Cia5dregulates a new fibroblast-like synoviocyte invasion-associated gene expression signature
© Laragione et al.; licensee BioMed Central Ltd. 2008
Received: 18 April 2008
Accepted: 15 August 2008
Published: 15 August 2008
The in vitro invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) have been shown to correlate with disease severity and radiographic damage. We recently determined that FLSs obtained from pristane-induced arthritis (PIA)-susceptible DA rats are also highly invasive in the same in vitro assay through Matrigel. The transfer of alleles derived from the arthritis-resistant F344 strain at the arthritis severity locus Cia5d (RNO10), as in DA.F344(Cia5d) congenics, was enough to significantly and specifically reduce the invasive properties of FLSs. This genetically controlled difference in FLS invasion involves increased production of soluble membrane-type 1 matrix metalloproteinase (MMP) by DA, and is dependent on increased activation of MMP-2. In the present study we aimed to characterize the pattern of gene expression that correlates with differences in invasion in order to identify pathways regulated by the Cia5d locus.
Synovial tissues were collected from DA and DA.F344(Cia5d) rats 21 days after the induction of PIA. Tissues were digested and FLSs isolated. After a minimum of four passages, FLSs were plated on Matrigel-covered dishes at similar densities, followed by RNA extraction. Illumina RatRef-12 expression BeadChip arrays were used. Expression data were normalized, followed by t-test, logistic regression, and cluster analysis. Real-time PCR was used to validate the microarray data.
Out of the 22,523 RefSeq gene probes present in the array, 7,665 genes were expressed by the FLSs. The expression of 66 genes was significantly different between the DA and DA.F344(Cia5d) FLSs (P < 0.01). Nineteen of the 66 differentially expressed genes (28.7%) are involved in the regulation of cell cycle progression or cancer-associated phenotypes, such as invasion and contact inhibition. These included Cxcl10, Vil2 and Nras, three genes that are upregulated in DA and known to regulate MMP-2 expression and activation. Nine of the 66 genes (13.6%) are involved in the regulation of estrogen receptor signaling or transcription. Five candidate genes located within the Cia5d interval were also differentially expressed.
We have identified a novel FLS invasion associated gene expression signature that is regulated by Cia5d. Many of the genes found to be differentially expressed were previously implicated in cancer cell phenotypes, including invasion. This suggests a parallel in the behavior of arthritis FLSs and cancer cells, and identifies novel pathways and genes for therapeutic intervention and prognostication.
Rheumatoid arthritis (RA) is a common chronic autoimmune disease that affects approximately 1% of the population . It is a complex trait, in which genetic and environmental factors mediate disease susceptibility and severity . Basic joint pathology in RA is characterized by pronounced synovial hyperplasia, also called 'pannus', which produces several proinflammatory cytokines and proteases and, like a malignant tumor, invades and destroys cartilage and bone [2–4].
The formation of the synovial pannus is regulated by complex interactions between synovial resident cells and infiltrating inflammatory cells [5, 6], and their production of paracrine and autocrine factors such as cytokines and growth factors [7–9], nuclear factor-kB activation , and angiogenesis . The fibroblast-like synoviocyte (FLS) is a key player in this process, and its numbers are markedly increased in the hyperplastic synovial pannus of RA and rodent models of arthritis . RA FLSs invade cartilage  and produce increased amounts of several proteolytic enzymes that further contribute to joint destruction [2, 3]. The invasive properties of RA FLSs have also been associated with radiographic damage in RA, a parameter of disease severity, which emphasizes their direct clinical relevance .
We have previously identified Cia5d as an arthritis severity locus and showed that DA.F344(Cia5d) rats congenic for this interval developed significantly milder arthritis, with nearly no pannus formation and neither bone nor cartilage destruction, as compared with highly susceptible DA rats . We also determined that Cia5d regulates the invasive properties of FLSs, thus providing an explanation for its role in joint damage . The arthritis gene located within Cia5d controls the FLS production of soluble membrane-type 1 (MT1)-matrix metalloproteinase (MMP) and activation of MMP-2 . This was the first time that FLS phenotypes were found to be genetically regulated.
In the present study we took advantage of this genetically regulated FLS invasive phenotype and compared highly invasive with minimally invasive cells' gene expression signatures using microarrays. The study of more than 22,000 genes identified a gene expression signature related to invasion that is differentially regulated between FLSs from DA and DA.F344(Cia5d) rats. The novel FLS invasion pathways described here resemble those described in cancer cell lines and have the potential to become novel targets for therapeutic intervention.
Materials and methods
Induction of PIA and arthritis scoring
Rats aged 8 to 12 weeks received 150 μl of pristane by intradermal injection divided into two sites at the base of the tail [14, 16]. The animals were scored on days 14, 18 and 21 after pristane induction using a previously described arthritis scoring system [17, 18]. On day 21 after injection, the animals were killed and synovial tissue was collected from the ankles for FLS isolation.
Isolation and culture of primary FLS
FLSs were isolated by enzymatic digestion of the synovial tissue. Briefly, tissues were minced and incubated with a solution containing DNase 0.15 mg/ml, hyaluronidase type I-S 0.15 mg/ml, and collagenase type IA 1 mg/ml (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco's modified Eagle's medium (DMEM; Gibco, Invitrogen Corporation, Carlsbad, CA, USA) for 1 hour at 37°C. Cells were washed and re-suspended in DMEM supplemented with 10% fetal bovine serum (Gibco), glutamine 30 mg/ml, amphotericin B 250 μg/ml (Sigma), and gentamicin 10 mg/ml (Gibco). After overnight culture, nonadherent cells were removed and adherent cells were cultured. All experiments were performed with cells after passage four (95% FLS purity).
Flow-cytometric characterization of FLSs
Freshly trypsinized FLSs (105) were re-suspended in phosphate-buffered saline with 0.02% azide (Sigma-Aldrich) and 1% bovine serum albumin (P Biomedicals, Aurora, OH, USA), and incubated with 1 μg anti-CD32 (Pharmingen, San Diego, CA, USA) to block Fcγ II receptors. Cells were stained with saturating concentrations of CD90 (OX-7; PerCP, Pharmingen) or isotype control. Stained cells were fixed with 1% paraformaldehyde in phosphate-buffered saline and analyzed by flow cytometry in a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA), using the BD Cell-Quest™ Pro version 4.0.1 software (Becton Dickinson).
FLS culture on Matrigel
We previously studied the invasive properties of FLSs through a collagen matrix (Matrigel). Cell interactions with the extracellular matrix are known to influence the expression of several genes, including activation of MMP-2 , which is a key mediator of the FLS invasive phenotype. Therefore, in order to study the gene expression signature of highly invasive and minimally invasive FLSs, cells were cultured under the same conditions as used in the invasion studies. Specifically, 100% confluent 75 cm2 FLS culture flasks were trypsinized (trypsin 0.25% with EDTA 0.1%). The rates of cellular proliferation differed among cell lines, and we previously showed that FLS proliferation does not correlate with the FLS invasive behavior. In order to have similar cell confluence at the time of FLS harvesting for RNA extraction, 10% to 50% of the high-density 75 cm2 cell culture flasks (depending on the cell line) were plated in Matrigel-coated 10 cm culture dishes (Becton Dickinson) with DMEM, 10% fetal bovine serum, antibiotics, and glutamine. Cell cultures were maintained at 37°C with 5% carbon dioxide for 24 hours. After 24 hours, FLSs were harvested using a cell scraper (Corning, Acton, MA, USA) followed by digestion of the Matrigel with 10 ml collagenase D 1 mg/ml (Roche Applied Science, Indianapolis, IN, USA) at 37°C for 10 minutes. FLSs were then collected by centrifugation, washed twice with ice-cold phosphate-buffered saline. Cell pellets were re-suspended in RLT lysis buffer (RNeasy Mini Kit; Qiagen, Valencia, CA, USA) with 1% (vol/vol) β-mercaptoethanol (Sigma). Cell-lysis buffer suspension was vortexed, frozen in liquid nitrogen and stored at -80°C until RNA extraction.
RNA extraction and quality assessment
Cells in RLT buffer were disrupted using QIAshredder spin columns (Qiagen), and total RNA was extracted using the RNeasy Mini Kit (Qiagen), in accordance with the manufacturer's instructions. Samples were digested with DNase (Qiagen) and eluted with 30 μl RNase-free water. RNAs were quantified and assessed for purity using a NanoDrop spectrophotometer (Rockland, DE, USA). RNA integrity was verified with a BioAnalyzer 2100 (Agilent, Palo Alto, CA, USA).
RNA preparation and microarray experiments
The RatRef-12 Expression BeadChip contains 22,524 probes for a total of 22,228 rat genes selected primarily from the NCBI RefSeq database (Release 16; Illumina, San Diego, CA, USA), and was used in accordance with the manufacturer's instructions. All reagents have been optimized for use with Illumina's Whole-Genome Expression platform. Total RNA 200 ng was used for cRNA in vitro transcription and labeling with the TotalPrep™ RNA Labeling Kit using Biotinylated-UTP (Ambion, Austin, TX, USA). Hybridization is carried out in Illumina Intellihyb chambers at 58°C for 18.5 hours, which is followed by washing and staining, in accordance with the Illumina Hybridization System Manual. The signal was developed by staining with Cy3-streptavidin. The BeadChip was scanned on a high resolution Illumina BeadArray reader, using a two-channel, 0.8 μm resolution confocal laser scanner.
Data extraction and normalization
The Illumina BeadStudio software (Version 2.0) was used to extract and normalize the expression data (fluorescence intensities) for the mean intensity of all 12 arrays. Genes expressed in all 12 arrays were selected for analyses. Normalized data were analyzed using the t-test and logistic regression.
Statistics and analyses
The t-test was used to compare means of the log-transformed and non-log-transformed data. Genes with a P value under 0.01 between DA and DA.F344(Cia5d) were considered significant and included in additional analysis. The logistic regression model fitting was carried out as previously described [20, 21] using the filtered gene list. The statistical significance of a logistic regression result was obtained by comparing the deviance with the 'null deviance'. This null deviance is the (-2)log-likelihood of a random model in which the probability for a sample to belong to a group (for example, DA) is equal to the proportion of DA samples in the dataset. The difference between the deviance and the null deviance follows the χ2 distribution with one degree of freedom by chance alone, and this χ2 distribution was used to determine the P value. The R statistical package  was used for t-test and logistic regression analyses.
The Ingenuity IPA 5.5.1 program (Ingenuity, Redwood City, CA, USA) and PubMed and GEO (Gene Expression Omnibus) searches were used for pathways detection. CLUSTER  and TREEVIEW  were used for cluster analysis and generation of a heat map.
Quantitative real-time PCR
Genes studied with QPCR for confirmatory studies, primers and probe sequences
Up-regulated in DA
Exiqon Universal probe 67
Exiqon Universal probe 1
Exiqon Universal probe 6
Exiqon Universal probe 97
Exiqon Universal probe 17
Down-regulated in DA
Exiqon Universal probe 68
Exiqon Universal probe 106
Exiqon Universal probe 25
Exiqon Universal probe 67
Exiqon Universal probe 94
Characterization of the FLS cell lines used
In previous studies we determined that DA FLSs were highly invasive, and that alleles derived from the arthritis-resistant strain F344 at the Cia5d interval, as in DA.F344(Cia5d) congenics (Figure 1), specifically reduced the invasive properties of FLSs. Additionally, FLSs from DA and DA.F344(Cia5d) strains expressed similar mRNA levels of transforming growth factor-β, tumor necrosis factor-α, IL-1β and IL-6, as well as MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, MT1-MMP and MT2-MMP . Both strains had similar collagenase and MMP-3 activity, but levels of soluble MT1-MMP and active MMP-2 were increased in DA. MMP-2 inhibition reduced DA FLS invasion to levels similar to those of DA.F344(Cia5d). Cytoskeleton characteristics were also similar in DA and DA.F344(Cia5d) FLSs .
In the present study FLSs were stained with CD90, a marker for FLS , and analyzed by flow cytometry. Comparable numbers of CD90+ cells were detected both in five different DA and five different DA.F344(Cia5d) rats (percentage of CD90+ cells [mean ± standard deviation]: DA 95.46 ± 8.9 and DA.F344 [Cia5d] 96.51 ± 5.9), demonstrating that the cell lines were homogeneously CD90+.
Genes expressed by FLSs and filtering criteria
A total of 7,665 genes out of 22,228 genes represented in the Illumina RatRef-12 BeadChip were expressed by both DA and DA.F344(Cia5d) FLSs. Log transformation did not significantly affect the list of differentially expressed genes, and therefore results are shown from analyses done with non-log-transformed data.
Genes differentially expressed between DA and DA.F344(Cia5d) FLSs
Genes with reduced expression in synovial fibroblasts from DA.F344 (Cia5d) compared with highly invasive DA, including those associated with cancer-phenotypes and estrogen signaling
Cancer, Cell Cycle, DNA replication, recombination and repair
Tripartite motif protein 16 (predicted)
Chemokine (C-X-C motif) ligand 10 f
Similar to Dynamin binding protein (Scaffold protein Tuba)
Villin 2 (Ezrin) f
Neuroblastoma RAS viral (v-ras) oncogene homologf
Breast cancer metastasis-suppressor 1-like (predicted)
Heterogeneous nuclear ribonucleoprotein D (AU-rich element RNA binding protein 1, 37 kDa)
Replication protein A2
Ubiquitin-conjugating enzyme E2D 3
LSM8 homolog, U6 small nuclear RNA associated (S. cerevisiae) (predicted)
Structural maintenance of chromosomes 1 like 1 (S. cerevisiae)
Replication protein A3 (predicted)
Stress-induced phosphoprotein 1 (Stip1)
Ubiquitin specific protease 24 (predicted)
STIP1 homology and U-Box containing protein 1 (predicted)
Ribosomal protein S6 (Rps6)
Similar to 40S ribosomal protein S9
Similar to 40S ribosomal protein S9
Transient receptor potential cation channel, subfamily V, member 2
GINS complex subunit 3 (Psf3 homolog)
Similar to cell division cycle associated 5
Similar to hypothetical protein FLJ33868 (predicted)
Telomeric repeat binding factor 1 (predicted)
Similar to hypothetical protein FLJ13188 (predicted)
Similar to RIKEN cDNA 0610040D20
Similar to F23N19.9 (predicted)
Similar to CDNA sequence BC028440
Acidic nuclear phosphoprotein 32 family, member B
RAN binding protein 6 (predicted)
Similar to RIKEN cDNA 6720467C03 (predicted)
Quinoid dihydropteridine reductase
Ring finger protein 134 (predicted)
Similar to hypothetical protein FLJ23017 (predicted)
Similar to hypothetical protein
Similar to RIKEN cDNA 4931400A14 (predicted)
Bridging integrator 2 (predicted)
Genes with increased expression in synovial fibroblasts from DA.F344 (Cia5d) compared with DA
Cancer, Cell Cycle, DNA replication, recombination and repair
Growth arrest and DNA-damage-inducible 45 beta
Glia maturation factor, gamma (Gmfg)
Pleckstrin homology domain containing, family G (with RhoGef domain) member 2 (predicted)
Similar to bromo domain-containing protein disrupted in leukemia (LOC317213)
Similar to anterior pharynx defective 1 homolog A (C. elegans)
Peroxisome biogenesis factor 19 (predicted)
FK506 binding protein 7 (predicted)
Nuclear receptor co-repressor 1
Transporter 1, ATP-binding cassette, sub-family B (MDR/TAP)
Frizzled homolog 4 (Drosophila)
H1 histone family, member 0
Hypothetical gene supported by NM_031819; Fath fat tumor suppressor homolog (Drosophila)
Collagen, type V, alpha 1 (Col5a1)
Gene trap locus F3b (predicted)
Olfactomedin-like 2B (predicted)
Gasdermin domain containing 1 (predicted)
Tripartite motif-containing 41 (predicted)
Hypothetical gene supported by AY771707
Similar to N-acetylneuraminate pyruvate lyase
SET domain, bifurcated 2 (predicted)
Similar to cDNA sequence BC013529 (predicted)
Similar to SERTA domain containing 4
ORM1-like 2 (S. cerevisiae) (predicted)
Similar to RIKEN cDNA 2310003P10 (LOC498067), mRNA.
Family with sequence similarity 18, member B (predicted)
UBX domain containing 2 (predicted)
Genes upregulated in the highly invasive DA FLSs and downregulated in DA.F344(Cia5d) include cancer-associated and invasion regulatory genes
Genes downregulated in the highly invasive DA FLSs and upregulated in DA.F344(Cia5d) include tumor suppressor and cell cycle check-point genes
The list of genes with reduced expression in DA, as compared with increased expression in DA.F344(Cia5d) congenics, included seven genes that are involved in tumor suppression-like activity and cell cycle check-points, such as Aph1a, Brwd3, Gadd45b, Gmfg, Lox, and Plekhg2 (Table 3). Gadd45b was chosen for quantitative real-time PCR confirmation (P < 0.05; Figure 4b). These observations, combined with the 11 cancer and invasion associated genes upregulated in DA, suggest an invasion-favoring profile similar to that described in cancer cells, characterized by reduced expression cell cycle check-point and tumor suppressor genes combined with increased expression of invasion genes.
Additional genes with reduced expression in DA FLSs
Additionally, Ubxd2, Fzd4, Fkbp7, Olfml2b, Gsdmdc1 and the transcriptional co-repressor Ncor1 were among the genes downregulated in DA and with increased expression in DA.F344(Cia5d). Gtlf3b (predicted), a gene trap fragment with unknown function, was among the most significantly differentially expressed genes (P = 0.000025; 2.2-fold difference; Table 3). The greater than twofold difference in expression of Olfml2b and Gsdmdc1 was confirmed with quantitative real-time PCR (Figure 4b).
Increased number of estrogen-inducible and ER signaling regulatory genes among the differentially expressed genes
Nine genes or 13.6% of the 66 differentially expressed genes were either estrogen-inducible genes, such as Cxcl10, Vil2, Trim16, Gins3 (predicted), and Gadd45b, or genes involved in modulating the estrogen receptor (ER) signaling such as Stub1 and Stip1. Ncor1 negatively regulates ER-mediated transcription and its levels were also reduced in DA, further suggesting unopposed ER-mediated transcription. The differential expression of Cxcl10, Vil2, Trim16, Gins3, and Gadd45b was confirmed with quantitative real-time PCR (Figure 4a, b). The ERs Esr1 and Esr2 were not differentially expressed in the microarray analysis, and those results were confirmed with quantitative real-time PCR (Figure 4b). There was a trend toward increased expression Esr2 in DA.F344(Cia5d), but that difference did not reach statistical significance (P = 0.093; Figure 4b). Taken together, this pattern of gene expression suggests that the invasive DA FLSs have an enhanced ER activity regulated at different levels that could include reduced degradation of the ER, reduced inhibition of the ER-mediated transcription, and increased levels of estrogen-inducible genes.
Five of the differentially expressed genes are located within the Cia5d interval
Differentially expressed genes located within the Cia5d interval on rat chromosome 10
Reduced levels in Cia5d
Tripartite motif protein 16 (predicted) (Trim16_predicted)
Transient receptor potential cation channel, subfamily V, member 2 (Trpv2)
Increased levels in Cia5d
Nuclear receptor co-repressor 1 (Ncor1)
Gene trap locus F3b (predicted) (Gtlf3b_predicted)
Tripartite motif-containing 41 (predicted)
A greater than expected number of genes located within the Cia5d interval were differentially expressed in FLSa
Genes located within Cia5d
Genes located outside Cia5d
Trim16, Trpv2, and Ncor1 are closely located on chromosome 10q23, raising the possibility that a polymorphism in a regulatory region or intron in this region, or even in one of these genes, could account for the difference in expression detected between the two strains.
RA histology is typically characterized by pronounced synovial hyperplasia, also called 'pannus'. The RA pannus produces proinflammatory cytokines and proteases, and invades cartilage and bone leading to joint destruction and deformities . The FLS is a key player in RA pannus and joint pathology, and has increased invasive properties, compared with osteoarthritis, even after several passages in vitro [12, 27]. Furthermore, the increased invasive properties of RA FLSs have been associated with increased radiographic joint destruction , underscoring the relevance of this in vitro phenotype to disease outcome.
We recently described the first evidence that the invasive properties of FLSs are genetically regulated . We determined that a gene located within the arthritis severity regulatory Cia5d interval specifically controls the invasive properties of FLSs via the regulation of the production of soluble MT1-MMP and activation of MMP-2 . Levels of active MMP-2 are also increased in the synovial fluid of patients with RA, and correlate with disease severity and radiographic damage . Therefore, understanding the regulation of cell invasion and MMP-2 activation is highly relevant to RA. In addition, several common cancers have increased levels of MMP-2, which correlates with worse prognosis [29–36], suggesting that identifying the Cia5d gene and the pathways controlled by it could potentially generate novel targets relevant to cancer treatment as well.
In the present study we used a novel strategy to identify differences in gene expression that correlate with the invasive properties of FLSs. First, two closely related strains were used. These strains have identical DA genomes, except that DA.F344(Cia5d) congenics have F344 arthritis-resistant alleles in a 37.2 megabase interval on chromosome 10. This strategy minimized noise related to allelic variations at other regions of the genome that are not related to the phenotype of interest. Second, instead of using synovial tissues, which have mixed cellularities that interfere with the interpretation of the results, we generate and used primary FLS cell lines. Third, FLSs from DA and DA.F344(Cia5d) differ in their invasive properties, thus providing a more precise phenotype. Finally, the cells used for RNA extractions were cultured on the same collagen matrix (Matrigel) used in the invasion experiments, hence recreating the same in vitro environment. This latter aspect is critical because extracellular matrix and cell influence processes that are central to cell invasion, such as the expression of adhesion molecules and MMP-2 activation , and are required for proper activation of the invasive phenotype, including gene transcription. This strategy led to the identification of new genes involved in FLS invasion.
A genome-wide analysis of gene expression conducted with RA FLSs suggested two patterns that correlated with increased or reduced inflammation in the tissues of origin . Those RA FLSs were not studied for invasion, and there was no control group without erosive changes for comparison. Furthermore, the RNA was obtained from cells cultured on plastic dishes and not on a collagen matrix such as Matrigel. Therefore, it was not surprising that using different methodologies to address a different question we detected a new FLS invasion signature that is different from the two RA FLS gene expression patterns previously reported.
A genome-wide microarray-based gene expression analysis was conducted to identify genes and pathways that are differentially expressed between highly invasive DA and minimally invasive DA.F344(Cia5d) FLSs. The analysis revealed that 66 genes out of the 7,665 genes expressed by FLSs were differentially expressed between DA and DA.F344(Cia5d) FLSs (P < 0.01). Nineteen of the 66 differentially expressed genes (28.7%) had previously been implicated in tumor suppression activity or other cancer cell phenotypes, but had not been implicated in the invasive properties of the FLSs. These cancer-related phenotypes include malignant transformation (Hnrpd) , tumor growth (Ach1a and Gfmg) [39, 40], oncogene-like activity (Plekgh2) , tumor apoptosis (Gadd45b) , tumor suppressor activity (Brwd3) , cancer cell growth arrest (Ube2d3) , contact inhibition (Gmfg) , and cell invasion (Lox, Ach1a, Cxcl10, Vil2, and Nras) [46–50]. Genetic variations in DNA synthesis gene Rpa3 have been associated with susceptibility to carcinomas , whereas increased cancer expression of Rpa2 is associated with adverse outcome in colon cancer . Some of these genes were found to be expressed in increased levels in certain cancers (Hnrpd and Lsm8) [53, 54], including highly invasive types . These observations suggest that FLSs derived from arthritis joints and cancer cells share common processes in the regulation of cell invasion, and that these processes are in part regulated by a gene located within the arthritis severity locus Cia5d.
In addition to the proinvasive and MMP-2 activating properties associated with Cxcl10 in FLSs, this chemokine can also attract C-X-C chemokine receptor (CXCR)3-expressing inflammatory cells such as memory T cells  and mast cells  into the joint, further contributing to disease severity. Indeed, recent studies that either targeted Cxcl10  or its receptor CXCR3  significantly ameliorated arthritis in rodents.
Cxcl10 , Vil2 , and Trim16  – three of the most significantly upregulated genes in DA – are known to be induced by estrogens (Figure 5). A complete analysis of all of the 66 differentially expressed genes revealed that nine of them (13.6%) were either regulated by estrogen (Cxcl10, Vil2, Trim16, Gins3, Gadd45b, and Gmfg)  or are involved in ER signaling (Stip1), ER ubiquitination (Stub1), or ER-mediated transcription (Ncor1). These observations suggested that abnormalities in the regulation of ER signaling and ER-mediated transcription could contribute to the invasive properties of DA FLSs. Indeed, estrogens have been shown to increase levels of active MMP-2 in various tissues and cell types [69–71], including breast cancers , and estrogen antagonists reversed that effect [71, 73]. Estrogens also increase the production of active MMP-2 and the in vitro invasive properties of RA FLSs  (Figure 5). Although estrogens are typically thought of as having anti-inflammatory properties , our observations suggest an intrinsic dysregulation in ER signaling in DA FLSs. This dysregulation in ER is controlled by the Cia5d gene, and could contribute to increased FLS invasion and cartilage and bone erosive changes.
Five of the differentially expressed genes were located within the Cia5d interval, and this number was greater than expected by chance. Three of these were upregulated in DA.F344(Cia5d) FLS (Ncor1, Trim41, and Gtlf3b) and two were downregulated in DA.F344(Cia5d) (Trpv2 and Trim16), raising the possibility that a polymorphism/mutation in one of these genes could explain the arthritis and FLS invasive phenotypes attributed to Cia5d. Specifically, a polymorphism in a regulatory element or intron in one of these genes, or in another gene in the region, could influence transcription, thus explaining differences in levels of mRNA and disease. This has been the case in studies of two other autoimmune or inflammatory diseases in which microarray analysis led to the identification of the disease-causing polymorphism [76, 77]. In the present study only Ncor1, a transcriptional repressor regulated by estrogens, appears to be an interesting candidate. Trpv2 is a cation channel ubiquitously expressed, and the other three genes (Trim16, Trim41, and Gtlf3b) have less clear functions. The Cia5d interval contains more than 100 genes, and not all were present in the Illumina microarray. It would be premature to exclude these genes at this point, and additional studies with recombinant subcongenic strains are under way.
We have identified a novel invasion-associated gene expression signature and evidence suggesting a dysregulation in ER signaling in arthritis FLSs, which are regulated by the arthritis severity locus Cia5d. It is anticipated that the specific identification of the Cia5d gene, and the continued characterization of processes regulated by this gene, will generate new targets for therapeutic intervention aimed at reducing cartilage and bone destruction, and new prognostic markers for RA. The parallels between our findings in FLSs and observations from cancer studies suggest that the Cia5d gene might be important for cancer biology as well.
This study was funded by National Institutes of Health grants R01-AR46213, R01-AR052439 (NIAMS), and R01-AI54348 (NIAID) to Dr P Gulko. The authors want to thank Franak Batliwalla and Aarti Damle, members of the Feinstein Institute's microarray core facility, for their assistance.
C-X-C chemokine receptor
Dulbecco's modified Eagle's medium
polymerase chain reaction
- Gregersen PK, Plenge RM, Gulko PS: Genetics of rheumatoid arthritis. Rheumatoid Arthritis. Edited by: Firestein G, Panayi G, Wollheim FA. 2006, New York, NY: Oxford University Press, 3-14. 2Google Scholar
- Okada Y, Morodomi T, Enghild JJ, Suzuki K, Yasui A, Nakanishi I, Salvesen G, Nagase H: Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. Eur J Biochem. 1990, 194: 721-730. 10.1111/j.1432-1033.1990.tb19462.x.View ArticlePubMedGoogle Scholar
- Hanemaaijer R, Sorsa T, Konttinen YT, Ding Y, Sutinen M, Visser H, van Hinsbergh VW, Helaakoski T, Kainulainen T, Rönkä H, Tschesche H, Salo T: Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells. Regulation by tumor necrosis factor-alpha and doxycycline. J Biol Chem. 1997, 272: 31504-31509. 10.1074/jbc.272.50.31504.View ArticlePubMedGoogle Scholar
- Gulko PS, Winchester RJ: Rheumatoid arthritis. Samter's Immunologic Diseases. Edited by: Austen KF, Frank MM, Atkinson JP, Cantor H. 2001, Baltimore, MD: Lippincott, Williams & Wilkins, II: 427-463. 6Google Scholar
- Lee DM, Kiener HP, Agarwal SK, Noss EH, Watts GF, Chisaka O, Takeichi M, Brenner MB: Cadherin-11 in synovial lining formation and pathology in arthritis. Science. 2007, 315: 1006-1010. 10.1126/science.1137306.View ArticlePubMedGoogle Scholar
- Yellin MJ, Winikoff S, Fortune SM, Baum D, Crow MK, Lederman S, Chess L: Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) up-regulation and IL-6 production and proliferation. J Leukoc Biol. 1995, 58: 209-216.PubMedGoogle Scholar
- Fava RA, Olsen NJ, Postlethwaite AE, Broadley KN, Davidson JM, Nanney LB, Lucas C, Townes AS: Transforming growth factor beta 1 (TGF-beta 1) induced neutrophil recruitment to synovial tissues: implications for TGF-beta-driven synovial inflammation and hyperplasia. J Exp Med. 1991, 173: 1121-1132. 10.1084/jem.173.5.1121.View ArticlePubMedGoogle Scholar
- Shealy DJ, Wooley PH, Emmell E, Volk A, Rosenberg A, Treacy G, Wagner CL, Mayton L, Griswold DE, Song XY: Anti-TNF-alpha antibody allows healing of joint damage in polyarthritic transgenic mice. Arthritis Res. 2002, 4: R7-10.1186/ar430.PubMed CentralView ArticlePubMedGoogle Scholar
- Bischof RJ, Zafiropoulos D, Hamilton JA, Campbell IK: Exacerbation of acute inflammatory arthritis by the colony-stimulating factors CSF-1 and granulocyte macrophage (GM)-CSF: evidence of macrophage infiltration and local proliferation. Clin Exp Immunol. 2000, 119: 361-367. 10.1046/j.1365-2249.2000.01125.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Miagkov AV, Kovalenko DV, Brown CE, Didsbury JR, Cogswell JP, Stimpson SA, Baldwin AS, Makarov SS: NF-kappaB activation provides the potential link between inflammation and hyperplasia in the arthritic joint. Proc Natl Acad Sci USA. 1998, 95: 13859-13864. 10.1073/pnas.95.23.13859.PubMed CentralView ArticlePubMedGoogle Scholar
- Storgard CM, Stupack DG, Jonczyk A, Goodman SL, Fox RI, Cheresh DA: Decreased angiogenesis and arthritic disease in rabbits treated with an alphavbeta3 antagonist. J Clin Invest. 1999, 103: 47-54. 10.1172/JCI3756.PubMed CentralView ArticlePubMedGoogle Scholar
- Muller-Ladner U, Kriegsmann J, Franklin BN, Matsumoto S, Geiler T, Gay RE, Gay S: Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice. Am J Pathol. 1996, 149: 1607-1615.PubMed CentralPubMedGoogle Scholar
- Tolboom TC, Helm-Van Mil van der AH, Nelissen RG, Breedveld FC, Toes RE, Huizinga TW: Invasiveness of fibroblast-like synoviocytes is an individual patient characteristic associated with the rate of joint destruction in patients with rheumatoid arthritis. Arthritis Rheum. 2005, 52: 1999-2002. 10.1002/art.21118.View ArticlePubMedGoogle Scholar
- Brenner M, Meng HC, Yarlett NC, Joe B, Griffiths MM, Remmers EF, Wilder RL, Gulko PS: The non-MHC quantitative trait locus Cia5 contains three major arthritis genes that differentially regulate disease severity, pannus formation, and joint damage in collagen- and pristane-induced arthritis. J Immunol. 2005, 174: 7894-7903.View ArticlePubMedGoogle Scholar
- Laragione T, Brenner M, Mello A, Symons M, Gulko PS: The arthritis severity locus Cia5d is a novel genetic regulator of the invasive properties of synovial fibroblasts. Arthritis Rheum. 2008, 58: 2296-2306. 10.1002/art.23610.PubMed CentralView ArticlePubMedGoogle Scholar
- Vingsbo C, Sahlstrand P, Brun JG, Jonsson R, Saxne T, Holmdahl R: Pristane-induced arthritis in rats: a new model for rheumatoid arthritis with a chronic disease course influenced by both major histocompatibility complex and non-major histocompatibility complex genes. Am J Pathol. 1996, 149: 1675-1683.PubMed CentralPubMedGoogle Scholar
- Gulko PS, Kawahito Y, Remmers EF, Reese VR, Wang J, Dracheva SV, Ge L, Longman RE, Shepard JS, Cannon GW, Sawitzke AD, Wilder RL, Griffiths MM: Identification of a new non-major histocompatibility complex genetic locus on chromosome 2 that controls disease severity in collagen-induced arthritis in rats. Arthrititis Rheum. 1998, 41: 2122-2131. 10.1002/1529-0131(199812)41:12<2122::AID-ART7>3.0.CO;2-#.View ArticleGoogle Scholar
- Remmers EF, Longman RE, Du Y, O'Hare A, Cannon GW, Griffiths MM, Wilder RL: A genome scan localizes five non-MHC loci controlling collagen-induced arthritis in rats. Nat Genet. 1996, 14: 82-85. 10.1038/ng0996-82.View ArticlePubMedGoogle Scholar
- Preaux AM, Mallat A, Nhieu JT, D'Ortho MP, Hembry RM, Mavier P: Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions. Hepatology. 1999, 30: 944-950. 10.1002/hep.510300432.View ArticlePubMedGoogle Scholar
- Batliwalla FM, Baechler EC, Xiao X, Li W, Balasubramanian S, Khalili H, Damle A, Ortmann WA, Perrone A, Kantor AB, Gulko PS, Kern M, Furie R, Behrens TW, Gregersen PK: Peripheral blood gene expression profiling in rheumatoid arthritis. Genes Immun. 2005, 6: 388-397. 10.1038/sj.gene.6364209.View ArticlePubMedGoogle Scholar
- Li W, Yang Y: Zipf's law in importance of genes for cancer classification using microarray data. J Theor Biol. 2002, 219: 539-551. 10.1006/jtbi.2002.3145.View ArticlePubMedGoogle Scholar
- R statistical package. [http://www.r-project.org/]
- Eisen MB, Spellman PT, Brown PO, Botstein D: Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA. 1998, 95: 14863-14868. 10.1073/pnas.95.25.14863.PubMed CentralView ArticlePubMedGoogle Scholar
- Treeview. [http://rana.lbl.gov]
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
- Zimmermann T, Kunisch E, Pfeiffer R, Hirth A, Stahl HD, Sack U, Laube A, Liesaus E, Roth A, Palombo-Kinne E, Emmrich F, Kinne RW: Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture: primary culture cells markedly differ from fourth-passage cells. Arthritis Res. 2001, 3: 72-76. 10.1186/ar142.PubMed CentralView ArticlePubMedGoogle Scholar
- Tolboom TC, Pieterman E, Laan van der WH, Toes RE, Huidekoper AL, Nelissen RG, Breedveld FC, Huizinga TW: Invasive properties of fibroblast-like synoviocytes: correlation with growth characteristics and expression of MMP-1, MMP-3, and MMP-10. Ann Rheum Dis. 2002, 61: 975-980. 10.1136/ard.61.11.975.PubMed CentralView ArticlePubMedGoogle Scholar
- Goldbach-Mansky R, Lee JM, Hoxworth JM, Smith D, Duray P, Schumacher RH, Yarboro CH, Klippel J, Kleiner D, El-Gabalawy HS: Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis. Arthritis Res. 2000, 2: 145-153. 10.1186/ar79.PubMed CentralView ArticlePubMedGoogle Scholar
- Azzam HS, Arand G, Lippman ME, Thompson EW: Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production. J Natl Cancer Inst. 1993, 85: 1758-1764. 10.1093/jnci/85.21.1758.View ArticlePubMedGoogle Scholar
- Abbas Abidi SM, Howard EW, Dmytryk JJ, Pento JT: Differential influence of antiestrogens on the in vitro release of gelatinases (type IV collagenases) by invasive and non-invasive breast cancer cells. Clin Exp Metastasis. 1997, 15: 432-439. 10.1023/A:1018458406797.View ArticlePubMedGoogle Scholar
- Garbisa S, Scagliotti G, Masiero L, Di Francesco C, Caenazzo C, Onisto M, Micela M, Stetler-Stevenson WG, Liotta LA: Correlation of serum metalloproteinase levels with lung cancer metastasis and response to therapy. Cancer Res. 1992, 52: 4548-4549.PubMedGoogle Scholar
- Gohji K, Fujimoto N, Fujii A, Komiyama T, Okawa J, Nakajima M: Prognostic significance of circulating matrix metalloproteinase-2 to tissue inhibitor of metalloproteinases-2 ratio in recurrence of urothelial cancer after complete resection. Cancer Res. 1996, 56: 3196-3198.PubMedGoogle Scholar
- Vaisanen A, Kallioinen M, Taskinen PJ, Turpeenniemi-Hujanen T: Prognostic value of MMP-2 immunoreactive protein (72 kD type IV collagenase) in primary skin melanoma. J Pathol. 1998, 186: 51-58. 10.1002/(SICI)1096-9896(199809)186:1<51::AID-PATH131>3.0.CO;2-P.View ArticlePubMedGoogle Scholar
- Nakada M, Okada Y, Yamashita J: The role of matrix metalloproteinases in glioma invasion. Front Biosci. 2003, 8: e261-269. 10.2741/1016.View ArticlePubMedGoogle Scholar
- Cockett MI, Murphy G, Birch ML, O'Connell JP, Crabbe T, Millican AT, Hart IR, Docherty AJ: Matrix metalloproteinases and metastatic cancer. Biochem Soc Symp. 1998, 63: 295-313.PubMedGoogle Scholar
- Davies B, Waxman J, Wasan H, Abel P, Williams G, Krausz T, Neal D, Thomas D, Hanby A, Balkwill F: Levels of matrix metalloproteases in bladder cancer correlate with tumor grade and invasion. Cancer Res. 1993, 53: 5365-5369.PubMedGoogle Scholar
- Kasperkovitz PV, Timmer TC, Smeets TJ, Verbeet NL, Tak PP, van Baarsen LG, Baltus B, Huizinga TW, Pieterman E, Fero M, Firestein GS, Pouw Kraan van der TC, Verweij CL: Fibroblast-like synoviocytes derived from patients with rheumatoid arthritis show the imprint of synovial tissue heterogeneity: evidence of a link between an increased myofibroblast-like phenotype and high-inflammation synovitis. Arthritis Rheum. 2005, 52: 430-441. 10.1002/art.20811.View ArticlePubMedGoogle Scholar
- Gouble A, Grazide S, Meggetto F, Mercier P, Delsol G, Morello D: A new player in oncogenesis: AUF1/hnRNPD overexpression leads to tumorigenesis in transgenic mice. Cancer Res. 2002, 62: 1489-1495.PubMedGoogle Scholar
- Paoni NF, Feldman MW, Gutierrez LS, Ploplis VA, Castellino FJ: Transcriptional profiling of the transition from normal intestinal epithelia to adenomas and carcinomas in the APCMin/+ mouse. Physiol Genomics. 2003, 15: 228-235.View ArticlePubMedGoogle Scholar
- Lim R, Liu YX, Zaheer A: Glia maturation factor beta regulates the growth of N18 neuroblastoma cells. Dev Biol. 1990, 137: 444-450. 10.1016/0012-1606(90)90269-O.View ArticlePubMedGoogle Scholar
- Himmel KL, Bi F, Shen H, Jenkins NA, Copeland NG, Zheng Y, Largaespada DA: Activation of clg, a novel dbl family guanine nucleotide exchange factor gene, by proviral insertion at evi24, a common integration site in B cell and myeloid leukemias. J Biol Chem. 2002, 277: 13463-13472. 10.1074/jbc.M110981200.View ArticlePubMedGoogle Scholar
- Qiu W, Zhou B, Chu PG, Luh F, Yen Y: The induction of growth arrest DNA damage-inducible gene 45 beta in human hepatoma cell lines by S-adenosylmethionine. Am J Pathol. 2007, 171: 287-296. 10.2353/ajpath.2007.070121.PubMed CentralView ArticlePubMedGoogle Scholar
- Muller P, Kuttenkeuler D, Gesellchen V, Zeidler MP, Boutros M: Identification of JAK/STAT signalling components by genome-wide RNA interference. Nature. 2005, 436: 871-875. 10.1038/nature03869.View ArticlePubMedGoogle Scholar
- Hattori H, Zhang X, Jia Y, Subramanian KK, Jo H, Loison F, Newburger PE, Luo HR: RNAi screen identifies UBE2D3 as a mediator of all-trans retinoic acid-induced cell growth arrest in human acute promyelocytic NB4 cells. Blood. 2007, 110: 640-650. 10.1182/blood-2006-11-059048.PubMed CentralView ArticlePubMedGoogle Scholar
- Lim R, Nakagawa S, Arnason BG, Turriff DE: Glia maturation factor promotes contact inhibition in cancer cells. Proc Natl Acad Sci USA. 1981, 78: 4373-4377. 10.1073/pnas.78.7.4373.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirschmann DA, Seftor EA, Fong SF, Nieva DR, Sullivan CM, Edwards EM, Sommer P, Csiszar K, Hendrix MJ: A molecular role for lysyl oxidase in breast cancer invasion. Cancer Res. 2002, 62: 4478-4483.PubMedGoogle Scholar
- Poola I, DeWitty RL, Marshalleck JJ, Bhatnagar R, Abraham J, Leffall LD: Identification of MMP-1 as a putative breast cancer predictive marker by global gene expression analysis. Nat Med. 2005, 11: 481-483. 10.1038/nm1243.View ArticlePubMedGoogle Scholar
- Zipin-Roitman A, Meshel T, Sagi-Assif O, Shalmon B, Avivi C, Pfeffer RM, Witz IP, Ben-Baruch A: CXCL10 promotes invasion-related properties in human colorectal carcinoma cells. Cancer Res. 2007, 67: 3396-3405. 10.1158/0008-5472.CAN-06-3087.View ArticlePubMedGoogle Scholar
- Alami J, Williams BR, Yeger H: Derivation and characterization of a Wilms' tumour cell line, WiT 49. Int J Cancer. 2003, 107: 365-374. 10.1002/ijc.11429.View ArticlePubMedGoogle Scholar
- Sizemore S, Cicek M, Sizemore N, Ng KP, Casey G: Podocalyxin increases the aggressive phenotype of breast and prostate cancer cells in vitro through its interaction with ezrin. Cancer Res. 2007, 67: 6183-6191. 10.1158/0008-5472.CAN-06-3575.View ArticlePubMedGoogle Scholar
- Michiels S, Danoy P, Dessen P, Bera A, Boulet T, Bouchardy C, Lathrop M, Sarasin A, Benhamou S: Polymorphism discovery in 62 DNA repair genes and haplotype associations with risks for lung and head and neck cancers. Carcinogenesis. 2007, 28: 1731-1739. 10.1093/carcin/bgm111.View ArticlePubMedGoogle Scholar
- Givalos N, Gakiopoulou H, Skliri M, Bousboukea K, Konstantinidou AE, Korkolopoulou P, Lelouda M, Kouraklis G, Patsouris E, Karatzas G: Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer. Mod Pathol. 2007, 20: 159-166. 10.1038/modpathol.3800719.View ArticlePubMedGoogle Scholar
- Waghray A, Schober M, Feroze F, Yao F, Virgin J, Chen YQ: Identification of differentially expressed genes by serial analysis of gene expression in human prostate cancer. Cancer Res. 2001, 61: 4283-4286.PubMedGoogle Scholar
- Chow LS, Lam CW, Chan SY, Tsao SW, To KF, Tong SF, Hung WK, Dammann R, Huang DP, Lo KW: Identification of RASSF1A modulated genes in nasopharyngeal carcinoma. Oncogene. 2006, 25: 310-316.PubMedGoogle Scholar
- Iacobuzio-Donahue CA, Argani P, Hempen PM, Jones J, Kern SE: The desmoplastic response to infiltrating breast carcinoma: gene expression at the site of primary invasion and implications for comparisons between tumor types. Cancer Res. 2002, 62: 5351-5357.PubMedGoogle Scholar
- Yoshida T, Hisamoto T, Akiba J, Koga H, Nakamura K, Tokunaga Y, Hanada S, Kumemura H, Maeyama M, Harada M, Ogata H, Yano H, Kojiro M, Ueno T, Yoshimura A, Sata M: Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors. Oncogene. 2006, 25: 6056-6066. 10.1038/sj.onc.1209635.View ArticlePubMedGoogle Scholar
- Thant AA, Sein TT, Liu E, Machida K, Kikkawa F, Koike T, Seiki M, Matsuda S, Hamaguchi M: Ras pathway is required for the activation of MMP-2 secretion and for the invasion of src-transformed 3Y1. Oncogene. 1999, 18: 6555-6563. 10.1038/sj.onc.1203049.View ArticlePubMedGoogle Scholar
- Pellegrino A, Antonaci F, Russo F, Merchionne F, Ribatti D, Vacca A, Dammacco F: CXCR3-binding chemokines in multiple myeloma. Cancer Lett. 2004, 207: 221-227. 10.1016/j.canlet.2003.10.036.View ArticlePubMedGoogle Scholar
- Ruschpler P, Lorenz P, Eichler W, Koczan D, Hanel C, Scholz R, Melzer C, Thiesen HJ, Stiehl P: High CXCR3 expression in synovial mast cells associated with CXCL9 and CXCL10 expression in inflammatory synovial tissues of patients with rheumatoid arthritis. Arthritis Res Ther. 2003, 5: R241-R252. 10.1186/ar783.PubMed CentralView ArticlePubMedGoogle Scholar
- Ueno A, Yamamura M, Iwahashi M, Okamoto A, Aita T, Ogawa N, Makino H: The production of CXCR3-agonistic chemokines by synovial fibroblasts from patients with rheumatoid arthritis. Rheumatol Int. 2005, 25: 361-367. 10.1007/s00296-004-0449-x.View ArticlePubMedGoogle Scholar
- Garcia-Vicuna R, Gomez-Gaviro MV, Dominguez-Luis MJ, Pec MK, Gonzalez-Alvaro I, Alvaro-Gracia JM, Diaz-Gonzalez F: CC and CXC chemokine receptors mediate migration, proliferation, and matrix metalloproteinase production by fibroblast-like synoviocytes from rheumatoid arthritis patients. Arthritis Rheum. 2004, 50: 3866-3877. 10.1002/art.20615.View ArticlePubMedGoogle Scholar
- Holse M, Assing K, Poulsen LK: CCR3, CCR5, CCR8 and CXCR3 expression in memory T helper cells from allergic rhinitis patients, asymptomatically sensitized and healthy individuals. Clin Mol Allergy. 2006, 4: 6-10.1186/1476-7961-4-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Salomon I, Netzer N, Wildbaum G, Schif-Zuck S, Maor G, Karin N: Targeting the function of IFN-gamma-inducible protein 10 suppresses ongoing adjuvant arthritis. J Immunol. 2002, 169: 2685-2693.View ArticlePubMedGoogle Scholar
- Mohan K, Issekutz TB: Blockade of chemokine receptor CXCR3 inhibits T cell recruitment to inflamed joints and decreases the severity of adjuvant arthritis. J Immunol. 2007, 179: 8463-8469.View ArticlePubMedGoogle Scholar
- Sentman CL, Meadows SK, Wira CR, Eriksson M: Recruitment of uterine NK cells: induction of CXC chemokine ligands 10 and 11 in human endometrium by estradiol and progesterone. J Immunol. 2004, 173: 6760-6766.View ArticlePubMedGoogle Scholar
- Smith PM, Heinrich CA, Pappas S, Peluso JJ, Cowan A, White BA: Reciprocal regulation by estradiol 17-beta of ezrin and cadherin-catenin complexes in pituitary GH3 cells. Endocrine. 2002, 17: 219-228. 10.1385/ENDO:17:3:219.View ArticlePubMedGoogle Scholar
- Liu HL, Golder-Novoselsky E, Seto MH, Webster L, McClary J, Zajchowski DA: The novel estrogen-responsive B-box protein (EBBP) gene is tamoxifen-regulated in cells expressing an estrogen receptor DNA-binding domain mutant. Mol Endocrinol. 1998, 12: 1733-1748. 10.1210/me.12.11.1733.View ArticlePubMedGoogle Scholar
- Moggs JG, Murphy TC, Lim FL, Moore DJ, Stuckey R, Antrobus K, Kimber I, Orphanides G: Anti-proliferative effect of estrogen in breast cancer cells that re-express ERalpha is mediated by aberrant regulation of cell cycle genes. J Mol Endocrinol. 2005, 34: 535-551. 10.1677/jme.1.01677.View ArticlePubMedGoogle Scholar
- Helvering LM, Adrian MD, Geiser AG, Estrem ST, Wei T, Huang S, Chen P, Dow ER, Calley JN, Dodge JA, Grese TA, Jones SA, Halladay DL, Miles RR, Onyia JE, Ma YL, Sato M, Bryant HU: Differential effects of estrogen and raloxifene on messenger RNA and matrix metalloproteinase 2 activity in the rat uterus. Biol Reprod. 2005, 72: 830-841. 10.1095/biolreprod.104.034595.View ArticlePubMedGoogle Scholar
- Paquette B, Bisson M, Therriault H, Lemay R, Pare M, Banville P, Cantin AM: Activation of matrix metalloproteinase-2 and -9 by 2- and 4-hydroxyestradiol. J Steroid Biochem Mol Biol. 2003, 87: 65-73. 10.1016/S0960-0760(03)00386-8.View ArticlePubMedGoogle Scholar
- Wingrove CS, Garr E, Godsland IF, Stevenson JC: 17beta-oestradiol enhances release of matrix metalloproteinase-2 from human vascular smooth muscle cells. Biochim Biophys Acta. 1998, 1406: 169-174.View ArticlePubMedGoogle Scholar
- Etique N, Grillier-Vuissoz I, Flament S: Ethanol stimulates the secretion of matrix metalloproteinases 2 and 9 in MCF-7 human breast cancer cells. Oncol Rep. 2006, 15: 603-608.PubMedGoogle Scholar
- Mitropoulou TN, Tzanakakis GN, Kletsas D, Kalofonos HP, Karamanos NK: Letrozole as a potent inhibitor of cell proliferation and expression of metalloproteinases (MMP-2 and MMP-9) by human epithelial breast cancer cells. Int J Cancer. 2003, 104: 155-160. 10.1002/ijc.10941.View ArticlePubMedGoogle Scholar
- Khalkhali-Ellis Z, Seftor EA, Nieva DR, Handa RJ, Price RH, Kirschmann DA, Baragi VM, Sharma RV, Bhalla RC, Moore TL, Hendrix MJ: Estrogen and progesterone regulation of human fibroblast-like synoviocyte function in vitro: implications in rheumatoid arthritis. J Rheumatol. 2000, 27: 1622-1631.PubMedGoogle Scholar
- Straub RH: The complex role of estrogens in inflammation. Endocr Rev. 2007, 28: 521-574. 10.1210/er.2007-0001.View ArticlePubMedGoogle Scholar
- Rozzo SJ, Allard JD, Choubey D, Vyse TJ, Izui S, Peltz G, Kotzin BL: Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus. Immunity. 2001, 15: 435-443. 10.1016/S1074-7613(01)00196-0.View ArticlePubMedGoogle Scholar
- Karp CL, Grupe A, Schadt E, Ewart SL, Keane-Moore M, Cuomo PJ, Köhl J, Wahl L, Kuperman D, Germer S, Aud D, Peltz G, Wills-Karp M: Identification of complement factor 5 as a susceptibility locus for experimental allergic asthma. Nat Immunol. 2000, 1: 221-226. 10.1038/79759.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.