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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Penuela, Silvia

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: 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.

Summary for Lay Audience

Pannexins 1 and 2 (PANX1, PANX2) are channel-forming proteins that participate in cell growth, death, and cell-to-cell communication processes. Both proteins are present in the skin, but only PANX1 has been thoroughly investigated and shown as essential for proper skin formation and wound healing. Here, we discovered that a PANX2 variant is present in the upper layers of the mouse skin regardless of age, and its levels increase during the maturation of specific skin cells called keratinocytes. Notably, we proved that PANX2 also speeds up death in keratinocytes exposed to ultraviolet light (UV), which could be a natural mechanism for eliminating cancerous cells in the outermost layers of skin after sunburn. We also showed that specialized enzymes cut PANX2 during this cell death process, but the consequences of this cut are still unknown. Furthermore, as PANX1 and PANX2 can exist simultaneously in the same cell, they have been shown to form mixed channels depending on the presence of sugars in the PANX1 molecular structure. However, we discovered that sugars in the PANX2 molecular structure are not required to interact with PANX1 but define PANX2 location within the cell. Furthermore, abnormal levels of PANX1 have been previously shown to assist the development of melanoma, a deadly type of skin cancer. We showed that in mice genetically engineered to develop melanomas, the absence of PANX1 does not diminish tumor formation or improve survival. Nevertheless, melanoma tumors without PANX1 had increased infiltration of cells responsible for the body's natural defenses against cancer. This study contributes to understanding the effect of chemical modifications on PANX2 distribution and function within the cell. Besides, we uncovered a new role for PANX2 in promoting skin cell death that may be essential to preserve skin health after harmful insults like UV exposure. Moreover, our work lays the foundation of the translational use of targeting PANX1 to enhance current therapies that fight melanoma tumors involving specific cells used by the body's natural defense system.

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