Intracellular RIG-I Signaling Regulates TLR4-Independent Endothelial Inflammatory Responses to Endotoxin.

Sepsis is a systemic inflammatory response to infections associated with organ failure that is the most frequent cause of death in hospitalized patients. Exaggerated endothelial activation, altered blood flow, vascular leakage, and other disturbances synergistically contribute to sepsis-induced organ failure. The underlying signaling events associated with endothelial proinflammatory activation are not well understood, yet they likely consist of molecular pathways that act in an endothelium-specific manner. We found that LPS, a critical factor in the pathogenesis of sepsis, is internalized by endothelial cells, leading to intracellular signaling without the need for priming as found recently in immune cells. By identifying a novel role for retinoic acid-inducible gene-I (RIG-I) as a central regulator of endothelial activation functioning independent of TLR4, we provide evidence that the current paradigm of TLR4 solely being responsible for LPS-mediated endothelial responses is incomplete. RIG-I, as well as the adaptor protein mitochondrial antiviral signaling protein, regulates NF-κB-mediated induction of adhesion molecules and proinflammatory cytokine expression in response to LPS. Our findings provide essential new insights into the proinflammatory signaling pathways in endothelial cells and suggest that combined endothelial-specific inhibition of RIG-I and TLR4 will provide protection from aberrant endothelial responses associated with sepsis.

Functional Validation of the Putative Oncogenic Activity of PLAU.

Plasminogen activator, urokinase (PLAU) is involved in cell migration, proliferation and tissue remodeling. PLAU upregulation is associated with an increase in aggressiveness, metastasis, and invasion of several cancer types, including breast cancer. In patients, this translates into decreased sensitivity to hormonal treatment, and poor prognosis. These clinical findings have led to the examination of PLAU as a biomarker for predicting breast cancer prognosis and therapy responses. In this study, we investigated the functional ability of PLAU to act as an oncogene in breast cancers by modulating its expression using CRISPR-deactivated Cas9 (CRISPR-dCas9) tools. Different effector domains (e.g., transcription modulators (VP64, KRAB)) alone or in combination with epigenetic writers (DNMT3A/3L, MSssI) were fused to dCas9 and targeted to the PLAU promoter. In MDA-MB-231 cells characterized by high PLAU expression downregulation of PLAU expression by CRISPR-dCas9-DNMT3A/3L-KRAB, resulted in decreased cell proliferation. Conversely, CRISPR-dCas9-VP64 induced PLAU upregulation in low PLAU expressing MCF-7 cells and significantly increased aggressiveness and invasion. In conclusion, modulation of PLAU expression affected metastatic related properties of breast cancer cells, thus further validating its oncogenic activity in breast cancer cells.

Endothelial Interferon Regulatory Factor 1 Regulates Lipopolysaccharide-Induced VCAM-1 Expression Independent of NFκB.

Sepsis is a severe systemic inflammatory response to infection. Endothelial activation and dysfunction play a critical role in the pathophysiology of sepsis and represent an important therapeutic target to reduce sepsis mortality. Interferon regulatory factor 1 (IRF-1) was recently identified as a downstream target of TNF-α-mediated signal transduction in endothelial cells. The aim of this study was to explore the importance of IRF-1 as a regulator of lipopolysaccharide (LPS)-induced endothelial proinflammatory activation. We found that renal IRF-1 was upregulated by LPS in vivo as well as in LPS-stimulated endothelial cells in vitro. Furthermore, we identified intracellular retinoic acid inducible gene-I (RIG-I) as a regulator of LPS-mediated IRF-1 induction. IRF-1 depletion specifically resulted in diminished induction of VCAM-1 in response to LPS, but not of E-selectin or ICAM-1, which was independent of NFκB signaling. When both IRF-1 and the RIG-I adapter protein mitochondrial antiviral signaling (MAVS) were absent, VCAM-1 induction was not additionally inhibited, suggesting that MAVS and IRF-1 reside in the same signaling pathway. Surprisingly, E-selectin and IL-6 induction were no longer inhibited by MAVS knockdown when IRF-1 was also absent, revealing a redundant endothelial activation pathway. In summary, we report an IRF-1-mediated proinflammatory signaling pathway that specifically regulates LPS-mediated VCAM-1 expression, independent of NFκB.