PFKFB3, a Direct Target of p63, Is Required for Proliferation and Inhibits Differentiation in Epidermal Keratinocytes  Robert B. Hamanaka, Gökhan M. Mutlu  Journal of Investigative Dermatology  Volume 137, Issue 6, Pages 1267-1276 (June 2017) DOI: 10.1016/j.jid.2016.12.020 Copyright © 2016 The Authors Terms and Conditions

Figure 1 p63 regulates PFKFB3 expression and glycolysis in nHEKs. (a) Medium lactate concentrations from control and p63-knockdown keratinocytes (mean ± standard error of the mean, n = 4). (b) Extracellular acidification rates (ECAR) of control and p63-knockdown keratinocytes subjected to a glycolysis stress test. (c) Relative glycolytic and maximum ECAR values from b (mean ± standard error of the mean, n = 6). (d) Heat map analysis of glycolytic enzyme mRNA expression in p63-knockdown cells relative to nontargeting control. (e) Relative p63 and PFKFB3 mRNA expression in control and p63-knockdown keratinocytes (mean ± standard error of the mean, n = 3 independent clones). (f) Western blot of p63 and PFKFB3 protein in control and p63-knockdown keratinocytes. (g) Chromatin immunoprecipitation analysis of the PFKFB3 gene. p63 consensus sequences are highlighted. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by Student t test. 2-DG, 2-deoxyglucose; A, adenine; C, cytosine; ECAR, extracellular acidification rates; G, guanine; h, hour; M, mol/L; min, minute; nHEK, neonatal human epidermal keratinocytes; NS, not significant; si, small interfering; T, thymine. Journal of Investigative Dermatology 2017 137, 1267-1276DOI: (10.1016/j.jid.2016.12.020) Copyright © 2016 The Authors Terms and Conditions

Figure 2 p63 promotes glycolysis in nHEKs through PFKFB3. (a) Medium lactate concentrations from control and PFKFB3-knockdown keratinocytes (mean ± standard error of the mean, n = 4). (b) Extracellular acidification rates (ECAR) in control and PFKFB3-knockdown keratinocytes subjected to a glycolysis stress test. (c) Relative glycolytic and maximum ECAR values from b (mean ± standard error of the mean, n = 5). (d) ECAR in keratinocytes infected with lentiviral constructs encoding either GFP (control) or PFKFB3, followed by transfection with siRNAs targeting p63 or nontargeting control. Cells were then subjected to a glycolysis stress test. (e) Relative glycolytic and maximum ECAR values from d (mean ± standard error of the mean, n = 4). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by (a, c) Student t test and (e) one-way analysis of variance with Newman-Keuls multiple comparison test. 2-DG, 2-deoxyglucose; ECAR, extracellular acidification rates; GFP, green fluorescent protein; hr, hour; M, mol/L; min, minute; nHEK, neonatal human epidermal keratinocytes; NS, not significant; si, small interfering. Journal of Investigative Dermatology 2017 137, 1267-1276DOI: (10.1016/j.jid.2016.12.020) Copyright © 2016 The Authors Terms and Conditions

Figure 3 PFKFB3 expression is required for keratinocyte proliferation. (a) Growth analysis of control and PFKFB3-knockdown keratinocytes grown in serum-free medium (mean ± standard deviation, n = 3). (b) Growth analysis of keratinocytes treated with the indicated doses of 3PO grown in serum free medium (mean ± standard deviation, n = 3). (c) Representative images of control and PFKFB3-knockdown keratinocytes grown on feeder fibroblasts. (d) Quantification of c indicated as average colony number per plate (mean ± standard error of the mean, n = 6). (e) Growth analysis of keratinocytes infected with lentiviral constructs encoding either GFP (control) or PFKFB3, followed by transfection with siRNAs targeting p63 or nontargeting control. Cells were grown in serum-free medium (mean ± standard deviation, n = 3). ∗∗∗P < 0.001 by Student t test. GFP, green fluorescent protein; M, mol/L; sh, short hairpin. Journal of Investigative Dermatology 2017 137, 1267-1276DOI: (10.1016/j.jid.2016.12.020) Copyright © 2016 The Authors Terms and Conditions

Figure 4 PFKFB3 expression inhibits keratinocyte differentiation. (a) Histological analysis of p63 and PFKFB3 expression in normal human epidermis. (b) Western blot analysis of keratinocytes induced to differentiate by elevation of extracellular calcium for the indicated intervals. (c) Representative Western blot analysis of GFP (control) and PFKFB3-expressing keratinocytes induced to differentiate for the indicated intervals. (d) Analysis of relative mRNA expression of Notch1, Hes1, and Hes5 (day 0) and keratin 1 and 10 (day 1) in GFP (control) or PFKFB3-expressing keratinocytes induced to differentiate (mean ± standard error of the mean, n = 3). (e) Representative histological analysis of GFP (control) and PFKFB3-expressing keratinocytes grown in three-dimensional organotypic cultures for 12 days. Scale bars = 50 μm. ∗P < 0.05, ∗∗∗P < 0.001 by Student t test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; H&E, hematoxylin and eosin; K, keratin; NS, not significant. Journal of Investigative Dermatology 2017 137, 1267-1276DOI: (10.1016/j.jid.2016.12.020) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Inhibition of PFKFB3 promotes keratinocyte differentiation. (a) Histological analysis of p63, PFKFB3, keratin 10, and loricrin expression in psoriatic epidermis. (b) Representative Western blot analysis of keratinocytes expressing short hairpin RNAs targeting PFKFB3 or nontargeting control. Cells were induced to differentiate by elevation of extracellular calcium for the indicated intervals. (c) Representative Western blot analysis of keratinocytes induced to differentiate by elevation of extracellular calcium for the indicated intervals. Cells were treated with 3PO (10 μmol/L) or left untreated starting on day 0. (d) Representative histological analysis of keratinocytes grown in three-dimensional organotypic cultures for 12 days at the air-medium interface. Cells were treated with 3PO (10 μmol/L) or left untreated starting on day 0. (a) Scale bar = 400 μm. (d) Scale bar = 50 μm. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H&E, hematoxylin and eosin; sh, short hairpin. Journal of Investigative Dermatology 2017 137, 1267-1276DOI: (10.1016/j.jid.2016.12.020) Copyright © 2016 The Authors Terms and Conditions