14-3-3η Promotes Invadosome Formation via the FOXO3-Snail Axis in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

Erosive destruction of joint structures is a critical event in the progression of rheumatoid arthritis (RA), in which fibroblast-like synoviocytes (FLS) are the primary effectors. We previously reported that the ability of RA FLS to degrade extracellular matrix (ECM) components depends on the formation of actin-rich membrane protrusions, called invadosomes, through processes that remain elusive. 14-3-3η belongs to a family of scaffolding proteins involved in a wide range of cellular functions, and its expression is closely related to joint damage and disease activity in RA patients. In this study, we sought to assess the role of 14-3-3η in joint damage by examining its contribution to the invadosome formation phenotype of FLS. Using human primary FLS, we show that 14-3-3η expression is closely associated with their ability to form invadosomes. Furthermore, knockdown of 14-3-3η using shRNAs decreases the level of invadosome formation in RA FLS, whereas addition of the recombinant protein to FLS from healthy individuals promotes their formation. Mechanistic studies suggest that 14-3-3η regulates invadosome formation by increasing Snail expression, a mechanism that involves nuclear exclusion of the transcription repressor FOXO3. Our results implicate the 14-3-3η-FOXO3-Snail axis in promoting the aggressive ECM-degrading phenotype of RA FLS, and suggest a role for this scaffolding protein in cartilage degradation.

14-3-3η Protein in serum and synovial fluid correlates with radiographic damage and progression in a longitudinal evaluation of patients with established rheumatoid arthritis.

Objectives: The aim of the study is to examine the association between 14-3-3η protein levels in both serum and synovial fluid (SF) with various parameters in a longitudinal cohort of patients with established rheumatoid arthritis (RA).Methods: Serum and SF samples were obtained from RA patients and 14-3-3η levels were measured. Radiological damage and progression were evaluated using Sharp/van der Heijde score (SHS) at study entry and at 2-years follow-up.Results: A total of 39 RA patients were included with a mean disease duration of 9.6 ± 8 years. Levels of 14-3-3η were two-fold higher in SF than in serum (mean of 3.7 versus 1.7 ng/mL, respectively). While no significant association was found between 14-3-3η levels with disease activity or other laboratory assessments, both serum and SF 14-3-3η levels positively correlated with radiographic damage at baseline (SHS; p < .001). SF, but not serum, 14-3-3η levels correlated with absolute progression (p < .03).Conclusion: 14-3-3η levels are significantly higher in RA SF than in serum in an established RA cohort. Serum and SF 14-3-3η levels correlate with radiographic damage at baseline and at 2-years follow-up. This study further substantiates the utility of 14-3-3η as a biomarker for mechanistic joint damage in established RA.

Differential targeting of protein kinase B in cell death induced by sulindac and its metabolite sulindac sulfide.

Non-steroidal anti-inflammatory drugs such as sulindac inhibit human colorectal carcinogenesis through a mechanism involving the direct inhibition of cyclooxygenase (Cox)-2. However, a wealth of recent evidence indicates that these agents might elicit their effects through mechanisms independently of Cox-2. In this study, we investigated the effects of sulindac and its metabolite, sulindac sulfide on modulation of the critical survival kinase, protein kinase B (PKB). Here, we demonstrate for the first time that treatment with either sulindac or sulindac sulfide results in a decrease in PKB activity, and we provide compelling evidence that this occurs through two distinct mechanisms. Additionally, we report that overexpression of, and conditional activation of PKB attenuates the apoptotic effects of sulindac, but not for sulindac sulfide - the metabolic metabolite of sulindac. We also demonstrate that treatment with sulindac sulfide, but not sulindac, results in a very early robust activation of both caspase-8 and -9. Furthermore, we show that the apoptotic effects of sulindac sulfide can be reverted by both the caspase-8 and -9 inhibitors. Evidence is provided to indicate that PKB is targeted by robust caspase activation due to sulindac sulfide. Hence, further investigation into the mechanisms regulating conversion of sulindac to sulindac sulfide (or direct use of the latter compound), may enhance our ability to target cancers with enhanced signaling through the growth factor-->phosphatidylinositol 3-kinase pathway.

A prospective cohort study of 14-3-3η in ACPA and/or RF-positive patients with arthralgia.

BACKGROUND: 14-3-3η (eta) is a novel serum/plasma protein biomarker involved in the upregulation of inflammatory and joint damage factors. We analysed the association of 14-3-3η with the development of clinically apparent arthritis in a cohort of subjects with arthralgia and positivity for at least one serologic marker: rheumatoid factor (RF) or anti-citrullinated protein antibody (ACPA). METHODS: Measurement of 14-3-3η in plasma collected on entry into the cohort. For this study, 144 subjects with a minimum of 2.5 (median and maximum 5) years of follow-up were available. The relationship between presence and levels of 14-3-3η and development of arthritis was investigated. RESULTS: Arthritis occurred in 43 (30 %) of the 144 subjects after a median of 15 months. 14-3-3η was detectable up to 5 years before onset of clinical arthritis and was present significantly more often (36 % versus 14 %; relative risk 2.5, 95 % confidence interval 1.2-5.6; p = 0.02) and at significantly higher levels (median 0.95 versus 0.28 ng/ml; p = 0.02) in subjects developing arthritis compared with those who did not. 14-3-3η levels/positivity and ACPA, but not RF, were univariately associated with the development of arthritis while generalized linear model analysis with RF and ACPA as obligatory factors could not return an incremental benefit with 14-3-3η. CONCLUSIONS: 14-3-3η was detectable prior to the onset of arthritis and was associated with arthritis development in arthralgia subjects pre-selected for positivity of RF or ACPA. Its power to predict onset of arthritis independent of ACPA and RF requires a new study in which patients are not pre-selected based on ACPA and/or RF seropositivity.

Serum levels of 14-3-3η protein supplement C-reactive protein and rheumatoid arthritis-associated antibodies to predict clinical and radiographic outcomes in a prospective cohort of patients with recent-onset inflammatory polyarthritis.

BACKGROUND: Age, C-Reactive Protein (CRP) and autoantibodies (Abs) are associated with worse prognosis in patients with recent-onset inflammatory polyarthritis (EPA). Serum 14-3-3η protein is a joint-derived biomarker that up-regulates cytokines and enzymes that perpetuate local and systemic inflammation and may contribute to joint damage. Our objective was to evaluate, over a 5-year prospective period of observation, the additional prognostic potential of serum 14-3-3η protein in EPA patients. METHODS: Clinical variables, serum and radiographs (scored according to the Sharp/van der Heijde (SvH) method) were collected serially. Relationships between serum 14-3-3η protein and other biomarkers were computed with Spearman correlations. Outcomes were Simple Disease Activity Index (SDAI) scores and joint damage progression: ΔSvH for SvH score and ΔErosion for its Erosive component. The additional predictive contribution of 14-3-3η was defined using generalized estimating equations (GEE) and generalized linear mixed models (GLMM). RESULTS: Among 331 patients, baseline 14-3-3η was ≥0.19 and ≥0.50 ng/ml in 153 (46.2 %) and 119 (36.0 %), respectively; CRP was >8.0 mg/L in 207 (62.5 %), and at least one Ab (Rheumatoid Factor, anti-CCP2 or anti-Sa/citrullinated vimentin) was positive in 170 (51.5 %). Elevated 14-3-3η levels moderately correlated with positive Abs, but not with elevated CRP. Baseline 14-3-3η ≥0.19 ng/ml was associated with more radiographic progression over 5 years. The optimal levels of baseline 14-3-3η to predict radiographic progression was defined by ROC curves at 0.50 ng/ml. Levels of 14-3-3η ≥0.50 ng/ml at baseline were associated with lower likelihoods of ever reaching SDAI remission (RR 0.79 (95 % CI 0.64-0.98), p = 0.03) and higher subsequent progression of Total and Erosion SvH scores. Elevated levels of 14-3-3η during follow-up also predicted higher subsequent progression, even in patients in SDAI remission. Decreases of 14-3-3η levels by at least 0.76 ng/ml and reversion to negative during follow-up associated with less subsequent radiographic progression. In multivariate models, elevated 14-3-3η interacted with positive Abs, elevated CRP and older age to predict subsequent radiographic progression. CONCLUSIONS: Levels of 14-3-3η protein ≥0.50 ng/ml predict poorer clinical and radiographic outcomes in EPA, both at baseline and after initiation of treatment, even in SDAI remitters. 14-3-3η, CRP, age and Abs represent independent predictors of subsequent joint damage. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT00512239 . Registered August 6, 2007.

Serum 14-3-3η level is associated with severity and clinical outcomes of rheumatoid arthritis, and its pretreatment level is predictive of DAS28 remission with tocilizumab.

INTRODUCTION: Treat-to-target strategies to achieve low disease activity or clinical remission are key in the treatment of rheumatoid arthritis (RA). 14-3-3η is a joint-derived biomarker that is expressed at significantly higher levels in patients with RA than in healthy subjects, other autoimmune diseases, or viral and bacterial arthritides. In this study, we sought to investigate the utility of pretreatment levels of 14-3-3η and serial measurement of 14-3-3η to inform therapeutic outcomes. METHODS: Serum 14-3-3η levels were measured in 149 Japanese patients with RA before the initiation of therapy and at 1-year follow-up. Patients were treated with either methotrexate (MTX), adalimumab (ADA), tocilizumab (TCZ), or tofacitinib (TOF). 14-3-3η positivity was defined as ≥0.19 ng/ml and at two times and four times this cutoff. In contingency analysis, we determined the association of 14-3-3η with disease severity. Wilcoxon matched-pairs test was used to evaluate the significance of pre- to post-treatment changes. Mann-Whitney U test was performed for differences between treatment response groups. Fisher's exact test was used to assess associations of 14-3-3η with a good response defined by European League Against Rheumatism criteria as well as remission defined by the Disease activity Score in 28 joints with erythrocyte sedimentation rate (DAS28-ESR) and the Clinical Disease Activity Index score. RESULTS: 14-3-3η-positive patients had more severe disease before the initiation of treatment. When combined with C-reactive protein (CRP), 14-3-3η positivity added significantly and incrementally to the identification of patients with high disease activity. 14-3-3η levels were significantly decreased at 1 year and were modifiable across all classes of therapeutics. Patients who reverted to negative 14-3-3η levels had better clinical response than patients who remained positive at 1 year or became positive. Pretreatment 14-3-3η levels informed 1-year DAS28-ESR remission in the TCZ-treated group, in contrast to the ADA, MTX, or TOF groups, while no differences in pretreatment 14-3-3η expression based on clinical response. CONCLUSIONS: 14-3-3η is a modifiable marker in identifying patients with RA in a high disease state. Patients who achieve a negative 14-3-3η status following 1-year of treatment do better clinically with pretreatment 14-3-3η informing response to TCZ.

14-3-3η Autoantibodies: Diagnostic Use in Early Rheumatoid Arthritis.

OBJECTIVE: To describe the expression and diagnostic use of 14-3-3η autoantibodies in early rheumatoid arthritis (RA). METHODS: 14-3-3η autoantibody levels were measured using an electrochemiluminescent multiplexed assay in 500 subjects (114 disease-modifying antirheumatic drug-naive patients with early RA, 135 with established RA, 55 healthy, 70 autoimmune, and 126 other non-RA arthropathy controls). 14-3-3η protein levels were determined in an earlier analysis. Two-tailed Student t tests and Mann-Whitney U tests compared differences among groups. Receiver-operator characteristic (ROC) curves were generated and diagnostic performance was estimated by area under the curve (AUC), as well as specificity, sensitivity, and likelihood ratios (LR) for optimal cutoffs. RESULTS: Median serum 14-3-3η autoantibody concentrations were significantly higher (p < 0.0001) in patients with early RA (525 U/ml) when compared with healthy controls (235 U/ml), disease controls (274 U/ml), autoimmune disease controls (274 U/ml), patients with osteoarthritis (259 U/ml), and all controls (265 U/ml). ROC curve analysis comparing early RA with healthy controls demonstrated a significant (p < 0.0001) AUC of 0.90 (95% CI 0.85-0.95). At an optimal cutoff of ≥ 380 U/ml, the ROC curve yielded a sensitivity of 73%, a specificity of 91%, and a positive LR of 8.0. Adding 14-3-3η autoantibodies to 14-3-3η protein positivity enhanced the identification of patients with early RA from 59% to 90%; addition of 14-3-3η autoantibodies to anticitrullinated protein antibodies (ACPA) and/or rheumatoid factor (RF) increased identification from 72% to 92%. Seventy-two percent of RF- and ACPA-seronegative patients were positive for 14-3-3η autoantibodies. CONCLUSION: 14-3-3η autoantibodies, alone and in combination with the 14-3-3η protein, RF, and/or ACPA identified most patients with early RA.

14-3-3η is a novel mediator associated with the pathogenesis of rheumatoid arthritis and joint damage.

INTRODUCTION: The aim of this study was to investigate whether 14-3-3η, a specific isoform of a family of proteins regulating processes such as cellular signalling, activates cell-signalling pathways and induces factors known to contribute to the pathophysiology of rheumatoid arthritis (RA). We also investigated whether 14-3-3η is associated with 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 of proinflammatory 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 the 3-year time point of the Behandel Strategieën study. The relationship between 14-3-3η titres and standard clinical variables was investigated by correlation analysis. The association with radiographic damage and radiographic progression 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 extracellular regulated kinase 1/2 and c-Jun kinase, but not p38MAPK. Activation by 14-3-3η, using levels spanning the concentration range found in RA patient serum, resulted in the induction of inflammatory transcripts such as interleukin 1 (IL-1) and IL-6 and factors linked to the joint damage process, such as receptor activator of nuclear factor κB ligand and matrix metalloproteinase 1. Serum 14-3-3η correlated significantly with rheumatoid factor (RF) (r = 0.43) and anticitrullinated protein antibodies (ACPAs) (r = 0.31) in the early RA cohort, but not with C-reactive protein (CRP) or the Disease Activity Score in 28 joints in either cohort. Serum 14-3-3η concentrations were significantly higher in patients with radiographically assessed joint damage and in those who had radiographic progression. By multivariate analysis, we show that 14-3-3η complemented markers such as CRP, RF and ACPA in informing RA radiographic status and/or progression. CONCLUSIONS: Extracellular 14-3-3η activates key signalling cascades and induces factors associated with the pathogenesis of RA at concentrations found in patients with RA, and its expression is higher in patients with radiographic damage and RA progression.

Serum 14-3-3η is a novel marker that complements current serological measurements to enhance detection of patients with rheumatoid arthritis.

OBJECTIVE: Serum 14-3-3η is a novel joint-derived proinflammatory mediator implicated in the pathogenesis of rheumatoid arthritis (RA). In our study, we assessed the diagnostic utility of 14-3-3η and its association with standard clinical and serological measures. METHODS: A quantitative ELISA was used to assess 14-3-3η levels. Early (n=99) and established patients with RA (n=135) were compared to all controls (n=385), including healthy subjects (n=189). The sensitivity, specificity, positive and negative predictive values of 14-3-3η, and the likelihood ratios (LR) for RA were determined through receiver-operator curve analysis. The incremental value of adding 14-3-3η to anticitrullinated protein antibody (ACPA) and rheumatoid factor (RF) in diagnosing early and established RA was assessed. RESULTS: Serum 14-3-3η differentiated established patients with RA from healthy individuals and all controls (p<0.0001). A serum 14-3-3η cutoff of ≥0.19 ng/ml delivered a sensitivity and specificity of 77% and 93%, respectively, with corresponding LR positivity of 10.4. At this cutoff in early RA, 64% of patients with early RA were positive for 14-3-3η, with a corresponding specificity of 93% (LR+ of 8.6), while 59% and 57% were positive for ACPA or RF, respectively. When ACPA, RF, and 14-3-3η positivity were used in combination, 77 of the 99 patients (78%) with early RA were positive for any 1 of the 3 markers. Serum 14-3-3η did not correlate with C-reactive protein, erythrocyte sedimentation rate, or Disease Activity Score, but patients who were 14-3-3η-positive had significantly worse disease. CONCLUSION: Serum 14-3-3η is a novel RA mechanistic marker that is highly specific, associated with worse disease, and complements current markers, enabling a more accurate diagnosis of RA.

Dysregulation of phosphatidylinositol 3-kinase and downstream effectors in human breast cancer.

Phosphatidylinositol 3-kinase (PI3-K) is a growth factor-activated transforming lipid (and protein) kinase, involved in cell motility and invasion, that has multiple effectors. Relatively little is known about its expression and enzymatic activity in human breast cancer. Since growth factor receptors are amplified in breast cancer, and the tumor suppressor PTEN may be mutated in human breast cancer, it was hypothesized that PI3-K and its downstream effectors would be activated in this disease. In 11 resected tumors analyzed for expression of this kinase, a mean 3-fold increase in protein expression was observed over the corresponding adjacent control tissue. Using an in vitro lipid kinase assay of the immunoprecipitated PI3-K protein, a greater than 2-fold increase in activation was observed. These changes were observed in the absence of an activation of either protein kinase B (PKB, akt1) or p70 S6 kinase (p70 S6K). However, p21-activated kinase (Pak), p38 mitogen-activated protein kinase (p38 MAPK) and mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK 2) were all overexpressed and demonstrated increased enzyme activity. It may be concluded that aberrant mitogenic signaling in human breast cancer in vivo involves Pak, p38 MAPK and MAPKAPK2 downstream of PI3-K, but neither of PKB or p70 S6K. It is proposed that this pathway may serve as a useful targeting nexus for investigation of small molecule inhibitors in human breast cancer.

Oxidized low density lipoprotein inhibits macrophage apoptosis by blocking ceramide generation, thereby maintaining protein kinase B activation and Bcl-XL levels.

Macrophages play a central role in the development and progression of atherosclerotic lesions. It is well known that oxidized low density lipoprotein (ox-LDL) promotes the recruitment of monocytes (which differentiate to macrophages) into the intima. We reported recently that ox-LDL blocks apoptosis in bone marrow-derived macrophages deprived of macrophage colony-stimulating factor (M-CSF) by a mechanism involving protein kinase B (PKB) (Hundal, R., Salh, B., Schrader, J., Gómez-Muñoz, A., Duronio, V., and Steinbrecher, U. (2001) J. Lipid Res. 42, 1483-1491). The aims of the present study were 1) to define the apoptotic pathway involved in the pro-survival effect of ox-LDL; 2) to determine which PKB target mediated this effect; and 3) to identify mechanisms responsible for PKB activation by ox-LDL. Apoptosis following M-CSF withdrawal was accompanied by activation of the caspase 9-caspase 3 cascade and cytochrome c release from mitochondria, but the caspase 8 pathway was unaffected. M-CSF withdrawal resulted in a marked and selective reduction in Bcl-XL protein and mRNA levels, and this decrease was prevented by ox-LDL. The ability of ox-LDL to preserve Bcl-XL levels was blocked by NFkappaB antagonists, thereby implicating IkappaB kinase as a key PKB target. M-CSF deprivation resulted in activation of acid sphingomyelinase and an increase in ceramide levels. Desipramine (a sphingomyelinase inhibitor) prevented the increase in ceramide and inhibited apoptosis after M-CSF deprivation. Ox-LDL completely blocked the increase in acid sphingomyelinase activity as well as the increase in ceramide after M-CSF deprivation. Pretreatment of macrophages with C2-ceramide reversed the effect of ox-LDL on PKB and macrophage survival. These results indicate that ox-LDL prevents apoptosis in M-CSF-deprived macrophages at least in part by inhibiting acid sphingomyelinase. This in turn prevents ceramide-induced down-regulation of PKB, the activity of which is required to maintain production of Bcl-XL.