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

Rafael E. Sanchez Pupo, The University of Western Ontario

Abstract

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: Braf^CA, Pten^loxP, Tyr::CreER^T2 (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.