Background 14-3-3η is a novel therapeutic target that stimulates specific cell lines (THP-1), activates disease-specific pathways (MAPK) and potentiates pathogenic factors (TNFα, IL-6). Clinically, 14-3-3η serum expression is higher in both erosive RA and PsA, correlates strongly with both synovial and serum levels of metalloproteinases (MMP-1 & -3), and predicts likelihood of ACR50 response in PsA and Good EULAR response in RA patients receiving TNFα therapy. Extracellular 14-3-3η’s involvement in disease-specific processes together with its clinical expression underscores its potential as a personalized medicine drug target.
Objectives To further characterize 14-3-3η’s role in disease processes by examining its effects on signaling pathways targeted by small molecule compounds, its potency versus TNFα on inflammatory factors, and the effectiveness of 14-3-3η antibodies at blocking its effects.
Methods Phosphoproteomic profiling was conducted using THP-1 cells stimulated with either 14-3-3η or TNFα, at the same dose, to examine activation of intracellular pathways. The evaluation included 25 distinct phosphorylation sites on 20 different targets. Staying within the 14-3-3η clinical serum expression range (median 1.12ng/ml and range of 0.12 to 20ng/ml), 3 downstream targets (IL-8, IL-6 and TNFα) were selected to evaluate if increases in mRNA reflected increased protein expression. 14-3-3η’s potency on inflammatory factors (IL-6, IL-8, MCP-1) was compared to TNFα at an 18- and 48-hour end-point in human macrophages. Responsiveness of PBMC and monocyte donor cells to 14-3-3η stimulation was also evaluated. Dose escalation studies with 14-3-3η-specific monoclonal antibodies were performed using primary human monocyte and macrophage donor cells.
Results The table below summarizes the findings of the phosphoproteomic analysis. 76% of the targets had similar changes in their phosphorylation status when stimulated with either TNFα and 14-3-3η, whereas 24% exhibited clear differences.
Human PBMCs, monocytes and macrophages were responsive to 14-3-3η and its potency in inducing IL-6, IL-8 and TNFα as measured by mRNA analysis corresponded with changes in protein expression. In a comparison of 14-3-3η’s inducing effects to those of TNFα, similar degrees of induction were observed for levels of IL-8 and MCP-1. In contrast, 14-3-3η was a more potent inducer of IL-6. Antibody targeting studies demonstrate that the potentiating effects of 14-3-3η can be blocked in a dose-dependent manner with as little as 80ng/ml in vitro using both human monocyte and macrophage donors.
Conclusions 14-3-3η activates key intracellular signaling pathways that are targeted by small molecule compounds. It potently induces inflammatory pathways, especially IL-6, and its effects can be blocked with 14-3-3η-specific monoclonal antibodies. These findings, together with its association with disease expression in RA and PsA may guide in vivo evaluation and underscores 14-3-3η as a personalized medicine target.
Disclosure of Interest A. Marotta Shareholder of: Co-founder, R. Kilani: None Declared, A. Ghahary: None Declared, W. Maksymowych: None Declared
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