N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1.

Pannexins (Panx1, 2, 3) are channel-forming glycoproteins expressed in mammalian tissues. We previously reported that N-glycosylation acts as a regulator of the localization and intermixing of Panx1 and Panx3, but its effects on Panx2 are currently unknown. Panx1 and Panx2 intermixing can regulate channel properties, and both pannexins have been implicated in neuronal cell death after ischemia. Our objectives were to validate the predicted N-glycosylation site of Panx2 and to study the effects of Panx2 glycosylation on localization and its capacity to interact with Panx1. We used site-directed mutagenesis, enzymatic de-glycosylation, cell-surface biotinylation, co-immunoprecipitation, and confocal microscopy. Our results showed that N86 is the only N-glycosylation site of Panx2. Panx2 and the N86Q mutant are predominantly localized to the endoplasmic reticulum (ER) and cis-Golgi matrix with limited cell surface localization was seen only in the presence of Panx1. The Panx2 N86Q mutant is glycosylation-deficient and tends to aggregate in the ER reducing its cell surface trafficking but it can still interact with Panx1. Our study indicates that N-glycosylation may be important for folding and trafficking of Panx2. We found that the un-glycosylated forms of Panx1 and 2 can readily interact, regulating their localization and potentially their channel function in cells where they are co-expressed

Inhibition of Pannexin 1 Reduces the Tumorigenic Properties of Human Melanoma Cells

Pannexin 1 (PANX1) is a channel-forming glycoprotein expressed in many tissues including the skin. PANX1 channels allow the passage of ions and molecules up to 1 kDa, including ATP and other metabolites. In this study, we show that PANX1 is highly expressed in human melanoma tumors at all stages of disease progression, as well as in patient-derived cells and established melanoma cell lines. Reducing PANX1 protein levels using shRNA or inhibiting channel function with the channel blockers, carbenoxolone (CBX) and probenecid (PBN), significantly decreased cell growth and migration, and increased melanin production in A375-P and A375-MA2 cell lines. Further, treatment of A375-MA2 tumors in chicken embryo xenografts with CBX or PBN significantly reduced melanoma tumor weight and invasiveness. Blocking PANX1 channels with PBN reduced ATP release in A375-P cells, suggesting a potential role for PANX1 in purinergic signaling of melanoma cells. In addition, cell-surface biotinylation assays indicate that there is an intracellular pool of PANX1 in melanoma cells. PANX1 likely modulates signaling through the Wnt/beta-catenin pathway, because beta-catenin levels were significantly decreased upon PANX1 silencing. Collectively, our findings identify a role for PANX1 in controlling growth and tumorigenic properties of melanoma cells contributing to signaling pathways that modulate melanoma progression

Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner

Background: Pannexin 3 (PANX3) is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggest that it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high-fat diet (HFD). Methods: C57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 to 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression. Results: In females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. In addition, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, multiple inflammatory genes were significantly different in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype in KO mice compared to WT. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs. Conclusion: PANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation

The regulation of Pannexin1 and Pannexin2 in the skin in health and disease

Pannexins (PANX1, 2, 3) are a family of channel-forming glycoproteins that mediate intracellular and paracrine signaling. In contrast to PANX2, PANX1 has been extensively investigated in the skin, modulating cell differentiation, wound healing, and melanoma development. PANX1 and PANX2 can co-exist in the same cell and form mixed channels where their glycosylation seems to regulate their intermixing. N-glycosylation and caspase cleavage have been proposed as modulators of the function of PANX1, but their effects on PANX2 are unknown. We explored the PANX2 expression in mouse skin and showed that a Panx2 splice variant (PANX2-202) is continuously expressed throughout aging skin. Furthermore, PANX2 was detected in keratinocytes and is upregulated during their in vitro differentiation. We showed that in UVB-induced apoptotic keratinocytes, caspase-3/7 cleaves the PANX2 C-terminus at residue D416. Notably, CRISPR-Cas9-mediated deletion of PANX2 delays but does not impair keratinocyte apoptosis, and its caspase-mediated cleavage is not required for this process. Thus, we propose that PANX2 promotes keratinocyte death after UVB, which may contribute to skin homeostasis. Moreover, we showed that N-glycosylation occurs at the N86 residue of PANX2, regulating folding and cell surface trafficking but not its interaction with PANX1. As PANX1 is known to modulate in vitro and ex vivo tumorigenicity of melanoma cells, we examined the effect of a germline deletion on in vivo melanoma progression by crossing Panx1 knockout (Panx1-/-) mice with the melanoma model: BrafCA, PtenloxP, Tyr::CreERT2 (BPC). We found that Panx1-deletion did not reduce melanoma formation or improve BPC-mice survival. However, tumors in BPC-Panx1-/- mice exhibited increased infiltration of CD4+, CD8+ T lymphocytes, and Granzyme B+ cells but not immunosuppressive FoxP3+ T cells. Remarkably, splenomegaly was also found in female BPC-Panx1-/- mice compared to males. Overall, this study revealed the location of two post-translational modifications in the PANX2 amino acid sequence modulating its localization and possibly its biological function. We provided further evidence that regulation of pannexins in the skin may influence cell death and the activity of the immune system during skin cancer conditions, which may have translational application in improving checkpoint inhibitor immunotherapy for melanoma

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