Pannexin 3 Deficiency in Mice Delays the Wound Healing Process
peipei zhang, M.D. Ph.D., Craig Rhodes, Ph.D., Andrew Doyle, Ph.D., Masaki Ishikawa, D.D.S. Ph.D, Tomoko Ikeuchi, D.D.S. Ph.D, Kuniyuki Nakamura, M.D. Ph.D., Yuta Chiba, D.D.S., Yoshihiko Yamada, Ph.D..
National Institute of Dental and Craniofacial Research, Bethesda, MD, USA.
PURPOSE:Pannexin 3 (Panx3) is a gap junction protein. We have previously shown that Panx3 plays multiple channel functions: 1) as a hemichannel to regulate intracellular ATP/cAMP levels between cells and the extracellular space, 2) as an ER calcium channel to regulate calcium flux within the cell, and 3) a gap junction to exchange ions and small molecules between cells. However, the role of Panx3 in skin tissue regeneration, proliferation, and/or differentiation is unclear. Here, we demonstrate that Panx3 plays a role in the skin wound healing process by controlling the inflammatory response, epidermal-mesenchymal transition (EMT), keratinocyte proliferation, and collagen deposition. METHODS: To identify Panx3 functions in the skin wound healing process, two 8 mm diameter full-thickness skin punches were made in Panx3 knockout (Panx3 -/-) mice and heterozygous knockout (Panx3 +/-) siblings as controls. The wound healing process was analyzed by evaluating the remaining wound area in a time course. The wound area skin samples were collected on the 5th and the 10th day post-surgery for histological and immunohistochemical analysis. The levels of inflammation, EMT and signaling pathway markers were analyzed by RT-PCR. To investigate whether Panx3 promotes keratinocyte differentiation, the human keratinocyte line HaCaT cells were transfected with a human Panx3 cDNA expression vector. Low (0.03mM) or high (1.2mM) Ca2+ concentrations in the medium were used to induce differentiation in mock- and Panx3-transfected HaCaT cells. Cell proliferation rates, cell cycle and keratinocyte differentiation markers were then analyzed. RESULTS: At 10 days post wound surgery, Panx3 -/- mice showed slower healing rates than control Panx3 +/-mice. The most obvious difference was on the 5th day post-surgery, which is associated with the inflammatory stage of wound healing. The cell infusion and collagen deposition rates were less in Panx3 -/- mice than in the Panx3 +/- mice. On the 5th day post-surgery, inflammatory markers including CD4, CD68, IL1-beta, IL1R1, IL6, and IL6R, as well as EMT markers such as MMP9, snail, and N-cadherin demonstrated a decrease in their expression levels in Panx3 -/- mice compared to Panx3 +/- mice. In addition, markers for several cell signaling pathways, such as Wnt, BMP2, BMP4, Shh and TGF-beta2, were reduced in Panx3 -/- mice. In contrast, TGF-beta1 was reduced in Panx3 +/- mice when compared with Panx3 -/- littermates. Using in vitro cell culture assays, we found that low concentrations of Ca2+ in Panx3-transfected HaCaT cells promoted cell proliferation. But high Ca2+ concentrations inhibited cell proliferation. Cell cycle analysis showed that Panx3 maintained HaCaT cells in the S-phase, indicating active DNA replication. Keratinocyte differentiation markers such as K10, K14, filaggrin, and involucrin were not altered by over-expressing Panx3 in HaCaT cells.CONCLUSION: Panx3 promotes keratinocyte proliferation but not differentiation. Panx3 deficiency reduces the expression of epithelial-mesenchymal transition markers, inflammatory markers and collagen deposition. This suggests Panx3 is involved in EMT and inflammatory processes. Panx3 plays a critical role in the skin wound healing process.
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