Telomerase activity in connective tissue diseases - Springer Link

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Oct 16, 2007 - Abstract Telomerase is a reverse transcriptase enzyme contributing to the maintenance of the telomeric structure by adding telomere repeat ...
Rheumatol Int (2008) 28:579–583 DOI 10.1007/s00296-007-0472-9

O R I G I N A L A R T I CL E

Telomerase activity in connective tissue diseases: elevated in rheumatoid arthritis, but markedly decreased in systemic sclerosis Figen Tarhan · Filiz Vural · Buket Kosova · Kenan Aksu · Ozgur Cogulu · Gokhan Keser · Cumhur Gündüz · Murat Tombuloglu · Gonca Oder · Emin Karaca · Eker Doganavsargil

Received: 25 September 2007 / Accepted: 3 October 2007 / Published online: 16 October 2007 © Springer-Verlag 2007

Abstract Telomerase is a reverse transcriptase enzyme contributing to the maintenance of the telomeric structure by adding telomere repeat sequences to chromosomal ends, thus compensating for its shortening. Telomerase activity which is common in cancers and human germ line tissue, may also be increased, although to a lesser extent, in systemic autoimmune diseases. We aimed to evaluate telomerase activity in a group of Turkish patients with various connective tissue diseases. In this cross sectional study, 19 patients with systemic sclerosis (SSc), 15 with systemic lupus erythematosus (SLE), 10 with rheumatoid arthritis (RA) and 14 with primary Sjögren’s syndrome (pSjS) were studied. As the control group, 29 healthy subjects were also included. Human telomerase-speciWc reverse transcriptase (hTERT) was measured in peripheral blood lymphocytes, using online real-time reverse-transcriptase polymerase chain reaction (PCR). We also investigated if hTERT values in each patient group were correlated with clinical parameters and disease activity. Highest hTERT values were observed in RA group (21.24 § 28.54), followed by SLE (13.38 § 26.05) and pSjS (11.73 § 10.59) groups.

F. Tarhan · K. Aksu · G. Keser · G. Oder · E. Doganavsargil Division of Rheumatology, Department of Internal Medicine, Ege University School of Medicine, 35100 Izmir, Turkey F. Vural · M. Tombuloglu Division of Haematology, Department of Internal Medicine, Ege University School of Medicine, 35100 Izmir, Turkey B. Kosova · O. Cogulu · C. Gündüz · E. Karaca Department of Medical Genetics, Ege University School of Medicine, 35100 Izmir, Turkey K. Aksu (&) 80. Sok. No:27/3, 35040 Bornova-Izmir, Turkey e-mail: [email protected]; [email protected]

Only hTERT values in RA group was signiWcantly higher than the healthy control group (7.62 § 4.21) (p < 0.05). Interestingly, hTERT values in SSc were very low (2.09 § 3.18), even less than the healthy control group. In consistent with previous studies, telomerase activity was increased in SLE and RA. Very low telomerase activity in SSc group was rather surprising. Since existing telomerase data in SSc was limited and telomere shortening was previously reported in SSc, our Wnding of low telomerase activity in SSc group deserves relevant discussion and further studies. Keywords

Telomerase · Rheumatic diseases

Introduction Telomeres which are essential structures for the stabilization of chromosomes are indeed specialized DNA protein complexes, located at the ends of the eukaryotic chromosomes. Since the telomere DNA is not replicated by the usual DNA polymerase, telomeres tend to shorten with each cell division. This shortening continues until a critical point, at which telomeres are no longer able to shorten and cells enter cellular senescence [1, 2]. Telomerase is a reverse transcriptase enzyme which contributes to the maintenance of the telomeric structure by adding telomere repeat sequences to the end of the chromosomes, thus compensating for its shortening [1, 2]. In cancer cells, telomerase activation occurs, resulting in the maintenance of telomere length despite cell division, thereby allowing them to divide indeWnitely [1–4]. However, telomerase activation also occurs in non-diseased cells, including stem cells, germline cells and lymphocytes [1, 5].

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Telomerase activity in peripheral blood lymphocytes (PBL) is low and varies with age. Since antigen processing and stimulation upregulate telomerase activity in PBL [5–8], telomerase activity may be a marker for PBL proliferation. Standing from this point, telomerase activation has attracted attention for autoimmunity pathogenesis and was studied in various autoimmune diseases, including systemic lupus erythematosus (SLE) [9–14], rheumatoid arthritis (RA) [15–17], systemic sclerosis (SSc) [9, 18, 19], mixed connective tissue disease (MCTD) [9] and primary Sjögren’s syndrome (pSjS) [9]. In the majority of these studies, patient populations were mostly from Japan [9–18] and telomeric repeat ampliWcation protocol (TRAP) [20] was used for assessing telomerase activity. In this study, we measured telomerase activity with a diVerent method, in a group of Turkish patients having various connective tissue diseases. Rather than TRAP, we used online real-time reverse-transcriptase polymerase chain reaction (PCR) to measure human telomerase-speciWc reverse transcriptase (hTERT). This method was previously shown to be comparable with the TRAP assay for evaluating telomerase activity [21–23]. We investigated whether hTERT values in our patients were correlated with clinical parameters and disease activity.

Materials and methods Study population In this cross-sectional study, we studied a total of 58 Turkish patients (19 SSc, 15 SLE, 10 RA, and 14 pSjS), all being followed up Ege University Rheumatology department. A control group including 29 healthy individuals with no history of any disease, and normal physical and routine laboratory examinations was also included. Both the patient groups and the healthy controls were Turkish, residing in Turkey for at least one generation. Informed consent was obtained from all individuals. The study protocol was approved by the Local Ethic Committee of Ege University. All patients fulWlled the diagnostic criteria for their respective disease [24–27]. In all patients, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), routine biochemical tests and complete blood count (CBC) were performed with standard method. In SSc patients, high resolution computed tomography (HRCT) of the lungs, carbon monoxide diVusion capacity (DLco), pulmonary artery systolic pressure measurement by echocardiography (ECHO), esophagus manometry were also made and anti-Scl 70 antibodies were tested. In RA patients, conventional hand and chest X-rays were evaluated; disease activity was measured using disease

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activity score (DAS28). DAS 28 values equal to or 3.2 were accepted as RA disease activity. In SjS patients, further investigations including pulmonary HRCT were made to detect extraglandular involvement. Anti-HIV and anti-HCV serology was also made in these patients. In SLE patients, disease activity was assessed using SLE disease activity index (SLEDAI). All patients were under speciWc treatment for their particular diseases. Among 19 SSc patients, 9 were receiving cyclophosphamide treatment for indications such as early progressive disease or active pulmonary involvement. Measurement of telomerase activity In order to detect telomerase activity, human telomerasespeciWc reverse transcriptase (hTERT) in peripheral blood (PB) lymphocytes was measured using online real-time reverse-transcriptase polymerase chain reaction PCR. In previous studies, this method has been shown to be comparable with the commonly used other method, namely telomeric repeat ampliWcation protocol (TRAP) [21–23]. We transferred 1.5 ml of peripheral blood into a tube containing 15 ml of RNA/DNA stabilization reactive and mRNA was extracted using an mRNA isolation kit (mRNA Isolation Kit for Blood/Bone Marrow Roche). hTERT was quantitatively detected by performing the commercially available LightCycler Telo TAGGG hTERT QuantiWcation Kit. It is speciWc for hTERT mRNA which encodes active telomerase protein and a housekeeping gene porphobilinogen deaminase (PBGD) mRNA. All subsequent steps were carried out according to the manufacturer’s instructions. The level of hTERT mRNA was determined as the relative ratio (RR), which was calculated by dividing the level of hTERT mRNA by the level of the porphobilinogen deaminase (PBGD) housekeeping gene in the same samples [1,000 £ (hTERT/PBGD)]. Statistical analysis Statistical analysis was made using the SPSS statistical package program. Regression analysis was used for the relationships between diVerent variables. Student’s t test was used to compare the means. The values of p < 0.05 were considered statistically signiWcant.

Results The demographic data, clinical features and laboratory parameters were delineated in Table 1. The mean age of the SLE group was younger than all the others.

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Table 1 Demographics, clinical and laboratory features of patients and controls Control n = 29

RA n = 10

SLE n = 15

SjS n = 14

Age (years) median § SD

47.17 § 9.28

52.0 § 7.75

35.73 § 8.16

51.9 § 6.33

47.68 § 11.03

Sex (F/M)

23/6

7/3

13/2

13/1

19/0

Disease duration (years) (median § SD)



10.3 § 4.87

6.5 § 5.1

3.5 § 2.0

10.21 § 7.78

Ages at diagnosis (median § SD)



41.7 § 7.38

28.4 § 7.94

48.29 § 6.21

37.58 § 12.83

Extraglandular involvement







5/14



Cyclophosphamide receiving patients





2/15



9/19

Lung involvement





2/15

1/14

8/19

Active joint involvement



7/10

9/15

4/14



Renal involvement





6/15



1/19

hTERT

7.62 § 4.21

21.24 § 28.54

13.38 § 26.05

11.73 § 10.59

2.08 § 3.17

hTERT values in RA group were highest (21.24 § 28.54), followed by SLE (13.38 § 26.05) and SjS (11.73 § 10.59) groups. hTERT values in RA group were signiWcantly higher than the healthy control group (7.62 § 4.21), (t = 2.070, p = 0.048). Interestingly, hTERT values in SSc group (2.08 § 3.17) were markedly low, even signiWcantly less than the control group (t = 4.568, p = 0.000). hTERT values were summarized in Fig. 1. In the next step, we investigated whether hTERT values in diVerent patient groups were correlated with clinical parameters and disease activity. Among patients with RA, hTERT values in clinically active group (DAS28 > 3.2) were signiWcantly higher than the clinically inactive group (DAS28 · 3.2); (26.35 § 29.95 vs. 0.76 § 1.07; t = 2,411, p = 0.046). Correlation between DAS28 scores and hTERT values were shown is Fig. 2. We found no statistically signiWcant diVerence between hTERT values and RA disease duration or RF titers. 35 *** 30 25 HTERT

* 20 ** 15 10 5 0

Control

RA

SLE

SjS

SSc n = 19

Fig. 2 The correlation between DAS28 scores and hTERT values

In SLE group, we found no correlation between SLEDAI scores and hTERT values. Among individual clinical parameters, hTERT values were signiWcantly higher in SLE subgroup with active arthritis (21.62 § 31.55), compared with those without active arthritis [1.02 § 1.57, (t = 1,955, p = 0.043)]. In SjS group, 5 out of 14 patients had extraglandular involvement (4 had arthritis, 1 had pulmonary involvement). hTERT values in those Wve patients with extraglandular involvement (15.15 § 8.89) were signiWcantly higher than the rest of the group [4.49 § 9.28, (t = 1,962, p = 0.035)]. In the whole group, there was no signiWcant diVerence between hTERT values and various parameters such as disease duration and autoantibody titers.

SSc

Fig. 1 Telomerase activities determined by the hTERT of the RA, SLE, SjS, SSc and control group. * SLE patients have statistically signiWcant higher hTERT levels than SSc patients. ** Primary SjS patients have statistically signiWcant higher hTERT levels than SSc patients. *** Highest hTERT levels were observed in RA and RA patients have statistically signiWcant higher hTERT levels than SSc patients

Discussion To our knowledge, in literature there are nearly ten studies investigating telomerase activity in connective tissue diseases, mostly performed in Japanese patients [9–18]. SLE

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is the most extensively studied disease, followed by RA. However, data regarding PBL telomerase activity in SSc and especially in pSjS was relatively scarce and limited only to the landmark study performed by Katayama and Kohriyama [9]. Standing from these observations, we investigated the telomerase activity in PBL using hTERT measurement in a Turkish group of patients with diVerent CTDs, including SSc and pSjS. We found highest hTERT values in RA patients, followed by SLE and pSjS patients. Although hTERT values in SLE and pSjS groups were also higher than healthy control group, only hTERT values in RA group reached signiWcance, compared with healthy control group. hTERT values in SSc group were surprisingly lowest, even signiWcantly less than the control group. Before discussing and comparing our results with the literature data, we acknowledge that the most important limitation of our study was the low number of patients in each group. The diVerent genetic background of our study patients, may possibly explain some of our controversial results, compared with the Japanese data. As suggested by Katayama and Kohriyama [9], we also believe that activated lymphocytes with high telomerase activity distribute systemically through the circulation and contribute disease activity in CTDs. The stimulatory eVect of estrogens on telomerase activity and female predominance in CTDs may contribute to high telomerase activity in these pathologies [28]. Among lymphocyte subgroups, CD4+ T cells exhibit the longest average telomeres throughout life, followed by CD19+ B cells and CD8+ T cells [29]. The importance of CD4+ T cells in the pathogenesis of RA, may possibly contribute to the highest telomerase activity which we found in the RA group. Indeed, our Wnding was in line with a previous study performed by Yudoh et al. [30] who found high level of telomerase activity both in the PBL and synovial inWltrating lymphocytes from RA patients. We also found that telomerase activity was positively correlated with disease activity in RA. Telomerase activity may play a role in the pathogenesis of the RA and may be a useful disease activity variable in patients with RA. Previous studies performed in SLE reported not only increased telomerase activity in peripheral blood B and T lymphocytes, but also a signiWcant correlation between lymphocytic telomerase activity and disease activity [9–14]. Although we also found high PBL telomerase activity in SLE group, we could not show a positive correlation between PBL telomerase activity and SLEDAI). On the other hand, according to Kurosaka et al. [14], only B cell telomerase activity was correlated with SLEDAI. The most interesting Wnding in our study deserving discussion was very low PBC hTERT levels in the SSc group. Although PBL telomerase activity gradually decreases with advancing age, the mean age of the SSc group was not the highest among the whole groups. In literature there was

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only one study investigating PBL telomerase activity in SSc, using TRAP [9]. In this study, telomerase activity in SSc group was lower compared with the SLE, MCTD and pSjS groups, and only insigniWcantly higher than the control group. If really there is a tendency for lower telomerase activity in SSc, this may be related with the telomere shortening reported by Artlett et al. [19]. This author hypothesized that the chromosomal instability observed in SSc patients may result from the loss of long stretches of the telomeric repeats. Telomeric shortening may be expected to stimulate telomerase activation; however, the reverse may also be true. In other words, since the expression of telomerase stabilizes telomere length and allows for continual replication, low telomerase activity may contribute to telomere shortening. Finally, treatment regimens might have contributed to very low hTERT values in SSc group. Nearly half of our SSc patients included in this study had been receiving cyclophosphamide treatment, which might have inhibited PBL telomerase activity. Besides PBL telomerase activity, Ohtsuka et al. [18] studied telomerase RNA component gene polymorphisms in the skin-derived Wbroblasts in patients with SSc. They reported that increase in telomerase activity might cause emergence of abnormal Wbroblast clones in SSc. With regard to PBL telomerase activity in pSjS, in literature, there is only one study performed by Katayama [9], and our pSjS telomerase data is similar with this study. In line with this study, we also found a positive correlation between telomerase activity and extraglandular involvement in pSjS. In conclusion, in line with previous studies, we also found increased telomerase activity in SLE and in RA groups. PBL telomerase activity may possibly be a useful disease activity variable in these diseases. Very low telomerase activity in SSc group was rather surprising. Since existing telomerase data in SSc was limited and telomere shortening was previously reported in SSc, our Wnding of low telomerase activity in SSc group deserves relevant discussion and further studies.

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