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14-3-3η is a novel mediator associated with the pathogenesis of rheumatoid arthritis and joint damage Maksymowych, Walter P; van der Heijde, Désirée; Allaart, Cornelia F; Landewé, Robert; Boire, Gilles; Tak, Paul P; Gui, Yuan; Ghahary, Aziz; Kilani, Ruhangiz; Marotta, Anthony Apr 21, 2014

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RESEARCH ARTICLE Open Access14-3-3η is a novel mediator associated with thepathogenesis of rheumatoid arthritis and jointdamageWalter P Maksymowych1*, Désirée van der Heijde2, Cornelia F Allaart2, Robert Landewé3, Gilles Boire4, Paul P Tak5,6,Yuan Gui7, Aziz Ghahary8, Ruhangiz Kilani8 and Anthony Marotta7AbstractIntroduction: The aim of this study was to investigate whether 14-3-3η, a specific isoform of a family of proteinsregulating processes such as cellular signalling, activates cell-signalling pathways and induces factors known tocontribute to the pathophysiology of rheumatoid arthritis (RA). We also investigated whether 14-3-3η is associatedwith more severe disease in both early and established RA.Methods: We investigated the effect of 14-3-3η on the activation of RA-relevant signalling cascades and induction ofproinflammatory mediators that contribute to the joint damage process. 14-3-3η titres from 33 patients with early RA(mean RA duration = 1.8 months) and from 40 patients with established RA were measured in serum drawn at the3-year time point of the Behandel Strategieën study. The relationship between 14-3-3η titres and standard clinicalvariables was investigated by correlation analysis. The association with radiographic damage and radiographicprogression over at least a 2-year period was investigated using univariate and multivariate regression analyses.Results: 14-3-3η activated selected members of the mitogen-activated protein kinase (MAPK) family, mainly extracellularregulated kinase 1/2 and c-Jun kinase, but not p38MAPK. Activation by 14-3-3η, using levels spanning the concentrationrange found in RA patient serum, resulted in the induction of inflammatory transcripts such as interleukin 1 (IL-1) andIL-6 and factors linked to the joint damage process, such as receptor activator of nuclear factor κB ligand and matrixmetalloproteinase 1. Serum 14-3-3η correlated significantly with rheumatoid factor (RF) (r = 0.43) and anticitrullinatedprotein antibodies (ACPAs) (r = 0.31) in the early RA cohort, but not with C-reactive protein (CRP) or the DiseaseActivity Score in 28 joints in either cohort. Serum 14-3-3η concentrations were significantly higher in patients withradiographically assessed joint damage and in those who had radiographic progression. By multivariate analysis, weshow that 14-3-3η complemented markers such as CRP, RF and ACPA in informing RA radiographic status and/orprogression.Conclusions: Extracellular 14-3-3η activates key signalling cascades and induces factors associated with thepathogenesis of RA at concentrations found in patients with RA, and its expression is higher in patients withradiographic damage and RA progression.* Correspondence: walter.maksymowych@ualberta.ca1Department of Medicine, University of Alberta, 562 Heritage MedicalResearch Building, Edmonton, AB T6G 2S2, CanadaFull list of author information is available at the end of the article© 2014 Maksymowych et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (, which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.Maksymowych et al. Arthritis Research & Therapy 2014, 16:R99 arthritis (RA) is a chronic autoimmunedisease characterised by inflammation of the synovium[1]. When left untreated, RA typically leads to severejoint destruction [2]. Early diagnosis, combined with anaccurate prognostic assessment at presentation, is a cen-tral tenet in the effective management of RA patients[3]. There is consensus amongst rheumatologists thatthe development of a risk stratification strategy to grouppatients into low-, medium- and high-risk categories forradiographic progression at presentation is a clinicalimperative [4]. Currently, this remains a major limitationin patient management because risk factors such asrheumatoid factor (RF), anticitrullinated protein anti-bodies (ACPAs; often detected by performing an anti–cyclic citrullinated peptide antibody test) and C-reactiveprotein (CRP) together account for only 32% of the totalvariance in predicting joint destruction, leaving 68% ofthe variance unaccounted for [4].Serum 14-3-3η, which was first described in 2007 [5]as being elevated in arthritis, has been reported to besignificantly higher in RA patients than in healthy indi-viduals and in various disease controls including, butnot limited to, osteoarthritis (OA), ankylosing spondylitis(AS) and gout [6,7]. On the basis of receiver operatingcharacteristic curve analyses defining optimal sensitivityand specificity, serum 14-3-3η positivity is denoted by aconcentration ≥0.19 ng/ml [6,7]. We have previouslyshown that (1) serum 14-3-3η expression is not stronglycorrelated with standard clinical and serological mea-sures, is detected in both early and established RA, withsensitivities of 60% to 82% and 78%, respectively, and (2)that 14-3-3η positivity adds incrementally to both RFand ACPA positivity for diagnostic sensitivity [8]. In aprevious study of an early RA cohort, we reported that60% of patients were positive for 14-3-3η, 32% for RF,44% for ACPA and 72% for at least one of those threemarkers [8].The 14-3-3 family of conserved regulatory proteinsconsists of seven isoforms: α/β, γ, δ/ζ, ε, η, θ/τ and σ.Under normal circumstances, these proteins exist asintracellular adapters that can either homo- or hetero-dimerise to form a cuplike structure known as theamphipathic groove, which allows them to interact withmore than 200 intracellular proteins to modulate theiractivities. Interactions include an array of biological pro-cesses, such as protein trafficking and cellular signalling.Externalisation of 14-3-3η, similarly to its process in RA[5], is believed to be mediated in part through an exoso-mal process in a like that of other intracellular proteins,such as the release of heat shock proteins (HSPs) fromhuman peripheral blood mononuclear cells (PBMCs) orB lymphocytes under heat stress [9,10]. Importantly,these two families of proteins—14-3-3 and HSPs—havebeen described as key components of exosomal microve-sicles [11], and it is now widely accepted that membersof the HSP superfamily, such as HSP60, behave as proin-flammatory factors priming the immune system to re-spond to a noxious agent [12].In 2007, we reported a strong correlation betweenthe expression of 14-3-3η and matrix metalloproteinases(MMPs) and demonstrated that extracellular 14-3-3ηpossesses MMP-1-inducing activity in vitro on the basisof 14-3-3η levels at the upper range of serum levels ob-served in a small subset of RA patients [5-7]. MMPs areserine proteases that play a critical role in maintainingtissue homeostasis, but, in the context of RA, the im-balance between expression of these proteolytic enzymesand their cognate inhibitors leads to the breakdown ofcartilage [13].MMP expression has been reported to be regulatedthrough the transcription factor activator protein 1 (AP-1),which resides downstream from intracellular signallingfactors such as mitogen-activated protein kinase (MAPK)[14-17]. The MAPK family, which includes extracellularregulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38MAPK, hasbeen investigated extensively in RA [18]. Recently, deLaunay et al. reported a specific increase in ERK and JNKactivation, but not in p38MAPK activation, in early RApatients with progressive joint destruction, underscoringtheir possible relevance to the aetiology of RA [19].In our present study, given the strong correlation of14-3-3η with MMP-1 and MMP-3 in synovial fluid andserum [5], we sought to advance the understanding ofthe role of 14-3-3η in RA by assessing whether 14-3-3η(1) possesses extracellular ligand properties leading toactivation of MAPK, (2) induces mediators of inflamma-tion and joint destruction and (3) contributes prognosticinformation in addition to clinical and serological mea-sures used in the management of RA.Methods14-3-3η effects on cell signalling and induction ofpro-inflammatory mediatorsTo examine whether 14-3-3η possesses extracellular lig-and properties relevant to RA, we investigated its effectson the activation of RA-relevant signalling cascades andthe induction of proinflammatory mediators that con-tribute to the joint damage process.Low-endotoxin human recombinant 14-3-3η (AugurexLife Sciences Corp, North Vancouver, BC, Canada) andhuman recombinant tumour necrosis factor α (TNFα)from (R&D Systems, Minneapolis, MN, USA) were usedfor cell stimulation. 14-3-3η and TNFα were diluted usingserum-free media whereby 5 μl of the 14-3-3η or TNFαstock were added to each millilitre of medium in wells toachieve the concentration used for cell stimulation dosing.Maksymowych et al. Arthritis Research & Therapy 2014, 16:R99 Page 2 of 11 dose selection study (0 to 100 ng/ml), coupled with a15-minute cell stimulation experiment, was performed toselect the doses and time points for assessment of cellsignalling pathways, which were analysed by immunoblot-ting with a phosphotyrosine antibody. On the basis of theresults of this study, we determined that, for cell signallingexperiments, a minimum dose of 12.5 ng/ml 14-3-3η andTNFα would be used in order to observe a visibly de-tectable difference in phosphorylation status above the un-stimulated control. This dose of extracellular 14-3-3ηreflects concentrations observed in patient sera (0.0to >20.0 ng/ml). Kilani et al. [5] reported that 14-3-3ηexpression detected by Western blot analysis was three tofive times higher in synovial fluid than in matched patientserum, supporting the 12.5 ng/ml cell stimulation doseas being consistent with the concentration of 14-3-3ηpresent in the joint. For transcriptional studies of proin-flammatory mediators, we analysed the impact of a rangeof 14-3-3η concentrations comparable to those detectedin RA patient serum (0.1 to 100 ng/ml) and found thatthey were well within the range found in synovial fluid.Cell cultureMonocyte lineage THP-1 cells were selected to investi-gate the stimulatory effects of 14-3-3η, as this cell linehas been used extensively in the field because of theimportance of this lineage of cells in the pathogenesis ofRA [20]. Briefly, THP-1 cells were cultured in RPMI1640 medium supplemented with 10% foetal bovineserum in a humidified chamber at 37°C in a 5% CO2atmosphere. Prior to being seeded, cells were centrifugedfor 5 minutes at 2,000 rpm. The cell pellet was thenwashed once gently with serum-free medium, centri-fuged and resuspended to 1 × 106 cells/ml.Activation of cell signalling pathwaysTo determine which cell signalling pathways are specific-ally activated by 14-3-3η, the THP-1 cells were seeded ata density of 2 × 106 cells per well and deprived of serumfor 6 hours prior to cell stimulation. Following cell star-vation, the cells were stimulated with 12.5 ng/ml of lig-and (14-3-3η or TNFα) up to 30 minutes. Cells wereharvested and lysed, and the phosphorylation status ofMAPK family members, specifically ERK, JNK/SAPKand p38MAPK, were evaluated using phosphorylatedMAPK and the corresponding pan-antibodies (CellSignaling Technology, Beverly, MA, USA). Changes inphosphorylation status were measured by densitometryusing ImageJ software (National Institutes of Health,Bethesda, MD, USA). Total protein levels were used as abackground control to ensure that changes were due toincreases in phosphorylation status rather than to in-creases in the respective kinases.Expression of proinflammatory mediatorsInduction of mediators of inflammation and destructionby a range of concentrations of 14-3-3η (0.1 to 100 ng/ml)were evaluated by mRNA analysis for interleukin 1β(IL-1β), IL-6, MMP-1, MMP-9, receptor activator of nu-clear factor κB ligand (RANKL) and TNFα. THP-1 cellswere resuspended in medium containing 0.1% foetal bo-vine serum, seeded at a density of 2 × 106 cells per wellinto a six-well dish and allowed to stand for 2 hours. Thecells were then stimulated with 14-3-3η (0.1 to 100 ng/ml)or TNFα (50 ng/ml) for 18 hours using 5 μl of ligand permillilitre of culture medium added to each well. Thereaction was terminated by the addition of ice-coldphosphate-buffered saline, and then the cells were centri-fuged. Following centrifugation, RNA was extracted fromthe cell pellets using the illustra RNAspin Mini RNAIsolation Kit (GE Healthcare Life Sciences, Pittsburgh, PA,USA). The cDNA was synthesised from purified totalRNA using the RevertAid™ H Minus First Strand cDNASynthesis Kit (Thermo Scientific, Pittsburgh, PA, USA).The PCR conditions for each primer were optimised inde-pendently. Primers for RT-PCR analyses were producedeither by Augurex Life Sciences Corp or purchased fromR&D Systems. The levels of the housekeeping geneGAPDH were evaluated to ensure that equal amounts ofcDNA were used for all samples.Rheumatoid arthritis cohortsCohort ACohort A comprised 33 patients with RA (defined ac-cording to the 1987 American Rheumatism classificationcriteria [19]) who were members of the Early Undifferen-tiated PolyArthritis (EUPA) Cohort of the University ofSherbrooke. Adult patients with synovitis affecting at leastthree joints for 1 to 12 months were followed longitu-dinally as previously described [21]. The mean age (SD) ofthe patients was 51.1 years (5.0), 23 (70%) of the patientswere female, the median (IQR) disease duration was1.8 months (1.5 to 2.8) and the median (IQR) DiseaseActivity Score in 28 joints (DAS28) was 5.9 (4.8 to 6.5).Serological assessment comprised erythrocyte sedimenta-tion rate (ESR), CRP, RF and ACPA, and radiographic as-sessments were performed using the Sharp/van der HeijdeScore (SHS). Radiographs obtained at baseline and at the30-month follow-up examination were available. Radio-graphic progression was determined based on the changein SHS at 30 months. All radiographs were read in knowntime sequence by one or two trained reviewers blindedto patient characteristics and treatment. At baseline,all patients were disease-modifying anti-rheumatic drug(DMARD)–naïve. Patients were treated with the objectiveof rapidly attaining a zero swollen joint count. During the30-month follow-up period, 29 patients (88%) receivedconventional DMARD therapy and 5 (15%) received oralMaksymowych et al. Arthritis Research & Therapy 2014, 16:R99 Page 3 of 11 therapy, and 4 patients were receiving anti-TNFtherapy at the time of the 30-month evaluation.Cohort BCohort B comprised 40 patients with established RA atthe year 3 time point of the Behandel Strategieën (BeSt)study [22]. The patients’ mean age (SD) was 50.9 years(12.2), and 30 (75%) of the patients were female. Themedian (IQR) DAS28 score was 1.9 (1.5 to 2.6). In theBeSt study, patients were assigned to one of four treat-ment strategies: sequential DMARD monotherapy(group 1), step-up combination therapy (group 2), initialcombination therapy with tapered high-dose prednisone(group 3) and initial combination therapy with the TNFinhibitor infliximab (group 4). The patients in ourpresent 14-3-3η study were selected from among groups1 through 3. Patients in group 4 were not included,because, as we have previously reported, 14-3-3η is aTNF-responsive gene whose levels are modifiable withTNF inhibitor therapy in both RA and psoriatic arthritispatients [6,23]. Treatment may therefore have a con-founding effect on the assessment of 14-3-3η’s asso-ciation with radiographic progression, even in groups 1,2 and 3.Serological measurements for CRP, RF and ACPA wereanalysed, and joint assessments performed, using theSHS as previously described [22,24]. Radiographs wereavailable for the 3- and 5-year time points of our presentstudy. Radiographic progression was determined basedon the change in the SHS at year 5.This study was performed in accordance with theDeclaration of Helsinki, and the study protocol was ap-proved by the Health Research Ethics Review Board ofthe University of Sherbrooke and the Ethics ReviewBoard of Leiden University. All patients provided theirwritten informed consent to participate in the study.Radiographic assessmentTo investigate 14-3-3η’s clinical association with radio-graphic joint damage and joint damage progression forboth the early and established RA cohorts, we comparedthe groups according to the presence or absence of jointdamage at the start of the study (SHS <1 or ≥1) and accor-ding to the presence or absence of radiographic progressionat the end of the follow-up period (ΔSHS <1 or ≥1).Serum 14-3-3η measurementsSerum 14-3-3η levels were measured using a 14-3-3ηenzyme-linked immunosorbent assay (Augurex LifeSciences Corp). Serum samples were diluted 1:20 usingthe supplied assay dilution buffer. A 100-μl volume ofeither the corresponding standard or sample was incu-bated in a 96-well plate shaker for 2 hours at 500 rpmand 27°C. Following incubation, the plates were washedfour consecutive times with wash buffer and then incu-bated with the prediluted horseradish peroxidase–conjugated antibody for 1 hour at room temperaturewithout shaking. The plates were washed four additionaltimes and subsequently incubated with 3,3′,5,5′-tetra-methylbenzidine substrate for 30 minutes, at which timethe reaction was terminated by the addition of 1 N HClstop solution. Serum 14-3-3η levels were quantifiedusing a four-parameter logistic regression curve gener-ated against the diluted standards. The accuracy of theresults were evaluated by examining the precision of theback-calculated concentrations of each of the standardsand the corresponding measurements of three qualitycontrol samples with known 14-3-3η levels within thelinear range of the assay. All clinical samples were runin duplicate. Samples with levels below the reportablerange were assigned a concentration of 0.0 ng/ml, andthose with levels above the upper limit were defined ashaving levels >20 ng/ml. Actual 14-3-3η concentrationsfor samples with values >20 ng/ml were determined byserial dilution and used for statistical analysis.Statistical analysis of clinical 14-3-3η expressionThe Mann–Whitney U test was used to compare me-dian differences between groups. A two-tailed Student’st-test with Welch’s correction was employed to accountfor significant variances between groups. Pearson pro-duct–moment correlations were calculated to examinethe relationship between 14-3-3η and clinical and sero-logical measurements, including DAS28 and titres ofCRP, RF and ACPA.Fisher’s exact test was used to compare the associationbetween 14-3-3η positivity, at various positivity cutpoints, of RF or ACPA with radiographic damage status(SHS <1 or ≥1) and radiographic progression (ΔSHS <1or ≥1). 14-3-3η positivity cutoffs were defined at thediagnostic positivity cutoff of ≥0.19 ng/ml, approxi-mately twice the diagnostic cutoff (0.4 ng/ml) and fourtimes the diagnostic cutoff (0.8 ng/ml). Univariate re-gression analyses were performed to identify which clin-ical and serological markers were associated with jointdamage status and radiographic progression in the twocohorts. Further regression analyses were performed toidentify variables associated with radiographic damagestatus and/or progression by combining only those vari-ables that had a P-value ≤0.10 in univariate analysis.The strength of the association is expressed using χ2likelihood ratios with 95% confidence intervals (CI), to-gether with the total variance, to inform the primaryoutcome (R2).All statistical analyses were performed using Prism 6software (GraphPad Software, La Jolla, CA, USA) andJMP9 software (SAS Institute, Cary, NC, USA). Resultswere considered significantly different at P < 0.05.Maksymowych et al. Arthritis Research & Therapy 2014, 16:R99 Page 4 of 11 pathophysiologic role for extracellular 14-3-3ηStimulation of THP-1 cells with recombinant 14-3-3η re-sulted in an increase in the phosphorylation of ERK andJNK/SAPK by approximately 87% and 227% above controlat 2 and 5 minutes, respectively (Figure 1A to 1C). Incontrast to TNFα, no phosphorylation of p38MAPK by14-3-3η was observed at any time point evaluated. Theseresults suggest that 14-3-3η and TNFα possess both com-mon and unique signalling effects.As depicted in Figure 1D and 1E, stimulation of THP-1cells with 14-3-3η resulted in the dose-dependent induc-tion of inflammatory transcripts and factors directly linkedto the joint damage process, such as IL-1β, IL-6, MMP-1,MMP-9 and RANKL. Similar effects were observed withTNFα when the same concentration was used. Inductionof transcripts by 14-3-3η was evident with concentrationsas little as 1.0 ng/ml across all of the factors tested(Figure 1E), which corresponds with levels reported inpatients with RA (Table 1). Certain transcripts, such asIL-1β and MMP-1, were determined to be more sensitiveto stimulation by 14-3-3η, which was evident even as lowas 0.1 ng/ml.Relationship of 14-3-3η with clinical and serologicalmeasuresTable 2 provides the correlations between the clinicaland serological variables. In both the early and esta-blished RA cohorts, 14-3-3η titres did not correlatesignificantly with DAS28 scores or CRP levels. Therewas a modest correlation with RF (r = 0.43, P < 0.01) andACPA (r = 0.31, P < 0.05) in the early RA cohort, but notin the established RA cohort.14-3-3η serum concentration and its association withradiographic damage and progression in rheumatoidarthritisTwenty (61%) of the thirty-three early RA patients with30-month follow-up data developed radiographic pro-gression and twenty (50%) of forty RA patients in theFigure 1 Extracellular 14-3-3η selectively activates extracellular signal-regulated kinase and c-Jun N-terminal kinase/stress-activatedprotein kinase and potentiates inflammatory and joint damage factors. (A) 14-3-3η leads to the phosphorylation and activation of extracellularsignal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). p-MEK, Phosphorylated mitogen-activated protein kinase kinase; p-SAPK, Phosphorylatedstress-activated protein kinase. (B) Equal protein loading. (C) The effects were specific for ERK and JNK because phosphorylation of p38 mitogen-activatedprotein kinase (p38MAPK) by 14-3-3η was not observed. (D) Similarly to tumour necrosis factor α (TNFα), 14-3-3η leads to the induction of inflammatoryand joint damage factors. IL, Interleukin; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; MMP, Matrix metalloproteinase; RANKL, Receptor activatorof nuclear factor κB ligand. (E) 14-3-3η induced effects observed with 14-3-3η concentrations that closely approximated the 14-3-3η median rheumatoidarthritis (RA) serum concentrations.Maksymowych et al. Arthritis Research & Therapy 2014, 16:R99 Page 5 of 11 cohort had radiographic progression. The character-istics of the patients in the two cohorts, who were groupedaccording to radiographic progression (ΔSHS <1 or ≥1),are provided in Table 1. Patients in both groups werewell-matched for age and sex. In the early RA patientgroup, the median baseline DAS28 score was higher inthe progression group than in the no-progression group(6.1 vs. 5.0; P = 0.04), whereas baseline DAS28 scores inthe established RA cohort did not differ significantly bet-ween the progression and no-progression groups (1.9 vs.1.9; P = 0.56). Median (IQR) baseline 14-3-3η levels weresignificantly higher in early RA patients with radiographicprogression compared with those whose RA did not pro-gress (2.68 ng/ml (0.12 to 15.94) vs. 0.09 ng/ml (0.06 to12.59); P = 0.006). 14-3-3η levels were also higher inestablished RA patients with radiographic progression.However, these levels were not significantly different(1.11 ng/ml (0.10 to 2.89) vs. 0.28 ng/ml (0 to 4.5);P = 0.16). The respective scatterplots for 14-3-3η serumconcentrations according to radiographic progression(ΔSHS <1 or ≥1) are provided in Figure 2.Univariate analysis revealed that the strength of theassociation of radiographic damage at baseline andradiographic damage progression increased with higher14-3-3η positivity cut points in both the early and estab-lished RA cohorts (Table 3). Using the diagnostic cutoffof ≥0.19 ng/ml, the odds ratio (OR) of radiographic dam-age progression in early RA was 6.2 (95% CI = 1.3 to 30.2;P = 0.022), which increased to 10.2 (95% CI = 1.7 to 59.7;P = 0.006) with cutoffs of ≥0.40 ng/ml and ≥0.80 ng/ml,respectively. RF positivity was also determined to be asso-ciated with radiographic progression in the early RA co-hort, with an OR of 9.1 (95% CI = 1.7 to 47.7; P = 0.008).In the established RA cohort, the OR of radiographicTable 1 Patient characteristics based on radiographic progression in two cohorts of rheumatoid arthritis patientsaEarly RA (n = 33) Established RA (n = 40)No progression(SHS <1)Progression(SHS ≥1)P-value(U-test)No progression(SHS <1)Progression(SHS ≥1)P-value(U-test)Patients, n 13 20 – 20 20 –DAS28 ESR 5.0 (4.4 to 5.7) 6.1 (5.3 to 6.5) 0.04 1.9 (1.4 to 2.3) 1.9 (1.6 to 2.7) 0.56Mean age (SD), yr 50.9 (5.2) 51.3 (5.0) 0.82 50.3 (10.6) 51.5 (13.9) 0.77Sex, n (% female) 10 (77%) 13 (65%) – 14 (70%) 16 (80%) –Baseline SHS 0 (0 to 0) 2.5 (1.0 to 6.8) 0.0002 1.0 (0 to 16) 38.7 (23.2 to 56.8) <0.0001CRP, mg/L 12.0 (6.0 to 22.5) 12.5 (6.5 to 24.5) 0.82 3.0 (2.0 to 6.0) 8.0 (5.3 to 22.0) 0.001RF, IU/ml 0 (0 to 160.0) 160.0 (40.0 to 320.0) 0.035 48.0 (2.0 to 68.0) 55.0 (19.5 to 223.8) 0.13ACPA, U/ml 12 (6.5 to 96.0) 101.0 (14.3 to 194.3) 0.18 59.5 (20.3 to 365.9) 75.1 (20.3 to 440.3) 0.77Mean 14-3-3η (SD), ng/ml 1.30 (3.51) 6.13 (8.33) 0.03 3.76 (6.92) 4.37 (8.02) 0.79Median 14-3-3η (IQR), ng/ml 0.09 (0.06 to 12.59) 2.68 (0.12 to 15.94) 0.006 0.28 (0 to 4.54) 1.11 (0.10 to 2.89) 0.16aACPA, Anticitrullinated protein antibody; SHS, Sharp/van der Heijde score; CRP, C-reactive protein; DAS28 ESR, Disease Activity Score in 28 joints with erythrocytesedimentation rate; RA, Rheumatoid arthritis; RF, Rheumatoid factor. P-values were calculated using the Mann–Whitney U test.Table 2 Correlation of 14-3-3η with clinical and serological measures in two cohorts of patients with rheumatoidarthritisaCohort Variable, score or titres DAS28 CRP RF ACPAA DAS28 Pearson product–moment correlation (r)CRP 0.12RF 0.22 −0.13ACPA −0.14 0.20 0.2714-3-3η 0.18 0.20 0.43b 0.31cBDAS28 Pearson product–moment correlation (r)CRP 0.46dRF −0.06 −0.02ACPA −0.17 −0.14 0.1714-3-3η −0.03 −0.09 0.03 −0.07aACPA, Anticitrullinated protein antibody; CRP, C-reactive protein DAS28: Disease Activity Score in 28 joints; RF: rheumatoid factor. bP < 0.01; cP ≤ 0.05; dP < 0.001.Cohort A is the University of Sherbrooke early rheumatoid arthritis cohort at the baseline time point (n = 33). Cohort B is the Behandel Strategieën (BeSt) studycohort of patients with established RA at the year 3 time point (n = 40).Maksymowych et al. Arthritis Research & Therapy 2014, 16:R99 Page 6 of 11 was similarly higher at the positivity cutoff of≥0.40 ng/ml (OR = 4.3 (95% CI = 1.2 to 16.3; P = 0.028)than at the cutoff of ≥0.19 ng/ml (OR = 2.5 (95% CI = 0.6to 9.4; P = 0.18). The CRP titre was the only other variabledetermined to be associated with radiographic progressionin the established RA cohort (OR = 1.1 (95% CI = 1.0 to1.4; P = 0.003).The models presented in Table 4 demonstrate that14-3-3η positivity adds incrementally to other significantclinical and serological measures in univariate analysiswith regard to informing radiographic status and progres-sion. In the early RA cohort, when 14-3-3η positivity (cut-off set at 0.19 ng/ml) is combined with CRP to informradiographic status, the contribution to the total varianceof the outcome (R2) increases from 0.065 to 0.154. Inestablished RA, when combined with CRP titres, togetherwith RF and ACPA positivity, the contribution to totalvariance (R2) increases from 0.376 to 0.425. With respectto radiographic RA progression, when 14-3-3η positivity(cutoff set at ≥0.40 ng/ml) is combined with DAS28 scoreand RF positivity, the contribution to the total variance inthe early RA cohort increases from 0.252 to 0.310. In theestablished RA cohort, when 14-3-3η positivity is com-bined with CRP titres, the contribution to the total vari-ance increases from 0.167 to 0.262.Discussion14-3-3η is one of seven members of the 14-3-3 familythat are preferentially expressed at higher concentrationsin certain tissues, underscoring the importance of spe-cific isoforms in the regulation of tissue-specific func-tions [25-27]. Data that we reported in 2007 indicatedthat 14-3-3η is expressed at significantly higher levelsthan the other isoforms in the synovial fluid of patientswith arthritis and that these levels were three to fivetimes higher than corresponding levels in the serum ofmatched donors, citing the joint as the likely source ofserum 14-3-3η [5]. Since that first report, we have de-monstrated that 14-3-3η is an RA-specific marker thatcomplements both RF and ACPA, increasing their diag-nostic value [6-8]. We also reported a positive asso-ciation between 14-3-3η and MMPs and suggested that14-3-3η may have a role in the pathogenesis of RA [28].To expand upon our understanding of the biologicalrelevance of extracellular 14-3-3η, we performed in vitrocell signalling studies to determine if 14-3-3η signalsthrough the MAPK family, as well as through whichfamily members. This family was selected because thetranscription factor AP-1, which resides downstream in0. Yes No YesEarly RA Established RA14-3-3ng/ml>Figure 2 Scatterplots illustrating serum 14-3-3η concentrationaccording to radiographic progression of rheumatoid arthritis.Radiographic progression of rheumatoid arthritis (RA) was defined asany change in Sharp/van der Heijde score <1 or ≥1 at the end ofthe follow-up period (30 months for early RA (n = 33) and 2 years forestablished RA (n = 40)). Triangle = patients with no erosion;Square = patients with erosions; Line = mean levels.Table 3 Univariate association of clinical and laboratory variables with damage status and radiographic outcomesaVariableEarly RA (n = 33) Established RA (n = 40)Baseline damage status Progression Baseline damage status ProgressionOR (95% CI) P-value OR (95% CI) P-value OR (95% CI) P-value OR (95% CI) P-valueDAS28 ESR 1.2 (0.7 to 2.3) 0.485 1.7 (0.9 to 3.6) 0.094 1.5 (0.5 to 4.9) 0.496 1.4 (0.6 to 3.5) 0.475Age 1.0 (0.8 to 1.1) 0.505 1.0 (0.9 to 1.2) 0.815 1.1 (1.0 to 1.2) 0.112 1.0 (1.0 to 1.1) 0.763Sex 2.8 (0.6 to 13.8) 0.192 1.8 (0.4 to 8.7) 0.462 1.0 (0.2 to 6.0) 0.660 1.7 (0.4 to 7.3) 0.358CRP 1.0 (1.0 to 1.1) 0.084 1.0 (1.0 to 1.0) 0.906 1.6 (1.1 to 3.1) 0.004 1.1 (1.0 to 1.4) 0.003ACPA + ve 1.1 (0.3 to 4.4) 0.580 2.2 (0.5 to 9.0) 0.239 7.7 (1.3 to 45.3) 0.022 2.2 (0.6 to 8.5) 0.214RF + ve 2.5 (0.6 to 11.3) 0.195 9.1 (1.7 to 47.7) 0.008 10.3 (1.7 to 62.7) 0.010 1.2 (0.3 to 4.5) 0.50014-3-3η+ve ≥0.19 ng/ml 4.0 (0.9 to 17.2) 0.057 6.2 (1.3 to 30.2) 0.022 9.0 (1.5 to 53.9) 0.014 2.5 (0.6 to 9.4) 0.160+ve ≥0.40 ng/ml 5.5 (1.2 to 24.8) 0.025 10.2 (1.7 to 59.7) 0.006 4.4 (0.8 to 25.2) 0.089 4.3 (1.2 to 16.3) 0.028+ve ≥0.80 ng/ml 5.5 (1.2 to 24.8) 0.025 10.2 (1.7 to 59.7) 0.006 7.9 (0.8 to 25.2) 0.044 3.5 (0.9 to 13.0) 0.055aACPA, Anticitrullinated protein antibody; CI, Confidence interval; CRP, C-reactive protein; DAS28, Disease Activity Score in 28 joints; ESR, Erythrocyte sedimentationrate; OR, Odds ratio; RA, Rheumatoid arthritis; RF, Rheumatoid factor; +ve, Dependent variable increase for increase in independent variable. Variables withcorresponding P-value ≤0.10 are presented in bold.Maksymowych et al. Arthritis Research & Therapy 2014, 16:R99 Page 7 of 11 MAPK signalling nexus, is a key regulator of MMPexpression. The data produced in this study indicate thatstimulation of cells with 14-3-3η, similarly to TNFα, re-sults in the phosphorylation of both ERK and JNK/SAPKin a time-dependent manner. However, unlike TNFα,14-3-3η had no impact on p38MAPK phosphorylation.The differential effects of 14-3-3η in relation to TNFαon the activation of MAPK family members indicatesthat these two ligands, which are present in the serum ofpatients with RA, are likely to signal by divergent mech-anisms. Phosphorylation of these two MAPK members,ERK and JNK/SAPK, together with the finding that 14-3-3η is present in the serum of 60% to 82% of early RApatients [8], is consistent with the observations repor-ted by de Launay et al.: that ERK and JNK, but notp38MAPK, are preferentially phosphorylated in early RA[19]. On the basis of these findings, we propose that therelationship between 14-3-3η expression and the phos-phorylation status of the MAPK family members, espe-cially in early RA, be investigated in human synovialtissue.It is well-established that phosphorylation of both ERKand JNK/SAPK at the TXY motif within the activationloop of these kinases is reflective of their activation. Inturn, these MAPKs directly and/or indirectly phospho-rylate the transcription factor c-Jun. Phosphorylation ofc-Jun results in its association with c-fos, the formationof the AP-1 transcription complex and the transactivationor switching-on of genes containing the AP-1 binding site[29]. In this head-to-head analysis, we demonstrate that14-3-3η at a concentration similar to that of TNFα, iscapable of inducing genes that have been described in theliterature as being TNFα-responsive. Our 14-3-3η dose es-calation studies demonstrate that these TNFα-responsivegenes are highly sensitive to 14-3-3η stimulation and canbe induced with as little as 1.0 ng/ml, a 14-3-3η con-centration that approximates median concentrations mea-sured in the serum of RA patients [6-8]. On the basis ofthese in vitro findings, it is evident that 14-3-3η may playa role in perpetuating inflammation through the inductionof factors such as IL-6 and by exacerbating joint des-truction via MMPs and RANKL. Examining 14-3-3η’sexpression in relation to clinical outcomes in RA patientswill be of utmost importance in understanding how 14-3-3η serum expression used as a diagnostic test can assistclinicians with patient management, to further substanti-ate its relevance to the pathophysiology of RA and to gaina better understanding of how 14-3-3η biomarker expres-sion can be aligned with the advancement of a 14-3-3η-targeted therapy to identify the subset of patients who aremore likely to respond to such an approach.High levels of other 14-3-3 isoforms have been associ-ated with more severe disease or worse outcomes in manypatient groups, including, but not limited to, carcinoma[30-33], Creutzfeldt-Jakob disease [34], Alzheimer’s dis-ease [35], multiple sclerosis [36], infarction [37] and HIV[38]. Analysis of serum 14-3-3η expression in relation tojoint damage and progression revealed significantly higherlevels of 14-3-3η in patients who already had radiographicevidence of damage at study baseline, as well as in thosewho developed progression by the end of the follow-upperiod. Amongst other laboratory variables tested, nonewere significantly associated with radiographic status atbaseline in the early RA cohort and only RF was signifi-cantly associated with radiographic progression. Neverthe-less, 14-3-3η contributed to the variance of a regressionmodel for radiographic progression (R2) that included RF.The association of 14-3-3η with radiographic status andTable 4 Multivariate analyses indicating contribution of 14-3-3η to total variance in radiographic damage status andradiographic progressionaCohort Outcome Without 14-3-3ηWith 14-3-3η≥0.2 ng/ml ≥0.4 ng/ml ≥0.8 ng/mlEarly RA Joint damage status Model LR 2.99 7.06 8.54 8.54p 0.084 0.029 0.014 0.014R2 0.065 0.154 0.187 0.187Radiographic progression Model LR 11.16 12.27 13.72 13.72P 0.004 0.007 0.003 0.003R2 0.252 0.273 0.310 0.310Established RA Joint damage status Model LR 12.35 13.94 12.36 12.76P 0.006 0.008 0.015 0.013R2 0.376 0.425 0.377 0.389Radiographic progression Model LR 8.78 9.71 13.78 13.13P 0.003 0.008 0.001 0.001R2 0.167 0.185 0.262 0.250aLR, Likelihood ratio; RA, Rheumatoid arthritis.Maksymowych et al. Arthritis Research & Therapy 2014, 16:R99 Page 8 of 11 was less prominent in the BeSt cohort than inthe early RA cohort, but DAS28 control was already verywell-established by year 3 in the majority of patients in theBeSt cohort (mean DAS28 = 1.9). Of the three laboratoryvariables examined—CRP, RF, and ACPA—only CRP wasassociated with radiographic progression. 14-3-3η contri-buted to the variance of a regression model for radio-graphic progression that included CRP.In this study, we observed that serum 14-3-3η did notcorrelate with disease activity (DAS28) or with titres ofCRP. We noted a modest correlation with ACPA and RFin the early RA cohort, but not in the established RAcohort. These findings indicate that 14-3-3η may act in-dependently of these measures, which is one of the keyrequirements proposed by the Outcome Measures inRheumatology (OMERACT) soluble biomarker workinggroup for a biomarker reflecting joint damage [39]. TheOMERACT guidelines state that, in order to be useful atinforming structural damage endpoints, a marker shouldbe independent of other commonly available variablesassociated with radiographic progression and shouldcomplement them in predictive models. Regression ana-lyses revealed that 14-3-3η added incrementally to thepredictive power of the model. On the basis of thesefindings, a prospective study designed to examine 14-3-3η’s capacity as an independent predictor of radiographicdamage status and progression is underway.As with proof-of-concept studies, one of the main li-mitations of the present study is the small sample sizefor both RA cohorts. In addition, relatively few patientsdemonstrated substantial joint damage progression. Theresults of this study are being used in the design of lar-ger studies to formally investigate the merits of 14-3-3ηin marking joint damage and predicting radiographicprogression in combination with other clinical variables.In addition, we have previously shown that 14-3-3η ismodifiable by both anti-TNF and standard DMARDtherapies [40,41]. Confounding by treatment is a signifi-cant limitation of studies in which the predictive role ofbiomarkers is assessed when only baseline biosamplesare available. It is to be anticipated that levels of 14-3-3ηare likely to change in both cohorts when treatmentchanges are driven by DAS28 targets, which will limitthe predictive capacity of biosample data that are avail-able only at baseline. This was particularly noteworthyin a previous report of BeSt study patients in whichbaseline levels of acute-phase reactants, RF and ACPAwere not shown to be predictive of radiographic progres-sion in the two patient groups undergoing intensivetherapy. However, RF and ACPA were predictive inpatients being treated with monotherapy or combinationtherapy with standard DMARDs [24]. Consequently,future studies should assess biosamples at multiple timepoints.ConclusionsIn our present study, we demonstrate that extracellular 14-3-3η is an activator of ERK and JNK, but not p38MAPK.These effects are comparable to, yet distinct from, TNFα.At concentrations present in the serum of RA patients,stimulation of monocytes with soluble 14-3-3η results inthe induction of genes that perpetuate inflammation anddrive joint damage. Our clinical biomarker results revealthat 14-3-3η expression is higher in the serum of patientswith radiographic evidence of damage and progression.The alignment between the in vitro findings and the clin-ical biomarker expression profile provide a strong rationalefor investigating 14-3-3η as both a marker of radiographicoutcome and possibly a new therapeutic target.AbbreviationsACPA: Anticitrullinated protein antibody; AS: Ankylosing spondylitis;CRP: C-reactive protein; DAS28: Disease Activity Score in 28 joints;DMARD: Disease-modifying antirheumatic drug; ERK: Extracellular signal-regulated kinase; HSP: Heat shock protein; JNK/SAPK: c-Jun N-terminalkinase/stress-activated protein kinase; LR: Likelihood ratio; MAPK: Mitogen-activated protein kinase; MMP: Matrix metalloproteinase; OA: Osteoarthritis;OR: Odds ratio; PBMC: Peripheral blood mononuclear cell; RA: Rheumatoidarthritis; RANKL: Receptor activator of nuclear factor κB ligand;RF: Rheumatoid factor; SHS: Sharp/van der Heijde score; TNFα: Tumournecrosis factor α.Competing interestsWPM is a coinventor of the 14-3-3η technology and Chief Medical Officer ofCaRE Arthritis. DvdH received consulting fees and/or research grants fromAbbVie, Amgen, AstraZeneca, Augurex Life Sciences Corp, Bristol-Myers Squibb,Centocor Biotech, Chugai Pharmaceutical Co, Daiichi Sankyo, Eli Lilly & Co,GlaxoSmithKline, Janssen Biologics, Merck & Co, Novartis, Novo Nordisk, OtsukaPharmaceutical, Pfizer, Roche, Sanofi-Aventis, Schering-Plough, UCB and VertexPharmaceuticals, and is Director of Imaging ǀ Rheumatology BV. CFA declaresno competing interests. RL has been a consultant to or participated on advisoryboards for Abbott/AbbVie, Ablynx, Amgen, AstraZeneca, Bristol-Myers Squibb,Centocor Biotech, GlaxoSmithKline, Novartis, Merck & Co, Pfizer, Roche,Schering-Plough, UCB and Wyeth; has received research grants from Abbott,Amgen, Centocor Biotech, Novartis, Pfizer, Roche, Schering-Plough, UCB andWyeth Pharmaceuticals; has received speaker fees from Abbott, Amgen,Bristol-Myers Squibb, Centocor Biotech, Merck & Co, Pfizer, Roche, Schering-Plough, UCB and Wyeth; and is Director of Rheumatology ǀ Consultancy BV; GBhas received research grants and/or speaker honoraria from, and has served onadvisory boards of, Abbott Canada, Amgen Canada, Aventis Canada,Bristol-Myers Squibb, Hoffmann-La Roche, Janssen, Novartis Canada, Pfizer,Servier and UCB Canada. PPT became an employee of GlaxoSmithKline afterthe completion of this study. YG and AM are employees of Augurex LifeSciences Corp. AG is a coinventor of the 14-3-3η technology. RK is a coinventorof the 14-3-3η technology.Authors’ contributionsWPM participated in the study design and the drafting and review of themanuscript. DvdH and CFA participated in the study design, provided RAtissue samples, collected and analysed data and participated in the draftingand review of the manuscript. RL participated in the study design, providedRA tissue samples, performed statistical analysis and presentation of theresults and participated in the drafting and review of the manuscript. GBprovided RA tissue samples as well as clinical and radiographic data,collected and analysed data and reviewed the manuscript. PPT participatedin the study design and the drafting and review of the manuscript. YG, AGand RK performed cell signalling and transcriptional studies and reviewedthe manuscript. AM participated in the study design, development of the14-3-3η test, quantification of 14-3-3η levels in patient tissue samples,performed cell signalling and transcriptional studies and participated in thedrafting and review of the manuscript. All authors read and approved thefinal manuscript.Maksymowych et al. Arthritis Research & Therapy 2014, 16:R99 Page 9 of 11’ informationPPT is currently with GlaxoSmithKline, Stevenage, UK.AcknowledgementsGrants and financial support were received from Augurex Life Sciences Corp,North Vancouver, BC, Canada.Author details1Department of Medicine, University of Alberta, 562 Heritage MedicalResearch Building, Edmonton, AB T6G 2S2, Canada. 2Department ofRheumatology, C1R, Leiden University Medical Center, PO Box 9600, 2300 RCLeiden, The Netherlands. 3Department of Clinical Immunology andRheumatology, Academic Medical Center, University of Amsterdam & AtriumMedical Center, Meibergdreef 9, Amsterdam 1105AZ, The Netherlands.4Service de rhumatologie, Département de médecine, Faculté de médecineet des sciences de la santém, Université de Sherbrooke, 3001-12e AvenueNord, Sherbrooke, Québec, J1H 5N4, Canada. 5Department of ClinicalImmunology and Rheumatology, Academic Medical Center, University ofAmsterdam & Atrium Medical Center, Meibergdreef 9, Amsterdam 1105AZ,The Netherlands. 6GlaxoSmithKline, Stevenage, UK. 7Augurex Life SciencesCorp, 887 Great Northern Way, Vancouver, BC V5T 4T5, Canada. 8Departmentof Surgery, University of British Columbia, 818 West 10th Avenue, Vancouver,BC V5Z 1M9, Canada.Received: 9 July 2013 Accepted: 25 March 2014Published: 21 April 2014References1. 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Marotta A, Kilani R, Ghahary A, Maksymowych WP: Extracellular 14-3-3ηrepresents a novel rheumatology biomarker and drug target forpersonalized medicine [Abstract AB0111]. Ann Rheum Dis 2012, 71:644.41. Britsemmer K, Maksymowych WP, van Schaardenburg D, Marotta A: 14-3-3ηis an early RA biomarker that is modifiable with standard DMARDs andcorresponds with improvement in clinical variables. Ann Rheum Dis 2013,72:388.doi:10.1186/ar4547Cite this article as: Maksymowych et al.: 14-3-3η is a novel mediatorassociated with the pathogenesis of rheumatoid arthritis and jointdamage. Arthritis Research & Therapy 2014 16:R99.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at et al. 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