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Pannexin 1 phosphorylation sites differentially modulate channel activity and physiological outcomes

ABSTRACT: Within the vasculature, pannexin 1 (PANX1) channels in smooth muscle cells (SMCs) regulate α-adrenergic constriction and blood pressure. PANX1 channel activity is regulated by phosphorylation at Y198, S205 and Y308 residues, but the physiological significance of these modifications is unknown. Here we utilize newly developed PANX1 Y198F, S205A and Y308F phospho-dead mutant mice to test physiological changes related to hemodynamics. Radiotelemetry-measured blood pressure was decreased in Y198F, increased in Y308F, but unchanged in S205A mice at baseline. During sympathetic-driven hypertension, only Y198F mice showed decreases in blood pressure, with reductions resembling control mice treated with the sympathetic inhibitor clonidine. Pressure myography revealed α-adrenergic contractile responses were decreased in Y198F, slightly enhanced in Y308F, but unchanged in S205A third-order mesenteric arteries; with Y198F responses mimicking control vessels treated with PANX1 inhibitors. To understand signaling changes driving these phenotypes, we performed mesenteric artery bulk RNA sequencing but found a minimal number of differentially expressed genes between phospho-dead mutant and controls. Similarly, co-immunoprecipitation-mass spectrometry of wildtype or phospho-dead mutant-expressing vascular SMCs revealed few interacting proteins distinct to each PANX1 variant. However, PANX1 channel activity assessments in HEK293T cells expressing the α1D-adrenergic receptor and each phospho-dead mutant PANX1 showed phenylephrine-induced ATP release from Y198F channels was significantly decreased compared to wildtype, but current was unaffected. Conversely, S205A and Y308F ion flux was reduced, with ATP release resembling controls. Taken together, these findings demonstrate that physiological outcomes are regulated by distinct PANX1 metabolite versus current conducting properties, which are ultimately controlled by PANX1 phosphorylation.

ORGANISM(S): Mus musculus

PROVIDER: GSE335251 | GEO | 2026/07/12

REPOSITORIES: GEO

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Project description:Glioblastoma (GBM) is the most common primary brain tumour, with a median survival of 12-18 months, highlighting a need for new treatment targets. We observed that pannexin 1 (PANX1), a channel-forming glycoprotein important in purinergic signalling, is upregulated in GBM compared to normal tissue and expressed throughout patient tumours. Western blot analysis of patient-derived GBM cell lines revealed significantly increased PANX1 expression in these primary lines compared to brain tissue and control glial cells. Immunofluorescent staining indicated mostly intracellular localization of PANX1 in GBM cells. Bulk RNA-sequencing compared the gene expression of GBM cells devoid of PANX1 via CRISPR/Cas9 deletion (PANX1-KO) compared to controls. Gene Ontology and KEGG gene set analyses revealed PANX1-KO in GBM cells affects cell surface and cell junction components, processes, and pathways, including the HIPPO pathway, in addition to critically downregulating β-catenin mRNA and other components of the Wnt pathway. The deletion of PANX1 resulted in a disruption of the β-catenin protein and a dramatic reduction in migration and cell growth. Pharmacological inhibition of PANX1 in GBM cells with Probenecid (PBN) and Spironolactone (SPIR) demonstrated a significant reduction in live cell numbers and migration via scratch assay. The intracellular PANX1 staining was unaffected by channel inhibition; however, both blockers dramatically decreased F-actin filament formation. PANX1 and b-actin protein levels remained unchanged with blocker treatment, but the cellular localization of b-catenin became more intracellular compared to controls upon drug treatments. Xenografted GBM tumours grown on a chick chorioallantoic membrane model showed a reduction in tumour cell viability by bioluminescent imaging and reduced hemorrhaging incidence when treated with PBN. PBN-treated tumours had reduced pro-inflammatory IL-8 mRNA expression, a cytokine involved in vascular leakage. We provide evidence that PANX1 is upregulated in GBM, and PANX1 inhibition or deletion reduces several tumorigenic properties of GBM cells across multiple model systems. These new insights support further investigation of PANX1 as a potential GBM therapeutic target and its role in multiple cancer signaling pathways that regulate this devastating disease.

Project description:BACKGROUND Enteric glia contribute to the pathophysiology of various intestinal immune-driven diseases, such as postoperative ileus (POI), a motility disorder and common complication after abdominal surgery. Enteric gliosis of the intestinal muscularis externa (ME) has been identified as part of POI development. However, the glia-restricted responses and activation mechanisms are poorly understood. The sympathetic nervous system becomes rapidly activated by abdominal surgery. It modulates intestinal immunity, innervates all intestinal layers, and directly interfaces with enteric glia. We hypothesized that sympathetic innervation controls enteric glia reactivity in response to surgical trauma. METHODS Sox10iCreERT2/Rpl22HA/+ mice were subjected to a mouse model of laparotomy or intestinal manipulation to induce POI. Histological, protein, and transcriptomic analyses were performed to analyze glia-specific responses. Interactions between the sympathetic nervous system and enteric glia were studied in mice chemically depleted of TH+ sympathetic neurons and glial-restricted Sox10iCreERT2/JellyOPfl/+/Rpl22HA/+ mice, allowing optogenetic stimulation of β-adrenergic downstream signaling and glial-specific transcriptome analyses. A laparotomy model was used to study the effect of sympathetic signaling on enteric glia in the absence of intestinal manipulation. Mechanistic studies included adrenergic receptor expression profiling in vivo and in vitro and adrenergic agonism treatments of primary enteric glial cell cultures to elucidate the role of sympathetic signaling in acute enteric gliosis and POI. RESULTS With ~4000 differentially expressed genes, the most substantial enteric glia response occurs early after intestinal manipulation. During POI, enteric glia switch into a reactive state and continuously shape their microenvironment by releasing inflammatory and migratory factors. Sympathetic denervation reduced the inflammatory response of enteric glia in the early postoperative phase. Optogenetic and pharmacological stimulation of β-adrenergic downstream signaling triggered enteric glia reactivity. Finally, distinct adrenergic agonists revealed β-1/2 adrenoceptors as the molecular targets of sympathetic–driven enteric glial reactivity. CONCLUSIONS Enteric glia act as early responders during post-traumatic intestinal injury and inflammation. Intact sympathetic innervation and active β-adrenergic receptor signaling in enteric glia is a trigger of the immediate glial postoperative inflammatory response. With immune-activating cues originating from the sympathetic nervous system as early as the initial surgical incision, adrenergic signaling in enteric glia presents a promising target for preventing POI development.

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Pannexin 1 phosphorylation sites differentially modulate channel activity and physiological outcomes

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