Abstract 11462: A Surprising Role of TNIK in Regulating Endothelial Interferon Signaling and Activation

Abstract

Background: Endothelial cells (ECs) predominantly govern vascular tone and homeostasis. Activation of ECs is the central pathophysiological process in the initiation of atherosclerosis (AS). Interferon signaling (IFN Type I and II) is activated in ECs under inflammatory conditions such as systemic lupus erythematosus and is implicated in EC activation. Yet, the underlying mechanisms are still unknown. Through RNA sequencing and pathway analysis we have identified, TNIK, a Ste20 family of MAP kinases (MAP4K) to regulate IFN signaling in ECs. Our study is the first to report the role and function of TNIK in IFN and EC activation. Hypothesis: TNIK regulates IFNs through focal adhesion kinase (FAK) mediated STAT signaling in EC activation (Fig 1A). Methods: Cultured human aortic endothelial cells (HAECs) were treated with control or TNIK-targeted siRNA. Later cells were subjected to static, laminar or disturbed flow conditions. Total RNA was isolated and then RNA-sequencing was performed. The differential expression data were analyzed using CLC genomics and Ingenuity Pathway Analysis (IPA). Western blotting and qRT-PCR were performed to validate STAT activation and interferon-stimulated genes (ISGs) Results: We found >450 genes differentially expressed in siTNIK vs siCTRL treated ECs (fold change>2, FDR<0.05). IPA revealed that under static (ST) and disturbed flow (DF) conditions TNIK depletion led to inhibition IFN (-log P-value >11) and upregulation of eukaryotic transcription elongation factor signaling (eIF2, -log P-value >7) (Fig 1B, C). In addition, under DF, TNIK depletion led to downregulation of hypercytokinemia/hyperchemokinemia (-log P-value >8, Fig 1B). Validation through western blotting and qRT-PCR (Fig 1D, E) confirmed that TNIK regulates FAK and STAT phosphorylation and IFN stimulated gene targets. Conclusions: Our study elucidates the unique role of TNIK in regulating FAK-STAT mediated IFN signaling and subsequently EC activation.