Pannexin Research Articles

Pannexin 1 and pannexin 3 differentially regulate the cancer cell properties of cutaneous squamous cell carcinoma.

Pannexin (PANX) channels are present in skin and facilitate the movement of signalling molecules during cellular communication. PANX1 and PANX3 function in skin homeostasis and keratinocyte differentiation but were previously reduced in a small cohort of human cutaneous squamous cell carcinoma (cSCC) tumours compared to normal epidermis. In our study we used SCC-13 cells, limited publicly available RNA-seq data and a larger cohort of cSCC patient-matched samples to analyse PANX1 and PANX3 expression and determine the association between their dysregulation and the malignant properties of cSCC. In a bioinformatics analysis, PANX1 transcripts were increased in cSCC and head and neck SCC tumours compared to normal tissues, but PANX3mRNA showed no differences. However, in our own cohort PANX3 transcripts were decreased in cSCC compared to patient-matched aged skin, whereas PANX1 protein was upregulated in cSCC. PANX1 localized to all regions within the cSCC tumour microenvironment, and increased levels were associated with larger tumour dimensions. To investigate PANX1 function in SCC-13 cells, we deleted PANX1 via CRISPR/Cas9 and treated with PANX1 inhibitors, which markedly reduced cell growth and migration. To assess PANX3 function in cutaneous carcinogenesis, we employed the 7,12-dimethylbenz(a)anthracene/12-otetradecanoylphorbol-13-acetate (DMBA/TPA) model using our global Panx3 knockout (KO) mice, where 60% of wild-type and 100% of KO mice formed precancerous papillomas. Average papilloma volumes at endpoint were significantly increased in KO mice and showed moderate evidence of increases in KO mice over time. Collectively, these findings suggest PANX1 and PANX3 dysregulation may have potential tumour-promoting and tumour-suppressive effects for keratinocyte transformation, respectively. KEY POINTS: Pannexin 1 (PANX1) and pannexin 3 (PANX3) are channel-forming proteins which are critical in the normal maintenance and function of keratinocytes in the skin but may become altered in cutaneous squamous cell carcinoma (cSCC) tumours. In this study we used a combination of culture models, mouse models and patient-derived tissues. We found PANX1 levels are increased in cSCC tumours and present in all tumour regions, functioning to promote cSCC cell growth and migration. Conversely, PANX3 levels are decreased in cSCC tumours, and this protein reduces the incidence and growth of precancerous lesions. Taken together our data indicate that in cSCC these pannexin family members seem to have opposite effects, in either promoting or restricting cancer cell properties. These results help us to better understand the mechanisms of malignant transformation of keratinocytes and offer a new potential therapeutic target for the treatment of advanced cSCC.

Pannexin-1 regulation of ATP release promotes the invasion of pituitary adenoma.

Pannexin-1 (PANX1) channel participates in the development and progression of many tumor types, however, its role of PANX1 in invasive pituitary adenoma (PA) remains unknown. The current study was designed to investigate the role of PANX1 in invasion of PA. We examined the expression of PANX1 in 116 surgical invasion and non-invasion PA samples (60 for bulk transcriptome and 56 for immunohistochemistry). The effects of PANX1 on PA growth were assessed in vitro and xenograft models. Meanwhile, the metabolism changes of PA cells are explored via transcriptomics and metabolomics using integration strategy. PANX1 is significantly upregulated in invasive PA compared with noninvasive PA and pituitary gland, and have a potential diagnostic signature for invasive PA. Accordingly, overexpression of PANX1 could promote the proliferation and invasion of GH3 and MMQ cell lines in vitro and in vivo. Further metabolomics results confirme that overexpression of PANX1 could trigger changes in several metabolic pathways of GH3 cells. Among the dysregulated cellular metabolites, decreased intracellular ATP suggeste that PANX1 may promote the invasion of PA through impacting extracellular ATP concentration. Mechanistically, extracellular ATP might promote Ca2+ influx and upregulated the expression of MMP2/9 by activating P2X7R. Additionally, PANX1-ATP-P2 X7R signaling pathway might enhance GH3 cell invasion by remodeling the actin cytoskeleton. Our findings point to a pivotal role of PANX1 in promoting PA invasion, which indicated a potential therapeutic target for invasive PA.

Research progress on biomarkers of sepsis-associated acute kidney injury

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis-associated acute kidney injury (SA-AKI) is a common organ dysfunction of sepsis, and its incidence and mortality are increasing,which brings heavy economic burden to patients and society. Early diagnosis and effective intervention can block the occurrence and progression of SA-AKI effectively, improve prognosis, and reduce medical costs. Diagnosis on SA-AKI relies on urine volume and serum creatinine, which has the disadvantages of being easily disturbed and delaying. The identification of biomarkers in blood and urine can facilitate diagnosis and provide targeted therapy to enhance the management of SA-AKI. This article reviews the characteristics of a variety of SA-AKI biomarkers that have been found and validated, including pre-damage biomarkers, damage biomarkers and functional biomarkers, and explore the clinical value of newly discovered biomarkers related to the diagnosis and treatment of SA-AKI, such as blood uncoupling protein 2 (UCP2), Sestrin 2 protein and pannexin 1 (PANX1), to provide reference for the early diagnosis and effective treatment of SA-AKI.

Dual role for pannexin 1 at synapses: regulating functional and morphological plasticity.

Pannexin 1 (PANX1) is an ion and metabolite membrane channel and scaffold protein enriched in synaptic compartments of neurons in the central nervous system. In addition to a well-established link between PANX1 and synaptic plasticity, we recently identified a role for PANX1 in the regulation of dendritic spine stability. Notably, PANX1 and its interacting proteins are linked to neurological conditions involving dendritic spine loss. Understanding the dual role of PANX1 in synaptic function and morphology may help to shed light on these links. We explore potential mechanisms, including PANX1's interactions with postsynaptic receptors and cytoskeleton regulating proteins. Finally, we contextualize PANX1's dual role within neurological diseases involving dendritic spine and synapse dysfunction.

Novel Pannexin 1 isoform is increased in cancer.

Pannexin 1 (PANX1) is upregulated in many cancers, where its activity and signalling promote tumorigenic properties. Here, we report a novel ∼25 kDa isoform of human PANX1 (hPANX1-25K) which lacks the N-terminus and was detected in several human cancer cell lines including melanoma, osteosarcoma, breast cancer and glioblastoma multiforme. This isoform was increased upon hPANX1 CRISPR/Cas9 deletion targeting the first exon near M1, suggesting a potential alternative translation initiation (ATI) site. hPANX1-25K was confirmed to be a hPANX1 isoform via mass spectrometry, can be N-linked glycosylated at N254, and can interact with both β-catenin and full length hPANX1. A double deletion of hPANX1 and hPANX1-25K reduces cell growth and viability in cancer cells. hPANX1-25K is prevalent throughout melanoma progression, and its levels are increased in squamous cell carcinoma cells and patient-derived tumours, compared to keratinocytes and normal skin, indicating that it may be differentially regulated in normal and cancer cells.

Pannexin 1 crosstalk with the Hippo pathway in malignant melanoma.

In this study, we explored the intricate relationship between Pannexin 1 (PANX1) and the Hippo signaling pathway effector, Yes-associated protein (YAP). Analysis of The Cancer Genome Atlas (TCGA) data revealed a significant positive correlation between PANX1 mRNA and core Hippo components, YAP, TAZ, and Hippo scaffold, IQGAP1, in invasive cutaneous melanoma and breast carcinoma. Furthermore, we demonstrated that PANX1 expression is upregulated in invasive melanoma cell lines and is associated with increased YAP protein levels. Notably, our investigations uncovered a previously unrecognized interaction between endogenous PANX1 and the Hippo scaffold protein IQGAP1 in melanoma cells. Moreover, our findings revealed that IQGAP1 exhibits differential expression in melanoma cells and plays a regulatory role in cellular morphology. Functional studies involving PANX1 knockdown provided compelling evidence that PANX1 modulates YAP protein levels and its co-transcriptional activity in both melanoma and breast carcinoma cells. Importantly, our study showcases the potential therapeutic relevance of targeting PANX1, as pharmacological inhibition of PANX1 using selective FDA-approved inhibitors or PANX1 knockdown reduced YAP abundance in melanoma cells. Furthermore, our Clariom™ S analysis unveiled key genes implicated in cell proliferation, such as neuroglin1 (NRG1), β-galactoside binding protein, galectin-3 (LGALS3), that are affected in PANX1-deficient cells. In summary, our investigation delves into the intricate interplay between PANX1 and YAP in the context of invasive melanoma, offering valuable insights into potential therapeutic strategies for effective treatment.

A novel heterozygous missense variant of PANX1 causes human oocyte death and female infertility

Pannexin1 (PANX1) is a highly glycosylated membrane channel-forming protein, which has been found to implicate in multiple physiological and pathophysiological functions. Variants in the PANX1 gene have been reported to be associated with oocyte death and recurrent in vitro fertilization failure. In this study, we identified a novel heterozygous PANX1 variant (NM_015368.4 c.410 C > T (p.Ser137Leu)) associated with the phenotype of oocyte death in a non-consanguineous family, followed by an autosomal dominant (AD) mode. We explored the molecular mechanism of the novel variant and the variant c.976_978del (p.Asn326del) that we reported previously. Both of the variants altered the PANX1 glycosylation pattern in cultured cells, led to aberrant PANX1 channel activation, affected ATP release and membrane electrophysiological properties, which resulted in mouse and human oocyte death in vitro. For the first time, we presented the direct evidence of the effect of the PANX1 variants on human oocyte development. Our findings expand the variant spectrum of PANX1 genes associated with oocyte death and provide new support for the genetic diagnosis of female infertility.

Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy.

PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1MyHC6). PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 (H9c2 rat myoblast cell line) cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45+), particularly neutrophils (CD11b+ [integrin subunit alpha M], Ly6g+ [lymphocyte antigen 6 family member G]), to the myocardium. Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure.

PANX1-mediated ATP release confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis

BackgroundExtracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet β cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed.Methodsdb/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein–protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays.ResultsIn livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers.ConclusionsPANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A’s suppressive effects on hepatic gluconeogenesis and lipogenesis.

Hyperactivation of SREBP induces pannexin-1-dependent lytic cell death

Sterol-regulatory element binding proteins (SREBPs) are a conserved transcription factor family governing lipid metabolism. When cellular cholesterol level is low, SREBP2 is transported from the endoplasmic reticulum to the Golgi apparatus where it undergoes proteolytic activation to generate a soluble N-terminal fragment, which drives the expression of lipid biosynthetic genes. Malfunctional SREBP activation is associated with various metabolic abnormalities. In this study, we find that overexpression of the active nuclear form SREBP2 (nSREBP2) causes caspase-dependent lytic cell death in various types of cells. These cells display typical pyroptotic and necrotic signatures, including plasma membrane ballooning and release of cellular contents. However, this phenotype is independent of the gasdermin family proteins or mixed lineage kinase domain-like (MLKL). Transcriptomic analysis identifies that nSREBP2 induces expression of p73, which further activates caspases. Through whole-genome CRISPR-Cas9 screening, we find that Pannexin-1 (PANX1) acts downstream of caspases to promote membrane rupture. Caspase-3 or 7 cleaves PANX1 at the C-terminal tail and increases permeability. Inhibition of the pore-forming activity of PANX1 alleviates lytic cell death. PANX1 can mediate gasdermins and MLKL-independent cell lysis during TNF-induced or chemotherapeutic reagents (doxorubicin or cisplatin)-induced cell death. Together, this study uncovers a noncanonical function of SREBPs as a potentiator of programmed cell death and suggests that PANX1 can directly promote lytic cell death independent of gasdermins and MLKL.

Human pannexin 1 channel is not phosphorylated by Src tyrosine kinase at Tyr199 and Tyr309.

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above-which were used to identify endogenous PANX1 phosphorylation at these two sites-are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Human pannexin 1 channel is not phosphorylated by Src tyrosine kinase at Tyr199 and Tyr309

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above—which were used to identify endogenous PANX1 phosphorylation at these two sites—are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

The small molecule raptinal can simultaneously induce apoptosis and inhibit PANX1 activity

Discovery of new small molecules that can activate distinct programmed cell death pathway is of significant interest as a research tool and for the development of novel therapeutics for pathological conditions such as cancer and infectious diseases. The small molecule raptinal was discovered as a pro-apoptotic compound that can rapidly trigger apoptosis by promoting the release of cytochrome c from the mitochondria and subsequently activating the intrinsic apoptotic pathway. As raptinal is very effective at inducing apoptosis in a variety of different cell types in vitro and in vivo, it has been used in many studies investigating cell death as well as the clearance of dying cells. While examining raptinal as an apoptosis inducer, we unexpectedly identified that in addition to its pro-apoptotic activities, raptinal can also inhibit the activity of caspase-activated Pannexin 1 (PANX1), a ubiquitously expressed transmembrane channel that regulates many cell death-associated processes. By implementing numerous biochemical, cell biological and electrophysiological approaches, we discovered that raptinal can simultaneously induce apoptosis and inhibit PANX1 activity. Surprisingly, raptinal was found to inhibit cleavage-activated PANX1 via a mechanism distinct to other well-described PANX1 inhibitors such as carbenoxolone and trovafloxacin. Furthermore, raptinal also interfered with PANX1-regulated apoptotic processes including the release of the ‘find-me’ signal ATP, the formation of apoptotic cell-derived extracellular vesicles, as well as NLRP3 inflammasome activation. Taken together, these data identify raptinal as the first compound that can simultaneously induce apoptosis and inhibit PANX1 channels. This has broad implications for the use of raptinal in cell death studies as well as in the development new PANX1 inhibitors.

Global pannexin 1 deletion increases tumor-infiltrating lymphocytes in the BRAF/Pten mouse melanoma model.

Immunotherapies for malignant melanoma seek to boost the anti-tumoral response of CD8+ T cells, but have a limited patient response rate, in part due to limited tumoral immune cell infiltration. Genetic or pharmacological inhibition of the pannexin 1 (PANX1) channel-forming protein is known to decrease melanoma cell tumorigenic properties in vitro and ex vivo. Here, we crossed Panx1 knockout (Panx1-/-) mice with the inducible melanoma model BrafCA, PtenloxP, Tyr::CreERT2 (BPC). We found that deleting the Panx1 gene in mice does not reduce BRAF(V600E)/Pten-driven primary tumor formation or improve survival. However, tumors in BPC-Panx1-/- mice exhibited a significant increase in the infiltration of CD8+ T lymphocytes, with no changes in the expression of early T-cell activation marker CD69, lymphocyte activation gene 3 protein (LAG-3) checkpoint receptor, or programmed cell death ligand-1 (PD-L1) in tumors when compared to the BPC-Panx1+/+ genotype. Our results suggest that, although Panx1 deletion does not overturn the aggressive BRAF/Pten-driven melanoma progression in vivo, it does increase the infiltration of effector immune T-cell populations in the tumor microenvironment. We propose that PANX1-targeted therapy could be explored as a strategy to increase tumor-infiltrating lymphocytes to boost anti-tumor immunity.

Apically localized PANX1 impacts neuroepithelial expansion in human cerebral organoids

Dysfunctional paracrine signaling through Pannexin 1 (PANX1) channels is linked to several adult neurological pathologies and emerging evidence suggests that PANX1 plays an important role in human brain development. It remains unclear how early PANX1 influences brain development, or how loss of PANX1 alters the developing human brain. Using a cerebral organoid model of early human brain development, we find that PANX1 is expressed at all stages of organoid development from neural induction through to neuroepithelial expansion and maturation. Interestingly, PANX1 cellular distribution and subcellular localization changes dramatically throughout cerebral organoid development. During neural induction, PANX1 becomes concentrated at the apical membrane domain of neural rosettes where it co-localizes with several apical membrane adhesion molecules. During neuroepithelial expansion, PANX1−/− organoids are significantly smaller than control and exhibit significant gene expression changes related to cell adhesion, WNT signaling and non-coding RNAs. As cerebral organoids mature, PANX1 expression is significantly upregulated and is primarily localized to neuronal populations outside of the ventricular-like zones. Ultimately, PANX1 protein can be detected in all layers of a 21–22 post conception week human fetal cerebral cortex. Together, these results show that PANX1 is dynamically expressed by numerous cell types throughout embryonic and early fetal stages of human corticogenesis and loss of PANX1 compromises neuroepithelial expansion due to dysregulation of cell-cell and cell-matrix adhesion, perturbed intracellular signaling, and changes to gene regulation.

Propofol Triggers Cell Death in Lung Cancer Cells by Increasing PANX1 Expression, Activating the Mitochondrial Cell Death Pathway, and Enhancing ROS Levels.

Lung cancer treatment remains a global challenge due to tumor cell resistance. Propofol, traditionally used as an anesthetic, has demonstrated potential anti-tumor properties. This study seeks to elucidate how propofol induces cell death in lung cancer cells by upregulating Pannexin 1 (PANX1) expression, activating the mitochondrial cell death pathway, and augmenting reactive oxygen species (ROS) production. In this study, the A549 lung cancer cell line was employed as the experimental model. Cells underwent exposure to varying propofol concentrations and were pre-treated with H2O2 and N-acetylcysteine (NAC) to simulate oxidative stress and antioxidant conditions. Various techniques, including 5-Ethynyl-2'-deoxyuridine (EdU), colony formation, Transwell, 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA), Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL), and JC-1 (5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide) probes, were employed to evaluate propofol's effects on lung cancer cell viability, growth, invasion, ROS levels, apoptosis, and mitochondrial membrane potential. Western blot analysis was used to measure PANX1, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, and Cytochrome C (Cyt C) protein levels. Additionally, PANX1's influence on propofol-induced apoptosis was investigated through siRNA interference. The experiment unveiled propofol's dose-dependent inhibition of A549 lung cancer cell growth, coupled with decreased cell proliferation and invasion attributable to heightened ROS production. Notably, propofol treatment significantly elevated mitochondrial membrane potential, signifying activation of the mitochondrial cell death pathway (p < 0.01). Furthermore, propofol upregulated PANX1 expression (p < 0.01), thereby intensifying apoptosis signaling, whereas PANX1 inhibition ameliorated propofol-induced apoptosis (p < 0.01). These findings underscore the pivotal role of PANX1 upregulation and ROS augmentation in propofol-induced apoptosis in lung cancer cells. This study provides evidence that propofol induces cell death in lung cancer cells by upregulating PANX1, activating the mitochondrial apoptosis pathway, and increasing ROS production. These findings suggest that targeting PANX1 and ROS could enhance the anti-cancer efficacy of propofol in lung cancer.

Anti-Inflammatory Properties of Eugenol in Lipopolysaccharide-Induced Macrophages and Its Role in Preventing β-Cell Dedifferentiation and Loss Induced by High Glucose-High Lipid Conditions.

Inflammation is a natural immune response to injury, infection, or tissue damage. It plays a crucial role in maintaining overall health and promoting healing. However, when inflammation becomes chronic and uncontrolled, it can contribute to the development of various inflammatory conditions, including type 2 diabetes. In type 2 diabetes, pancreatic β-cells have to overwork and the continuous impact of a high glucose, high lipid (HG-HL) diet contributes to their loss and dedifferentiation. This study aimed to investigate the anti-inflammatory effects of eugenol and its impact on the loss and dedifferentiation of β-cells. THP-1 macrophages were pretreated with eugenol for one hour and then exposed to lipopolysaccharide (LPS) for three hours to induce inflammation. Additionally, the second phase of NLRP3 inflammasome activation was induced by incubating the LPS-stimulated cells with adenosine triphosphate (ATP) for 30 min. The results showed that eugenol reduced the expression of proinflammatory genes, such as IL-1β, IL-6 and cyclooxygenase-2 (COX-2), potentially by inhibiting the activation of transcription factors NF-κB and TYK2. Eugenol also demonstrated inhibitory effects on the levels of NLRP3 mRNA and protein and Pannexin-1 (PANX-1) activation, eventually impacting the assembly of the NLRP3 inflammasome and the production of mature IL-1β. Additionally, eugenol reduced the elevated levels of adenosine deaminase acting on RNA 1 (ADAR1) transcript, suggesting its role in post-transcriptional mechanisms that regulate inflammatory responses. Furthermore, eugenol effectively decreased the loss of β-cells in response to HG-HL, likely by mitigating apoptosis. It also showed promise in suppressing HG-HL-induced β-cell dedifferentiation by restoring β-cell-specific biomarkers. Further research on eugenol and its mechanisms of action could lead to the development of therapeutic interventions for inflammatory disorders and the preservation of β-cell function in the context of type 2 diabetes.

Role of cAMP in Cardiomyocyte Viability: Beneficial or Detrimental?

3', 5'-cyclic AMP (cAMP) regulates numerous cardiac functions. Various hormones and neurotransmitters elevate intracellular cAMP (i[cAMP]) in cardiomyocytes through activating GsPCRs (stimulatory-G-protein-coupled-receptors) and membrane-bound ACs (adenylyl cyclases). Increasing evidence has indicated that stimulating different GsPCRs and ACs exhibits distinct, even opposite effects, on cardiomyocyte viability. However, the underlying mechanisms are not fully understood. We used molecular and pharmacological approaches to investigate how different GsPCR/cAMP signaling differentially regulate cardiomyocyte viability with in vitro, ex vivo, and in vivo models. For prodeath GsPCRs, we explored β1AR (beta1-adrenergic receptor) and H2R (histamine-H2-receptor). We found that their prodeath effects were similarly dependent on AC5 activation, ATP release to the extracellular space via PANX1 (pannexin-1) channel, and extracellular ATP (e[ATP])-mediated signaling involving in P2X7R (P2X purinoceptor 7) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). PANX1 phosphorylation at Serine 206 by cAMP-dependent-PKA (protein-kinase-A) promoted PANX1 activation, which was critical in β1AR- or H2R-induced cardiomyocyte death in vitro and in vivo. β1AR or H2R was localized proximately to PANX1, which permits ATP release. For prosurvival GsPCRs, we explored adenosine-A2-receptor (A2R), CGRPR (calcitonin-gene-related-peptide-receptor), and RXFP1 (relaxin-family peptide-receptor 1). Their prosurvival effects were dependent on AC6 activation, cAMP efflux via MRP4 (multidrug resistance protein 4), extracellular cAMP metabolism to adenosine (e[cAMP]-to-e[ADO]), and e[ADO]-mediated signaling. A2R, CGRPR, or RXFP1 was localized proximately to MRP4, which enables cAMP efflux. Interestingly, exogenously increasing e[cAMP] levels by membrane-impermeable cAMP protected against cardiomyocyte death in vitro and in ex vivo and in vivo mouse hearts with ischemia-reperfusion injuries. Our findings indicate that the functional diversity of different GsPCRs in cardiomyocyte viability could be achieved by their ability to form unique signaling complexes (signalosomes) that determine the fate of cAMP: either stimulate ATP release by activating PKA or directly efflux to be e[cAMP].

Overlap in synaptic neurological condition susceptibility pathways and the neural pannexin 1 interactome revealed by bioinformatics analyses

ABSTRACT Many neurological conditions exhibit synaptic impairments, suggesting mechanistic convergence. Additionally, the pannexin 1 (PANX1) channel and signaling scaffold is linked to several of these neurological conditions and is an emerging regulator of synaptic development and plasticity; however, its synaptic pathogenic contributions are relatively unexplored. To this end, we explored connections between synaptic neurodevelopmental disorder and neurodegenerative disease susceptibility genes discovered by genome-wide association studies (GWASs), and the neural PANX1 interactome (483 proteins) identified from mouse Neuro2a (N2a) cells. To identify shared susceptibility genes, we compared synaptic suggestive GWAS candidate genes amongst autism spectrum disorders, schizophrenia, Parkinson’s disease, and Alzheimer’s disease. To further probe PANX1 signaling pathways at the synapse, we used bioinformatics tools to identify PANX1 interactome signaling pathways and protein–protein interaction clusters. To shed light on synaptic disease mechanisms potentially linking PANX1 and these four neurological conditions, we performed additional cross-analyses between gene ontologies enriched for the PANX1 synaptic and disease-susceptibility gene sets. Finally, to explore the regional specificity of synaptic PANX1-neurological condition connections, we identified brain region-specific elevations of synaptic PANX1 interactome and GWAS candidate gene set transcripts. Our results confirm considerable overlap in risk genes for autism spectrum disorders and schizophrenia and identify potential commonalities in genetic susceptibility for neurodevelopmental disorders and neurodegenerative diseases. Our findings also pinpointed novel putative PANX1 links to synaptic disease-associated pathways, such as regulation of vesicular trafficking and proteostasis, warranting further validation.

Pannexin 1 targets mitophagy to mediate renal ischemia/reperfusion injury

Renal ischemia/reperfusion (I/R) injury contributes to the development of acute kidney injury (AKI). Kidney is the second organ rich in mitochondrial content next to the heart. Mitochondrial damage substantially contributes for AKI development. Mitophagy eliminates damaged mitochondria from the cells to maintain a healthy mitochondrial population, which plays an important role in AKI. Pannexin 1 (PANX1) channel transmembrane proteins are known to drive inflammation and release of adenosine triphosphate (ATP) during I/R injury. However, the specific role of PANX1 on mitophagy regulation in renal I/R injury remains elusive. In this study, we find that serum level of PANX1 is elevated in patients who developed AKI after cardiac surgery, and the level of PANX1 is positively correlated with serum creatinine and urea nitrogen levels. Using the mouse model of renal I/R injury in vivo and cell-based hypoxia/reoxygenation (H/R) model in vitro, we prove that genetic deletion of PANX1 mitigate the kidney tubular cell death, oxidative stress and mitochondrial damage after I/R injury through enhanced mitophagy. Mechanistically, PANX1 disrupts mitophagy by influencing ATP-P2Y-mTOR signal pathway. These observations provide evidence that PANX1 could be a potential biomarker for AKI and a therapeutic target to alleviate AKI caused by I/R injury.