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The Journal of Immunology, 2007, 179, 5553 -5563
Copyright © 2007 by The American Association of Immunologists, Inc.
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*HYDRALAZINE HYDROCHLORIDE
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*Lupus

Impaired T Cell Protein Kinase C{delta} Activation Decreases ERK Pathway Signaling in Idiopathic and Hydralazine-Induced Lupus1

Gabriela Gorelik*, Jing Yuan Fang{ddagger}, Ailing Wu*, Amr H. Sawalha§ and Bruce Richardson2,*,{dagger}

* Department of Medicine, University of Michigan, and {dagger} Ann Arbor Veterans Affairs Medical Center, Ann Arbor, MI 48109; {ddagger} 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 Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cells from patients with lupus or treated with the lupus-inducing drug hydralazine have defective ERK phosphorylation. The reason for the impaired signal transduction is unknown but important to elucidate, because decreased T cell ERK pathway signaling causes a lupus-like disease in animal models by decreasing DNA methyltransferase expression, leading to DNA hypomethylation and overexpression of methylation-sensitive genes with subsequent autoreactivity and autoimmunity. We therefore analyzed the PMA stimulated ERK pathway phosphorylation cascade in CD4+ T cells from patients with lupus and in hydralazine-treated cells. The defect in these cells localized to protein kinase C (PKC){delta}. Pharmacologic inhibition of PKC{delta} or transfection with a dominant negative PKC{delta} mutant caused demethylation of the TNFSF7 (CD70) promoter and CD70 overexpression similar to lupus and hydralazine-treated T cells. These results suggest that defective T cell PKC{delta} activation may contribute to the development of idiopathic and hydralazine-induced lupus through effects on T cell DNA methylation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human systemic lupus erythematosus (SLE)3 is a chronic autoimmune disease characterized by abnormal T signaling and the presence of autoantibodies that cause multiple pathologic abnormalities, including glomerulonephritis and vasculitis. The cause of human lupus remains unknown.

T cell DNA hypomethylation has been implicated in the pathogenesis of idiopathic and drug-induced human lupus (1, 2). DNA methylation is one mechanism regulating gene expression, and hypomethylation of regulatory sequences correlates with active transcription, whereas hypermethylation suppresses expression (3). Our group has demonstrated that treating human or murine cloned or polyclonal CD4+ T cells with DNA methylation inhibitors including 5-azacytidine, procainamide, and hydralazine demethylates the DNA and makes the cells become autoreactive, and injecting the autoreactive murine T cells into syngeneic recipients causes a lupus-like disease (4, 5, 6, 7). Overexpression of the adhesion molecule LFA-1 (CD11a/CD18) contributes to the autoreactivity (8, 9), whereas abnormal perforin expression contributes to the cytotoxic potential of the autoreactive CD4+ cells, (10, 11) and increased CD70 expression to B cell overstimulation (12). The same abnormalities in LFA-1, perforin, and CD70 expression are found in CD4+ T cells from patients with active lupus; all three genes are overexpressed and display demethylation of the same regulatory sequences as in T cells treated with DNA methylation inhibitors (11, 13, 14). Together these results indicate a relationship involving DNA hypomethylation, methylation-sensitive immune gene overexpression, and T cell autoreactive responses in the pathogenesis of both idiopathic and drug-induced lupus.

DNA methylation patterns are maintained by DNMT1 (DNA (cytosine-5-) methyltransferase 1), which is regulated in part by the ERK signaling pathway (15, 16, 17). T cells from patients with lupus exhibit lower DNMT1 expression due to decreased ERK activity (18). Furthermore, hydralazine, a lupus-inducing drug, similarly inhibits T cell DNA methylation by decreasing DNMT1 expression through inhibition of ERK pathway signaling, and T cells treated with ERK pathway inhibitors become autoreactive in vitro 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 overall decreases in total ERK protein (18). The mechanisms causing decreased ERK activation in both systems are unknown.

Because DNA hypomethylation due to decreased ERK pathway signaling is common to idiopathic and hydralazine-induced lupus, and may contribute to autoimmunity, we characterized the defect causing decreased ERK pathway signaling T cells from patients with idiopathic lupus and in hydralazine-treated T cells. The results indicate that impaired protein kinase C (PKC){delta} phosphorylation is responsible for the decreased ERK signaling in SLE patient T cells and hydralazine-treated T cells. Because mice with genetic PKC{delta} deficiency develop lupus (20), these studies suggest that impaired PKC{delta} phosphorylation may contribute to the development of idiopathic and hydralazine-induced lupus through DNA methylation inhibition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

Rottlerin was obtained from Calbiochem and PD98059 was from Cell Signaling Technology. Hydralazine was purchased from VWR and PMA from Sigma-Aldrich.

Subjects

Groups of patients with lupus consisted of eight women and three men with a mean SLE disease activity index (21) of 4.7, range 2–6, and average age of 48 years (range 30–67 years). All were on low-dose prednisone (mean 6.1 mg, range 2–10 mg), and one was on mycophenolate mofetil and two on azathioprine. Groups of patients with rheumatoid arthritis (RA) consisted 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 patients were receiving TNF antagonists, two patients were receiving hydroxychloroquine, and one receiving sulfasalazine and one leflunomide. All the patients with lupus met the American College of Rheumatology criteria for patients with SLE (22) and patients with RA met criteria from The American Rheumatism Association (23). The patients were recruited from the outpatient rheumatology clinics and inpatient services at the University of Michigan (Ann Arbor, MI). Healthy controls were recruited by advertising. These studies were reviewed and approved by the University of Michigan Institutional Review Board for Human Subject Research.

T cell isolation

PBMC were isolated from venous blood of healthy donors and patients with SLE or RA using density gradient centrifugation as previously described (1). CD4+ cells were then isolated by negative selection using magnetic beads (CD4+ T cell isolation kit; Miltenyi Biotec), according to the 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 or stimulated with 50 ng/ml PMA for 15 min at 37°C. Where indicated, cells were incubated in the presence or absence of hydralazine (10 µM), rottlerin (10 µM), or PD98059 (50 µM) 60 min before stimulation. Following stimulation, the cells were centrifuged, resuspended in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.25% deoxycholic acid, 1% Nonidet P-40, 1 mM EDTA, 100 µg/ml PMSF, 100 µM sodium orthovanadate, 1 mM DTT) and a protease inhibitor cocktail (Roche), and rotated at 4°C for 30 min. Insoluble material was removed by centrifugation at 16,000 x g for 30 min and the supernatant saved as whole cell lysate. When experiments required cytosolic or membrane fractions, treated cells were resuspended in lysis buffer (20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 5 mM EGTA, 10 mM 2-ME, 100 µg/ml PMSF, 100 µM sodium orthovanadate, 1 mM DTT and protease inhibitor cocktail) and incubated for 30 min at 4°C and vortexed each 10 min. The lysate was centrifuged at 16,000 x g for 30 min and the cytoplasmic protein containing supernatant saved for analysis. The pellet was resuspended in lysis buffer containing 1% Triton X-100, incubated for 30 min on ice then vortexed, centrifuged, and the particulate fraction saved. The amount of cellular protein present in the whole cell lysate or in each of the fractions was measured using the BCA Protein 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. In brief, CD4+ T cell lysates were incubated with an agarose-immobilized GST fusion protein containing the RBD of Raf-1 to precipitate GTP-bound Ras. Precipitated (GTP-bound) and soluble (GDP-bound) Ras were determined by immunoblotting with an Ab recognizing and precipitating all isoforms of Ras (clone Ras 10; Upstate Biotechnology).

Kinase activity measurement

A total of 20 µg of isolated proteins were diluted in Laemmli loading buffer and denatured by boiling for 5 min followed by electrophoresis in 10–12% SDS-polyacrylamide gels. The fractionated proteins were then electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell) and stained with Ponceau S (Sigma-Aldrich) to verify equal amounts of protein between lanes before Western blot analyses. Membranes 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 with the 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 incubated with a HRP-linked secondary Ab for 1 h. After three washes with TBS containing 0.1% Tween 20, the membranes were treated with ECL detection system (Amersham Biosciences), exposed to x-ray film (Kodak) and developed to visualize the labeled protein bands. Molecular mass was estimated by comparison of sample bands with prestained molecular mass marker (Bio-Rad). For quantitative studies, the bands on x-ray films were scanned using a photodocumentation system (Alpha Innotech) and analyzed with ImageQuant 5.2 software (Amersham Bioscience). Where indicated, blots were stripped and reblotted with the corresponding Ab. Values were normalized respect to beta-actin as indicated.

Ab products

The following primary Abs were used: rabbit polyclonal anti-phospho-PKC{alpha} (Thr638/641), anti-phospho-PKC{theta} (Thr538), anti-phospho-PKC{delta} (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 Promega, and anti-total PKC{alpha} (1.5 mg/ml) and anti-total PKC{delta} (1.5 µg/ml) were from Upstate Biotechnology. Monoclonal mouse anti-total PKC{theta} (1/250; BD Transduction Laboratory) was also used. Secondary Abs included: anti-rabbit IgG HRP (1/2000; Cell Signaling Technology) and anti-mouse IgG HRP (1/4000; Amersham Biosciences).

RNA isolation

PBMC from normal donors were cultured in RPMI 1640 supplemented with 10% FCS and antibiotics as described, then stimulated with 1 µg/ml PHA for 18 h. CD4+ T cells were then bead purified and cultured in the same medium supplemented with IL-2 at a density of 1 x 106 cells/ml as previously described (24) for an additional 72 h in the presence or absence of rottlerin (10 µM). Following incubation, total RNA was isolated using an RNA isolation kit (Qiagen) according to the manufacturer’s instruction.

RT-PCR

RNA was digested with 2 U of DNaseI (Ambion), and 2.5 µg were used for reverse transcription reactions in a total volume of 20 µl using reverse transcriptase (Qiagen). Then, 2 µl of cDNA in a mixture with 2.5 mM MgCl2, 0.2 mM dNTP (Promega), 1 µM each primer, and 2.5 U of TaqDNA polymerase (Promega) in a volume of 50 µl were amplified under the following conditions: denaturation at 95°C for 5 min, amplification at 95°C for 1 min, 60°C for 1 min, 72°C for 1 min for a total of 35 cycles followed by a final extension at 72°C for 5 min. Amplification for beta-actin was also performed as a loading and RNA quality control. The following primers were used: CD70 forward 5'–TGCTTTGGTCCCATTGGTCG-3' and reverse 5'-TCCTGCTGAGGTCCTGTGTGATTC-3'; beta-actin forward 5'-GGACTTCGAGCAAGAGATGG-3' and reverse 5'-AGCACTGTGTTGGCGTACAG-3'. The PCR products were run on a 2% agarose gel and stained with ethidium bromide.

RNA quantification by real-time RT-PCR

A total of 200 ng of RNA was converted to cDNA and amplified in one step using Quanti-Tect SYBR Green RT-PCR kit (Qiagen). CD70 transcripts were quantitated by real-time semiquantitative RT-PCR using a Rotor-Gene 3000 (Corbett Research) and previously published protocols (12). The following amplification conditions were used: reverse transcription at 50°C for 30 min, denaturation at 95°C for 15 min, amplification at 94°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 dilutions of one RNA sample were also included to generate a standard curve, and this was used to obtain relative concentrations of the transcript of interest in each of the RNA samples. In each experiment, water was included as a negative control to rule out primer dimer formation. Amplification of beta-actin was performed to confirm that equal amounts of total RNA were added for each sample and that the RNA was intact and equally amplifiable among all samples. The primers used were the same as for RT-PCR, as described.

Methylation-specific PCR

Genomic T cell DNA from cells cultured and treated as for RT-PCR was isolated using a DNeasy isolation kit (Qiagen), according to the manufacturer’s instructions, and bisulfite-treated as previously described (25). Briefly, 2–20 µg of purified DNA was treated with 350 mM sodium hydroxide 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 M sodium hydroxide and 3 M ammonium acetate, then precipitated with glycogen and 95% ethanol at –80°C. The precipitate was washed with 70% ethanol, dried by vacuum, and redissolved in double-distilled H2O. Semiquantitative PCR was then used to determine the methylation status of CG pairs within the TNFSF7 (CD70) promoter regulatory region (from –660 to –466 bp (14)), using 5 µg of bisulfite-treated DNA. The cycling conditions 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 the product were determined, a standard curve was generated using one DNA sample, and water was included as negative control. The primers designed to hybridize with methylated CG pairs were forward 5'-CGA GGT TTA GAT AGG AGA ATC GT–3' (interrogating two CG pairs) and reverse 5'-TAA AAA TAC TCC CCA AAT ATT CGT-3' (interrogating one CG pair). Loading controls consisted of primers designed to avoid CG pairs and were forward 5'-GGGTGG ATT ATT TAA GGT TAG GAGT-3' and reverse 5'-AAT CTC CCT CTA TCA CCC AAA CTA-3'. The amplified fragment was cloned, and five fragments were sequenced by the University of Michigan DNA Sequencing Core (Ann Arbor, MI). In studies involving the PKC{delta} mutant, a third set of primers were used to hybridize with unmethylated CG pairs, and the methylation index was calculated with the following equation: (fraction methylated)/(fraction methylated + fraction unmethylated). The values are relative to p-EGFP-N1 considered as 1. The unmethylated primers were forward 5'–GTG AGG TTT AGA TAG GAG AAT TGT–3' and reverse 5'–TAA AAA TAC TCC CCA AAT ATT CAT A–3'.

Expression plasmids

A plasmid expressing a dominant negative form of mouse PKC{delta} (PKC{delta}K376R-GFP fusion protein) was a gift from Dr. S. H. Yuspa (National Cancer Institute Bethesda, MD) (26). To generate the GFP control vector without PKC{delta}K376R, the PKC{delta}K376R-GFP construct was digested with BglII and BamHI (New England Biolabs and Roche, respectively). This method excised PKC{delta}K376R from the PKC{delta}K376R-GFP construct in two pieces. The remainder of the construct (~4.5 kb) was extracted using a gel extraction kit (Qiagen) and self-ligated at 14°C overnight using T4 DNA ligase (Promega).

Transient transfections

CD4+ T cells were obtained from normal donors as previously described and immediately transfected with 5 µg of the dominant negative PKC{delta} mutant or with the empty vector pEGFP-N1 using Amaxa nucleofection technology, according to the manufacturer’s instructions. After 6 h the medium was changed, and after 72 h the cells were lysed to obtain protein, total RNA, and genomic DNA as described. As the vector includes the GFP, transfection efficiency was assessed by fluorescence microscopy, and was 65 ± 5% of total cell number (fluorescent and nonfluorescent cells). Every transfection was confirmed by verifying expression of the transfected protein by Western blot and Abs against phospho-PKC{delta}Thr505 using protein lysates from cells transfected with the PKC{delta} mutant and stimulated with PMA for 15 min. Band intensity was compared with cells transfected with the empty vector, which is considered as controls.

Statistical analysis

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Defective ERK pathway signaling in T cells from patients with active lupus

Our group previously demonstrated that PMA stimulates ERK phosphorylation to a lesser extent in CD4+ lupus T cells than controls. This decrease in phospho-ERK was not due to lower ERK protein levels because kinase protein expression was similar in control and lupus T cells (18). To elucidate where in the signaling cascade the defect lies, we analyzed PMA-induced activation of the molecules upstream of ERK (PKC -> Raf -> MEK1/2 -> ERK1/2) in CD4+ T cells from patients with lupus. PKC enzymes are activated directly by PMA, then each subsequent molecule in this pathway acquires protein kinase activity after phosphorylation, and is subsequently able to phosphorylate and activate the next member of the signaling cascade. Phospho-ERK translocates to the nucleus, providing a direct link with an extracellular signal, an internal pathway, and the genetic response (27).

Purified CD4+ T cells from patients with lupus and healthy donors were stimulated with PMA for 15 min and ERK pathway signaling was compared by immunoblot using specific Abs. Fig. 1A shows a representative immunoblot using whole cell extracts from unstimulated or stimulated CD4+ T cells processed immediately after isolation from a healthy donor or two different patients with lupus. The protein extracts were fractionated by SDS-PAGE, transferred to nitrocellulose membranes then probed with the relevant Abs. The membrane was stripped and reblotted with Abs to the next signaling molecule. beta-actin was used as loading control. Phosphorylation of ERK1/2 was detected with an Ab against the active dually phosphorylated form of ERK1/2. To detect MEK1/2, the blot was probed with an anti-phospho-MEK1/2 Ab that recognizes MEK1/2 when phosphorylated by Raf at Ser217 and Ser221; both are sites of phosphorylation that result in its activation (28). Phosphorylation of Raf was tested using an Ab to Raf-1 phosphorylated at Ser338, localized in the catalytic domain (29). Decreased levels of phospho-ERK1/2, phospho-MEK1/2, and phospho-Raf-1 are seen in PMA-stimulated CD4+ lupus T cells compared with normal controls. These results suggest an overall lower ERK pathway signaling in lupus T cells, and that the defect may lie upstream of Raf at PKC. Fig. 1B summarizes three to six serial repeats of the experiments shown in Fig. 1A. PMA-stimulated phosphorylation of ERK, MEK, and Raf is significantly reduced in CD4+ T cells from patients with lupus.


Figure 1
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  		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 different patients with SLE (L1, L2) were isolated and immediately stimulated with 50 ng/ml PMA for 15 min or not stimulated as described in Materials and Methods. Proteins from whole cell lysates were fractionated by SDS-PAGE, transferred to nitrocellulose membranes and probed with a polyclonal Ab against the active dually phosphorylated form of ERK1/2. The membrane was stripped and reblotted sequentially for phospho-MEK1/2 Ser217/221 and Raf-1 p-Ser338. beta-actin was used as loading control. B, Quantitative immunoblot analysis of phospho-ERK, phospho-MEK, and phospho-Raf in CD4+ T cells from three to six different patients with active lupus treated as in A and compared with normal donors. Values were normalized to beta-actin. The relative phosphorylation 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.

 
PKC{delta} 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 cell activation (31) and is able to activate ERK (32), and as a generalized PKC defect in lupus T cells has been reported (33), we examined PKC isoform activation in lupus T cells. The PKC isoforms activated by PMA belong to two different subfamilies (34); the conventional that are calcium-dependent ({alpha}, beta and {gamma}), and the novel or calcium-independent PKC isoforms ({delta}, {epsilon}, {eta}, {theta}, and µ). A third subfamily comprises the atypical isoforms, but the members are insensitive to PMA (34) and thus were not studied. PKC{alpha} is one of the most abundant isoenzymes in human and murine T lymphocytes (35, 36). PKC{theta} expression is tissue restricted and is most highly expressed in T lymphocytes playing an important role in their activation (37), whereas PKC{delta} exhibits the highest homology to PKC{theta} (38). We therefore examined PMA-stimulated phosphorylation of {alpha}, {theta}, and {delta} in lupus T cells.

CD4+ T cells, isolated from a normal donor and two different patients with lupus, were cultured alone or with PMA for 15 min, and PKC{alpha}, {theta}, and {delta} phosphorylation analyzed by immunoblotting with Abs to PKC{alpha} phospho-Thr638/641, PKC{theta} phospho-Thr538, and PKC{delta} phospho-Thr505, respectively (Fig. 2A). PKC{alpha} Thr638/641 is a C-terminal autophosphorylation site that plays a unique role in preventing degradation and is required for its catalytic activity (39). PKC{theta} activation loop phosphorylation at Thr538 results in significant kinase activity (40). PKC{delta}-Thr505 is an activation loop site, its phosphorylation is induced by PMA and is an indicator of PKC{delta} activation (41). The phosphorylation of PKC{alpha} and PKC{theta} did not differ significantly between lupus and normal cells. In contrast, PKC{delta} phosphorylation was reduced in CD4+ lupus T cells. Because the Ab used binds to PKC{delta} when phosphorylated at Thr505, these results suggest lower PKC{delta} activity in CD4+ lupus T cells. In contrast, PMA-stimulated PKC{delta} phosphorylation in CD8+ T cells from patients with lupus did not differ significantly from phosphorylation in cells from normal donors (mean percentage ± SD: 0.89 ± 0.20, n = 3 patients with lupus, normalized to total PKC{delta} and expressed relative to normal cells), in agreement with previous reports showing decreased ERK activity in CD4+ lupus T cells (18).


Figure 2
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  		FIGURE 2. Impaired PKC{delta} 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{theta}, PKC{alpha}, and PKC{delta} was then similarly compared by immunoblotting using Abs against phosphorylated forms of the enzymes. beta-actin was used as a loading control. B, PKC{delta} phosphorylation in unstimulated (–) or PMA-stimulated (+) CD4+ T cells from two different patients with lupus (L1, L2) was similarly compared with CD4+ T cells from a normal control by immunoblot analysis using an Ab against PKC{delta} phospho-T505. Total PKC{delta} protein expression is shown in the stripped and reprobed membrane. beta-actin was used as loading control. Results are representative of five independent experiments. C, CD4+ T cells from 11 patients with active lupus were similarly stimulated with PMA, and lysates were subjected to SDS-electrophoresis, transferred, and blotted with specific Abs against PKC{delta} phospho-T505. Bands were scanned and quantified by densitometry. Blots were stripped and reblotted with PKC{delta} and values were normalized to total PKC{delta} levels. The relative phosphorylation of PKC{delta} 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.

 
We considered the possibility that the decrease in phospho-PKC{delta} was due to decreased PKC{delta} protein. However, total PKC{delta} was not decreased in lupus T cells (Fig. 2B). Serial repeats of this experiment demonstrated that the ratio of total PKC{delta} of patients with lupus to total PKC{delta} 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 immunoblots from 11 different patients with lupus was quantitated by densitometry and data are shown in Fig. 2C. PMA-induced phosphorylation of PKC{delta} is significantly reduced in CD4+ T cells from patients with lupus respect to normal donors, whereas the total kinase expression is unaltered. Because PMA directly activates the calcium-independent PKC{delta} isoenzyme, these data suggest that the defective ERK pathway signaling in lupus T cells previously reported by our group (18) is a consequence of impaired PMA-stimulated PKC{delta} phosphorylation. We then studied PMA-induced activation of PKC{delta} in CD4+ T cells from seven patients with RA. No significant differences in PKC{delta} phosphorylation were observed between CD4+ T cells from patients with RA vs cells from healthy controls as measured by immunoblotting (mean ± SD: 4.1 ± 1.5 vs 4.8 ± 1.0, respectively, expressed in arbitrary units and normalized to total PKC{delta} expression in the same subject). The differences in PKC{delta} activation between patients with lupus and patients with RA could not be attributed to differences in age, gender, or medications received (see Materials and Methods). These results are consistent with our previous data showing a significant decrease in ERK phosphorylation only in patients with lupus when compared with phosphorylation in patients with RA and in healthy controls, for which effects due to age, gender, and medications were similarly excluded (18).

Hydralazine inhibits PKC{delta} phosphorylation

Where hydralazine inhibits ERK pathway signaling is unknown. Because impaired PKC{delta} activation contributes to decreased T cell ERK pathway signaling in lupus, and the PKC{delta} knockout mouse develops lupus (20), we hypothesized that hydralazine may also inhibit T cell PKC{delta} activation. Fig. 3 shows a representative immunoblot comparing the effects in idiopathic lupus by 10 µM hydralazine, 10 µM rottlerin, on PMA-stimulated PKC{delta} phosphorylation. Rottlerin inhibits protein kinases with specificity for PKC and is able to differentiate PKC isoenzymes with IC50 values for PKC{delta} more than two orders of magnitude lower than for other isoenzymes (42). Both drugs decreased PKC{delta} phosphorylation in normal CD4+ T cells, similar to the decrease observed in lupus T cells, although hydralazine appears to be somewhat less potent at this concentration. Fig. 4 shows the kinetics of PMA-stimulated PKC{delta} phosphorylation inhibition by hydralazine and rottlerin. Fig. 4A shows a representative immunoblot, and Fig. 4B the densitometric analysis plotted against time. The kinetics were similar for both inhibitors with maximal inhibition at 1 h of incubation. Based on these results, cells were treated with hydralazine or rottlerin for 1 h in all additional experiments. Fig. 4C shows the mean percentage ± SD of densitometric analyses from seven independent experiments comparing rottlerin and hydralazine on PKC{delta} stimulation. PMA-induced phosphorylation was inhibited ~33% by hydralazine (p = 0.02) and ~65% by rottlerin (p = 0.001). This finding suggests that hydralazine inhibits PMA-stimulated PKC{delta} phosphorylation.


Figure 3
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  		FIGURE 3. Hydralazine inhibits PMA-induced PKC{delta} phosphorylation. CD4+ T cells from normal controls or patients with lupus were stimulated for 15 min with 50 ng/ml PMA as indicated. Where indicated CD4+ T cells from the normal controls were also treated with hydralazine or the PKC{delta}-specific inhibitor rottlerin for 1 h before stimulation with PMA. Lysates were subjected to immunoblot analysis and PKC{delta} phosphorylation was measured using anti-phospho-PKC{delta} T505 as before. The same blot was stripped and reprobed with Abs anti-beta-actin as a loading control. Results are representative of three independent experiments.

 

Figure 4
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  		FIGURE 4. Inhibition of PKC{delta} phosphorylation by hydralazine. A, CD4+ T cells from a normal donor were untreated or treated with 10 µM hydralazine ({blacksquare}) or 10 µM rottlerin ({diamondsuit}) for the indicated times and then stimulated with PMA for 15 min. PKC{delta} phosphorylation was analyzed by immunoblot using anti-phospho-PKC{delta} as before, and a representative Western blot is shown. beta-actin was used as a loading control. B, Bands from two different experiments performed as in A were quantitated by densitometry and phospho-PKC{delta} levels normalized to beta-actin. Dashed line represents PKC{delta} phosphorylation in PMA-stimulated cells in the absence of inhibitors. Results represent the mean ± SEM. C, Immunoblot analysis of PMA-stimulated PKC{delta} phosphorylation after treatment with hydralazine or rottlerin for 1 h. The mean levels of PKC{delta} phosphorylation ± SD for seven independent experiments were calculated by normalization to beta-actin expression. The relative PKC{delta} phosphorylation in PMA-stimulated cells in the absence of inhibitors was considered as 100%. *, p ≤ 0.02; **, p ≤ 0.001.

 
To more rigorously test whether other PKC isozymes are affected, we further analyzed PKC{alpha} and PKC{theta} activation with and without hydralazine. CD4+ T cells were treated with hydralazine and stimulated with PMA. Whole cell lysates were fractionated by SDS-PAGE, then transferred to filters as before. The filters were then probed using Abs specifically binding the active PKC{alpha} and PKC{theta} phosphoproteins. In two additional experiments, neither PKC{alpha} nor PKC{theta} showed differences in band intensity in PMA-stimulated cells in the absence or presence of hydralazine (for PKC{alpha} mean intensity ± SD: 1.05 ± 0.12; for PKC{theta}: 0.99 ± 0.13), with values normalized to beta-actin and expressed relative to PMA-stimulated cells in absence of hydralazine. These results were confirmed by measuring PKC translocation. PMA stimulates the translocation of active PKC from the cytosol to the membrane. Abs against total specific isoforms were used. In untreated cells PKC{alpha} and PKC{theta} were present in both the cytosolic and membrane fraction (Fig. 5, A and B), which was as described by others (43). PMA stimulated PKC{alpha} and PKC{theta} translocation to the membrane fraction, and they were not affected by hydralazine. In contrast, PKC{delta} 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{delta} was observed. These results thus further indicate that hydralazine affects PKC{delta} but not PKC{alpha} and PKC{theta} isoenzymes.


Figure 5
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  		FIGURE 5. Subcellular distribution of PKC isoenzymes in CD4+ T cells. CD4+ T cells from normal donors were treated for 1 h in the absence or presence of hydralazine or rottlerin then stimulated with PMA as described. Cytosolic ({square}) and membrane (Figure 5) fractions of 1 x 106 cell equivalents were analyzed by SDS-PAGE and immunoblotting using Abs against PKC{alpha} (A), PKC{theta} (B), and PKC{delta} (C). Band intensity was quantitated relative to beta-actin and is expressed in arbitrary units. Results represent the mean percentage ± SD of two independent experiments.

 
ERK pathway inhibition by hydralazine

To confirm that hydralazine affects activation of the signaling molecules downstream of PKC, we determined whether hydralazine also inhibits activation of Ras, Raf, MEK, and ERK. Ras was examined first. PKC activates ERK in both a Ras-dependent (44) and Ras-independent mechanism by directly activating Raf (45) or MEK (46). GTP-bound Ras is active, whereas GDP-bound Ras is inactive. A fusion protein containing the RBD of Raf-1, which binds Ras-GTP, was used to immunoprecipitate active Ras from PMA-stimulated CD4+ T cells with and without hydralazine or rottlerin 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 approximately one-half the Ras is activated within 15 min of PMA stimulation. Both hydralazine and rottlerin inhibit Ras activation, indicating not only that hydralazine inhibits PMA-Ras activation, but also confirming that Ras is a signaling molecule downstream of PKC{delta} in T cells.


Figure 6
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  		FIGURE 6. PMA-stimulated Ras activation is PKC{delta}-dependent. CD4+ T cells from normal controls were treated with hydralazine or rottlerin and stimulated with PMA as described. The cells were then lysed, and Ras was precipitated using the RBD of Raf (Raf-1-RBD) conjugated to beads. Precipitated proteins binding Ras-GTP (active, Figure 6) or the supernatants (inactive, {square}) were fractionated by electrophoresis, transferred to membranes, and immunoblotted for Ras. Results are normalized to beta-actin expression in supernatants and presented as the mean ± SD of two independent experiments.

 
The effect of hydralazine and rottlerin on Raf, MEK, and ERK activation were tested using the approach described for the patients with lupus. Fig. 7A shows a representative blot comparing the drug effects on Raf activation, and Fig. 7B shows the mean ± SD of the densitometric analyses from two independent experiments. Hydralazine and rottlerin appear to cause approximately equal inhibition. Similarly, Fig. 7C shows a representative immunoblot comparing the effects of hydralazine and the MEK inhibitor PD98059 (47) on MEK activation. Fig. 7D shows the mean ± SD of three similar, independent experiments. Hydralazine inhibits MEK activation to the same degree as PD98059. In these experiments an Ab to total MEK was used as loading control and confirms that MEK protein expression does not change with the treatment. Fig. 7E shows a representative immunoblot comparing the effects of hydralazine and rottlerin on ERK phosphorylation, and Fig. 7F the mean ± SD of three to five independent experiments. Hydralazine inhibits ERK phosphorylation, as previously described by our group (19), and rottlerin causes a similar inhibition. Together these results indicate that hydralazine inhibits PMA-stimulated ERK pathway signaling in normal CD4+ T cells and that its effects are mediated at least in part through PKC{delta} inhibition, resembling those observed in CD4+ lupus T cells. However, it is also worth noting that rottlerin is somewhat more potent than hydralazine in inhibiting PKC{delta}, but has equal effects on the downstream signaling molecules. This suggests that hydralazine may have an additional effect on downstream molecules, or reflect differential mechanisms of how hydralazine and rottlerin inhibit PKC{delta} activation.


Figure 7
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  		FIGURE 7. Hydralazine inhibits PMA-stimulated activation of Raf-1, MEK1/2, and ERK1/2. CD4+ T cells from normal controls were treated with hydralazine, rottlerin, or PD98059 stimulated with PMA and lysates subjected 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. beta-actin or total MEK expression used as loading controls are indicated. B, D, and F show the mean ± SD of the densitometric analyses relative to beta-actin or MEK of the indicated number of independent experiments performed as in A, C, and E, respectively.

 
Inhibition of PKC{delta} results in CD70 overexpression and promoter demethylation

Additional experiments confirmed that inhibiting PKC{delta} activation with rottlerin causes demethylation and overexpression of methylation-sensitive T cell genes, similar to that found in lupus T cells and hydralazine-treated T cells. One of the methylation-sensitive genes overexpressed in lupus and hydralazine-treated cells is CD70 (12), a costimulatory ligand for B cell CD27. PBMC were stimulated with PHA then treated with rottlerin using the same protocols used for DNA methylation inhibitors and hydralazine (12). Fig. 8A shows increased CD70 mRNA in cells pretreated with rottlerin. This experiment was confirmed using semiquantitative RT-PCR. Fig. 8B shows a slightly greater than 2-fold increase in CD70 mRNA in rottlerin-treated cells.


Figure 8
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  		FIGURE 8. PKC{delta} inhibition increases CD70 mRNA. A, CD4+ T cells from two healthy donors (donors 1 and 2) were stimulated with PHA for 18 h and left untreated (–) or treated (+) with rottlerin for 72 h. Total cellular RNA was extracted and reverse-transcribed, and CD70 transcripts were amplified as described in Materials and Methods. PCR products were fractionated by agarose gel electrophoresis and stained with ethidium bromide. RT-PCR for beta-actin transcripts was used as a control. B, CD70 transcripts from untreated ({square}) or rottlerin-treated (Figure 8) cells were quantitated relative to beta-actin by real-time RT-PCR as indicated in Materials and Methods. Results represent the mean ± SD of seven different experiments, normalized to untreated control. *, p < 0.02.

 
The possibility that PKC{delta} inhibition causes CD70 overexpression as a consequence of DNA hypomethylation was tested by analyzing the methylation status of the CD70 promoter in rottlerin-treated cells using bisulfite-treated DNA. Bisulfite deaminates cytosine bases to form uracil but does not affect methylcytosine. PCR amplification using primers specific for methylated or unmethylated CG pairs, or amplification followed by sequencing of bisulfite-treated DNA permits quantification of the methylation status of a specific sequence (48).

Fig. 9 shows the methylation status of the CD70 promoter gene as determined by methylation-specific PCR. Rottlerin decreased the cytosine methylation in the CD70 promoter relative to untreated cells, similar to our previous results using hydralazine-treated T cells and CD4+ T cells from patients with lupus (14). These results were confirmed by bisulfite sequencing. For each CpG pair in the methylation-sensitive region (14), the methylation status was assessed in five cloned fragments from stimulated T cells cultured in the absence (Fig. 10A) or presence (Fig. 10B) of rottlerin. Only six CG pairs were methylated in this region in rottlerin-treated cells compared with 18 pairs in untreated cells. Because we previously reported that methylation of this region suppresses CD70 promoter function (14), these results suggest that decreased PKC{delta} activity increases CD70 gene expression through DNA hypomethylation.


Figure 9
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  		FIGURE 9. Rottlerin decreases CD70 promoter methylation. Genomic DNA was isolated from CD4+ T cells that were untreated ({square}) or treated (Figure 9) with rottlerin for 72 h as described in Fig. 8. The DNA was treated with bisulfite then subjected to semiquantitative PCR using methylation sensitive primers interrogating the CG pairs described in Materials and Methods, and the methylation status of each sample was calculated relative to untreated cells. Amplification of a sequence lacking CG pairs was used as a loading control. Results represent the mean ± SD of two independent experiments.

 

Figure 10
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  		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 DNA was isolated from untreated or treated cells. Cells treated with bisulfite were amplified by PCR, and five fragments were cloned and sequenced from untreated (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 methylation status of the corresponding CG pair from each cloned fragment is shown on the y-axis, represented as methylated deaminate cytosine bases (•) or unmethylated bases ({circ}).

 
Transfection with a dominant negative PKC{delta} reproduces the effects of rottlerin, hydralazine, and lupus on a methylation-sensitive T cell gene

To confirm the role of PKC{delta} in the CD70 methylation defect observed in lupus and hydralazine-treated cells, we examined ERK phosphorylation, CD70 mRNA levels, and CD70 promoter methylation in normal T cells transfected with a dominant negative PKC{delta} mutant lacking kinase activity.

CD4+ T cells from a healthy donor were transfected with the dominant negative PKC{delta} cloned into pEGFP-N1 or the empty vector. T cells transfected with the dominant negative PKC{delta} showed decreased PKC{delta} phosphorylation in response to PMA compared with T cells transfected with pEGFP-N1 alone (Fig. 11A). In serial repeats of this experiment, a 45 ± 6% decrease in PKC{delta} phosphorylation (n = 4 donors, p ≤ 0.05) was seen in PKC{delta}K376R-transfected cells by Western blot analysis using Abs recognizing phospho-PKC{delta}Thr505. Total PKC{delta} was not affected by the transfection (Fig. 11A). Cells transfected with the dominant negative PKC{delta} also had decreased PMA-stimulated ERK phosphorylation compared with cells transfected with the empty vector (Fig. 11A). These studies confirm observations made with rottlerin, and further support involvement of PKC{delta} in the ERK signaling pathway.


Figure 11
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  		FIGURE 11. Transfection with a dominant negative PKC{delta} inhibits T cell ERK phosphorylation, demethylates the CD70 promoter and causes CD70 overexpression. CD4+ T cells from a healthy donor were transfected with 5 µg of a PKC-{delta}K376R-GFP fusion protein construct or with the empty vector pEGFP-N1. A, PKC{delta} inactivation decreases ERK phosphorylation. Transfected cells were cultured for 72 h then stimulated with PMA for 15 min. Lysates were analyzed by SDS-PAGE and immunoblotted for phosphorylated forms of PKC{delta} and ERK. The blots were stripped and reprobed for total PKC{delta}. beta-actin is shown as an additional loading control. Blots are representative of four independent experiments. B, The PKC{delta} 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 dominant negative PKC{delta} ({blacksquare}) or the empty vector ({square}). Transcripts were quantitated relative to beta-actin as described in Materials and Methods. Results are expressed relative to empty vector and represent the mean ± SD of three independent experiments run in duplicate. *, p ≤ 0.01. C, Transfection with a dominant negative PKC{delta} decreases CD70 promoter methylation. Genomic DNA was isolated from CD4+ T cells transfected with empty vector ({square}) or the dominant negative PKC{delta} ({blacksquare}) and cultured 72 h as in B. Purified DNA was treated with bisulfite then subjected to semiquantitative PCR using methylation-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.

 
We next determined whether a decrease in PKC{delta} activity, caused by transfection with the dominant negative, results in demethylation and overexpression of methylation-sensitive genes as found in lupus T cells and hydralazine-treated T cells (11, 13, 14). CD4+ T cells were transiently transfected with the PKC{delta} dominant negative and cultured for 72 h as before. The cells were harvested, and RNA and DNA were isolated. Fig. 11B shows that cells transfected with the dominant negative PKC{delta} express greater amounts of CD70 compared with cells transfected with the empty vector. Fig. 11C shows demethylation of the CD70 promoter gene as determined by methylation-specific PCR. Together these results confirm our observation that decreased PKC{delta} activation decreases ERK pathway signaling, resulting in hypomethylation of DNA and increased expression of methylation-sensitive immune-sensitive genes, as observed in idiopathic and drug-induced lupus (11, 13, 14).

IFN-{alpha} does not explain decreased PKC{delta} phosphorylation in lupus

The mechanism by which PKC{delta} phosphorylation is impaired in idiopathic lupus is unknown. Because IFN-{alpha} is markedly increased in the serum of patients with active SLE and induces autoimmunity (49), we considered the possibility that IFN-{alpha} inhibited PKC{delta} activation. CD4+ T cells were treated with IFN-{alpha} concentrations similar to those found in patients with lupus and the cells were stimulated with PMA as before. IFN-{alpha} had no significant effect on PKC{delta} phosphorylation as measured by immunoblotting analysis (PMA vs IFN-{alpha}: 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 obtained for ERK1/2 phosphorylation (PMA vs IFN-{alpha}: 34.4 ± 11.3 vs 30.3 ± 9.2 (100 U/ml) and 38.3 ± 5.4 (1000 U/ml), mean ± SD, n = 3). These results suggest that IFN-{alpha} is not responsible for the decreased ERK pathway signaling in lupus T cells.

To test the possibility that lupus serum factors other than IFN-{alpha} inhibit ERK pathway signaling, normal CD4+ T cells were cultured in medium supplemented with 10% SLE serum or 10% FBS for 24 h. SLE serum did not affect PMA-stimulated PKC{delta} phosphorylation as determined by Western blot analysis (mean percentage ± SD: 0.98 ± 0.15, n = 3 patients, normalized to beta-actin). Similarly, culturing normal PBMC for 24 h in lupus or FBS-containing medium, then measuring PKC{delta} activation in purified CD4+ T cells also revealed equivalent levels of PKC{delta} phosphorylation following PMA stimulation, indicating that in the absence or presence of accessory cells, SLE serum did not alter PKC{delta} phosphorylation in CD4+ T cells (data not shown).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These experiments implicate abnormal PKC{delta} activation in the pathogenesis of idiopathic and hydralazine-induced lupus. Abnormal T cell signaling plays a central role in the pathogenesis of SLE, and defects in the proximal, middle, and distal signal transduction pathways have been implicated in the T cell dysfunction (50). The proximal defects include abnormalities in TCR {zeta}-chain expression and Ca2+ fluxes, whereas the middle defects include protein kinase A and mitochondrial abnormalities (50). The distal abnormalities include a decrease in ERK activity in CD4+ T cells from patients with lupus, which is proportional to disease activity; signaling through the JNK and p38 pathways is intact (12, 18). The ERK pathway defect is likely involved in disease pathogenesis because inhibiting T cell ERK pathway signaling with hydralazine or U0126, a MEK inhibitor, causes autoreactivity in vitro and a lupus-like disease in animal models (19). The relationship between the ERK pathway defect and the more proximal defects is unknown.

Although PKC{delta} substrates are not completely characterized, others have shown activation of the ERK pathway in a PKC{delta}-dependent fashion in different cell systems and mediated by a variety of stimuli (44, 51, 52). The results from the present study showed significantly lower PKC{delta}-Raf-MEK1/2-ERK1/2 signaling in CD4+ lupus T cells, consistent with our earlier report of decreased ERK phosphorylation (18). Similar to the defect in ERK phosphorylation (18) the decrease in phospho-PKC{delta} levels was also restricted to CD4+ lupus T cells because PKC{delta} phosphorylation in PMA-stimulated CD4+ T cells from patients with RA was not significantly different from healthy donors and was not due to a decrease in total PKC{delta} protein expression. PMA stimulates PKC{delta} translocation to the cytoplasmic membrane where it is phosphorylated by phosphoinositide-dependent kinase-1 (41). Activation of T cells through the TCR complex using anti-CD3 alone or in combination with anti-CD28 resulted in lesser PKC{delta} activation (data not shown), consistent with reports showing minimally modified distribution of PKC{delta} after stimulation of T cells with anti-CD3 alone (53). Thus the abnormality may lie in PKC{delta}, the translocation or the activation by phosphoinositide-dependent kinase-1. The PKC{delta} gene resides at 3p21.31, which is not a susceptibility region identified in studies of familial lupus (54), suggesting that the defect is not genetic. The nature of the defect remains uncertain, but in these experiments did not appear to be a direct effect of IFN-{alpha} because IFN-{alpha} did not inhibit PKC{delta}-ERK1/2 signaling. The IFN-{alpha} concentrations used were 100 and 1000 U/ml, and the highest serum IFN-{alpha} level in patients with SLE at flare was <300 U/ml with a mean of 30.3 U/ml (55). It is also interesting to note that although IFN-{alpha} therapy has occasionally been associated with lupus, the most common autoimmune manifestation induced by IFN-{alpha} therapy is thyroid dysfunction (56). Additionally, SLE serum did not affect PKC{delta} phosphorylation in normal CD4+ T cells, suggesting that the defect is not due to serum factors in patients with lupus.

A defect in lupus T cell PKC activity has been reported by other research. Tada et al. (33) found decreased total PKC activity without differentiation of isozymes. Biro et al. (57) reported lower protein expression of several isoforms including PKC{delta} that was restored with corticosteroids. In our study, all of the patients were receiving corticosteroid treatment, and none had decreased total PKC{delta} levels. Decreased PKC{delta} activation may contribute to lupus pathogenesis. PKC{delta} is expressed ubiquitously among cells and tissues and is distinguished from the other PKC isoforms because is involved in negative regulation of cellular functions. For example, transgenic mice overexpressing PKC{delta} are resistant to tumor formation by phorbol esters (58). In contrast, mice deficient in PKC{delta} demonstrate increased B lymphocyte proliferation and develop a lupus-like autoimmune disease with anti-chromatin Abs and an immune complex glomerulonephritis (20). Recent studies have demonstrated that PKC{delta}-deficient T cells have a reduced threshold for activation by cell-bound allogeneic MHC stimulation, and map PKC{delta} to a signaling pathway that is necessary for T cell attenuation (59). The reduced threshold for activation is similar to the response to subthreshold stimulation by demethylated T cells reported by our group (4). All these observations are consistent with our findings of diminished PKC{delta} activity in lupus, a disease characterized by exaggerated cellular and humoral immune responses.

Our previous work also showed that the lupus-inducing drug hydralazine decreased ERK phosphorylation in a selective manner because p38 and JNK signaling pathways were unaffected (19), thus resembling the defect seen in idiopathic lupus. The same work also showed that T cells treated with hydralazine or U0126, another ERK pathway inhibitor, induced a lupus-like disease when injected in syngeneic mice. We therefore asked whether hydralazine inhibits ERK pathway signaling at the same point as occurs in idiopathic lupus.

We found that hydralazine selectively inhibits PMA-stimulated PKC{delta}, but not PKC{alpha} and PKC{theta}, phosphorylation, resembling the results seen in idiopathic lupus. These results were confirmed by demonstrating that hydralazine selectively inhibits PMA stimulated PKC{delta} translocation to the cytosolic membrane. We also found that PKC{alpha} and PKC{theta} were associated with the membrane fraction in resting T cells, coincident with the presence of phospho-PKC isoforms in total lysates from untreated normal and lupus T cells. This may be due to a preactive state of these molecules in freshly isolated cells because in lymphocytes isolated and cultured under serum-starved conditions for 24 h, PKC-phosphorylated forms were almost undetectable in whole cell lysates (data not shown). In contrast, PKC{delta} was only present in the cytosolic fraction under unstimulated conditions, indicating the presence of inactive enzyme coincident with the absence of phosphorylated isoforms in resting cells.

We had previously reported that hydralazine could demethylate the CD70 promoter and cause CD70 overexpression (14). Inhibition of PKC{delta} with rottlerin was also sufficient to demethylate the CD70 promoter and cause CD70 overexpression in CD4+ T cells, further supporting a commonality of effects between rottlerin and hydralazine. It is also worth noting that the same sequence demethylates in lupus T cells, and that the increment in CD70 mRNA levels induced by rottlerin was 2-fold with respect to untreated cells, similar to that observed in lupus T cells (14).

The observation that cells transfected with a dominant negative PKC{delta} express lower levels of phosphorylated ERK confirms a signaling link between PKC{delta} and ERK in CD4+ T cells. Cells transfected with the dominant negative PKC{delta} also demethylated the TNFSF7 (CD70) promoter and increased CD70 expression similar to that reported in lupus and hydralazine-treated T cells (14). The PKC{delta} antagonist rottlerin had the same effect on TNFSF7 promoter methylation and expression. Together these results strongly suggest that a defect in PKC{delta} activation is responsible for impaired ERK pathway signaling in lupus T cells, and is sufficient to cause overexpression of methylation-sensitive immune genes through regulatory element hypomethylation, reproducing effects observed in dominant negative transfected T cells, hydralazine-treated T cells and by rottlerin inhibition of PKC{delta} in T cells.

In conclusion, our results localize the lupus and hydralazine-induced ERK pathway signaling defect to PKC{delta} phosphorylation. This defect causes an increase in CD70 expression that correlates with 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. Because mice with genetic PKC{delta} deficiency develop a lupus-like disease (20), and cells transfected with a dominant negative PKC{delta} reproduce abnormalities seen in lupus, our results strongly indicate that a PKC{delta} signaling abnormality may contribute to the development of idiopathic and hydralazine-induced lupus through DNA methylation inhibition. Additional experiments are necessary to elucidate the mechanism causing the PKC{delta} abnormality in lupus and hydralazine-treated CD4+ T cells.


   Acknowledgment
 
We thank Cindy Bourke for expert secretarial assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Public Health Service Grants AR42525, AG25877, and ES15214 and a Merit grant from the Department of Veterans Affairs. Back

2 Address correspondence and reprint requests to Dr. Bruce Richardson, 3007 Biomedical Science Research Building, Department of Medicine, University of Michigan, Ann Arbor, MI 48109-2200. E-mail address: brichard{at}umich.edu Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; PKC, protein kinase C; DNMT1, DNA (cytosine-5-) methyltransferase 1; RA, rheumatoid arthritis; RBD, Ras binding domain. Back

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


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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