Hydration Status Regulates Sodium Flux and Inflammatory Pathways through Epithelial Sodium Channel (ENaC) in the Skin  Wei Xu, Seok Jong Hong, Michael Zeitchek, Garry Cooper, Shengxian Jia, Ping Xie, Hannan A. Qureshi, Aimei Zhong, Marshall D. Porterfield, Robert D. Galiano, D James Surmeier, Thomas A. Mustoe  Journal of Investigative Dermatology  Volume 135, Issue 3, Pages 796-806 (March 2015) DOI: 10.1038/jid.2014.477 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of cyclooxygenase 2 (COX-2) was increased in the reduced hydration status. (a) Establishment of human ex vivo skin culture (HESC). (b) COX-2 mRNA expressions in HESCs (n=4). (c) Western blot (WB) analysis for expression of COX-2 in HESC. (d) Schematic drawing of stratified HKCs. (e) WB analysis for HKCs. The stratified HKCs (i, ii, iii, and iv) were treated with different humidity conditions for 16 hours. (c, d) WB was analyzed with the NIH ImageJ program (n=4). **P<0.01; Student’s t-test. (f–k) Immunofluorescent staining. Expressions of PGE2 were measured in the HESC (f–h) and HKCs (i–k) that were treated with control, RHC, or HSC for 16 hours. CHS, control hydration status; HKC, human keratinocyte culture; HSC, high sodium concentration (165 mM); PGE2, prostaglandin E2; RHS, reduced hydration status. Journal of Investigative Dermatology 2015 135, 796-806DOI: (10.1038/jid.2014.477) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Sodium flux in keratinocytes is regulated by epithelial sodium channel (ENaC). (a–d) Sodium flux measurement using scanning ion-selective electrode technique (SIET). (a) Sodium flux in human ex vivo skin cultures (HESCs) culture with reduced hydration condition. (b) Sodium flux in stratified HKCs culture with high sodium condition. (c) Sodium flux in amiloride treated stratified human keratinocyte culture (HKC) culture with reduced hydration status. (d) Sodium flux in ENaC-α knockdown stratified HKC culture with reduced hydration status or high sodium concentration. Positive values of ion flux suggest a sodium influx to the cell. (e, f) Whole-cell patch clamp. (e) Whole-cell patch clamp on a single HaCaT cell elicited from -120 to 0 mV with blockade or knockdown of ENaC-α (ENaC-α KD). (f) Corresponding current–voltage curves for the patch clamp readings on HKCs with different treatments. The slope of each curve reflects the sodium current. Bigger slope represents bigger sodium flux (n=3∼4). *P<0.05, **P<0.01. WT, wild type. Journal of Investigative Dermatology 2015 135, 796-806DOI: (10.1038/jid.2014.477) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Regulation of cyclooxygenase 2 (COX-2) and PGE2 by sodium flux is mediated by ENaC. (a) COX-2 mRNA expression in stratified keratinocytes. (b) Prostaglandin E2 (PGE2) protein expression using ELISA on monolayer wild type or epithelial sodium channel (ENaC)-knockdown keratinocytes (n=4). (c) Western blot analysis (n=4) on stratified keratinocytes with antibodies against phosphorylated acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) and Akt. (d) p-AKT western blot normalized by β-actin was quantified using ImageJ. (e, f) Effect of the inhibition of the PI3K/Akt signaling pathway on COX-2 expression. LY294002, a specific inhibitor for phosphatidylinositol 3-kinase (PI3K), was treated (10 μg ml-1) in keratinocytes with reduced hydration status (e) or high sodium concentration (f), as well as control. COX-2 mRNA (e) and PGE2 (f) protein expression were analyzed by RT-qPCR and ELISA. *P<0.05, **P<0.01. High sodium concentration, 165 mM; control sodium concentration, 150 mM; HKC, human keratinocyte culture. Journal of Investigative Dermatology 2015 135, 796-806DOI: (10.1038/jid.2014.477) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Downstream pathway of epithelial sodium channels (ENaC) upon reduced hydration condition. Stratified human keratinocytes (HKs) and human dermal fibroblasts (HDFs) were cocultured in transwell plates and subjected to reduced hydration status and high sodium concentration for 16 hours. Expression of α-SMA and pro-Col I was detected in HDF with their specific antibodies and visualized with fluorescence labeled secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (a–d) Stratified HKs and HDFs in control hydration status. (e–h) Stratified HKs and HDFs in reduced hydration status. (i–l) Stratified ENaC-α knockdown HKs and HDFs in control hydration status. (m–p) Stratified ENaC-α knockdown HKs and HDFs in reduced hydration status. (q, r) The quantification of expression levels of α-SMA and pro-Col I by ImageJ (n=4). Scale bar=100 μm. *P<0.05, **P<0.01. K/D, knockdown; WT, wild type. Journal of Investigative Dermatology 2015 135, 796-806DOI: (10.1038/jid.2014.477) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Reduction in hypertrophic scar formation by pharmacological inhibition of epithelial sodium channels (ENaC) and cyclooxygenase 2 (COX-2) in vivo. (a–d) Representative pictures of wound histology at POD28. Rabbit ear wounds with Amiloride treatment (b) and control (a) and with Celecoxib treatment (d) and its control (c) are shown. (e) The scar elevation index was calculated and used to evaluate the scar formation. (f, g) SEI. Twelve wounds for each dose of treatment and 12 controls were used for the analysis. (h) Expression of COX-2 in rabbit ear incisional grids with the topical treatment of 1% amiloride at post epithlialization day 3. n=5. *P<0.05. (i) A putative mechanism of activation of fibroblast through ENaC by reduced hydration. Scale bar=1,000 μm. **P<0.01. POD, post operation day; SEI, scar elevation index. Journal of Investigative Dermatology 2015 135, 796-806DOI: (10.1038/jid.2014.477) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions