathic and some forms of drug-induced rtTA double-transgenic mice were incubated with dox- lupus. ycycline, without doxycycline or with the MEK inhibitor T-cell DNA demethylation in idiopathic lupus may be PD 98059 for 24 h. CD3 þ cells incubated with doxycy- due to decreased ERK pathway signaling. As noted cline demonstrated reduced ERK signaling as measured above, inhibiting ERK pathway signaling decreases T-cell by phosphorylated ERK (active MAPK), upon stimula- Dnmt1 expression, causing DNA demethylation and tion with phorbol myristate acetate (Figure 2). methylation-sensitive gene overexpression.8,23,28 Lupus T cells display similar decreases in ERK pathway signaling DnMEK/CD2-rtTA double-transgenic mice demonstrate and Dnmt1 levels, and the degree of impairment is reduced Dnmt1 expression and the overexpression of T-cell directly proportional to disease activity.29 Also, murine methylation-sensitive genes CD4 þ T cells treated with hydralazine or the MEK To test whether decreased ERK signaling in T cells inhibitor U0126 demethylate, overexpress methylation- decreases Dnmt1 expression, double-transgenic mice sensitive T-cell genes and cause anti-DNA antibodies were given doxycycline (2 mg/ml) in their drinking when injected into syngeneic mice.23 water for 1 week, then treated and untreated mice were Based on these observations, we hypothesized that killed and the spleens extracted. RNA was isolated from acquired ERK pathway signaling defects, occurring in the whole spleen, and Dnmt1 mRNA levels were mature T cells, may be fundamental to the development measured by real-time reverse transcription (RT)-PCR. of lupus-like autoimmunity. To test this hypothesis, we Dnmt1 transcripts were reduced by B50% with doxycy- generated transgenic mice with an inducible ERK path- cline treatment (Figure 3a, P ¼ 0.02). This confirms earlier way signaling defect. We report that inducing an ERK reports that ERK signaling pathway is involved in pathway signaling defect in mature T cells is sufficient to regulating Dnmt1 expression.29 We also measured decrease Dnmt1 expression, cause overexpression of a Dnmt1 expression in vitro in stimulated T cells from methylation-sensitive T-cell genes, and induce interfer- dnMEK/CD2-rtTA mice as compared to CD2-rtTA mice on-regulated gene expression in a non-autoimmune treated with doxycycline (2 mgmlÀ1). There was a prone mouse strain. significant reduction in Dnmt1 mRNA levels in stimulated T cells from dnMEK/CD2-rtTA (0.40±0.05 versus 1.36±0.28 (mean±s.e.m.), P ¼ 0.028, n ¼ 3) (Figure 3b). Results To assess if reduced Dnmt1 expression could cause overexpression of methylation-sensitive genes in vivo, Generation of transgenic mice double-transgenic mice were given doxycycline (4 mg/ml) MEK is a protein kinase, which upon phosphorylation by for 2 weeks, then killed, and transcripts of the methylation- Raf, results in ERK phosphorylation and therefore sensitive genes CD11a and CD70 were quantitated in the activation of the ERK signaling pathway. We generated spleen by real-time RT–PCR. Both genes were over- a double-transgenic mouse (dnMEK/CD2-rtTA) that expressed in doxycycline-treated double-transgenic mice, inducibly expresses a dominant-negative MEK selec- as predicted (Figure 4). The expression of CD11a has

Genes and Immunity ERK pathway signaling and epigenetic regulation in lupus AH Sawalha et al 370

CD2 rtTA T cell specific expression

Doxycycline

TRE dnMEK TRE dnMEK

120

100 Doxycycline No doxycycline 80

40 Dominant negative MEK Relative expression 20

0 Lymph Heart Liver Spleen Thymus Brain Lung Kidney nodes Tissue Figure 1 Conditional expression of a dnMEK in T cells. (a) A transgenic mouse strain was generated by subcloning a dnMEK cDNA downstream of a Tet-responsive promoter. This promoter contains the Tet response element (TRE), consisting of seven copies of the 42-bp tet operator sequence (tetO). Breeding with another transgenic mouse containing the reverse tetracycline-regulated transactivator (rtTA) under the control of the CD2 promoter yielded a double-transgenic mouse (dnMEK/CD2-rtTA) capable of T-cell specific expression of the dnMEK. When doxycycline is provided, rtTA binds to the tetO in the Tet-responsive promoter and activates the transcription of the dnMEK. (b)A representative of two independent experiments showing the expression of dominant-negative MEK (dnMEK) in various tissues in dnMEK/ CD2-rtTA double-transgenic mice assessed by real-time reverse transcription (RT)–PCR. The expression of the transgene is restricted to the lymph nodes, spleen and thymus and is induced by doxycycline (2 mg mlÀ1 in drinking water). (c) dnMEK expression is restricted to CD3 þ splenocytes. CD3 þ and CD3À splenocytes were isolated and incubated for 24 h with doxycycline at a concentration of 1 mgmlÀ1. RNA was isolated and dnMEK expression was assessed by real-time RT-PCR and normalized to GAPDH (representative of two experiments).

previously been reported to be methylation-sensitive in Defective ERK signaling in T cells induces anti-dsDNA both mouse and human T cells.15,23,31 To confirm that the antibody production and differential expression expression of CD70 is also methylation-sensitive in of interferon-regulated genes mouse T cells, wild-type mouse spleen cells were We then compared the development of anti-dsDNA stimulated with Con-A overnight, and then incubated antibodies in three dnMEK/CD2-rtTA mice receiving with or without the DNA methylation inhibitor 5- doxycycline (4 mg/ml) and three not, administered azacytidine (5 mM) in interleukin 2 (IL-2; 50 U mlÀ1) continuously over 12 weeks. As tetracyclines can cause containing culture media for an additional 72 h. T cells a lupus-like disease in some people,32 six wild-type mice were isolated and the expression of CD70 measured by were also given doxycycline as a control. In addition, real-time RT-PCR. We found that CD70 expression anti-dsDNA antibodies were measured in seven wild- increased with 5-azacytidine treatment in wild-type type mice without doxycycline treatment. Figure 5a mouse T cells (1.18±0.26 to 5.12±0.84 (mean±s.e.m.), shows the kinetics of the anti-dsDNA response in a P ¼ 0.011, n ¼ 3) confirming that CD70 is a methylation- representative double-transgenic mouse receiving dox- sensitive gene in mouse T cells. ycycline, and a double-transgenic mouse not receiving We next measured cytokine production in vitro in the drug. Anti-ds DNA antibodies appear between 4 and stimulated T cells from dnMEK/CD2-rtTA mice as 8 weeks, and increase through 12 weeks. Figure 5b shows compared to CD2-rtTA mice treated with doxycycline the mean anti-dsDNA titers in the four groups, measured (2 mgmlÀ1) using a bead-based immunofluorescence after 12 weeks of doxycycline or control drinking water. assay (Luminex Inc., Austin, TX, USA). Multiplex Only the double-transgenic mice given doxycycline were cytokine reagents were purchased from Invitrogen positive for anti-dsDNA antibodies (Figure 5). (Carlsbad, CA, USA). Following red blood cell lysis, Expression microarray experiments were performed to splenocytes (3 Â 106 cells per ml) were stimulated with detect differential gene expression in the spleens of four Con-A overnight and cytokine levels were measured in dnMEK/CD2-rtTA mice and four CD2-rtTA controls the supernatants. We found no difference in the produc- after two weeks of doxycycline treatment (4 mg/ml). We tion of the cytokines measured including IFN-g, IL-12, identified 60 genes that were differentially expressed by IL-6, GM-CSF and TNF-a. at least twofold between the dnMEK/CD2-rtTA mice

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Double Wild-type transgenic

Doxycycline - + --+- PD-98056 - -+ --+

p-ERK 1/2

t-ERK 1/2

Wild-type Double transgenic 1.1 1.1 1.0 1.0 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 p-ERK/t-ERK 0.3 p-ERK/t-ERK 0.3 0.2 0.2 0.1 0.1 0.0 0.0 Doxycycline - + - - +- PD-98056 --+ --+ Figure 2 (a) A representative western blot demonstrating extracellular signal-regulated kinase (ERK) signaling as measured by phosphorylated ERK 1 and 2 (p-ERK 1/2) compared to total ERK (t-ERK 1/2). CD3 þ cells isolated from spleens of double-transgenic mice and wild-type mice were incubated with doxycycline (1 mgmlÀ1), without doxycycline or with the MEK inhibitor PD 98059 (50 mM) for 24 h. CD3 þ cells from double-transgenic mice incubated with doxycycline demonstrated reduced ERK signaling upon stimulation with phorbol myristate acetate. (b) A relative reduction in p-ERK in double-transgenic mice with versus without doxycycline (0.52±0.08 versus 1±0.00 (mean±s.e.m.), P ¼ 0.0032, n ¼ 3). No reduction in p-ERK was observed in wild-type mice with doxycycline (n ¼ 2).

1.3 and CD2-rtTA mice (Figure 6). Of interest, several 1.2 differentially expressed genes are interferon-regulated 1.1 1.0 genes (Figure 7a). Isg15 (G1p2) and Isg20 are part of the 0.9 ‘interferon signature’ genes described in lupus patients 0.8 and scleroderma patients respectively.33,34 We validated 0.7 0.6 the microarray results by confirming the expression of 0.5 four transcripts differentially expressed in dnMEK/CD2- 0.4 Tk1 Isg15 Isg20 0.3 rtTA mice using real-time RT-PCR ( , , and

Dnmt1 Relative Expression 0.2 Il7r; Figure 7b). 0.1 Using Ingenuity Pathway Analysis software (Ingenu- 0.0 ity Systems Inc., Redwood City, CA, USA), two interest- Doxycycline No Doxycycline ing biological networks were identified that incorporated differentially expressed genes in dnMEK/CD2-rtTA mice 1.75 (Figure 8). Network ‘A’ is particularly interesting, as it 1.50 reveals the central role for interferon-g in the regulation 1.25 of several differentially expressed genes. Network ‘B’ identifies a central role for both b-estradiol and TGFb1in 1.00 some of the differentially expressed genes in dnMEK/ 0.75 CD2-rtTA mice. 0.50 Dnmt1 Relative Expression 0.25 Discussion 0.00 dnMEK/CD2-rtTA CD2-rtTA This is a novel mouse model demonstrating that Figure 3 (a) Dnmt1 expression in the spleen of dnMEK/CD2-rtTA induction of a T-cell ERK pathway signaling defect mice. Double-transgenic mice given doxycycline and thus expres- results in changes consistent with human lupus. This sing the dnMEK gene show decreased Dnmt1 expression as indicates an important role for defective T-cell ERK compared to double-transgenic mice without doxycycline signaling in the pathogenesis of lupus. The production of (0.678±0.132 versus 1.194±0.098 (mean±s.e.m.); P ¼ 0.02, n ¼ 4). anti-dsDNA antibodies in this animal model can be (b) Reduced Dnmt1 expression in vitro in doxycycline-treated T cells induced by inhibiting T-cell ERK signaling. Indeed, from dnMEK/CD2-rtTA mice as compared to CD2-rtTA controls. Dnmt1 mRNA levels were determined by real-time RT-PCR and disease activity in lupus patients correlates well with normalized to GAPDH (n ¼ 3, P ¼ 0.028). both the degree of T-cell ERK signaling defect as well as the anti-dsDNA antibody titer.29,35 Inducing a T-cell ERK

Genes and Immunity ERK pathway signaling and epigenetic regulation in lupus AH Sawalha et al 372 4 +/+ with doxycycline Doxycycline 0.35 +/+ without doxycycline 3.5 No doxycycline

3 0.3

2.5 0.25 2 0.2 1.5 CD11a Relative expression 1 0.15

0.5

Anti-dsDNA antibody (Absorbance) 0.1 0

0.05 2.5 Doxycycline No doxycycline 0 2 08412 Weeks

P=0.04 1.5 0.4

0.3 1 CD70 Relative expression 0.2 0.5

0.1 Anti-dsDNA antibody (Absorbance) 0 0.0 Figure 4 Expression of methylation-sensitive genes in dnMEK/ CD2-rtTA mice. CD11a and CD70 expression were determined in dnMEK/rtTA +/+ +/+ -/- -/- the spleen by real-time reverse transcription (RT)–PCR and normal- Doxycycline + -+ - ized to GAPDH. (a) Increased expression of the methylation- sensitive gene Itgal (CD11a) in double-transgenic mice with as Figure 5 Development of anti-dsDNA antibodies in dnMEK/CD2- compared to without doxycycline (P ¼ 0.036, n ¼ 7). (b) Increased rtTA mice. (a) An example showing serial anti-dsDNA antibody expression of the methylation-sensitive gene Tnfsf7 (CD70) in titer in two littermate mice; double transgenic with and without double-transgenic mice with as compared to without doxycycline doxycycline. (b) Anti-dsDNA antibody titer at 12 weeks following (P ¼ 0.032, n ¼ 10). doxycycline treatment. Sera were collected from double-transgenic mice as well as wild-type mice with and without doxycycline. Double-transgenic mice with, but not without, doxycycline develop anti-dsDNA antibodies specific for lupus (0.319±0.039 versus signaling defect, resulted in reduced expression of 0.156±0.040 (mean absorbance±s.e.m.); P ¼ 0.04). The cutoff for Dnmt1, supporting an important role for ERK signaling positive result in this assay was 0.253 (dotted line). in regulating Dnmt1 expression.29 As expected, the expression of T-cell methylation-sensitive genes was increased in the spleen when ERK signaling defect was could be involved in the pathogenesis of lupus.38 In induced. The overexpression of these methylation- lupus mouse models, however, a more prominent role sensitive genes such as CD11a, and CD70 is also for interferon-g rather than interferon-a has been observed in patients with active lupus as well as in reported.39,40 A prominent role for interferon-g is also human T cells treated with DNA methylation inhibi- seen in our mouse model as suggested by the differen- tors.15,18 tially expressed gene profiles (Figure 8). In the MRL/lpr Microarray expression experiments performed on lupus-prone mouse, 24 interferon-regulated genes spleen RNA from mice with T-cell ERK signaling defect are differentially expressed in the spleen relative to revealed differential expression of 60 genes. Interestingly, MRL þ / þ controls.41 The finding that interferon-g eight genes that are differentially expressed are inter- expression progressively increased in the spleens of feron-regulated genes (Figure 7). The increased level and MRL/lpr mice with time, whereas the levels of inter- activity of interferon-a is well described in lupus feron-a and interferon-b remained stable, suggest an patients.36 Indeed, a number of interferon-regulated ‘interferon-g signature’ in the MRL/lpr lupus-prone genes are differentially expressed in peripheral blood mouse.41 Interestingly, MRL/lpr mice also have T-cell mononuclear cells from lupus patients, constituting what DNA methylation defect as demonstrated by reduced is referred to as the ‘interferon signature’ in lupus.33,37 T-cell Dnmt1 expression, and hypomethylation with Recent data implies a correlation between high inter- overexpression of methylation-sensitive genes such as feron-a activity and anti-dsDNA antibody production in CD70,42 similar to what we observed in the dnMEK/ lupus patients suggesting that the interferon pathway CD2-rtTA mouse model.

Genes and Immunity ERK pathway signaling and epigenetic regulation in lupus AH Sawalha et al 373

6.5 13.6 Log2 Intensity -Sparc -1300014I06Rik -LOC243423 -Ifi47 -E330016A09Rik -1200006G13Rik -Cops8 -LOC331139 -LOC243451 -Erdr1 -Hist1h4m -Tubb5 -LOC380799 -LOC380803 -LOC232067 -Tm4sf3 -2410076I21Rik -4933404M02Rik -LOC384538 -2510042H12Rik -Fech -Kif21b -Isq20 -LOC238427 -Piqt -Fzr1 -G1p2 -Brm1 -Cdca8 -Tk1 -2410030K01Rik -Metap2 -Rad23a -Lmna -Bc034054 -Tpx2 -Diap3 -Ccnb1 -Spag5 -AI449441 -PIk1 -Kif22 -Dedc1b -Dfy -Icam4 -Cdkn3 -Hagh -SIc43a1 -BC022224 -Prdx2 -Ctse -Car2 -Agp1 -Hmbs -E2f2 -Hist1h2ae -Abcb10 -LOC243461 -AI324046 -Il7r 12341234

dnMEK/CD2-rtTA CD2-rtTA Figure 6 Hierarchical Clustering of microarray data for 60 genes that are differentially expressed between dnMEK/CD2-rtTA mice and CD2-rtTA mice (n ¼ 4). Both mice groups received doxycycline. Red indicates higher gene expression levels and green indicates lower expression levels.

The mouse model described herein reproduces several suggests that episodic environmental exposures that abnormalities that exist in lupus patients. These include decrease ERK signaling in T cells can trigger autoimmu- a defective Dnmt1 expression, overexpression of CD11a nity. However, genetic susceptibility is crucial for and CD70, the production of anti-dsDNA antibodies and developing clinical pathology. In human lupus these the overexpression of interferon-regulated genes. How- environmental agents can be certain drugs such as ever, the lack of a lupus clinical phenotype indicates the hydralazine and procainamide, in the case of drug- importance of genetics in lupus susceptibility. Our model induced lupus, or environmental triggers that are yet to

Genes and Immunity ERK pathway signaling and epigenetic regulation in lupus AH Sawalha et al 374

Gene Description Fold difference* p value

Ifi47 Interferon gamma inducible protein, 47 kDa 4.18 0.0000001 Tk1 Thymidine kinase-1 2.66 0.0030 Isg15 Interferon-induced 15 kDa protein 2.44 0.0014 Prdx2 Peroxiredoxin 2 2.22 0.00090 Sparc Secreted acidic cysteine rich glycoprotein 2.17 0.0014 Metap2 Methionine aminopeptidase 2 2.03 0.0026 Isg20 interferon stimulated gene 20 kDa 2.01 0.00024 Il7r Interleukin 7 receptor 0.55 0.00041

* Ratio between geometric mean of expression in dnMEK/CD2-rtTA mice to geometric mean of expression in the CD2-rtTA mice.

2 Relative expression (RT PCR) 1

0 +/+ -/+ +/+ -/+ +/+ -/+ +/+ -/+ Tk1 Isg15 Il7r Isg20

Figure 7 (a) Interferon-regulated genes differentially expressed between dnMEK/CD2-rtTA and CD2-rtTA mice. (b) The differential expression of four representative genes was confirmed by real-time reverse transcription (RT)–PCR and normalized to GAPDH.

be identified, in the case of idiopathic lupus. In either tetracycline transactivator (rtTA) under the control of a case, genetic susceptibility is necessary but not sufficient CD2 promoter allowing for lymphoid specific expres- to cause disease in the vast majority of lupus patients. sion.43 Screening for the double-transgenic mice (dnMEK/ CD2-rtTA) was by PCR on tail-biopsy DNA. The following genotyping primers were used: dnMEK Materials and methods forward, 50-ACGCCATCCACGCTGTTTTG-30; reverse, Generation and breeding of transgenic mice 50-TTCGCTGCTGCTCATCAAGC-30; rtTA forward, 50- A human dominant negative MEK1 cDNA (hereafter GTGATTAACAGCGCATTA-30; reverse, 50-ATCAATTC referred to as dnMEK)30 was excised from the parent AAGGCCGAAT-30. vector pCMV-dnMEK with the restriction enzyme BamHI In order to generate wild-type littermate controls, (Roche, Basel, Switzerland) and subcloned into the double-transgenic mice were bred with wild-type multiple cloning site of pTRE-2 vector (Clontech Labora- C57BL/6 mice. Mice were maintained in specific patho- tories, Palo Alto, CA, USA) at the BamHI restriction site gen-free environment and all our protocols and proce- via T4 DNA ligase (Promega, Madison, WI, USA). The dures were approved by the Institutional Animal Care orientation and integrity of the dnMEK insert was and Use Committee at the Oklahoma Medical Research determined by direct sequencing. The resulting trans- Foundation. Animals were given doxycycline in the gene, TRE2-dnMEK, was excised from the vector by the drinking water at a concentration of 2 or 4 mg per ml in restriction enzymes NotI and HindIII (Roche) and was different experiments. Doxycycline solution was made in purified prior to microinjection into C57BL/6 Â (C57BL/ 5% sucrose to cover the bitter taste of doxycycline, and in 6 Â SJL) F1 mouse eggs implanted into psuedopregnant all in-vivo experiments, double-transgenic control mice females by personnel at the University of Michigan were given 5% sucrose without doxycycline. Animal Model Core. Founders were identified by PCR on tail biopsy DNA, and the founders backcrossed onto a RNA isolation C57BL/6 background. Double-transgenic mice were Both Trizol (Invitrogen, Carlsbad, CA, USA) and RNeasy generated by crossing TRE2-dnMEK transgenic mice kits (Qiagen, Valencia, CA, USA) were used to isolate with CD2-rtTA mice. CD2-rtTA mice express the reverse tissue RNA. Briefly, tissues are first homogenized in

Genes and Immunity ERK pathway signaling and epigenetic regulation in lupus AH Sawalha et al 375

Figure 8 Networks of genes that are deferentially expressed in the dnMEK/CD2-rtTA mice compared to CD2-rtTA controls. Networks were identified using the Ingenuity software. (a) Cell death and immunological disease network, and (b) Connective tissue development network. Key: Solid lines indicate direct interaction, dotted lines indicate indirect interaction. An arrow from (a)to(b) indicates that (a) acts on (b), a line without an arrowhead indicates binding only and a line with a small vertical line at the end from (a)to(b) indicates (a) inhibits (b). Red indicates genes that are upregulated, green indicates genes that are downregulated and white indicates genes that are not user specified but incorporated into the network through relationships with other genes. Node shapes are: square, cytokine; diamond (vertical), enzyme; diamond (horizontal), peptidase; dotted rectangular (vertical), ion channel; solid rectangular (vertical), G-protein coupled receptor; triangle, kinase; oval (horizontal), transcription regulator; oval (vertical), transmembrane receptor; trapezoid, transporter; circle, other.

Genes and Immunity ERK pathway signaling and epigenetic regulation in lupus AH Sawalha et al 376 Trizol, chloroform added and then the aqueous layer Microarrays are washed under high stringency, labeled mixed with an equal volume of 70% ethanol and loaded with streptavidin-Cy3, and scanned with an Illumina onto the RNeasy columns. The remainder of the RNA BeadStation 500 scanner. isolation was according to the RNeasy kit manufacturer’s instructions. DNA digestion was performed using Turbo Analysis. Illumina BeadChip arrays (Illumina Inc., San DNA-free (Ambion Inc., Austin, TX, USA) following the Diego, CA, USA) were scanned with the Illumina Bead manufacturer’s instructions. Array Reader and bead intensities were quantitated with the Illumina BeadStudio software. Raw values were Real-time RT-PCR quantile-normalized using MATLAB software (The One-step real-time RT-PCR was performed using the MathWorks Inc., Natick, MA, USA) and the resulting QuantiTect SYBR Green RT-PCR kit (Qiagen) and the normalized values were imported into BRB ArrayTools Rotor-Gene 3000 real-time thermocycler (Corbett Re- (Biometric Research Branch, National Cancer Institute) search, Australia). A total of 150 ng of RNA was used for where they were then log transformed. Genes were each reaction. The following amplification conditions filtered using the ‘Log Expression Variation Filter’ to were used: 30 min at 50 1C, 15 min at 95 1C, 54 cycles of screen out genes that are not likely to be informative, 15 s at 94 1C and 20 s at 56 1C and 30 s at 72 1C. based on the variance of each gene across the arrays. In Quantitation was performed by comparison with inter- this case, the filter was set to exclude genes that fell nal standards prepared by serial dilutions. Various below the fiftieth percentile of gene variance. We transcript expression levels were always normalized for identified genes that were differentially expressed the housekeeping gene GAPDH. The following primers between the two classes by using a multivariate were used: mouse GAPDH forward, 50-CAACGACC permutation test.45 We used the multivariate permuta- CCTTCATTGACCTC-30; reverse, 50-GCCTCACCCCATT tion test to provide a median false discovery rate (FDR) TGATGTTAGTG-30; mouse CD11a forward, 50-CAGATT of 10% (80% confidence). The test statistics used were GAAGATGGGGTTGTCG-30; reverse, 50-CGGGACGAT random variance t-statistics for each gene.46 Although t- TTTGTAACATAGGTC-30; mouse CD70 forward, 50-TGG statistics were used, the multivariate permutation test is CTGTGGGCATCTGCTC-30; reverse, 50-ACATCTCCGT nonparametric and does not require the assumption of GGACCAGGTATG-30; mouse Dnmt1 forward, 50-GG Gaussian distributions. Data were exported to Excel AAGGCTACCTGGCTAAAGTCAAG-30; reverse, 50-ACT (Microsoft, Redmond, WA, USA) where averages of the GAAAGGGTGTCACTGTCCGAC-30, mouse Tk1 forward, classes were used to calculate expression ratios. Genes 50-GCAGATTCAGGTGATTCTCGGG-30; reverse, 50-GCA that were differentially expressed (o10% FDR) and TACTTGATGACCAGGCACTTG-30; mouse Isg15 forward, simultaneously had ratios twofold or larger were used 50-ACGGTCTTACCCTTTCCAGTC-30; reverse, 50-CCCC in further analyses. Pathways were explored by placing TTTCGTTCCTCACCAG-30; mouse Isg20 forward, the only genes that passed our significance/ratio thresh- 50-GTCACGCCTCAGCACATGGT-30; reverse, 50-CCAC old into Ingenuity Pathways Analysis (Ingenuity Sys- CAGCTTGCCTTTCAGAA-30; mouse Il7r forward, 50- tems, www.ingenuity.com). TATGTGGGGCTCTTTTACGAGT-30; reverse, 50-GCCTC GGCTTTAACTATTGTGT-30. All primers were purchased Cell separation and culture from Integrated DNA Technologies Inc. (Coralville, IA, CD3 þ lymphocytes were isolated from the spleens of USA). double-transgenic mice via magnetic bead separation using indirect labeling (Miltenyi Biotec, Auburn, CA, Expression microarray USA) following the manufacturer’s protocol. Cell purity RNA. RNA was isolated from the spleens using RNeasy was routinely checked by flow cytometry. Where kit (Qiagen) following the manufacturer’s protocol. RNA indicated, cells were cultured in RPMI 1640 with 10% concentration was determined with a Nanodrop scan- fetal calf serum and were incubated with doxycycline, ning spectrophotometer (Nanodrop Technologies, Wil- without doxycycline or with the MEK inhibitor PD 98059 mington, DE, USA), and then qualitatively assessed for (50 mM) for 24 h. Phorbol myristate acetate (50 ng mlÀ1) degradation using the ratio of 28:18 rRNA using a was added and cells were harvested after 15 min. Where capillary gel electrophoresis system (Agilent 2100 Bio- indicated, mouse spleen cells were incubated with con-A analyzer, Agilent Technologies, Santa Clara, CA, USA). (5 mgmlÀ1) overnight for T-cell stimulation.

Labeling and hybridization. Biotinylated amplified RNA Protein isolation and western blot was produced from 200 ng total RNA per sample using a CD3 þ T cells were harvested, centrifuged and resus- modification of the Eberwine protocol,44 as described in pended in radioimmunoprecipitation assay buffer the Illumina TotalPrep RNA Amplification Kit from (50 mM Tris-HCL, pH 7.4, 150 mM NaCl, 0.25% deoxy- Ambion Inc. Briefly, RNA was reverse transcribed with cholic acid, 1% NP-40, 1 mM EDTA, 100 mgmlÀ1 phenyl- oligo(dT) primer containing a T7 promoter. RNA methylsulfonyl fluoride, 100 mM sodium orthovanadate, containing biotin-UTP ribonucleotides is amplified by 1mM dithiothreitol and a protease inhibitor cocktail in-vitro transcription to generate anti-sense RNA. This (Roche)). The suspension was rotated at 4 1C for 30 min, RNA is hybridized overnight at 58 1C to Sentrix Mouse-6 insoluble material removed by centrifugation at 16 000 g v1.1 Expression BeadChip microarrays obtained from for 30 min and the supernatant saved as whole cell Illumina Corp. (San Diego, CA, USA). The arrays have lysate. The amount of cellular protein present in the 46 657 genes, splice variants and control beads. These whole cell lysate was measured using the bicinchoninic arrays contain 50-mer oligonucleotides coupled to beads acid (BCA) protein assay (Pierce, Rockford, IL, USA). that are mounted on glass slides. Each bead has Isolated proteins (20 mg) were diluted in Laemmli approximately 20- to 30-fold redundancy per microarray. loading buffer and denatured by boiling for 5 min

Genes and Immunity ERK pathway signaling and epigenetic regulation in lupus AH Sawalha et al 377 followed by electrophoresis in 10–12% SDS–polyacryla- 2 Sarzi-Puttini P, Atzeni F, Iaccarino L, Doria A. Environment mide gels. The fractionated proteins were then electro- and systemic lupus erythematosus: an overview. Autoimmu- phoretically transferred to nitrocellulose membranes nity 2005; 38: 465–472. (Schleicher and Schuell, Keene, NH, USA) and stained 3 James JA, Kaufman KM, Farris AD, Taylor-Albert E, Lehman with Ponceau S (Sigma-Aldrich, St Louis, MO, USA) to TJ, Harley JB. An increased prevalence of Epstein-Barr verify equal amounts of protein between lanes prior to virus infection in young patients suggests a possible etiology western blot analyses. Membranes were blocked for 2 h for systemic lupus erythematosus. J Clin Invest 1997; 100: in Tris-buffered saline (TBS) containing 0.1% Tween 3019–3026. 4 Sawalha AH, Richardson BC. DNA methylation in the (Sigma-Aldrich) and 5% nonfat dry milk (Bio-Rad pathogenesis of systemic lupus erythematosus. Curr Pharma- Laboratories, Hercules, CA, USA). After a 16-h incuba- cogen 2005; 3: 73–78. tion with a rabbit polyclonal anti-active MAPK (1:5000; 5 Richardson B, Yung R. Role of DNA methylation in the Promega) in TBS, 0.1% Tween, 5% nonfat dry milk, blots regulation of cell function. J Lab Clin Med 1999; 134: 333–340. were washed three times with TBS containing 0.1% 6 Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Tween and incubated with a horseradish peroxidase- Dnmt3a and Dnmt3b are essential for de novo methylation and linked anti-rabbit antibody (1:2000; Cell Signaling Tech- mammalian development. Cell 1999; 99: 247–257. nologies, Danvers, MA, USA) for 1 h. After three washes 7 Bird AP. Use of restriction enzymes to study eukaryotic DNA with TBS–0.1% Tween the membranes were treated with methylation: II. The symmetry of methylated sites supports ECL chemiluminiscence detection system (Amersham, semi-conservative copying of the methylation pattern. JMol Biol 1978; 118: 49–60. Piscataway, NJ, USA), exposed to X-ray film (Kodak, 8 Deng C, Yang J, Scott J, Hanash S, Richardson BC. Role of the Rochester, NY, USA) and developed to visualize the ras-MAPK signaling pathway in the DNA methyltransferase labeled protein bands. Molecular mass was estimated by response to DNA hypomethylation. Biol Chem 1998; 379: comparison of sample bands with prestained molecular 1113–1120. mass marker (Bio-Rad Laboratories). Blots were stripped 9 MacLeod AR, Rouleau J, Szyf M. Regulation of DNA and reblotted with a rabbit anti-ERK antibody (1:5000; methylation by the Ras signaling pathway. J Biol Chem 1995; Promega). Values were normalized to total ERK. 270: 11327–11337. 10 Lu R, Wang X, Chen ZF, Sun DF, Tian XQ, Fang JY. Inhibition Statistical analysis of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway decreases DNA methylation in colon For continuous variables, differences between groups cancer cells. J Biol Chem 2007; 282: 12249–12259. were tested using Student’s t-test. P-values of o0.05 11 Richardson B. Effect of an inhibitor of DNA methylation on T were considered statistically significant. cells. II. 5-Azacytidine induces self-reactivity in antigen- specific T4+ cells. Hum Immunol 1986; 17: 456–470. 12 Richardson B, Powers D, Hooper F, Yung RL, O’Rourke K. Lymphocyte function-associated antigen 1 overexpression and Acknowledgements T cell autoreactivity. Arthritis Rheum 1994; 37: 1363–1372. We are indebted to Dr Kun-Liang Guan for donating the 13 Lu Q, Ray D, Gutsch D, Richardson B. Effect of DNA methylation and chromatin structure on ITGAL expression. dominant-negative MEK1 mutant, and to Dr Rose Blood 2002; 99: 4503–4508. Zamoyska and Dr Avery August for providing the 14 Yung R, Powers D, Johnson K, Amento E, Carr D, Laing T et al. CD2-rtTA mice. We are thankful to Dr John Attwood Mechanisms of drug-induced lupus. II. T cells overexpressing for assistance and discussion. We are also thankful to lymphocyte function-associated antigen 1 become autoreac- Maggie Van Keuren and Dr Thomas L Saunders at the tive and cause a lupus like disease in syngeneic mice. J Clin University of Michigan Transgenic Animal Model Core Invest 1996; 97: 2866–2871. for generating the TRE2-dnMEK transgenic mouse and 15 Richardson BC, Strahler JR, Pivirotto TS, Quddus J, Bayliss for very helpful discussion. We are indebted to the GE, Gross LA et al. Phenotypic and functional similarities Microarray Research Facility at OMRF and Dr Mark between 5-azacytidine-treated T cells and a T cell subset in Barton Frank, Dr Michael Centola, Dr R Craig Cadwell patients with active systemic lupus erythematosus. Arthritis 35 and Dr Jonathan D Wren for analyzing the microarray Rheum 1992; : 647–662. 16 Yung R, Kaplan MJ, Ray D, Schneider K, Mo R-R, Johnson K et al. data. The microarray core receives funding from the NIH Autoreactive murine Th1 and Th2 cells kill syngeneic macro- grants number P20RR016478, P20RR020143, phages and induce autoantibodies. Lupus 2001; 10: 539–546. P20RR017703, P20RR15577, and U19AI062629. This pub- 17 Kaplan MJ, Lu Q, Wu A, Attwood J, Richardson B. lication was made possible by NIH Grant Number P20- Demethylation of promoter regulatory elements contributes RR015577 from the National Center for Research Re- to perforin overexpression in CD4+ lupus T cells. J Immunol sources (AHS), PHS grants AR42525, AG25877, and 2004; 172: 3652–3661. ES015214, and a Merit grant from the Department of 18 Oelke K, Lu Q, Richardson D, Wu A, Deng C, Hanash S et al. Veterans Affairs (BR). Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors. Arthritis Rheum 2004; 50: 1850–1860. 19 Lu Q, Wu A, Tesmer L, Ray D, Yousif N, Richardson B. Conflict of interest Demethylation of CD40LG on the inactive X in T cells from None. women with lupus. J Immunol 2007; 179: 6352–6358. 20 Cornacchia E, Golbus J, Maybaum J, Strahler J, Hanash S, Richardson B. Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity. J Immunol 1988; References 140: 2197–2200. 21 Scheinbart LS, Johnson MA, Gross LA, Edelstein SR, Richard- 1 Sestak AL, Nath SK, Sawalha AH, Harley JB. Current status of son BC. Procainamide inhibits DNA methyltransferase in a lupus genetics. Arthritis Res Ther 2007; 9: 210. human T cell line. J Rheumatol 1991; 18: 530–534.

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