Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium

Abstract

<p class="first" id="P3">The proinflammatory cytokine IL-1β is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1β expression and release are tightly controlled by changes in intracellular Ca ²⁺ [Ca ²⁺] _i which has been associated with ATP release and purinergic signaling. Despite this, the mechanisms that regulate these changes have not been identified. The pannexin 1 (Panx1) channels have canonically been implicated in ATP release, especially during inflammation. We examined Panx1 in human umbilical vein endothelial cells (HUVECs) following treatment with the pro-inflammatory cytokine tumor necrosis alpha (TNF). Analysis by whole transcriptome sequencing (RNA-seq) and immunoblot, identified a dramatic increase in Panx1 mRNA and protein expression that is regulated in an NFκβ-dependent manner. Furthermore, genetic inhibition of Panx1 reduced the expression and secretion of IL-1β. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL-1β expression was not altered after direct ATP stimulation in HUVECs. Because Panx1 forms a large pore channel, we hypothesized it may permit Ca ²⁺ diffusion into the cell to regulate IL-1β. High-throughput flow cytometric analysis demonstrated that TNF treatments lead to elevated [Ca ²⁺] _i corresponding with Panx1 membrane localization. Genetic or pharmacological inhibition of Panx1 reduced TNF-associated increases in [Ca ²⁺] _i, blocked phosphorylation of the NFκβ-p65 protein and reduced IL-1β transcription. Taken together, our study provides the first evidence that [Ca ²⁺] _i regulation via the Panx1 channel induces a feed-forward effect on NFκβ to regulate IL-1β synthesis and release in endothelium during inflammation. </p>