of July 13, 2018.This information is current as

Idiopathic and Hydralazine-Induced LupusDecreases ERK Pathway Signaling in

ActivationδImpaired T Cell Protein Kinase C

Sawalha and Bruce RichardsonGabriela Gorelik, Jing Yuan Fang, Ailing Wu, Amr H.

http://www.jimmunol.org/content/179/8/5553doi: 10.4049/jimmunol.179.8.5553

2007; 179:5553-5563; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Impaired T Cell Protein Kinase C� Activation Decreases ERKPathway Signaling in Idiopathic and Hydralazine-Induced Lupus1

Gabriela Gorelik,* Jing Yuan Fang,‡ Ailing Wu,* Amr H. Sawalha,§ and Bruce Richardson2*†

T cells from patients with lupus or treated with the lupus-inducing drug hydralazine have defective ERK phosphorylation. Thereason for the impaired signal transduction is unknown but important to elucidate, because decreased T cell ERK pathwaysignaling causes a lupus-like disease in animal models by decreasing DNA methyltransferase expression, leading to DNA hypom-ethylation and overexpression of methylation-sensitive genes with subsequent autoreactivity and autoimmunity. We thereforeanalyzed the PMA stimulated ERK pathway phosphorylation cascade in CD4� T cells from patients with lupus and in hydral-azine-treated cells. The defect in these cells localized to protein kinase C (PKC)�. Pharmacologic inhibition of PKC� or trans-fection with a dominant negative PKC� mutant caused demethylation of the TNFSF7 (CD70) promoter and CD70 overexpressionsimilar to lupus and hydralazine-treated T cells. These results suggest that defective T cell PKC� activation may contribute to thedevelopment of idiopathic and hydralazine-induced lupus through effects on T cell DNA methylation. The Journal of Immunol-ogy, 2007, 179: 5553–5563.

H uman systemic lupus erythematosus (SLE)3 is a chronicautoimmune disease characterized by abnormal T sig-naling and the presence of autoantibodies that cause

multiple pathologic abnormalities, including glomerulonephritisand vasculitis. The cause of human lupus remains unknown.

T cell DNA hypomethylation has been implicated in the patho-genesis of idiopathic and drug-induced human lupus (1, 2). DNAmethylation is one mechanism regulating gene expression, and hy-pomethylation of regulatory sequences correlates with active tran-scription, whereas hypermethylation suppresses expression (3).Our group has demonstrated that treating human or murine clonedor polyclonal CD4� T cells with DNA methylation inhibitors in-cluding 5-azacytidine, procainamide, and hydralazine demethyl-ates the DNA and makes the cells become autoreactive, and in-jecting the autoreactive murine T cells into syngeneic recipientscauses a lupus-like disease (4–7). Overexpression of the adhesionmolecule LFA-1 (CD11a/CD18) contributes to the autoreactivity(8, 9), whereas abnormal perforin expression contributes to thecytotoxic potential of the autoreactive CD4� cells, (10, 11) andincreased CD70 expression to B cell overstimulation (12). Thesame abnormalities in LFA-1, perforin, and CD70 expressionare found in CD4� T cells from patients with active lupus; all

three genes are overexpressed and display demethylation of thesame regulatory sequences as in T cells treated with DNA meth-ylation inhibitors (11, 13, 14). Together these results indicate arelationship involving DNA hypomethylation, methylation-sen-sitive immune gene overexpression, and T cell autoreactive re-sponses in the pathogenesis of both idiopathic and drug-inducedlupus.

DNA methylation patterns are maintained by DNMT1 (DNA(cytosine-5-) methyltransferase 1), which is regulated in part bythe ERK signaling pathway (15–17). T cells from patients withlupus exhibit lower DNMT1 expression due to decreased ERKactivity (18). Furthermore, hydralazine, a lupus-inducing drug,similarly inhibits T cell DNA methylation by decreasing DNMT1expression through inhibition of ERK pathway signaling, and Tcells treated with ERK pathway inhibitors become autoreactive invitro and induce autoimmunity in vivo, similar to hydralazine-treated cells (19). In both idiopathic lupus and in hydralazine-treated T cells, decreased phospho-ERK levels did not reflect over-all decreases in total ERK protein (18). The mechanisms causingdecreased ERK activation in both systems are unknown.

Because DNA hypomethylation due to decreased ERK pathwaysignaling is common to idiopathic and hydralazine-induced lupus,and may contribute to autoimmunity, we characterized the defectcausing decreased ERK pathway signaling T cells from patientswith idiopathic lupus and in hydralazine-treated T cells. The re-sults indicate that impaired protein kinase C (PKC)� phosphory-lation is responsible for the decreased ERK signaling in SLE pa-tient T cells and hydralazine-treated T cells. Because mice withgenetic PKC� deficiency develop lupus (20), these studies suggestthat impaired PKC� phosphorylation may contribute to the devel-opment of idiopathic and hydralazine-induced lupus through DNAmethylation inhibition.

Materials and MethodsMaterials

Rottlerin was obtained from Calbiochem and PD98059 was from Cell Sig-naling Technology. Hydralazine was purchased from VWR and PMA fromSigma-Aldrich.

*Department of Medicine, University of Michigan, and †Ann Arbor Veterans AffairsMedical Center, Ann Arbor, MI 48109; ‡Shanghai Institute of Digestive Disease,Shanghai Jiao Tong University School of Medicine, Renji Hospital, Shanghai, China;and §U.S. Department of Veterans Affairs Medical Center, Department of Medicine,University of Oklahoma Health Sciences Center, and Arthritis and Immunology Pro-gram, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104

Received for publication July 12, 2007. Accepted for publication August 7, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Public Health Service Grants AR42525, AG25877, andES15214 and a Merit grant from the Department of Veterans Affairs.2 Address correspondence and reprint requests to Dr. Bruce Richardson, 3007 Bio-medical Science Research Building, Department of Medicine, University of Michi-gan, Ann Arbor, MI 48109-2200. E-mail address: brichard@umich.edu3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; PKC, proteinkinase C; DNMT1, DNA (cytosine-5-) methyltransferase 1; RA, rheumatoid arthritis;RBD, Ras binding domain.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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Subjects

Groups of patients with lupus consisted of eight women and three men witha mean SLE disease activity index (21) of 4.7, range 2–6, and average ageof 48 years (range 30–67 years). All were on low-dose prednisone (mean6.1 mg, range 2–10 mg), and one was on mycophenolate mofetil and twoon azathioprine. Groups of patients with rheumatoid arthritis (RA) con-sisted of four women and three men, average age 48 years (range 29–59).Four of the patients with RA were receiving prednisone (mean 10.6 mg,range 5–20 mg), two patients were receiving methotrexate, two patientswere receiving TNF antagonists, two patients were receiving hydroxychlo-roquine, and one receiving sulfasalazine and one leflunomide. All the pa-tients with lupus met the American College of Rheumatology criteria forpatients with SLE (22) and patients with RA met criteria from The Amer-ican Rheumatism Association (23). The patients were recruited from theoutpatient rheumatology clinics and inpatient services at the University ofMichigan (Ann Arbor, MI). Healthy controls were recruited by advertising.These studies were reviewed and approved by the University of MichiganInstitutional Review Board for Human Subject Research.

T cell isolation

PBMC were isolated from venous blood of healthy donors and patientswith SLE or RA using density gradient centrifugation as previously de-scribed (1). CD4� cells were then isolated by negative selection usingmagnetic beads (CD4� T cell isolation kit; Miltenyi Biotec), according tothe manufacturer’s instructions, and used immediately.

T cell stimulation and protein isolation

CD4� T cells were resuspended in RPMI 1640 supplemented with 10%FCS, 2 mM glutamine and penicillin/streptomycin then left unstimulated orstimulated with 50 ng/ml PMA for 15 min at 37°C. Where indicated, cellswere incubated in the presence or absence of hydralazine (10 �M), rottlerin(10 �M), or PD98059 (50 �M) 60 min before stimulation. Following stim-ulation, the cells were centrifuged, resuspended in RIPA buffer (50 mMTris-HCl (pH 7.4), 150 mM NaCl, 0.25% deoxycholic acid, 1% NonidetP-40, 1 mM EDTA, 100 �g/ml PMSF, 100 �M sodium orthovanadate, 1mM DTT) and a protease inhibitor cocktail (Roche), and rotated at 4°C for30 min. Insoluble material was removed by centrifugation at 16,000 � gfor 30 min and the supernatant saved as whole cell lysate. When experi-ments required cytosolic or membrane fractions, treated cells were resus-pended in lysis buffer (20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 5 mMEGTA, 10 mM 2-ME, 100 �g/ml PMSF, 100 �M sodium orthovanadate,1 mM DTT and protease inhibitor cocktail) and incubated for 30 min at4°C and vortexed each 10 min. The lysate was centrifuged at 16,000 � gfor 30 min and the cytoplasmic protein containing supernatant saved foranalysis. The pellet was resuspended in lysis buffer containing 1% TritonX-100, incubated for 30 min on ice then vortexed, centrifuged, and theparticulate fraction saved. The amount of cellular protein present in thewhole cell lysate or in each of the fractions was measured using the BCAProtein Assay (Pierce).

Ras activation assays

Ras activity was measured using a Raf-1 Ras binding domain (RBD) kit(Upstate Biotechnology) following the manufacturer’s instructions. Inbrief, CD4� T cell lysates were incubated with an agarose-immobilizedGST fusion protein containing the RBD of Raf-1 to precipitate GTP-boundRas. Precipitated (GTP-bound) and soluble (GDP-bound) Ras were deter-mined by immunoblotting with an Ab recognizing and precipitating allisoforms of Ras (clone Ras 10; Upstate Biotechnology).

Kinase activity measurement

A total of 20 �g of isolated proteins were diluted in Laemmli loading bufferand denatured by boiling for 5 min followed by electrophoresis in 10–12%SDS-polyacrylamide gels. The fractionated proteins were then electro-phoretically transferred to nitrocellulose membranes (Schleicher andSchuell) and stained with Ponceau S (Sigma-Aldrich) to verify equalamounts of protein between lanes before Western blot analyses. Mem-branes were blocked for 2 h in TBS containing 0.1% Tween 20 (Sigma-Aldrich) and 5% nonfat dry milk (Bio-Rad). After an 16-h incubation withthe kinase specific Ab in TBS, 0.1% Tween 20, and 5% nonfat dry milk,blots were washed three times with TBS containing 0.1% Tween and in-cubated with a HRP-linked secondary Ab for 1 h. After three washes withTBS containing 0.1% Tween 20, the membranes were treated with ECLdetection system (Amersham Biosciences), exposed to x-ray film (Kodak)and developed to visualize the labeled protein bands. Molecular mass wasestimated by comparison of sample bands with prestained molecular massmarker (Bio-Rad). For quantitative studies, the bands on x-ray films were

scanned using a photodocumentation system (Alpha Innotech) and ana-lyzed with ImageQuant 5.2 software (Amersham Bioscience). Where in-dicated, blots were stripped and reblotted with the corresponding Ab. Val-ues were normalized respect to �-actin as indicated.

Ab products

The following primary Abs were used: rabbit polyclonal anti-phospho-PKC� (Thr638/641), anti-phospho-PKC� (Thr538), anti-phospho-PKC�(Thr505), anti-phospho-Raf (Ser338), anti-phospho-MEK1/2 (Ser217/221),and anti MEK1/2 used at 1/1000 dilution (Cell Signaling Technology).Rabbit polyclonal anti-active MAPK (1/5000) was purchased from Pro-mega, and anti-total PKC� (1.5 mg/ml) and anti-total PKC� (1.5 �g/ml)were from Upstate Biotechnology. Monoclonal mouse anti-total PKC� (1/250; BD Transduction Laboratory) was also used. Secondary Abs included:anti-rabbit IgG HRP (1/2000; Cell Signaling Technology) and anti-mouseIgG HRP (1/4000; Amersham Biosciences).

RNA isolation

PBMC from normal donors were cultured in RPMI 1640 supplementedwith 10% FCS and antibiotics as described, then stimulated with 1 �g/mlPHA for 18 h. CD4� T cells were then bead purified and cultured in thesame medium supplemented with IL-2 at a density of 1 � 106 cells/ml aspreviously described (24) for an additional 72 h in the presence or absenceof rottlerin (10 �M). Following incubation, total RNA was isolated usingan RNA isolation kit (Qiagen) according to the manufacturer’s instruction.

RT-PCR

RNA was digested with 2 U of DNaseI (Ambion), and 2.5 �g were usedfor reverse transcription reactions in a total volume of 20 �l using reversetranscriptase (Qiagen). Then, 2 �l of cDNA in a mixture with 2.5 mMMgCl2, 0.2 mM dNTP (Promega), 1 �M each primer, and 2.5 U ofTaqDNA polymerase (Promega) in a volume of 50 �l were amplified underthe following conditions: denaturation at 95°C for 5 min, amplification at95°C for 1 min, 60°C for 1 min, 72°C for 1 min for a total of 35 cyclesfollowed by a final extension at 72°C for 5 min. Amplification for �-actinwas also performed as a loading and RNA quality control. The followingprimers were used: CD70 forward 5�–TGCTTTGGTCCCATTGGTCG-3�and reverse 5�-TCCTGCTGAGGTCCTGTGTGATTC-3�; �-actin forward5�-GGACTTCGAGCAAGAGATGG-3� and reverse 5�-AGCACTGTGTTGGCGTACAG-3�. The PCR products were run on a 2% agarose gel andstained with ethidium bromide.

RNA quantification by real-time RT-PCR

A total of 200 ng of RNA was converted to cDNA and amplified in onestep using Quanti-Tect SYBR Green RT-PCR kit (Qiagen). CD70 tran-scripts were quantitated by real-time semiquantitative RT-PCR using aRotor-Gene 3000 (Corbett Research) and previously published protocols(12). The following amplification conditions were used: reverse transcrip-tion at 50°C for 30 min, denaturation at 95°C for 15 min, amplification at94°C for 15 s, 56°C for 20 s, and 72°C for 30 s for a total of 54 cycles.Product quality was determined by melting curves. A series of five dilu-tions of one RNA sample were also included to generate a standard curve,and this was used to obtain relative concentrations of the transcript ofinterest in each of the RNA samples. In each experiment, water was in-cluded as a negative control to rule out primer dimer formation. Amplifi-cation of �-actin was performed to confirm that equal amounts of totalRNA were added for each sample and that the RNA was intact and equallyamplifiable among all samples. The primers used were the same as forRT-PCR, as described.

Methylation-specific PCR

Genomic T cell DNA from cells cultured and treated as for RT-PCR wasisolated using a DNeasy isolation kit (Qiagen), according to the manufac-turer’s instructions, and bisulfite-treated as previously described (25).Briefly, 2–20 �g of purified DNA was treated with 350 mM sodium hy-droxide for 20 min then with 1.7 M sodium bisulfite at 55°C overnight.Samples were desalted using the Wizard DNA clean-up system (Promega).After washing with 80% ethanol, DNA was eluted, treated with 0.3 Msodium hydroxide and 3 M ammonium acetate, then precipitated with gly-cogen and 95% ethanol at �80°C. The precipitate was washed with 70%ethanol, dried by vacuum, and redissolved in double-distilled H2O. Semi-quantitative PCR was then used to determine the methylation status of CGpairs within the TNFSF7 (CD70) promoter regulatory region (from �660to �466 bp (14)), using 5 �g of bisulfite-treated DNA. The cycling con-ditions were 94°C for 5 min followed by 70 cycles of 94°C for 15 min,55°C for 15 s, and 72°C for 20 s. As before, the melting characteristics of

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the product were determined, a standard curve was generated using oneDNA sample, and water was included as negative control. The primersdesigned to hybridize with methylated CG pairs were forward 5�-CGAGGT TTA GAT AGG AGA ATC GT–3� (interrogating two CG pairs) andreverse 5�-TAA AAA TAC TCC CCA AAT ATT CGT-3� (interrogatingone CG pair). Loading controls consisted of primers designed to avoid CGpairs and were forward 5�-GGGTGG ATT ATT TAA GGT TAGGAGT-3� and reverse 5�-AAT CTC CCT CTA TCA CCC AAA CTA-3�.The amplified fragment was cloned, and five fragments were sequenced bythe University of Michigan DNA Sequencing Core (Ann Arbor, MI). Instudies involving the PKC� mutant, a third set of primers were used tohybridize with unmethylated CG pairs, and the methylation index wascalculated with the following equation: (fraction methylated)/(fractionmethylated � fraction unmethylated). The values are relative to p-EGFP-N1considered as 1. The unmethylated primers were forward 5�–GTG AGGTTT AGA TAG GAG AAT TGT–3� and reverse 5�–TAA AAA TAC TCCCCA AAT ATT CAT A–3�.

Expression plasmids

A plasmid expressing a dominant negative form of mouse PKC�(PKC�K376R-GFP fusion protein) was a gift from Dr. S. H. Yuspa (NationalCancer Institute Bethesda, MD) (26). To generate the GFP control vectorwithout PKC�K376R, the PKC�K376R-GFP construct was digested withBglII and BamHI (New England Biolabs and Roche, respectively). Thismethod excised PKC�K376R from the PKC�K376R-GFP construct in twopieces. The remainder of the construct (�4.5 kb) was extracted using a gelextraction kit (Qiagen) and self-ligated at 14°C overnight using T4 DNAligase (Promega).

Transient transfections

CD4� T cells were obtained from normal donors as previously describedand immediately transfected with 5 �g of the dominant negative PKC�

mutant or with the empty vector pEGFP-N1 using Amaxa nucleofectiontechnology, according to the manufacturer’s instructions. After 6 h themedium was changed, and after 72 h the cells were lysed to obtain protein,total RNA, and genomic DNA as described. As the vector includes theGFP, transfection efficiency was assessed by fluorescence microscopy, andwas 65 � 5% of total cell number (fluorescent and nonfluorescent cells).Every transfection was confirmed by verifying expression of the trans-fected protein by Western blot and Abs against phospho-PKC�Thr505 usingprotein lysates from cells transfected with the PKC� mutant and stimulatedwith PMA for 15 min. Band intensity was compared with cells transfectedwith the empty vector, which is considered as controls.

Statistical analysis

The significance of the difference between mean values was determinedusing Student’s t test. Values of p � 0.05 were considered significant.

ResultsDefective ERK pathway signaling in T cells from patients withactive lupus

Our group previously demonstrated that PMA stimulates ERKphosphorylation to a lesser extent in CD4� lupus T cells thancontrols. This decrease in phospho-ERK was not due to lowerERK protein levels because kinase protein expression was similarin control and lupus T cells (18). To elucidate where in the signalingcascade the defect lies, we analyzed PMA-induced activation of themolecules upstream of ERK (PKC3Raf3MEK1/23 ERK1/2) inCD4� T cells from patients with lupus. PKC enzymes are activated

FIGURE 1. Decreased PMA-Raf-1-Erk pathway phosphorylation in lupus T cells. A, CD4� T cells from a healthy control (N) or from two differentpatients with SLE (L1, L2) were isolated and immediately stimulated with 50 ng/ml PMA for 15 min or not stimulated as described in Materials andMethods. Proteins from whole cell lysates were fractionated by SDS-PAGE, transferred to nitrocellulose membranes and probed with a polyclonal Abagainst the active dually phosphorylated form of ERK1/2. The membrane was stripped and reblotted sequentially for phospho-MEK1/2 Ser217/221 and Raf-1p-Ser338. �-actin was used as loading control. B, Quantitative immunoblot analysis of phospho-ERK, phospho-MEK, and phospho-Raf in CD4� T cellsfrom three to six different patients with active lupus treated as in A and compared with normal donors. Values were normalized to �-actin. The relativephosphorylation of the different kinases in PMA-stimulated CD4� lupus T cells was compared with normal-treated T cells arbitrarily considered as 100%.Results shown are the mean percentage of phosphorylation � SD of the indicated number of independent experiments.

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directly by PMA, then each subsequent molecule in this pathway ac-quires protein kinase activity after phosphorylation, and is subse-quently able to phosphorylate and activate the next member of thesignaling cascade. Phospho-ERK translocates to the nucleus, provid-ing a direct link with an extracellular signal, an internal pathway, andthe genetic response (27).

Purified CD4� T cells from patients with lupus and healthydonors were stimulated with PMA for 15 min and ERK pathwaysignaling was compared by immunoblot using specific Abs. Fig.1A shows a representative immunoblot using whole cell extractsfrom unstimulated or stimulated CD4� T cells processed im-mediately after isolation from a healthy donor or two differentpatients with lupus. The protein extracts were fractionated bySDS-PAGE, transferred to nitrocellulose membranes thenprobed with the relevant Abs. The membrane was stripped andreblotted with Abs to the next signaling molecule. �-actin wasused as loading control. Phosphorylation of ERK1/2 was de-tected with an Ab against the active dually phosphorylated formof ERK1/2. To detect MEK1/2, the blot was probed with ananti-phospho-MEK1/2 Ab that recognizes MEK1/2 when phos-phorylated by Raf at Ser217 and Ser221; both are sites of phos-phorylation that result in its activation (28). Phosphorylation ofRaf was tested using an Ab to Raf-1 phosphorylated at Ser338,localized in the catalytic domain (29). Decreased levels of phos-pho-ERK1/2, phospho-MEK1/2, and phospho-Raf-1 are seen inPMA-stimulated CD4� lupus T cells compared with normalcontrols. These results suggest an overall lower ERK pathway

signaling in lupus T cells, and that the defect may lie upstreamof Raf at PKC. Fig. 1B summarizes three to six serial repeats ofthe experiments shown in Fig. 1A. PMA-stimulated phosphor-ylation of ERK, MEK, and Raf is significantly reduced in CD4�

T cells from patients with lupus.

PKC� phosphorylation is decreased in lupus T cells

Because PMA binds PKC and induces its phosphorylation (30),and because PKC is one of the key signaling molecules in T cellactivation (31) and is able to activate ERK (32), and as a gener-alized PKC defect in lupus T cells has been reported (33), weexamined PKC isoform activation in lupus T cells. The PKC iso-forms activated by PMA belong to two different subfamilies (34);the conventional that are calcium-dependent (�, � and �), and thenovel or calcium-independent PKC isoforms (�, �, , �, and �). Athird subfamily comprises the atypical isoforms, but the membersare insensitive to PMA (34) and thus were not studied. PKC� isone of the most abundant isoenzymes in human and murine Tlymphocytes (35, 36). PKC� expression is tissue restricted and ismost highly expressed in T lymphocytes playing an important rolein their activation (37), whereas PKC� exhibits the highest homol-ogy to PKC� (38). We therefore examined PMA-stimulated phos-phorylation of �, �, and � in lupus T cells.

CD4� T cells, isolated from a normal donor and two differentpatients with lupus, were cultured alone or with PMA for 15 min,and PKC�, �, and � phosphorylation analyzed by immunoblottingwith Abs to PKC� phospho-Thr638/641, PKC� phospho-Thr538,

FIGURE 2. Impaired PKC� phosphorylation in lupus T cells. A, CD4� T cells from a normal subject and from two different patients with lupus (L1,L2) were treated (�) or not (�) with PMA during 15 min as described in Fig. 1. Phosphorylation of PKC�, PKC�, and PKC� was then similarly comparedby immunoblotting using Abs against phosphorylated forms of the enzymes. �-actin was used as a loading control. B, PKC� phosphorylation in unstimu-lated (�) or PMA-stimulated (�) CD4� T cells from two different patients with lupus (L1, L2) was similarly compared with CD4� T cells from a normalcontrol by immunoblot analysis using an Ab against PKC� phospho-T505. Total PKC� protein expression is shown in the stripped and reprobed membrane.�-actin was used as loading control. Results are representative of five independent experiments. C, CD4� T cells from 11 patients with active lupus weresimilarly stimulated with PMA, and lysates were subjected to SDS-electrophoresis, transferred, and blotted with specific Abs against PKC� phospho-T505.Bands were scanned and quantified by densitometry. Blots were stripped and reblotted with PKC� and values were normalized to total PKC� levels. Therelative phosphorylation of PKC� in PMA-stimulated CD4� lupus T cells was compared with normal-stimulated T cells arbitrarily considered as 100%.Results shown are the mean percentage � SD of 11 different patients with SLE. �, p � 0.001.

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and PKC� phospho-Thr505

, respectively (Fig. 2A). PKC� Thr638/641

is a C-terminal autophosphorylation site that plays a unique role inpreventing degradation and is required for its catalytic activity(39). PKC� activation loop phosphorylation at Thr538 results insignificant kinase activity (40). PKC�-Thr505 is an activation loopsite, its phosphorylation is induced by PMA and is an indicator ofPKC� activation (41). The phosphorylation of PKC� and PKC�did not differ significantly between lupus and normal cells. In con-trast, PKC� phosphorylation was reduced in CD4� lupus T cells.Because the Ab used binds to PKC� when phosphorylated atThr505, these results suggest lower PKC� activity in CD4� lupusT cells. In contrast, PMA-stimulated PKC� phosphorylation inCD8� T cells from patients with lupus did not differ significantlyfrom phosphorylation in cells from normal donors (mean percent-age � SD: 0.89 � 0.20, n � 3 patients with lupus, normalized tototal PKC� and expressed relative to normal cells), in agreementwith previous reports showing decreased ERK activity in CD4�

lupus T cells (18).We considered the possibility that the decrease in phospho-

PKC� was due to decreased PKC� protein. However, total PKC�was not decreased in lupus T cells (Fig. 2B). Serial repeats of thisexperiment demonstrated that the ratio of total PKC� of patientswith lupus to total PKC� of controls was 1.00 � 0.18 (total � SD,n � 8 patients with lupus), indicating that the defect is in PMA-stimulated phosphorylation. Band intensity of the immunoblotsfrom 11 different patients with lupus was quantitated by densitom-etry and data are shown in Fig. 2C. PMA-induced phosphorylationof PKC� is significantly reduced in CD4� T cells from patientswith lupus respect to normal donors, whereas the total kinase ex-pression is unaltered. Because PMA directly activates the calcium-independent PKC� isoenzyme, these data suggest that the defec-tive ERK pathway signaling in lupus T cells previously reportedby our group (18) is a consequence of impaired PMA-stimulatedPKC� phosphorylation. We then studied PMA-induced activationof PKC� in CD4� T cells from seven patients with RA. No sig-nificant differences in PKC� phosphorylation were observed be-tween CD4� T cells from patients with RA vs cells from healthycontrols as measured by immunoblotting (mean � SD: 4.1 � 1.5vs 4.8 � 1.0, respectively, expressed in arbitrary units and nor-malized to total PKC� expression in the same subject). The dif-ferences in PKC� activation between patients with lupus and pa-tients with RA could not be attributed to differences in age, gender,or medications received (see Materials and Methods). These re-sults are consistent with our previous data showing a significantdecrease in ERK phosphorylation only in patients with lupus whencompared with phosphorylation in patients with RA and in healthy

controls, for which effects due to age, gender, and medicationswere similarly excluded (18).

Hydralazine inhibits PKC� phosphorylation

Where hydralazine inhibits ERK pathway signaling is unknown.Because impaired PKC� activation contributes to decreased T cellERK pathway signaling in lupus, and the PKC� knockout mousedevelops lupus (20), we hypothesized that hydralazine may alsoinhibit T cell PKC� activation. Fig. 3 shows a representative im-munoblot comparing the effects in idiopathic lupus by 10 �M hy-dralazine, 10 �M rottlerin, on PMA-stimulated PKC� phosphor-ylation. Rottlerin inhibits protein kinases with specificity for PKCand is able to differentiate PKC isoenzymes with IC50 values forPKC� more than two orders of magnitude lower than for otherisoenzymes (42). Both drugs decreased PKC� phosphorylation innormal CD4� T cells, similar to the decrease observed in lupus Tcells, although hydralazine appears to be somewhat less potent atthis concentration. Fig. 4 shows the kinetics of PMA-stimulated

FIGURE 3. Hydralazine inhibits PMA-induced PKC� phosphorylation.CD4� T cells from normal controls or patients with lupus were stimulatedfor 15 min with 50 ng/ml PMA as indicated. Where indicated CD4� T cellsfrom the normal controls were also treated with hydralazine or the PKC�-specific inhibitor rottlerin for 1 h before stimulation with PMA. Lysateswere subjected to immunoblot analysis and PKC� phosphorylation was mea-sured using anti-phospho-PKC� T505 as before. The same blot was strippedand reprobed with Abs anti-�-actin as a loading control. Results are represen-tative of three independent experiments.

FIGURE 4. Inhibition of PKC� phosphorylation by hydralazine. A,CD4� T cells from a normal donor were untreated or treated with 10 �Mhydralazine (f) or 10 �M rottlerin (�) for the indicated times and thenstimulated with PMA for 15 min. PKC� phosphorylation was analyzed byimmunoblot using anti-phospho-PKC� as before, and a representativeWestern blot is shown. �-actin was used as a loading control. B, Bandsfrom two different experiments performed as in A were quantitated bydensitometry and phospho-PKC� levels normalized to �-actin. Dashed linerepresents PKC� phosphorylation in PMA-stimulated cells in the absenceof inhibitors. Results represent the mean � SEM. C, Immunoblot analysisof PMA-stimulated PKC� phosphorylation after treatment with hydral-azine or rottlerin for 1 h. The mean levels of PKC� phosphorylation � SDfor seven independent experiments were calculated by normalization to�-actin expression. The relative PKC� phosphorylation in PMA-stimulatedcells in the absence of inhibitors was considered as 100%. �, p � 0.02;��, p � 0.001.

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PKC� phosphorylation inhibition by hydralazine and rottlerin. Fig.4A shows a representative immunoblot, and Fig. 4B the densito-metric analysis plotted against time. The kinetics were similar forboth inhibitors with maximal inhibition at 1 h of incubation. Basedon these results, cells were treated with hydralazine or rottlerin for1 h in all additional experiments. Fig. 4C shows the mean per-centage � SD of densitometric analyses from seven independentexperiments comparing rottlerin and hydralazine on PKC� stimu-lation. PMA-induced phosphorylation was inhibited �33% by hy-dralazine ( p � 0.02) and �65% by rottlerin ( p � 0.001). Thisfinding suggests that hydralazine inhibits PMA-stimulated PKC�phosphorylation.

To more rigorously test whether other PKC isozymes are af-fected, we further analyzed PKC� and PKC� activation with andwithout hydralazine. CD4� T cells were treated with hydralazineand stimulated with PMA. Whole cell lysates were fractionated bySDS-PAGE, then transferred to filters as before. The filters werethen probed using Abs specifically binding the active PKC� andPKC� phosphoproteins. In two additional experiments, neitherPKC� nor PKC� showed differences in band intensity in PMA-stimulated cells in the absence or presence of hydralazine (forPKC� mean intensity � SD: 1.05 � 0.12; for PKC�: 0.99 � 0.13),with values normalized to �-actin and expressed relative to PMA-stimulated cells in absence of hydralazine. These results were con-firmed by measuring PKC translocation. PMA stimulates the trans-location of active PKC from the cytosol to the membrane. Absagainst total specific isoforms were used. In untreated cells PKC�and PKC� were present in both the cytosolic and membrane frac-tion (Fig. 5, A and B), which was as described by others (43). PMAstimulated PKC� and PKC� translocation to the membrane frac-

tion, and they were not affected by hydralazine. In contrast, PKC�is largely confined to the cytosolic fraction in unstimulated cells,translocates to the membrane fraction following PMA stimulation,and its translocation is inhibited by both hydralazine and rottlerin(Fig. 5C). No effect of hydralazine on total PKC� was observed.These results thus further indicate that hydralazine affects PKC�but not PKC� and PKC� isoenzymes.

ERK pathway inhibition by hydralazine

To confirm that hydralazine affects activation of the signaling mol-ecules downstream of PKC, we determined whether hydralazinealso inhibits activation of Ras, Raf, MEK, and ERK. Ras wasexamined first. PKC activates ERK in both a Ras-dependent (44)and Ras-independent mechanism by directly activating Raf (45) orMEK (46). GTP-bound Ras is active, whereas GDP-bound Ras isinactive. A fusion protein containing the RBD of Raf-1, whichbinds Ras-GTP, was used to immunoprecipitate active Ras fromPMA-stimulated CD4� T cells with and without hydralazine orrottlerin treatment. Fig. 6 shows the densitometric analyses(mean � SD for n � 2 determinations, expressed in arbitrary units)of Ras in the immunoprecipitates (active) and in the supernatants(inactive). Unstimulated cells lack activated Ras but approximatelyone-half the Ras is activated within 15 min of PMA stimulation.Both hydralazine and rottlerin inhibit Ras activation, indicating notonly that hydralazine inhibits PMA-Ras activation, but also con-firming that Ras is a signaling molecule downstream of PKC� in Tcells.

The effect of hydralazine and rottlerin on Raf, MEK, and ERKactivation were tested using the approach described for the patientswith lupus. Fig. 7A shows a representative blot comparing the drugeffects on Raf activation, and Fig. 7B shows the mean � SD of thedensitometric analyses from two independent experiments. Hy-dralazine and rottlerin appear to cause approximately equal inhi-bition. Similarly, Fig. 7C shows a representative immunoblot com-paring the effects of hydralazine and the MEK inhibitor PD98059(47) on MEK activation. Fig. 7D shows the mean � SD of threesimilar, independent experiments. Hydralazine inhibits MEK acti-vation to the same degree as PD98059. In these experiments an Abto total MEK was used as loading control and confirms that MEKprotein expression does not change with the treatment. Fig. 7Eshows a representative immunoblot comparing the effects of hy-dralazine and rottlerin on ERK phosphorylation, and Fig. 7F themean � SD of three to five independent experiments. Hydralazineinhibits ERK phosphorylation, as previously described by ourgroup (19), and rottlerin causes a similar inhibition. Together theseresults indicate that hydralazine inhibits PMA-stimulated ERK

FIGURE 5. Subcellular distribution of PKC isoenzymes in CD4� Tcells. CD4� T cells from normal donors were treated for 1 h in the absenceor presence of hydralazine or rottlerin then stimulated with PMA as de-scribed. Cytosolic (�) and membrane (p) fractions of 1 � 106 cell equiv-alents were analyzed by SDS-PAGE and immunoblotting using Absagainst PKC� (A), PKC� (B), and PKC� (C). Band intensity was quanti-tated relative to �-actin and is expressed in arbitrary units. Results repre-sent the mean percentage � SD of two independent experiments.

FIGURE 6. PMA-stimulated Ras activation is PKC�-dependent. CD4�

T cells from normal controls were treated with hydralazine or rottlerin andstimulated with PMA as described. The cells were then lysed, and Ras wasprecipitated using the RBD of Raf (Raf-1-RBD) conjugated to beads. Pre-cipitated proteins binding Ras-GTP (active, p) or the supernatants (inac-tive, �) were fractionated by electrophoresis, transferred to membranes,and immunoblotted for Ras. Results are normalized to �-actin expressionin supernatants and presented as the mean � SD of two independentexperiments.

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pathway signaling in normal CD4� T cells and that its effects aremediated at least in part through PKC� inhibition, resemblingthose observed in CD4� lupus T cells. However, it is also worthnoting that rottlerin is somewhat more potent than hydralazine ininhibiting PKC�, but has equal effects on the downstream signal-ing molecules. This suggests that hydralazine may have an addi-tional effect on downstream molecules, or reflect differential mech-anisms of how hydralazine and rottlerin inhibit PKC� activation.

Inhibition of PKC� results in CD70 overexpression andpromoter demethylation

Additional experiments confirmed that inhibiting PKC� activationwith rottlerin causes demethylation and overexpression of methy-lation-sensitive T cell genes, similar to that found in lupus T cellsand hydralazine-treated T cells. One of the methylation-sensitivegenes overexpressed in lupus and hydralazine-treated cells isCD70 (12), a costimulatory ligand for B cell CD27. PBMC werestimulated with PHA then treated with rottlerin using the sameprotocols used for DNA methylation inhibitors and hydralazine(12). Fig. 8A shows increased CD70 mRNA in cells pretreatedwith rottlerin. This experiment was confirmed using semiquanti-tative RT-PCR. Fig. 8B shows a slightly greater than 2-fold in-crease in CD70 mRNA in rottlerin-treated cells.

The possibility that PKC� inhibition causes CD70 overexpres-sion as a consequence of DNA hypomethylation was tested byanalyzing the methylation status of the CD70 promoter in rottlerin-treated cells using bisulfite-treated DNA. Bisulfite deaminates cy-tosine bases to form uracil but does not affect methylcytosine. PCRamplification using primers specific for methylated or unmethyl-ated CG pairs, or amplification followed by sequencing of bisul-

fite-treated DNA permits quantification of the methylation statusof a specific sequence (48).

Fig. 9 shows the methylation status of the CD70 promotergene as determined by methylation-specific PCR. Rottlerin de-creased the cytosine methylation in the CD70 promoter relativeto untreated cells, similar to our previous results using hydral-azine-treated T cells and CD4� T cells from patients with lupus(14). These results were confirmed by bisulfite sequencing. Foreach CpG pair in the methylation-sensitive region (14), the meth-ylation status was assessed in five cloned fragments from stimu-lated T cells cultured in the absence (Fig. 10A) or presence (Fig.10B) of rottlerin. Only six CG pairs were methylated in this regionin rottlerin-treated cells compared with 18 pairs in untreated cells.Because we previously reported that methylation of this regionsuppresses CD70 promoter function (14), these results suggest thatdecreased PKC� activity increases CD70 gene expression throughDNA hypomethylation.

FIGURE 7. Hydralazine inhibits PMA-stimulated activation of Raf-1,MEK1/2, and ERK1/2. CD4� T cells from normal controls were treatedwith hydralazine, rottlerin, or PD98059 stimulated with PMA and lysatessubjected to SDS-electrophoresis and immunoblot analysis as described. A,C, and E show representative blots of the phosphorylated forms of Raf-1,MEK1/2, and ERK1/2, respectively. �-actin or total MEK expression usedas loading controls are indicated. B, D, and F show the mean � SD of thedensitometric analyses relative to �-actin or MEK of the indicated numberof independent experiments performed as in A, C, and E, respectively.

FIGURE 8. PKC� inhibition increases CD70 mRNA. A, CD4� T cellsfrom two healthy donors (donors 1 and 2) were stimulated with PHA for18 h and left untreated (�) or treated (�) with rottlerin for 72 h. Totalcellular RNA was extracted and reverse-transcribed, and CD70 transcriptswere amplified as described in Materials and Methods. PCR products werefractionated by agarose gel electrophoresis and stained with ethidium bro-mide. RT-PCR for �-actin transcripts was used as a control. B, CD70transcripts from untreated (�) or rottlerin-treated (`) cells were quanti-tated relative to �-actin by real-time RT-PCR as indicated in Materials andMethods. Results represent the mean � SD of seven different experiments,normalized to untreated control. �, p � 0.02.

FIGURE 9. Rottlerin decreases CD70 promoter methylation. GenomicDNA was isolated from CD4� T cells that were untreated (�) or treated(`) with rottlerin for 72 h as described in Fig. 8. The DNA was treated withbisulfite then subjected to semiquantitative PCR using methylation sensi-tive primers interrogating the CG pairs described in Materials and Meth-ods, and the methylation status of each sample was calculated relative tountreated cells. Amplification of a sequence lacking CG pairs was used asa loading control. Results represent the mean � SD of two independentexperiments.

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Transfection with a dominant negative PKC� reproducesthe effects of rottlerin, hydralazine, and lupus on amethylation-sensitive T cell gene

To confirm the role of PKC� in the CD70 methylation defect ob-served in lupus and hydralazine-treated cells, we examined ERKphosphorylation, CD70 mRNA levels, and CD70 promoter meth-ylation in normal T cells transfected with a dominant negativePKC� mutant lacking kinase activity.

CD4� T cells from a healthy donor were transfected with thedominant negative PKC� cloned into pEGFP-N1 or the empty vec-tor. T cells transfected with the dominant negative PKC� showeddecreased PKC� phosphorylation in response to PMA comparedwith T cells transfected with pEGFP-N1 alone (Fig. 11A). In serialrepeats of this experiment, a 45 � 6% decrease in PKC� phos-phorylation (n � 4 donors, p � 0.05) was seen in PKC�K376R-transfected cells by Western blot analysis using Abs recognizingphospho-PKC�Thr505. Total PKC� was not affected by the trans-fection (Fig. 11A). Cells transfected with the dominant negativePKC� also had decreased PMA-stimulated ERK phosphorylationcompared with cells transfected with the empty vector (Fig. 11A).These studies confirm observations made with rottlerin, and furthersupport involvement of PKC� in the ERK signaling pathway.

We next determined whether a decrease in PKC� activity,caused by transfection with the dominant negative, results in de-methylation and overexpression of methylation-sensitive genes asfound in lupus T cells and hydralazine-treated T cells (11, 13, 14).CD4� T cells were transiently transfected with the PKC� domi-nant negative and cultured for 72 h as before. The cells were har-vested, and RNA and DNA were isolated. Fig. 11B shows thatcells transfected with the dominant negative PKC� express greateramounts of CD70 compared with cells transfected with the emptyvector. Fig. 11C shows demethylation of the CD70 promoter geneas determined by methylation-specific PCR. Together these resultsconfirm our observation that decreased PKC� activation decreasesERK pathway signaling, resulting in hypomethylation of DNA andincreased expression of methylation-sensitive immune-sensitivegenes, as observed in idiopathic and drug-induced lupus (11, 13, 14).

IFN-� does not explain decreased PKC� phosphorylationin lupus

The mechanism by which PKC� phosphorylation is impaired inidiopathic lupus is unknown. Because IFN-� is markedly increasedin the serum of patients with active SLE and induces autoimmunity(49), we considered the possibility that IFN-� inhibited PKC�

FIGURE 10. Methylation pattern clonality. CD4� T cells from a healthy donor were treated with rottlerin for 72 h as described in Fig. 9, then DNAwas isolated from untreated or treated cells. Cells treated with bisulfite were amplified by PCR, and five fragments were cloned and sequenced fromuntreated (A) or treated (B) preparations. The location of the CG pairs is indicated on the x-axis relative to the transcription start site, and the methylationstatus of the corresponding CG pair from each cloned fragment is shown on the y-axis, represented as methylated deaminate cytosine bases (F) orunmethylated bases (E).

FIGURE 11. Transfection with a dominant negative PKC� inhibits T cell ERK phosphorylation, demethylates the CD70 promoter and causes CD70overexpression. CD4� T cells from a healthy donor were transfected with 5 �g of a PKC-�K376R-GFP fusion protein construct or with the empty vectorpEGFP-N1. A, PKC� inactivation decreases ERK phosphorylation. Transfected cells were cultured for 72 h then stimulated with PMA for 15 min. Lysateswere analyzed by SDS-PAGE and immunoblotted for phosphorylated forms of PKC� and ERK. The blots were stripped and reprobed for total PKC�.�-actin is shown as an additional loading control. Blots are representative of four independent experiments. B, The PKC� mutant increases CD70 mRNA.RNA was extracted from transfected CD4� T cells and cultured for 72 h. CD70 transcripts were measured in cells transfected with the dominantnegative PKC� (f) or the empty vector (�). Transcripts were quantitated relative to �-actin as described in Materials and Methods. Results areexpressed relative to empty vector and represent the mean � SD of three independent experiments run in duplicate. �, p � 0.01. C, Transfection witha dominant negative PKC� decreases CD70 promoter methylation. Genomic DNA was isolated from CD4� T cells transfected with empty vector (�) orthe dominant negative PKC� (f) and cultured 72 h as in B. Purified DNA was treated with bisulfite then subjected to semiquantitative PCR usingmethylation-sensitive primers interrogating the CG pairs described in Materials and Methods, and the methylation index of each sample was calculated,relative to empty vector and considered as 1. Amplification of a sequence lacking CG pairs was used as a loading control. Results represent the mean �SD of three independent experiments. �, p � 0.023.

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activation. CD4� T cells were treated with IFN-� concentrationssimilar to those found in patients with lupus and the cells werestimulated with PMA as before. IFN-� had no significant effect onPKC� phosphorylation as measured by immunoblotting analysis(PMA vs IFN-�: 52.7 � 12.1 vs 48.2 � 7.3 (100 U/ml) and 56.5 �10.5 (1000 U/ml), mean � SD, n � 3). Similar results were ob-tained for ERK1/2 phosphorylation (PMA vs IFN-�: 34.4 � 11.3vs 30.3 � 9.2 (100 U/ml) and 38.3 � 5.4 (1000 U/ml), mean �SD, n � 3). These results suggest that IFN-� is not responsible forthe decreased ERK pathway signaling in lupus T cells.

To test the possibility that lupus serum factors other than IFN-�inhibit ERK pathway signaling, normal CD4� T cells were cul-tured in medium supplemented with 10% SLE serum or 10% FBSfor 24 h. SLE serum did not affect PMA-stimulated PKC� phos-phorylation as determined by Western blot analysis (mean percent-age � SD: 0.98 � 0.15, n � 3 patients, normalized to �-actin).Similarly, culturing normal PBMC for 24 h in lupus or FBS-con-taining medium, then measuring PKC� activation in purifiedCD4� T cells also revealed equivalent levels of PKC� phosphor-ylation following PMA stimulation, indicating that in the absenceor presence of accessory cells, SLE serum did not alter PKC�phosphorylation in CD4� T cells (data not shown).

DiscussionThese experiments implicate abnormal PKC� activation in thepathogenesis of idiopathic and hydralazine-induced lupus. Abnor-mal T cell signaling plays a central role in the pathogenesis ofSLE, and defects in the proximal, middle, and distal signal trans-duction pathways have been implicated in the T cell dysfunction(50). The proximal defects include abnormalities in TCR -chainexpression and Ca2� fluxes, whereas the middle defects includeprotein kinase A and mitochondrial abnormalities (50). The distalabnormalities include a decrease in ERK activity in CD4� T cellsfrom patients with lupus, which is proportional to disease activity;signaling through the JNK and p38 pathways is intact (12, 18). TheERK pathway defect is likely involved in disease pathogenesisbecause inhibiting T cell ERK pathway signaling with hydralazineor U0126, a MEK inhibitor, causes autoreactivity in vitro and alupus-like disease in animal models (19). The relationship betweenthe ERK pathway defect and the more proximal defects isunknown.

Although PKC� substrates are not completely characterized,others have shown activation of the ERK pathway in a PKC�-dependent fashion in different cell systems and mediated by a va-riety of stimuli (44, 51, 52). The results from the present studyshowed significantly lower PKC�-Raf-MEK1/2-ERK1/2 signalingin CD4� lupus T cells, consistent with our earlier report of de-creased ERK phosphorylation (18). Similar to the defect in ERKphosphorylation (18) the decrease in phospho-PKC� levels wasalso restricted to CD4� lupus T cells because PKC� phosphory-lation in PMA-stimulated CD4� T cells from patients with RAwas not significantly different from healthy donors and was not dueto a decrease in total PKC� protein expression. PMA stimulatesPKC� translocation to the cytoplasmic membrane where it is phos-phorylated by phosphoinositide-dependent kinase-1 (41). Activa-tion of T cells through the TCR complex using anti-CD3 alone orin combination with anti-CD28 resulted in lesser PKC� activation(data not shown), consistent with reports showing minimally mod-ified distribution of PKC� after stimulation of T cells with anti-CD3 alone (53). Thus the abnormality may lie in PKC�, the trans-location or the activation by phosphoinositide-dependent kinase-1.The PKC� gene resides at 3p21.31, which is not a susceptibilityregion identified in studies of familial lupus (54), suggesting thatthe defect is not genetic. The nature of the defect remains uncer-

tain, but in these experiments did not appear to be a direct effect ofIFN-� because IFN-� did not inhibit PKC�-ERK1/2 signaling.The IFN-� concentrations used were 100 and 1000 U/ml, and thehighest serum IFN-� level in patients with SLE at flare was �300U/ml with a mean of 30.3 U/ml (55). It is also interesting to notethat although IFN-� therapy has occasionally been associated withlupus, the most common autoimmune manifestation induced byIFN-� therapy is thyroid dysfunction (56). Additionally, SLE se-rum did not affect PKC� phosphorylation in normal CD4� T cells,suggesting that the defect is not due to serum factors in patientswith lupus.

A defect in lupus T cell PKC activity has been reported by otherresearch. Tada et al. (33) found decreased total PKC activity with-out differentiation of isozymes. Biro et al. (57) reported lower pro-tein expression of several isoforms including PKC� that was re-stored with corticosteroids. In our study, all of the patients werereceiving corticosteroid treatment, and none had decreased totalPKC� levels. Decreased PKC� activation may contribute to lupuspathogenesis. PKC� is expressed ubiquitously among cells andtissues and is distinguished from the other PKC isoforms becauseis involved in negative regulation of cellular functions. For exam-ple, transgenic mice overexpressing PKC� are resistant to tumorformation by phorbol esters (58). In contrast, mice deficient inPKC� demonstrate increased B lymphocyte proliferation and de-velop a lupus-like autoimmune disease with anti-chromatin Absand an immune complex glomerulonephritis (20). Recent studieshave demonstrated that PKC�-deficient T cells have a reducedthreshold for activation by cell-bound allogeneic MHC stimula-tion, and map PKC� to a signaling pathway that is necessary for Tcell attenuation (59). The reduced threshold for activation is sim-ilar to the response to subthreshold stimulation by demethylated Tcells reported by our group (4). All these observations are consis-tent with our findings of diminished PKC� activity in lupus, adisease characterized by exaggerated cellular and humoral immuneresponses.

Our previous work also showed that the lupus-inducing drughydralazine decreased ERK phosphorylation in a selective mannerbecause p38 and JNK signaling pathways were unaffected (19),thus resembling the defect seen in idiopathic lupus. The same workalso showed that T cells treated with hydralazine or U0126, an-other ERK pathway inhibitor, induced a lupus-like disease wheninjected in syngeneic mice. We therefore asked whether hydral-azine inhibits ERK pathway signaling at the same point as occursin idiopathic lupus.

We found that hydralazine selectively inhibits PMA-stimulatedPKC�, but not PKC� and PKC�, phosphorylation, resembling theresults seen in idiopathic lupus. These results were confirmed bydemonstrating that hydralazine selectively inhibits PMA stimu-lated PKC� translocation to the cytosolic membrane. We alsofound that PKC� and PKC� were associated with the membranefraction in resting T cells, coincident with the presence of phos-pho-PKC isoforms in total lysates from untreated normal and lupusT cells. This may be due to a preactive state of these molecules infreshly isolated cells because in lymphocytes isolated and culturedunder serum-starved conditions for 24 h, PKC-phosphorylatedforms were almost undetectable in whole cell lysates (data notshown). In contrast, PKC� was only present in the cytosolicfraction under unstimulated conditions, indicating the presenceof inactive enzyme coincident with the absence of phosphory-lated isoforms in resting cells.

We had previously reported that hydralazine could demethylatethe CD70 promoter and cause CD70 overexpression (14). Inhibi-tion of PKC� with rottlerin was also sufficient to demethylate theCD70 promoter and cause CD70 overexpression in CD4� T cells,

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further supporting a commonality of effects between rottlerin andhydralazine. It is also worth noting that the same sequence de-methylates in lupus T cells, and that the increment in CD70 mRNAlevels induced by rottlerin was 2-fold with respect to untreatedcells, similar to that observed in lupus T cells (14).

The observation that cells transfected with a dominant negativePKC� express lower levels of phosphorylated ERK confirms asignaling link between PKC� and ERK in CD4� T cells. Cellstransfected with the dominant negative PKC� also demethylatedthe TNFSF7 (CD70) promoter and increased CD70 expressionsimilar to that reported in lupus and hydralazine-treated T cells(14). The PKC� antagonist rottlerin had the same effect on TNFSF7promoter methylation and expression. Together these results stronglysuggest that a defect in PKC� activation is responsible for impairedERK pathway signaling in lupus T cells, and is sufficient to causeoverexpression of methylation-sensitive immune genes through reg-ulatory element hypomethylation, reproducing effects observed indominant negative transfected T cells, hydralazine-treated T cells andby rottlerin inhibition of PKC� in T cells.

In conclusion, our results localize the lupus and hydralazine-induced ERK pathway signaling defect to PKC� phosphorylation.This defect causes an increase in CD70 expression that correlateswith a decreased methylation of its promoter in normal T cells,similar to what is observed in lupus and hydralazine-treated cells.Other methylation-sensitive genes may be similarly affected. Be-cause mice with genetic PKC� deficiency develop a lupus-likedisease (20), and cells transfected with a dominant negative PKC�reproduce abnormalities seen in lupus, our results strongly indicatethat a PKC� signaling abnormality may contribute to the devel-opment of idiopathic and hydralazine-induced lupus through DNAmethylation inhibition. Additional experiments are necessary toelucidate the mechanism causing the PKC� abnormality in lupusand hydralazine-treated CD4� T cells.

AcknowledgmentWe thank Cindy Bourke for expert secretarial assistance.

DisclosuresThe authors have no financial conflict of interest.

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