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Prolonged Activation of STAT3 Mediates the IL6‐Induced Loss of Stress Fibers and Increase in Endothelial Permeability

Interleukin 6 (IL6) promotes endothelial barrier breakdown in vitro, as well as, in vivo. However, the signaling mechanisms that mediate this response are not fully understood. IL6 trans‐signaling promotes a prolonged activation of JAK/STAT3 in human umbilical vein endothelial cells (HUVECs) that lasts for at least 24 hours, leading to a sustained increase in endothelial permeability. IL6‐induced STAT3 phosphorylation and loss of barrier function is prevented when cells are pre‐treated with the JAK inhibitor, ruxolitinib, suggesting that JAK activity is required for these responses. Furthermore, siRNA‐mediated STAT3 knockdown attenuates the loss of IL6‐induced loss of barrier function, demonstrating a critical role for STAT3 in this response. Immunofluorescence analysis showed that this change in barrier function correlates with a marked loss of junctional ZO‐1 (but not changes in ZO‐1 protein expression). Phalloidin staining demonstrates that IL‐6 also promotes a JAK, and STAT3, dependent loss of actin stress fibers and a marked reduction of the cortical actin signal. A prolonged activation of STAT3 is required for this response, because treatment of HUVECs with ruxolitinib up to 4 hours after activation of IL6 trans‐signaling, completely reverses the IL6‐induced loss of barrier function. STAT3 activation kinetics are regulated by a negative feedback loop that is mediated by the suppressor of cytokine signaling 3 (SOCS3). Consistently, siRNA‐mediated SOCS3 knockdown increases IL6‐induced STAT3 phosphorylation in HUVECs and exacerbates the IL6‐induced increase in permeability. Collectively, our data shows that prolonged activation of STAT3 is required for the IL6‐induced barrier breakdown, and loss of junctional ZO‐1, cortical actin and actin stress fibers in endothelial cells.