Estradiol upregulates calcineurin expression via overexpression of estrogen receptor alpha gene in systemic lupus erythematosus

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ORIGINAL ARTICLE

Estradiol upregulates calcineurin expression via

overexpression of estrogen receptor alpha gene in

systemic lupus erythematosus

全身性紅斑狼瘡之二羥雌激素調升蛋白質磷酸酶 3 CA之表現是經由雌

激素受體a基因之過度表現

Hui-Li Lin

a

, Jeng-Hsien Yen

b

, Shi-Shin Chiou

c

, Wen-Chan Tsai

b

, Tsan-Teng Ou

d

,

Cheng-Chin Wu

d

, Hong-Wen Liu

b,

*

林慧麗a_, _顏正賢b_,_邱世欣c_, _蔡文展b_,_歐燦騰d_, _吳正欽d_,_劉宏文b,_* a

Department of Food and Nutrition, Meiho University, Pingtung, Taiwan b

Division of Allergy, Immunology, and Rheumatology, Department of Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

c

Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

d

Division of Allergy, Immunology, and Rheumatology, Department of Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

Received 1 October 2010; accepted 4 November 2010 Available online 26 February 2011

KEYWORDS

Calcineurin; Estrogen;

Estrogen receptor alpha gene;

Systemic lupus erythematosus

關鍵詞

蛋白質磷酸酶3;

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease primarily affecting women (9:1 compared with men). To investigate the influence of female sex hormone estrogen on the development of female-biased lupus, we compared the expression of estrogen receptor alpha (ERa) gene and protein levels as well as expression of T-cell activation gene calcineurin in response to estrogen in peripheral blood lymphocytes (PBLs) from SLE patients and normal controls. PBLs were isolated from 20 female SLE patients and 6 normal female controls. The amount of ER_{a protein in PBL was measured by flow cytometry. The expression of ERa and} cal-cineurin messenger RNA was measured by semi-quantitative reverse transcription-polymerase chain reaction. Calcineurin phosphatase activity was measured by calcineurin assay kit. The expression of ERa messenger RNA and ERa protein was significantly increased (p Z 0.001 and p_{Z 0.023, respectively) in PBL from SLE patients compared with that from normal controls.}

* Corresponding author. Division of Allergy, Immunology, and Rheumatology, Department of Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 100 Zihyou 1st_{Road, Kaohsiung 807, Taiwan.}

E-mail address:liuhow@kmu.edu.tw(H.-W. Liu).

a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : h t t p : / / w w w . k j m s - o n l i n e . c o m

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雌激素;

雌激素受體a基因; 全身性紅斑狼瘡

In addition, the basal calcineurin in PBL from SLE patients was significantly higher (pZ 0.000) than that from normal controls, and estrogen-induced expression of calcineurin was increased (pZ 0.007) in PBL from SLE patients compared with that from normal controls, a 3.15-fold increase. This increase was inhibited by the ER_{a antagonism ICI 182,780. The effects of ER} antagonism were also found in calcineurin activity. These data suggest that overexpression of ERa gene and enhanced activation of calcineurin in response to estrogen in PBL may contribute to the pathogenesis of female dominant in SLE.

摘摘要要全身性紅斑狼瘡是一種自體免疫疾病，主要侵犯女性為主 (男女比為 1:9)。為探討女性雌激素如何影响紅斑狼瘡偏向女性發生而研究狼瘡病人與正常人週邊血液淋巴球之雌激素受體a 基因與蛋白的表現。我們收集了20 位女性之紅斑狼瘡病人與6位正常女性志願者為研究對象。雌激素受體a蛋白的表現是以流式細胞測定儀定量之。雌激素受體a 和蛋白質磷酸酶 3CA 信息核醣核酸之測定是以半定量之逆轉錄鏈聚合反應測定之。蛋白質磷酸酶 3CA 之活度是以蛋白質磷酸酶分析用成套工具測定之。結果發現紅斑狼瘡周邊血淋巴球雌激素受體a之信息核醣核酸及蛋白的表現比正常人有意義增加，其 p 值分別為 0.001和 0.023。此外基礎之蛋白質磷酸酶 3CA 表現在紅斑狼瘡淋巴球比正常人有意義的增加，p 值Z0.000。雌激素誘發之蛋白質磷酸酶 3CA 表現在紅斑狼瘡淋巴球比正常人有意義的增加 (pZ0.007), 其增加為 3.15 倍, 此增加可被雌激素受體拮抗者ICI 182,780 抑制。雌激素受體拮抗現象亦發生於蛋白質磷酸酶 3CA 之磷酸酶活度。由以上結果顯示雌激素受體 a 基因的過度表現以及淋巴球因雌激素之刺激所導致蛋白質磷酸酶 3CA 之高度活化結果對狼瘡病人之偏向女性之致病機轉有所貢獻。

Introduction

Women have a higher incidence of autoimmune disorders, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis[1], but the mechanism for this higher incidence is still unknown. Multiple mechanisms may contribute to the female bias in autoimmune diseases[2]. Several lines of evidence have indicated that sex hormone estrogens or their metabolites contribute to the female predilection or to exacerbation of SLE[3e6]. Estrogen functions as a tran-scriptional regulator by activating estrogen receptors (ERs), estrogen receptor alpha (ERa), and estrogen receptor beta (ERb), which then translocate to the nucleus, where they bind to estrogen response elements in gene promoters. Although both ERs are expressed in most immune cells, ERa is shown to be predominantly expressed[1]. Results from human studies indicate that estrogens enhance lupus development, but concrete evidence implicating the ER in promoting SLE is lacking. Recent murine studies using

ER-selective agonists in New Zealand Black New Zealand

White F1 female mice have suggested that activation of ERa, but not ERb, promotes SLE[2]. On the contrary, ER-deficient New Zealand Black New Zealand White F1 and NZM2419 mice with disruption of ERa attenuate glomeru-lonephritis and increase survival[7,8]. One of these studies has further indicated that ERa promotes lupus by inducing interferon-gamma, an estrogen-regulated cytokine that impacts this disease[8]. These data suggest that ERa plays a major role in mediating the effects of endogenous estrogens by promoting both loss of tolerance and the development of pathogenic autoantibodies [8]; however, studies of ERa in mediating estrogen’s effect on human SLE are limited. Although the peripheral blood mononuclear cells (PBMCs) of SLE patients have been reported to express wild-type ERa and ERb like those of normal individuals

[9,10], a recent report observed that expression of ERa,

but not ERb, was increased in PBMC from SLE patients

compared with normal controls [11]. These conflicting results prompted us to perform a quantitative analysis of ERa gene as well as protein expression by peripheral blood lymphocytes (PBLs) from SLE patients and a comparison with normal controls.

Materials and methods

Human subjects and cell culture

Approval for this study was obtained from Kaohsiung Medical University Hospital Human Ethics Committee. Twenty female patients with SLE and six healthy female controls were recruited in this study. The lupus patients

were between 19 and 48 years of age [mean standard

deviation (SD)Z38.2 8.73 years]. All had regular

menstrual cycle. They were selected randomly from outpatient services and met the American College of Rheumatology criteria for classification of SLE[12]. Disease duration varied from 4 years to 25 years with a mean SD of 12.2 6.29 years. Most of our patients were in remission state, their disease activity varying from none to active with SLE disease activity index scores of between 0 and 11 (mean 4.89 3.48). Four patients were not taking pred-nisolone. Two patients were receiving prednisolone at doses of 35 mg/d and 20 mg/d, respectively. Two patients were receiving 7.5 mg/d. The other 12 patients were receiving 5 mg/d. Nineteen patients were on hydroxy-chloquine (100e200 mg/d), one was on cyclophosphamide 50 mg/d and none were on azathiopurine. The female controls were six healthy volunteers selected from the laboratory staff aged between 25 and 35 years (mean

33.3 1.37 years) with normal menstrual cycle and no

history of collagen vascular disease. PBMCs were isolated by using Ficoll-Paque cushion centrifugation gradient. Cells were harvested and cultured in petri dishes for 2 hours.

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Nonadherent cells (lymphocyte-rich cells; PBL) were har-vested and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Biological Industries, Beit Haemek, Israel) until use. Estradiol (Sigma Chemical Co., St. Louis, MO, USA) at the concentration of 0.2mg/mL was used to measure dose-dependent response. ER antagonist ICI 182,780 (Sigma Chemical Co) at the concentration of 4mg/mL was used in ERa antagonism.

Flow cytometry analysis

Immunostaining of ERa was performed as described previ-ously with modification [13]. Harvested cells were first fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes on ice followed by washing and resuspension in PBS, and then they were permeabilized with 0.1% Triton X-100 at 4C for 5 minutes. Cells were stained with rabbit anti-human ERa antibody (Upstate Biotechnology, Charlotteswille, VA, USA) for 30 minutes and goat anti-rabbit IgG antibody-conjugated fluorescein isothiocyanate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 minutes followed by washing and resuspension in PBS. The positive cell count and the mean fluorescent intensity were analyzed by Coulter Epics XL Flow Cytom-eter (Beckman Coulter, Ramsey, MN, USA). The data were processed using EXPO32 ADC Software (Beckman Coulter, Ramsey, MN, USA).

Semi-quantitative reverse transcription-polymerase chain reaction

Cells were cultured with or without estradiol for 24 hours. Total RNA was extracted using REzol TM C & T (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Reverse transcription (RT) was performed with 1mg RNA, 20 mL of reaction reagent containing 5 mM MgCl2, 1 mM dNTP mixture, 62.5 U RNase inhibitor, 0.5mg

oligo(dT)15 primer, and 15 U avian myeloblastosis virus reverse transcriptase in RT buffer [10 mM TriseHCl (pH 9.0 at 25C), 50 mM KCl, and 0.1% Triton X-100]. The mixture was incubated at 42C for 15 minutes, followed by 99C for 5 minutes and 4C for 5 minutes. After RT, 0.625 U Taq DNA polymerase, 1.5 mM MgCl2, 0.8 mM dNTP, and 50 pM of each

primer in polymerase chain reaction (PCR) buffer were added to the complementary DNA mixture, the volume being 50mL. After denaturation at 95C for 5 minutes, 30 cycles ofb-actin amplification were performed at 94C for 45 seconds, 60C for 45 seconds, and 72C for 1.5 minutes/ cycle; ERa, at 94_{C for 45 seconds, 50}_{C for 45 seconds, and}

72C for 1.5 minutes/cycle. The primers for b-actin

message amplification were (forward) 50-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-30and (reverse) 50-CGT CAT

ACT CCT GCT TGC TGA TCC ACA TAT GC-30; ERa were

(forward) 50-TGC CAA GGA GAC TCG CTA CT-30(904e923)

and (reverse) 50-CTG GCG CTT GTG TTT CAA C-30

(1180e1162); calcineurin were (forward) 50_{-TTG ATT GCC}

ACT GTA GTT TGG T-30(2888e2909) and (reverse) 50-CAG

CGG CCC ATG CAT GGA AAT TT-30. The PCR products of

b-actin, ERa, and calcineurin were electrophoresed on a 2% agarose minigel at 100 V for 30 minutes and visualized with ethidium bromide staining under UV illumination.

Multimers of 100-bp DNA ladder plus were used as makers (MBI Fermentas, Hanover, MD, USA). The density of the bands was measured by densitometry, and the background was subtracted; subsequently, the ratios of ERa and cal-cineurin tob-actin mRNA were calculated, respectively.

Calcineurin phosphatase activity assay

Calcineurin enzymatic activity was measured using a calci-neurin assay kit (Biomol, Polymouth Meeting, PA, USA). The activity was measured as the phosphorylation rate of a synthetic phosphopeptite substrate (RII pt peptite) with the Biomol Green reagent and read optical density 620 nm on a microplate reader.

ERa antagonism

PBLs from lupus patients were cultured for 24 hours in medium alone, medium plus estradiol (0.2mg/mL), medium plus ER antagonist ICI 182,780 (4mg/mL), and medium plus estradiol (0.2mg/mL) and ICI 182,780 (4 mg/mL). Total RNA was isolated, and calcineurin mRNA was measured using semi-quantitative RT-PCR. Calcineurin phosphatase activity was measured by calcineurin assay kit in cells using the same culture condition.

Statistical analysis

Data were expressed as the mean SD and mean standard error of the mean. Comparisons of numerical data between groups were performed by Student t test. Values of p< 0.05 were considered statistically significant.

Results

ERa gene expression was upregulated in PBL from SLE patients

To investigate if the expression of the ERa gene was upre-gulated in patients with SLE, we analyzed PBL from patients with SLE and healthy controls using semi-quantitative RT-PCR. For internal control of the RNA, the expression of b-actin was also examined and the amount of ERa gene was

normalized to the endogenous reference b-actin. The

normalized ER-a gene expression (^Cr) of SLE patients was then related to the ^Cr of healthy controls for their relative expression levels. The results showed that the expression of ERa gene in SLE patients was significantly higher (p < 0.001) compared with that in healthy controls (Fig. 1). These observations indicate that ERa gene expression is upregu-lated in patients with SLE.

ERa protein was also upregulated in PBL from SLE

We further investigated the ERa protein expression using

flow cytometry. As shown in Fig. 2, the ERa protein

expression in SLE was significantly increased (pZ 0.023) compared with that in healthy controls. These results confirmed the observation obtained from semi-quantitative RT-PCR that ERa was indeed upregulated in SLE.

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Dose-dependent response of PBL to estradiol

The concentration of estradiol in the plasma of normal women is less than 0.5mg/mL (<2 nM). In most studies, estradiol has been used in concentrations (micromolar levels) hundreds of fold higher than normal plasma level

[14,15]. To investigate the concentrations of estradiol that increase calcineurin expression, we cultured PBL from lupus patients without estradiol and in medium containing estradiol of 0.2, 2.0, and 20mg/mL for 24 hours. The mean calcineurin expression in SLE cells cultured at concentration of 0.2, 2.0, and 20mg/mL of estradiol was 0.487, 0.407, and 0.417, respectively (Fig. 3). Hence, estradiol concentration at 0.2mg/mL was used to measure the expression of calci-neurin in response to estrogen.

Estradiol increased calcineurin and phosphatase activity in PBL from SLE patients

We have shown previously that ERa mRNA expression and protein level are increased in lupus patients compared with normal controls. However, how overexpressed ERa mediates the female sex hormone estrogen is unknown. In this study, we showed that the basal levels of calcineurin expression were significantly increased (pZ 0.000) in PBL from SLE patients compared with PBL from normal controls. The increase was 4.03-fold. When estradiol was added to cultures, the calcineurin expression from normal controls was not increased significantly (pZ 0.103) while that from

SLE patients increased significantly (pZ 0.017). The calci-neurin in SLE was 3.15-fold higher than in normal controls (Fig. 4). Similar effects were found in phosphatase activity of the protein that exerts biological effects (Fig. 5). Varia-tion of phosphatase activity was not prominent in cells of normal controls cultured with and without estradiol, whereas the phosphatase activity in PBL of lupus patients cultured with estradiol increased significantly (pZ 0.005) compared with that cultured without estradiol. This increase was inhibited by the ER antagonist ICI 182,780. Taken together, these data indicate that the increase of calcineurin expression in response to estrogen appeared to be limited to lupus patients.

ERa mediates the estradiol-dependent increase in calcineurin mRNA

To investigate if the estrogen-dependent increase in calci-neurin was mediated by the ERa, PBLs from lupus patients were cultured in medium alone, medium containing estra-diol, medium containing ER antagonist ICI 182,780, and medium containing estradiol plus ICI 182,780 for 24 hours. Total RNA was isolated, and the expression of calcineurin mRNA was measured using semi-quantitative RT-PCR. As shown in Fig. 6, PBL from SLE cultured with estradiol expressed significantly more calcineurin mRNA compared with PBL cultured in medium alone, and this increase was inhibited by the ER antagonist ICI 182,780. Taken together, Figure 1. ERa mRNA in peripheral blood lymphocyte of

SLE patients is increased compared with normal controls. (A) Representative experiments showing the ERa and b-actin mRNA levels in PBL from three normal controls and three SLE patients. (B) The results are mean_{standard deviation of ERa} tob-actin mRNA ratio (*p Z 0.001). ERa Z estrogen receptor alpha; SLE_{Z systemic lupus erythematosus.}

Figure 2. Intracellular ER_{a protein expression in peripheral} blood lymphocyte of SLE patients is increased compared with normal controls. (A) Representative histograms of ER_{a are} shown. The percentage displays the results of ERa-positive cells. (B) Statistical histograms of ERa. Data are mean standard deviation percentage of ER_{a-positive cells} (*pZ 0.023). ERa Z estrogen receptor alpha; SLE Z systemic lupus erythematosus.

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these results show that ER antagonist ICI 182,780 inhibited the estrogen-dependent upregulation of both calcineurin, indicating that ER directly mediates the action of estradiol on PBL.

Discussion

Recent studies have made it clear that activation of ERa but not ERb, at least in mice, promotes the development of SLE. However, studies directed toward the role of ERa in human SLE were limited. In the present study, we examined the ERa expression levels in SLE patients using semi-quan-titative RT-PCR and explored its role in SLE. Interestingly, we found that not only the ERa gene expression was upre-gulated in cells of SLE patients but also the ERa protein expression was significantly increased. Our observation is consistent with a recent study[11], which showed signifi-cant increased expression of ERa mRNA in PBMC from SLE patients compared with that from normal controls, but which did not explore ERa protein level and its role in mediating estrogen function. Other studies found no significant difference in the number and affinity of ER estimated by tritiated-estradiol binding assay and immu-noblotting between SLE patients and normal controls

[16,17]. There are two possible reasons for this discrep-ancy: (1) ER is a protein associated with the nucleus, and staining of ER requires membrane penetration by anti-ER antibodies and (2) epitopes recognized by some anti-ER antibodies in a denatured stage suitable for Western blot-ting may be sterically hindered in intact cells from binding by anti-ER antibodies[13].

The increase of the ER expression in PBL of SLE patients could reflect a factor predisposing to the disease, or it could be a consequence of the disease state. Recent studies involving various mouse models of lupus have suggested a prominent role for ERa in the development of lupus disease[18e22].

Alternatively, it is possible that the high level of ER expression in PBL of SLE patients could be because of the components of the lupus environment that are able to influence ER expression. Nancy and Berrih-Aknin[23]have shown that activation of normal thymocytes by inflamma-tory cytokines increases the level of ER expression in

β

Figure 3. Effect of nonphysiological concentrations of estradiol on calcineurin gene expression. Total RNA was iso-lated from PBL of three SLE patients treated for 24 hours with varying concentrations of E2. The RNA was analyzed by semi-quantitative reverse transcription-polymerase chain reaction. Data are mean_{standard error of the mean of CN to b-actin} mRNA ratios. CNZ calcineurin; E2 Z estradiol; mRNA Z mes-senger RNA; PBLZ peripheral blood lymphocyte; SLE Z syste-mic lupus erythematosus.

Figure 4. Calcineurin mRNA in PBL of SLE patients treated with and without estradiol compared with normal controls. Total RNA was isolated from none or E2-treated PBLs that were prepared from SLE patient and normal controls. The RNA was analyzed by semi-quantitative reverse transcription-poly-merase chain reaction. Data are mean standard deviation of CN to _{b-actin mRNA ratios (*p Z 0.000; **p Z 0.017;}

#

pZ 0.007). CN Z calcineurin; E2 Z estradiol; mRNA Z mes-senger RNA; PBL_{Z peripheral blood lymphocyte; SLE Z} syste-mic lupus erythematosus.

Figure 5. Comparison of calcineurin phosphatase activity in PBL between SLE patients and normal controls. PBLs were isolated from SLE patients and normal controls and treated with none, E2, or E2 plus ICI 182,780 (E2þ ICI). Calcineurin activity was measured using a calcineurin assay kit. Data are mean standard deviation of phosphatase released (nM) (*pZ 0.001; **p Z 0.005;

#_p_{Z 0.000). E2 Z estradiol; PBL Z peripheral blood lymphocyte;}

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thymocytes especially CD4þ subset in vitro, suggesting that an excess of proinflammatory cytokines could increase the expression of ERs on myasthenia gravis lymphocytes; however, data of ERa expression in PBL by inflammatory cytokines were lacking. This problem is under investigation in our laboratory. Although murine models of lupus have suggested a prominent role for ERa in the development of lupus disease, studies of ERa in mediating estrogen’s effects on human SLE are limited. Rider and colleagues

[17,24] in previous studies found increased expression of activation genes calcineurin in SLE T cells cultured in medium containing ERa agonist but did not demonstrate a higher amount of ERa in the SLE T cells compared with normal T cells. They hypothesized that the absence of either ERa or ERb subtype mRNA cannot account for the differential action of estrogen in women with lupus. These findings contradicted those of our study and others [11], which showed a higher expression of ERa mRNA and lower

expression of ERb mRNA by PBMC from SLE patients

compared with that from normal controls. In the present study, we observed a significantly higher expression of ERa gene and protein level in SLE patients than in normal controls. In addition, we observed that ERa mediates the estradiol-dependent increase in calcineurin. Such high expression of ERa may promote the influence of estrogen on PBL in lupus patients.

The pathogenesis of upregulation of ERa gene in lupus patients remains unclear. We hypothesize that genetic susceptibility and an environmental (epigenetic) trigger are two responsible contributory factors. Numerous studies, to the best of our knowledge, suggest that genetic factors have little influence on this female predilection [25e29]. Our other study intriguingly found that unmethylation in prox-imal promoter region of ERa gene enhanced ERa expression in SLE (prepare for submission), suggesting that an epige-netic factor is associated with upregulation of ERa gene in SLE. A recent study has indicated that female sex hormone and male sex hormone through ERa and androgen receptor differentially regulate the expression of the interferon-inducible lupus susceptibility (Ifi202) gene in immune cells, and female sex hormone-dependent increased levels of p202

protein in B cells by increasing cell survival contribute to sex bias in lupus susceptibility [6]. Evidence for the role of estrogen and progesterone in sex differences of SLE are derived from murine models although these hormone levels are normal in most SLE patients of either sex[30]. Clinical research provides support for a more estrogenic environ-ment in patients with SLE of both sexes. A preferential hydroxylation of estrone and increased oxidation of testos-terone in patients with SLE maximize the effects of estro-gens on T-cell functions [31]. Although these changes are found in males and females, they are most marked in females. These findings and the present work suggest that female sex hormone estrogen plays a key role in contributing to female sex bias in lupus susceptibility through increased expression of ERa.

In conclusion, our study indicates that ERa gene was upregulated in SLE patients. This overexpressed ERa in turn mediates the female sex hormone estrogen and evokes a direct increase in calcineurin expression in PBL, which could at least in part contribute to female bias in the development of SLE.

Acknowledgment

This work was supported by grants from Taiwan NSC 96-2314-B-037-017.

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Figure 6. Estrogen receptor alpha mediates the estradiol-dependent increase in calcineurin. Total RNA was isolated from none, E2, ICI 182,780 (ICI) or E2 plus ICI-treated peripheral blood lymphocytes that were prepared from two patients with systemic lupus erythematosus. The RNA was analyzed by semi-quantitative reverse transcription-polymerase chain reaction. Representative experiments showing the CN andb-actin mRNA levels are shown on the top. The ratio of CN/b-actin mRNA is shown on the bottom. CNZ calcineurin; E2 Z estradiol; mRNA Z messenger RNA.

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