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DNA Research 2006 13(4):169-183; doi:10.1093/dnares/dsl006
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© The Author 2006. Kazusa DNA Research Institute
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Isolation and Expression Profiling of Genes Upregulated in Bone Marrow-Derived Mononuclear Cells of Rheumatoid Arthritis Patients

Nobuo Nakamura1 {dagger}, Yasunori Shimaoka2 {dagger}, Takahiro Tougan3, Hiroaki Onda3,4, Daisuke Okuzaki3, Hanjun Zhao3, Azumi Fujimori3, Norikazu Yabuta3, Ippei Nagamori3, Akie Tanigawa4, Jun Sato3, Takenori Oda5, Kenji Hayashida6, Ryuji Suzuki7, Masao Yukioka2, Hiroshi Nojima3,4,* and Takahiro Ochi7

1 Center of Arthroplasty, Kyowakai Hospital Suita, Japan
2 Yukioka Hospital Osaka, Japan
3 Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University 3-1 Yamadaoka, Suita, Osaka 562-0031, Japan
4 Innovation Plaza Osaka Izumi, Japan
5 Department of Rheumatology, NHO Osaka-Minami Medical Center Kawachinagano, Japan
6 Hoshigaoka Kosei-Nenkin Hospital Hirakata, Japan
7 Clinical Research Center for Allergy and Rheumatology, National Sagamihara Hospital 18-1 Sakura-dai, Sagamihara, Kanagawa 228-8522, Japan

Received 25 July 2006; revised 21 August 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Patients, Materials and...
 3. RESULTS
 4. Discussion
 Acknowledgements
 References
 
We have comprehensively identified the genes whose expressions are augmented in bone marrow-derived mononuclear cells (BMMC) from patients with Rheumatoid Arthritis (RA) as compared with BMMCs from Osteoarthritis (OA) patients, and named them AURA after augmented in RA. Both stepwise subtractive hybridization and microarray analyses were used to identify AURA genes, which were confirmed by northern blot analysis and/or reverse transcription polymerase chain reaction (RT–PCR). We also assessed their expression levels in individual patients by quantitative real-time RT–PCR. Of 103 AURA genes we have identified, the mRNA levels of the following 10 genes, which are somehow related to immune responses, were increased in many of the RA patients: AREG (=AURA9), FK506-binding protein 5 (FKBP5 = AURA45), C-type lectin superfamily member 9 (CLECSF9 = AURA24), tyrosylprotein sulfotransferase 1 (TPST1 = AURA52), lymphocyte G0/G1 switch gene (G0S2 = AURA8), chemokine receptor 4 (CXCR4 = AURA86), nuclear factor-kappa B (NF-{kappa}B = AURA25) and two genes of unknown function (FLJ11106 = AURA1, BC022398 [GenBank] = AURA2 and XM_058513 = AURA17). Since AREG was most significantly increased in many of the RA patients, we subjected it to further analysis and found that AREG-epidermal growth factor receptor signaling is highly activated in synovial cells isolated from RA patients, but not in OA synoviocytes. We propose that the expression profiling of these AURA genes may improve our understanding of the pathogenesis of RA.

Key words: stepwise subtraction; microarray; RA; OA; amphiregulin; synoviolin


   1. Introduction
Top
 Abstract
 1. Introduction
2. Patients, Materials and...
 3. RESULTS
 4. Discussion
 Acknowledgements
 References
 
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by arthritis that predominantly results in chronic inflammation of systemic joints associated with the overgrowth of synovial cells. This induces progressive cartilage and bone destruction in the joint and subsequent disability. Since RA pathogenesis is likely to involve genetic elements, a number of groups have subjected samples from healthy and affected individuals to DNA microarray analyses for a broad-scale comparison. These studies have provided significant insights into RA pathogenesis.1Go,2Go The first samples tested were synovial specimens,3Go–8Go and peripheral blood mononuclear cells (PBMC),9Go from RA and osteoarthritis (OA) patients, and cluster analysis of the resulting microarray gene-expression data revealed some candidate genes that may play a specific role in RA pathogenesis.

In other studies searching for key factors in RA pathogenesis, immunoscreening by using an antirheumatoid synovial cell antibody identified synoviolin/Hrd1 to be a highly expressed enzyme (E3 ubiquitin ligase) in the rheumatoid synovium.10Go Synoviolin appears to be a pathogenic factor for RA because mice overexpressing this enzyme developed spontaneous arthropathy, while heterozygous knockdown results in increased synovial cell apoptosis and resistance to collagen-induced arthritis.11Go It was proposed that the excess elimination of unfolded proteins due to synoviolin overexpression triggers synovial cell overgrowth.12Go Thus, synoviolin may play a pivotal role in the pathogenesis of arthropathy due to its functions in the quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system; its elevated expression may therefore have an anti-apoptotic effect that causes synovial hyperplasia.

Bone marrow-derived mononuclear cells (BMMC) are another target for analyses aiming to identify the key genes that participate in RA pathogenesis because accumulating evidence suggests that BMMC cell abnormalities may contribute to the pathogenesis of RA and experimental arthritis models.13Go–17Go Moreover, RA patients suffer from defective central and peripheral B-cell tolerance checkpoints,18Go the first of which occurs in the bone marrow between the early immature and immature B-cell stages (the second counter selection step of autoantibody-expressing B cells takes place in the periphery, when the new emigrant becomes a mature naive B cell).18Go,19Go In addition, inflammatory changes similar to those found in RA synovium seem to occur in the subchondral bone marrow of the involved RA joint,20Go and synovial inflammatory tissue can reach the adjacent bone marrow by fully breaking the cortical barrier.21Go Thus, BMMC cells are an interesting subject for studies seeking to identify specific genes involved in RA pathogenesis.

To identify the genes whose expressions are dramatically induced or reduced in the pooled BMMC mRNAs of 50 RA patients as compared with 50 OA patients, we here subjected these pooled mRNAs to stepwise subtraction, which is a unique technique that we have developed previously.22Go This method permitted the comprehensive identification of those genes that are specifically up- or down-regulated during RA pathogenesis. In addition, we also used microarray analysis, since DNA microarray analyses on the BMMC of RA patients have not been described previously. As a control, we also subjected the BMMC RNA from OA patients to stepwise subtraction and microarray analysis to identify the genes that are specifically involved in OA pathogenesis. These analyses together resulted in the isolation of 103 RA-upregulated genes, of which amphiregulin (AREG) was revealed by quantitative real-time RT–PCR (QRT–PCR) to be the most conspicuously induced gene in RA patients. Interestingly, we also show here that AREG operates upstream of synoviolin in isolated synovial cells through an epidermal growth factor receptor (EFGR) signaling pathway. We discuss how AREG upregulation could contribute to RA pathogenesis.


   2. Patients, Materials and Methods
Top
 Abstract
 1. Introduction
 2. Patients, Materials and...
3. RESULTS
 4. Discussion
 Acknowledgements
 References
 
2.1. Human subjects and ethical considerations
All RA patients satisfied the 1987 revised diagnostic criteria of the American College of Rheumatology (ACR: formerly the American Rheumatism Association).23Go All OA patients fulfilled the ACR criteria for hip or knee OA.24Go The RA and OA patient groups were largely matched in terms of their average age and sex (Supplementary Figure S1A and B). This study was reviewed and approved by the Internal Review Board of the Research Institute for Microbial Diseases, Osaka University. Accordingly, a written informed consent was obtained from each participant before obtaining human tissues.

2.2. Cell proliferation assay
The synovial cells from each patient were seeded onto uncoated 35 mm tissue culture plates at 1 x 105 cells/well and cultured in 5% FBS/DMEM. After 12 h, the cells were incubated in fresh 5% FBS/DMEM with (100 ng/ml) or without AREG (Sigma-Aldrich, A 7080). Four photos were taken from fixed areas in four quadrants near the central area of each plate at the 0, 1, 3 and 4 day time points. The cells at each time point were counted from these four photos and expressed as mean ± standard error (SE).

2.3. Statistical analysis
Significant differences were determined using the Spearman's rank correlation (Supplemenatry Figure S4) or the Mann–Whitney U-test (Figs 2, 4 and Supplementary Figure S3). The data are expressed as means ± SE. P < 0.05 or P < 0.01 was considered to be statistically significant.


   3. RESULTS
Top
 Abstract
 1. Introduction
 2. Patients, Materials and...
 3. RESULTS
4. Discussion
 Acknowledgements
 References
 
3.1. Identification of RA- or OA-specific genes by stepwise subtraction and DNA microarray analysis
To isolate the putative RA-specific genes that are upregulated in BMMC of RA patients relative to those that are upregulated in OA patients, we first used our stepwise subtractive hybridization method. Briefly, we prepared a cDNA library from the pooled mRNA from the BMMC of 50 RA patients (Supplementary Figure S1A) by the linker-primer method using a pAP3neo vector.25Go Stepwise subtractive hybridization was then performed with the biotinylated pooled mRNA from the BMMC of 50 OA patients (Supplementary Figure S1A) to select candidate genes that may show upregulation in RA BMMC only as described previously.22Go To examine if the candidate genes are actually upregulated in RA but not OA BMMC, we performed northern blot analysis and/or RT–PCR using the pooled mRNA from the BMMC of 50 RA and 50 OA patients (Fig. 1). To reduce the possibility of missing important RA-specific pathogenic genes by this method, we also performed a genome-wide complementary DNA microarray analysis using the Agilent Hu44K array with the same pooled RNA samples obtained from the BMMC of RA and OA patients that were described above. When we tested top 70 genes from the microarray list of RA-upregulated genes by northern blot analysis and/or RT–PCR as described above, we found that only 20 genes really displayed RA-upregulated expressions. Thus, we identified 103 RA-upregulated genes (Fig. 1) and named them AURA (augmented in RA). As shown in Table 1, 15 AURA genes (AURA1~AURA7 and AURA10~AURA17) are uncharacterized novel genes.


Figure 1
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  		Figure 1. Northern blot or RT–PCR analysis of individual AURA cDNA clones to compare the expression levels of the genes in the BMMC of 50 RA patients and 50 OA patients (see Table 1 for their gene names). A northern blot or RT–PCR for GAPDH is also shown as a loading control. Left row: expression levels as detected by RT–PCR or northern blot analysis (denoted as n above each picture). Right row: confirmation of the expression level of each gene as determined by RT–PCR. The annealing temperature and amplification cycles for RT–PCR were always 50°C and 40 cycles, respectively, with the exception of the reactions denoted by a (50°C and 35 cycles, respectively), b (50°C and 30 cycles, respectively), c (55°C and 35 cycles, respectively), d (55°C and 40 cycles, respectively), and e (60°C and 40 cycles, respectively).

 

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  		Table 1. List of AURA genes

 
We also performed similar experiments to obtain candidate OA-upregulated genes by generating a cDNA library from the pooled mRNA from the BMMC of 50 OA patients (Supplementary Figure S1A) and then using biotinylated pooled mRNA from the BMMC of 50 RA patients for subtraction (Supplementary Figure S1A). DNA microarray analysis also yielded a number of candidate OA-specific genes, as described above. However, when we checked whether these candidate genes are truly specifically up-regulated in OA BMMCs by northern blot analysis and/or RT–PCR, we could confirm this for only two genes (Supplementary Figure S2). These two OA-upregulated genes encode nuclear receptor coactivator 1 and a hypothetical protein (FLJ20581). This result suggests that the gain of function due to the enhanced expression of the RA-upregulated candidate genes is important in the pathogenesis of RA. Thus, we subsequently concentrated our study on the RA-upregulated genes.

3.2. Expression profiles of RA-upregulated genes in individual RA or OA patients
To determine whether the upregulation of the 103 RA-specific candidate genes is widespread in many RA patients or occurs in only a few patients, we performed QRT–PCR using individually prepared RNA samples from the BMMC or PBMC of RA patients. Of the 103 candidate genes, 5 genes whose functions are unknown and 12 genes that may be related to growth regulation or immune response were analyzed by QRT–PCR. OA patients were also examined as negative controls. In every QRT–PCR, a standard RNA from the PBMC of a healthy volunteer (male, age 52) was used (denoted as normal with a relative intensity of 1.0). This allowed us to compare the expression profiles of the genes tested in this study. In addition, since we used this control, we could also compare the expression profiles of the genes in this study with those of other genes tested in our previous reports on other autoimmune diseases.26Go

Of the 17 tested AURA genes (denoted x in Table 1), AREG (AURA9) was the most conspicuously upregulated in the BMMC of many of the RA patients, while in contrast OA BMMCs invariably expressed this gene at very low levels (Fig. 2A). Similarly, the PBMC of many RA patients strongly expressed AREG, while only very low expression was detected in the PBMC of the OA patients (Fig. 2A). AREG is one of the EGF-like growth factors that stimulate cell growth by activating the EGF receptor (EGFR) signaling of the target cells in an autocrine/juxtacrine fashion.27Go


Figure 2
Figure 2
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  		Figure 2. Expression levels of AURA genes in individual RA and OA patients. QRT–PCR analyses show that the mRNA levels of (A) AREG, (B) AURA1, (C) FKBP5, (D) CLECSF9, (E) TPST1 and (F) AURA2 are conspicuously upregulated in RA patient BMMC (and sometimes PBMC), while the BMMC and PBMC of OA patients show negligible upregulation. Expression levels in the BMMC for 50 RA patients (from #1 to 50) are arranged in the denoted order. The inset in (B) shows that the thioesterase domain occupies most of the Aura1 protein. The mean values of the samples analyzed in triplicate from each individual RA BMMC, RA PBMC, OA BMMC and OA PBMC are indicated by filled circles, open squares, x's, or filled triangles, respectively. The average values for the RA patient group are shown by the horizontal arrows. The bar graphs in the right panels show the average ± SE values of these measurements using the RA or OA BMMC or PBMC. All measurements are statistically significant when RA and OA are compared (P < 0.01).

 
AURA1 was the next most conspicuously upregulated gene in the BMMC of many RA patients, while the BMMC of all OA patients showed only very low expression of this gene (Fig. 2B). However, unlike AREG, the PBMC of RA patients showed negligible enhancement in the expression of AURA1. AURA1 encodes an uncharacterized protein containing a thioesterase domain (Fig. 2B inset) that may cleave thioester bonds of an unknown target.

The gene encoding FK506 (tacrolimus)-binding protein 5 (FKBP5 = AURA45) also showed enhanced expression in nearly half of the RA patient BMMC samples, while no such increase was observed in the OA patient BMMC samples or in the PBMC of the RA patients (Fig. 2C). FKBP5 is a cellular receptor for FK506 and has an immunosuppressive effect on activated T cells because it inhibits the protein phosphatase calcineurin.28Go

Nearly half of the RA patient BMMC samples showed 5- to 50-fold greater expression of CLECSF9 (=AURA24), TPST1 (=AURA52) and AURA2 than the normal control PBMC sample (Fig. 2D–F). No such increase was observed in the BMMC of OA patients or in the PBMC of the RA patients. CLECSF9 encodes a macrophage-inducible C-type lectin (Mincle) that harbors a calcium-dependent carbohydrate-recognition domain. TPST1 is one of the two Golgi tyrosylprotein sulfotransferases (TPST1 and TPST2) that mediate the post-translational modification tyrosine O-sulfation.

G0S2 (=AURA8), chemokine receptor 4 (CXCR4 = AURA86), nuclear factor-kappa B (NF-{kappa}B = AURA25) and AURA17 showed augmented expression in both the BMMC and PBMC of some of the RA patients when compared to the expression in the BMMC and PBMC of the OA patients, although the differences between the RA and OA samples are not as significant as for the previously discussed genes (Supplementary Figure S3A–D). G0S2 is one of the G0/G1 switch (G0S) genes that are differentially expressed in lymphocytes during their lectin-induced switch from the G0 to the G1 phases of the cell cycle.29Go CXCR4, the receptor for a chemokine called stromal cell-derived factor-1 (SDF-1/CXCL12), is important in the migration, homing and survival of hematopoietic stem cells. SDF-1, which is secreted by ischemic myocardium, is involved in the homeostatic and inflammatory traffic of leukocytes, and is highly expressed in the synovial tissues of RA patients.30Go NF-{kappa}B is a transcription factor that resides in the cytoplasm of every cell and translocates to the nucleus when activated by a wide variety of agents, including cytokines.31Go AURA17 is an uncharacterized novel gene that encodes a large protein with 8 leucine rich repeats, Mitochondrial Rho (Miro) motif and protein tyrosine kinase domain (Supplementary Figure S3D inset).

We also tested seven other genes in RA and OA BMMC and PBMC samples by QRT–PCR, but none showed a widespread and conspicuous increase in expression in the RA BMMC samples (data not shown). Consequently, these genes appear to play a less significant role in RA pathogenesis. Since these experiments and those described above consumed almost all BMMC and PBMC samples from the RA and OA patients, the remaining AURA genes will have to be tested in the future with another RA patient set.

3.3. Expression pattern of AURA genes in PBMC
To determine whether the AURA genes are expressed in particular human blood cells, we performed RT–PCR on multiple tissue cDNA panels (MTC) from Clontech (Palo Alto, CA). As shown in Fig. 3, RT–PCR detected AREG mRNA in both monocytes (lane 4) and T and B cells (lanes 2–4), in particular in activated CD4+ T cells (lane 8). AURA1 is detected predominantly in resting CD4+ (T helper/inducer; lane 3) and activated CD4+ T (lane 8) cells. CLECSF9 is expressed in most cell types except for activated CD19+ T cells (lane 6), while G0S2 is found primarily in monocytes (lanes a and 4). FKBP5, TPST1, CXCR4, AURA2 and NF{kappa}B are ubiquitously expressed in most cell types. Thus, the analysis of the functions these AURA genes, apart from AURA1 and G0S2, play in specific blood cells will not be easy because they are already expressed in normal blood. However, the function of AURA1 can be studied by using CD4+ T cells of RA and OA patients. In this study, however, we could not perform this analysis because of the low amounts of BMMC that we could obtain from the RA patients.


Figure 3
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  		Figure 3. Determination by RT–PCR of the human blood cells that express AREG, AURA1, FKBP5, CLECSF9, TPST1, AURA2, G0S2, CXCR4 and NF{kappa}B. RT–PCR was performed using the multiple tissue cDNA panel for human blood fractions (MTC, Clontech). GAPDH was also amplified as a loading control. PCR amplifications were conducted at 55°C and over 30 cycles except as indicated on the right of the panels: 55°C and 35 cycles (#), 55°C and 27 cycles (*) or 53°C and 25 cycles ($). Lane 1, mononuclear cells (B, T cells and monocytes). Lane 2, resting CD8+ cells (T-suppressor/cytotoxic cells). Lane 3, resting CD4+ cells (T-helper/inducer). Lane 4, resting CD14+ cells (monocytes). Lane 5, resting CD19+ cells (B cells). Lane 6, activated mononuclear cells. Lane 7, activated CD4+ cells. Lane 8, activated CD8+ cells. Lane 9, activated CD19+ cells. Lane 10, human placenta control cDNA served as a DNA size marker.

 
3.4. AREG stimulates the growth of synovial cells
Since AREG appears to be the most conspicuously unpregulated gene in many RA patients, we subjected it to further analysis. We first examined its ability to stimulate the growth of isolated synovial cells because AREG is one of the ligands of EGFR and is known to induce cell growth. Thus, we isolated synovial cells from synovial tissues that were obtained from five RA and three OA patients during joint reconstructive surgery. In the absence of AREG in the culture medium, the synovial cells from both the RA and OA patients grew at a similar rate (Fig. 4A and B). However, when AREG was present, the synovial cells from RA patients appeared to grow slightly faster than the synovial cells from OA patients, which is statistically significant (P < 0.05) (Fig. 4A).


Figure 4
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  		Figure 4. The effect of AREG on the proliferation of synoviocytes from RA and OA patients. The synovial cells from three RA patients (RA1, RA2 and RA3) (A) and three individual OA patients (OA1, OA2 and OA3) were counted on days 0, 1, 3 and 4 after incubation with or without AREG. The cell counts on days 1, 3 and 4 are expressed relative to 0 day. Statistically significant measurements are indicated (P < 0.05).

 
To examine if this phenomenon is reflected in the signal transduction machinery of synovial cells, we investigated the activation of the EGFR signaling pathway in the AREG-treated and untreated RA synoviocytes. We first examined the phosphorylation of the extracellular signal-regulated kinases (ERK1/2) at Thr202 and Tyr204 by western blot analysis. ERK1/2 phosphorylation indicates the activation of the EGFR signaling pathway.32Go As shown in Fig. 5A, the phosphorylated ERK1/2 bands in the RA synoviocytes showed an increase in intensity when the cells had been treated with AREG; this effect peaked 8–12 h after AREG treatment but continued for 2–3 days. In contrast, the ERK1/2 protein levels remained largely unaffected by AREG treatment.


Figure 5
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  		Figure 5. Western blot analysis of RA and OA synovial cells incubated in the presence or absence of AREG. (A) Expression levels of ERK1/2 and its phospho-form (P-ERK1/2) that is phosphorylated at Thr202 and Tyr204. Pooled synovial cells from five RA patients were incubated with (100 ng/ml) or without AREG for varying periods ranging from 0 h to 3 days. (B) Expression levels of EGFR1, ERK1/2, P-ERK1/2, Grp94, Grp78 and synoviolin in synovial cells from individual RA and OA patients that were incubated with or without AREG (100 ng/ml) for 8 h. Alpha-tubulin served as a loading control. (C) Relative optical densities of the western blot bands in (B) to determine P-ERK1/2 expression relative to ERK1/2 expression. (D) Relative optical densities of the western blot bands in (B) to determine Grp94 expression relative to alpha-tubulin expression.

 
To compare the activation of EGFR signaling between RA and OA patients, we examined the activation of the EGFR signaling pathway in the synoviocytes from the five RA and three OA patients (Fig. 5B). We thus assessed the phosphorylated ERK1/2 expression levels by western blot analysis and expressed the results quantitatively by measuring the intensity of the lower phosphorylated band by densitometry and comparing it with the ERK1/2 band intensity (Fig. 5C). We found that the synoviocytes from the RA and OA patients expressed equivalent levels of EGFR and ERK1/2 proteins, regardless of AREG treatment. In contrast, AREG treatment upregulated the phosphorylated ERK1/2 expression levels much more strongly in the synoviocytes from RA2, RA3 and RA4 than in the synoviocytes of any of the OA patients. RA1 is an exception to this pattern as its limited phosphorylated ERK1/2 expression levels were similar to those in OA1–3. The AREG-induced upregulation of ERK1/2 phosphorylation was less apparent in the RA5 synovial cells because ERK1/2 was already activated in the absence of AREG.

Synoviolin plays a role in the synovial hyperplasia of RA by controlling the ERAD system.10Go To determine if the RA synovial cells have an abnormal ERAD system, we measured their levels of the ER stress proteins GRP78/BiP and GRP94, which protect cells from the stress-induced ER dysfunction that could lead to the accumulation of unfolded proteins.33Go We found that while the synovial cells of the RA and OA patients have similar levels of GRP78/BiP (Fig. 5B and D), the RA synoviocytes show enhanced levels of GRP94, irrespective of whether they have been stimulated with AREG. This suggests that at least part of the ER-stress responsive pathway, namely, that mediated by GRP94, is more activated in RA synoviocytes than in OA synoviocytes. Thus, the ERAD pathway does appear to be abnormally upregulated in RA synoviocytes. We confirmed by QRT–PCR that the BMMC and PBMC cells of RA patients RA1–5 show enhanced AREG mRNA levels, unlike the BMMC and PBMC of OA patients OA1–3 (Supplementary Figure S5A). Thus, chronic activation of AREG/EGFR signaling appears to be augmented in RA patients. Since AREG is expressed as transmembrane precursors that are cleaved in the extracellular domain to release soluble growth factor,34Go we speculated that the sera (PB) and bone marrow fluid (BM) of RA1–5 may show enhanced levels of cleaved AREG compared to the equivalent fluids of OA1–3. We tested this by enzyme-linked immunosorbent assay but found only one patient, RA2, showed levels of cleaved AREG that exceeded the detection level of the assay (Supplementary Figure S5B). Thus, it is not clear whether RA patients indeed secrete higher AREG levels than OA patients.

We also examined whether RA synoviocytes expressed higher synoviolin mRNA levels than OA synoviocytes in the presence or absence of AREG. However, we could not detect any significant differences between the RA and OA patients in this regard (Supplementary Figure S5C). It is not clear whether the synovial tissues of the patients would, like their cultured derivatives, show a similar lack of synoviolin upregulation.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Patients, Materials and...
 3. RESULTS
 4. Discussion
Acknowledgements
 References
 
In this study, we report our comprehensive isolation of AURA genes that show augmented mRNA expression in the BMMC of RA patients as compared to their expression in OA patient BMMC (Fig. 1 and Table 1). Since RA patients suffer from defective central and peripheral B-cell tolerance checkpoints, and often display unusual immunoglobulin light chain repertoires that suggest impaired secondary recombination regulation,18Go we had expected that many immune response genes would be identified as AURA genes. Indeed, >10% of the AURA genes are directly related to immune responses; moreover, while the other AURA genes may seem at first glance to be unrelated to immune responses, many of these can also be linked to immune responses (Table 1). QRT–PCR analysis on individual patient samples revealed that the AURA genes discussed below are significantly increased in the BMMC of many of the 50 RA patients we tested (Fig. 2). Thus, the identification of these genes may help us to understand the pathogenesis of RA.

FKBP5, one of the cellular receptors for the immunosuppressant FK506, was expressed at higher mRNA levels in many RA patients than in the OA patients; this was true for the BMMC of the RA patients but not for their PBMC (Fig. 2C). FK506 has been suggested to be an effective drug for reducing the pain associated with RA.35Go This is because it can suppress inflammation by inhibiting the production by synovial cells of prostaglandin E2; it does so by suppressing the IL-1ß production by leukocytes.36Go The enhanced FKBP5 expression in RA BMMC is not due to FK506 treatment since at the time of this study, treatment with FK506 was not permitted in Japan; consequently, none of the patients tested here have ever received FK506. In addition, the enhanced FKBP5 expression by RA BMMC does not correlate with therapeutic treatment using steroids. It remains possible, however, that the increased FKBP5 mRNA levels in the BMMC of RA patients may be due to treatment with other drugs. Alternatively, it may reflect genuine and spontaneous pathological events. Nevertheless, regardless of the cause of its elevated expression, the augmented FKBP expression may strongly inhibit the phosphatase activity of calcineurin, which could increase the dephosphorylation and thus inactivation of various substrates, including the NFAT family proteins and cytokines that are required for the expression of immunoregulatory molecules.

TPST1 mediates tyrosine sulfation within the trans-Golgi system, which affects 1% of all tyrosines in eukaryotic cells. It has been previously suggested that this post-translational modification may play an important role in the pathogenesis of autoimmune diseases because it regulates mononuclear cell function at various stages of the immune response by enhancing interactions between ligands and receptors.37Go Notably, of the 62 identified target proteins of tyrosine sulfation, nine are cell adhesion molecules and chemokine receptors, which are both central players in leukocyte trafficking. Thus, the augmented expression of TPST1 in RA patients may elevate the sulfation of crucial tyrosine residues in chemokine receptors that could constitutively increase their binding affinities with their ligands (e.g. the binding of CXCL12–CXCR4).

CLECSF9 belongs to the macrophage-inducible C-type lectin that serves multiple functions by recognizing carbohydrate chains; it plays important roles in macrophage function. Notably, a C-type lectin called DC-specific intercellular adhesion molecule 3-grabbing non-integrin is also highly expressed by macrophages in the synovium of RA patients.38Go However, the HH mRNA expression of macrophage-inducible C-type lectins is strongly induced in response to several inflammatory stimuli. Thus, the augmented expression of CLECSF9 in the BMMC of RA patients may simply be due to the inflammation in the joint.

Unlike FKBP5 and TPST1 genes, the mRNA levels of G0S2, CXCR4 and NF-{kappa}B are increased in both the BMMC and PBMC of RA patients (Fig. 2 and Supplementary Figure S3). We previously showed that the PBMC of both systemic lupus erythematosus (SLE) patients and healthy young females express enhanced levels of G0S2 mRNA.26Go Thus, G0S2 may not actually be involved in the pathogenesis of RA. With regard to the chemokine receptor CXCR4, it was also identified as a inflammation-related gene that is upregulated in synovial cells of patients with pigmented villonodular synovitis (PVNS), which is a joint problem that usually affects the hip or knee and involves the lining of the joint becoming swollen and growing.8Go The enhanced tyrosine sulfation of CXCR4 by augmented TPST1 activity, as described above, may also activate CXCR4, thereby elevating the ability of the CXCR4 ligand to induce the migration of bone marrow cells that could enhance the growth of synovial cells.39Go CXCR4 expression is also upregulated in the spinal cord of animals with experimental autoimmune encephalomyelitis, which is an animal model of autoimmune central nervous system inflammation.40Go With regard to NF-{kappa}B, this molecule along with the receptor activator of NF-{kappa}B (RANK) and its ligand RANKL have been found to play pivotal roles in the pathophysiological process of RA.41Go Thus, the increased mRNA levels of NF-{kappa}B in both the BMMC and PBMC of RA patients may contribute to the bone destruction mediated by activated NF-{kappa}B signaling pathway.42Go

AURA1 encodes a novel protein that is similar to thioesterase. Since the thioesterase homologs are widespread, functions of thioesterase vary in the human genome.43Go Thus, the physiological function of AURA1 remains unknown. A possible role that it could play in RA pathogenesis is suggested by the following observations. First, the stable overexpression of acyl-CoA thioesterase III in human and murine T-cell lines increased both peroxisome numbers and lipid droplet formation, which suggests that it participates in the metabolic regulation of peroxisome proliferation in T cells.44Go Second, altered immune responsiveness is observed in mice deficient in palmitoyl protein thioesterase (PPT1) gene that is mutated in infantile neuronal ceroid lipofuscinosis.45Go Third, CD4+ T cells are the prime mediators of RA in a mouse model SKG strain,46Go and AURA1 expression is detected predominantly in resting and activated CD4+ T cells (Fig. 3).

AREG is not directly related to immune responses but of all the genes examined, it showed the most conspicuously enhanced expression in both the BMMC and PBMC of many RA patients (Fig. 2A). We also found that the synovial cells of RA patients showed higher sensitivity to AREG, in terms of proliferation, than those of OA patients (Fig. 4). This is not due to augmented expression of EFGR (Fig. 5B, uppermost pane), but due to elevated activation of EGFR signaling pathway because the phosphorylation of ERK1/2 was more enhanced in AREG-treated RA patient synovial cells than that of AREG-treated OA patient synovial cells (Fig. 5). We here present a working hypothesis to explain how augmented AREG expression in BMMC and PBMC of RA patients and subsequent activation of EGFR signaling pathway lead to hyperproliferation of synovial cells in the joints of the RA patients (Fig. 6). Namely, this enhanced phosphorylation of ERK1/2 elevates the expression of many downstream target genes, which may also require the activation of the ERAD system. 12Go Given that the Ets-binding site (EBS) of the proximal promoter of the synoviolin gene is responsible for its expression,47Go and that EBS-carrying genes are also activated by signaling events from the ERK pathway,48Go it is possible that the enhanced activation of EGFR signaling induced by AREG may directly activate the expression of synoviolin as well as that of other genes, thereby inducing the hyperproliferation of synovial cells. Thus, it is possible that the ERAD system in RA patients is hyperactivated by synoviolin because of augmented AREG expression in blood cells, possibly in the macrophages that occur in the vicinity of the synovial cells of RA patients, releasing augmented amount of AREG. This hypothesis should be tested more rigourously in vivo in the future because the experiments using the isolated synoviocyte cells in tissue culture medium may display distinct response to AREG. Likewise, examination of other EGF family proteins in vivo can also be interesting future subjects.


Figure 6
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  		Figure 6. A working hypothesis to explain how augmented level of AREG in BMMC of RA patients may lead to hyperproliferation of synovial cells. Putative macrophages with enhanced expression of AREG precursor (ProAR) may approach to the synovial cells of the joint through blood flow, where they release AREG and activate the EGFR signaling pathway of synovial cells. Since Ets-binding site (ETS-1) of the proximal promoter of the synoviolin gene is one of the downstream targets of ERK pathway, the enhanced activation of EGFR signaling may directly activate the expression of synoviolin gene. The enhanced level of synoviolin activates the ERAD system, which may lead to hyperproliferation of synovial cells.

 
Overexpression of AREG has been linked to psoriasis in mice and humans.49Go,50Go Psoriasis is characterized by the hyperproliferation of keratinocytes and the loss of epidermal barrier function that leads to the infiltration of inflammatory cells into the epidermis and dermis.51Go AREG is also upregulated in a synoviocyte cell line derived from an RA patient in which the wild type and a dominant negative form of the orphan nuclear receptor Nurr1 were overexpressed.52Go Interestingly, AREG overexpression in the basal epidermis of transgenic mice induces a phenotype that is associated with synovial membrane inflammation.49Go Moreover, we showed previously that AREG expression is also enhanced in the PBMC of SLE and idiopathic thrombocytopenic purpura patients,26Go which suggests that AREG overexpression may also be associated with other autoimmune diseases. Notably, metalloprotease-mediated AREG shedding and the subsequent activation of EGFR appears to play a critical role in the secretion of IL-8 by the human airway epithelium-like NCI-H292 cells that is induced by tumor necrosis factor-{alpha} (TNF-{alpha}), a potent multifunctional cytokine that plays a central role in the pathogenesis of many inflammatory diseases like RA.53Go Since TNF-{alpha}-induced IL-8 secretion was completely inhibited by the neutralizing antibody against AREG,53Go this antibody could constitute a novel therapeutic tool for RA. Taken together, we propose that enhanced expression of AREG in BMMC and PMBC may play a pivotal role in the pathogenesis of RA.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Patients, Materials and...
 3. RESULTS
 4. Discussion
 Acknowledgements
References
 
We thank the patients and healthy volunteers who participated in this study. We also thank Ms Tomoko Motoyama, Ms Kumiko Ikeue, Ms Maki Masuda, and Ms Yuki Hamada for technical assistance and Dr Patrick Hughes for critically reading the manuscript. This work was primarily supported by a grant-in-aid from the Health Science Research grant from the Ministry of Health and Welfare of Japan. This work was also supported in part by Innovation Plaza Osaka of the Japan Science and Technology Agency (JST), and by grants-in-aid for Scientific Research on Priority Areas "Applied Genomics", Scientific Research (S), Exploratory Research, and the Science and Technology Incubation Program in Advanced Regions, from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Supplementary Data: Supplementary data are available online at www.dnaresearch.oxfordjournals.org.


   Footnotes
 
*To whom correspondence should be addressed. Tel. +81-6-6875-3980, Fax. +81-6-6875-5192, E-mail: snj-0212{at}biken.osaka-u.ac.jp

Communicated by Mitsuo Oshimura

{dagger}These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Patients, Materials and...
 3. RESULTS
 4. Discussion
 Acknowledgements
 References
 

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