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bioRxiv · 10.1101/750323

Endothelial pannexin 1 channels control inflammation by regulating intracellular calcium

Yang, Y.·Delalio, L. J.·Best, A. K.·Macal, E. H.·Milstein, J. A.·Donnelly, I.·Miller, A. M.·McBride, M.·Shu, X. H.·Koval, M.·Isakson, B. E.·Johnstone, S. R.
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Abstract

In BriefInterleukine-1 beta (IL-1{beta}) has been identified as a critical factor that contributes to the inflammatory response in cardiovascular disease (e.g., atherosclerosis). Pannexin 1 (Panx1) channel activity in endothelial cells regulates localized inflammatory cell recruitment. In response to prolonged tumor necrosis factor alpha (TNF) treatment, Yang et al. found that the Panx1 channel is targeted to the plasma membrane, where it facilitates an increase in intracellular calcium to control the production and release of cytokines including IL-1{beta}.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC=\"FIGDIR/small/750323v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (46K):\norg.highwire.dtl.DTLVardef@59e72borg.highwire.dtl.DTLVardef@57bd5corg.highwire.dtl.DTLVardef@50ed73org.highwire.dtl.DTLVardef@1151f56_HPS_FORMAT_FIGEXP M_FIG C_FIG AbstractThe proinflammatory cytokine IL-1{beta} is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1{beta} expression and release are tightly controlled by changes in intracellular Ca2+. In addition, purinergic signaling through ATP release has also been reported to promote IL-1{beta} production. Despite this, the mechanisms that control IL-1{beta} synthesis and expression have not been identified. The pannexin 1 (Panx1) channel has canonically been implicated in ATP release, especially during inflammation. However, resolution of purinergic signaling occurs quickly due to blood flow and the presence of ectonucleotidases. We examined Panx1 in human endothelial cells following treatment with the pro-inflammatory cytokine tumor necrosis alpha (TNF). In response to long-term TNF treatment, we identified a dramatic increase in Panx1 protein expression at the plasma membrane. Analysis by whole transcriptome sequencing (RNA-seq), qPCR, and treatment with specific kinase inhibitors, revealed that TNF signaling induced NF{kappa}{beta}-associated Panx1 transcription. Genetic inhibition of Panx1 reduced the expression and secretion of IL-1{beta}. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL1-{beta} expression was not altered after direct ATP stimulation, following degradation of ATP by apyrase, or after pharmacological blockade of P2 receptors. These data suggest that non-purinergic pathways, involving Panx1, control IL-1{beta} production. Because Panx1 forms a large pore channel, we hypothesized it may act to passively diffuse Ca2+ into the cell upon opening to regulate IL-1{beta}. High-throughput flow cytometric analysis demonstrated that TNF treatments lead to elevated intracellular Ca2+. Genetic or pharmacological inhibition of Panx1 reduced TNF-associated increases in intracellular Ca2+, and IL-1{beta} transcription. Furthermore, we found that the Ca2+-sensitive NF{kappa}{beta}-p65 protein failed to localize to the nucleus after genetic or pharmacological block of Panx1. Taken together, our study provides the first evidence that intracellular Ca2+ regulation via the Panx1 channel induces a feed-forward effect on NF{kappa}{beta} to regulate IL-1{beta} synthesis and release in endothelium during inflammation.

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Yang, Y., Delalio, L. J., Best, A. K., Macal, E. H., Milstein, J. A., Donnelly, I., Miller, A. M., McBride, M., Shu, X. H., Koval, M., Isakson, B. E., Johnstone, S. R.. 2019-08-29. Endothelial pannexin 1 channels control inflammation by regulating intracellular calcium. https://doi.org/10.1101/750323

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