The Characterization of Pannexin 1 and Pannexin 3 Channels and their Isoforms in Skin Health and Keratinocytic Cancers
Abstract
The skin contains the channel-forming glycoproteins pannexins (PANX) which facilitate cellular communication. PANX1 and PANX3 function in wound repair and keratinocyte differentiation, but unlike PANX1 which is abundant in young skin to establish proper skin architecture, the role of PANX3 in skin homeostasis and aging is unknown. Additionally, PANX1 promotes melanoma carcinogenesis, but little is known about PANX1 and PANX3 in the keratinocytic cancer cutaneous squamous cell carcinoma (cSCC). Here, we used global Panx3 knockout (KO) mice and congenic wildtype controls to investigate PANX3 in the skin. We determined PANX3 was undetectable in wildtype newborn dorsal skin but became upregulated from postnatal day 2-4, where its levels remained consistently high up to 18 months. Panx3 KO mice had thinner skin, a compromised epidermal barrier as well as increased dermatitis incidence and epidermal inflammatory signalling. Furthermore, KO primary keratinocytes showed adhesion deficits most likely due to reduced Wnt signalling. In human cSCC tumours, PANX1 was upregulated but PANX3 mRNA was downregulated compared to patient-matched skin. PANX1 was present in all cSCC tumour regions, where it promoted carcinoma cell growth and migration through its channel activity. Contrarily, using Panx3 KO mice in a chemical carcinoma model, we determined PANX3 reduces the incidence and volumes of pre-cancerous papillomas, highlighting its anti-tumorigenic effects in the skin. Lastly, when surveying human cancer cell lines we discovered a highly abundant ~25 kDa isoform of PANX1 (PANX1-25K) which is increased in cSCC compared to normal skin and promotes cancer cell survival and growth. We determined PANX1-25K lacks the N-terminus of PANX1 and becomes increased with CRISPR/Cas9 ablation of the initial methionine start site of PANX1, but does not result from alternative promotor usage or splicing, suggesting instead that it may be generated through alternative translation initiation. Moreover, PANX1-25K resembles PANX1 biochemically, being glycosylated on novel asparagine residues, phosphorylated and interacting with β-catenin, but exhibits an intracellular localization where it interacts with PANX1. Collectively, in this thesis we identified a new PANX1 isoform and illustrated that PANX1, PANX3 and their isoforms are key regulators of skin homeostasis and aging which become dysregulated in the context of cSCC.