Endothelial pannexin 1 channels control inflammation by regulating intracellular calcium

Abstract

25inflammation, endothelial cell, smooth muscle cell 4 to prolonged tumor necrosis factor alpha (TNF) treatment, Yang et al. found that the Panx1 5 channel is targeted to the plasma membrane, where it facilitates an increase in intracellular 6 calcium to control the production and release of cytokines including IL-1β. 7 8 GRAPHICAL ABSTRACT 9 10 Graphical Abstract: The inflammatory process in endothelial cells is controlled by Ca 2+ entry through Pannexin 1 membrane channels. 3 Abstract 1The proinflammatory cytokine IL-1β is a significant risk factor in cardiovascular disease 2 that can be targeted to reduce major cardiovascular events. IL-1β expression and release 3 are tightly controlled by changes in intracellular Ca 2+ . In addition, purinergic signaling 4 through ATP release has also been reported to promote IL-1β production. Despite this, 5 the mechanisms that control IL-1β synthesis and expression have not been identified. 6The pannexin 1 (Panx1) channel has canonically been implicated in ATP release, 7 especially during inflammation. However, resolution of purinergic signaling occurs quickly 8 due to blood flow and the presence of ectonucleotidases. We examined Panx1 in human 9 endothelial cells following treatment with the pro-inflammatory cytokine tumor necrosis 10 alpha (TNF). In response to long-term TNF treatment, we identified a dramatic increase 11 in Panx1 protein expression at the plasma membrane. Analysis by whole transcriptome 12 sequencing (RNA-seq), qPCR, and treatment with specific kinase inhibitors, revealed that 13 TNF signaling induced NFκβ-associated Panx1 transcription. Genetic inhibition of Panx1 14 reduced the expression and secretion of IL-1β. We initially hypothesized that increased 15Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. 16However, our data demonstrate that IL1-β expression was not altered after direct ATP 17 stimulation, following degradation of ATP by apyrase, or after pharmacological blockade 18 of P2 receptors. These data suggest that non-purinergic pathways, involving Panx1, 19 control IL-1β production. Because Panx1 forms a large pore channel, we hypothesized it 20 may act to passively diffuse Ca 2+ into the cell upon opening to regulate IL-1β. High-21 throughput flow cytometric analysis demonstrated that TNF treatments lead to elevated 22 intracellular Ca 2+ . Genetic or pharmacological inhibition of Panx1 reduced TNF-23 associated increases in intracellular Ca 2+ , and IL-1β transcription. Furthermore, we found 24 that the Ca 2+ -sensitive NFκβ-p65 protein failed to localize to the nucleus after genetic or 25 pharmacological block of Panx1. Taken together, our study provides the first evidence 26 that intracellular Ca 2+ regulation via the Panx1 channel induces a feed-forward effect on 27NFκβ to regulate IL-1β synthesis and release in endothelium during inflammation. 28 4 (Tumor Necrosis Factor)-Triggered Smooth Muscle Cell Inflammation and Attenuates

Consideration of Pannexin 1 channels in COVID-19 pathology and treatment

“…There are several lines of evidence demonstrating that PANX1 channel opening (and release of ATP) enhances inflammatory responses, including in the systemic endothelium (lung microvasculature), lung epithelium, olfactory epithelium, and the parenchyma of several tissues throughout the body ( 13 , 17 , 22 , 29 , 31 , 35 , 51 ). Multiple studies have shown that PANX1 signaling exacerbates inflammatory responses through: being activated and enhanced by TNFα-receptor signaling, being implicated in the inflammasome, involvement in leukocyte recruitment, and playing a role in the production and secretion of proinflammatory cytokines such as IL-1β and IL-6 by endothelial cells and other cell types ( 12 , 22 , 24 , 35 , 41 , 51 , 63 ). Given that disruption (both deletion and inhibition) of PANX1 in endothelial cells significantly reduces inflammation in several injury models ( 22 , 28 , 51 , 59 ), PANX1 represents a potential target in reducing inflammatory burden and the damaging effects of the cytokine storm in COVID-19 patients.…”

“…Finally, if PANX1 is found to be involved in the primary regulation of SARS-CoV-2 infectivity and inflammatory responses, it may also be worth considering direct targeting of the channel functions. Currently there are no FDA-approved PANX1-specific blockers, although a PANX1-specific inhibitor peptide, pannexin intracellular loop 2 peptide (PxIL2P), has shown promise in reducing inflammatory responses in vitro and in vivo ( 35 , 63 ). PxIL2P contains a short mimetic sequence for the IL2 region of PANX1 attached to an HIV-TAT transactivation protein ( 5 ) that binds to the second intracellular loop of PANX1 and blocks channel release of ATP, altering intracellular Ca 2+ flux ( 35 , 63 ).…”

“…Currently there are no FDA-approved PANX1-specific blockers, although a PANX1-specific inhibitor peptide, pannexin intracellular loop 2 peptide (PxIL2P), has shown promise in reducing inflammatory responses in vitro and in vivo ( 35 , 63 ). PxIL2P contains a short mimetic sequence for the IL2 region of PANX1 attached to an HIV-TAT transactivation protein ( 5 ) that binds to the second intracellular loop of PANX1 and blocks channel release of ATP, altering intracellular Ca 2+ flux ( 35 , 63 ). Using PxIL2P in cultured endothelial cells blocks PANX1-regulated expression and release of cytokines including IL-1β and CxCL10 and limits monocyte adhesion in the vasculature ( 35 , 63 ).…”

“…PxIL2P contains a short mimetic sequence for the IL2 region of PANX1 attached to an HIV-TAT transactivation protein ( 5 ) that binds to the second intracellular loop of PANX1 and blocks channel release of ATP, altering intracellular Ca 2+ flux ( 35 , 63 ). Using PxIL2P in cultured endothelial cells blocks PANX1-regulated expression and release of cytokines including IL-1β and CxCL10 and limits monocyte adhesion in the vasculature ( 35 , 63 ). Thus direct targeting of the PANX1 channel may have functionality in reducing SARS-CoV-2 infectivity and vascular inflammatory responses in COVID19 patients.…”

P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases

“…Additionally, these inflammatory conditions promoted VSMC proliferation, resulting in neo-intimal thickening, hyperplasia of VSMCs, and monocyte adhesion to EC. P2Y2 activity amplifies the release of IL-1 by a mechanism involving NFκB and Panx1 channels [ 65 ]. While overall P2Y2 effects are pro-inflammatory, it can initiate a negative feedback mechanism, upon which it down-regulates the ICAM-1 to limit inflammation in a miR-22 dependent manner [ 66 ].…”