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First published online 2 January 2007
doi: 10.1242/jcs.03329

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Role for WNT16B in human epidermal keratinocyte proliferation and differentiation

¹ Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, Blizard Building, 4 Newark Street, London, E1 2AT, UK
² Centre for Clinical and Diagnostic Oral Sciences, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, Blizard Building, 4 Newark Street, London, E1 2AT, UK
³ Bioinformatics Discovery and Analysis, GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park (North) Third Avenue, Harlow, CM19 5AW, UK

View larger version (77K): [in this window] [in a new window]   Fig. 1. Genomic structure of the human WNT16A and WNT16B isoforms. (A) WNT16A and WNT16B isoforms are produced by alternative expression of the first exon (1A or 1B) controlled by independent promoters (Fear et al., 2000). (B) First exon amino acid sequence alignment of the two WNT16 isoforms showing unique amino acid sequences encoded by each of the two first exons. (C-K) Protein and mRNA expression in normal human epidermis and basal cell carcinoma (BCC). (C) RT-PCR gel electrophoresis detection of WNT16B mRNA expression in normal human skin (NS), primary normal human keratinocyte culture (K), hairy human scalp skin (HS), primary dermal papillae culture (DP), BCC tumour samples (3 of 6 patients are shown here), blank (H2O), positive controls (plasmids WNT16A/B). WNT16B primers do not detect WNT16A template. M, molecular mass markers. GAPDH was used as a housekeeping gene control for all samples. (D) Gel densitometry quantification of WNT16B mRNA levels of NS, K, HS, DP and BCC samples. Each bar represents fold WNT16B activation (over NS as control) of mean ± s.e.m. with n number of samples indicated next to each bar. *P<0.05 and ***P<0.001 indicate statistically significant WNT16B mRNA activation over NS. (E) Immunoreactivity of WNT16 protein expression in normal human interfollicular epidermis showing cytoplasmic suprabasal staining pattern. (F) High power magnification of a portion within E showing WNT16 protein expression throughout the suprabasal layer (ss, stratum spinosum and sg, stratum granulosum) but discretely excluded the basal cell layer (sb, stratum basalae). (G-K) Immunoreactivity of WNT16 protein expression in BCCs and adjacent hair follicles. (H) High power magnification of a portion within G showing cytoplasmic WNT16 protein expression within BCC tumour mass (*) with increased WNT16 expression at the peripheral palisading advancing edge (arrowheads) of the tumour. WNT16 protein was also detected within the suprabasal layer in the epidermis above the BCC tumour. (I) Expression of WNT16 protein in a hair follicle adjacent to BCC tumours. Increased WNT16 protein expression was detected within the bulge region (b; J,K) and dermal papillae (dp; K) of the hair follicle.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Expression and detection of WNT16A and WNT16B isoforms in cultured human keratinocytes. (A) Immunocytochemistry using WNT16 antibody on retrovirally transduced primary NHEKs expressing EGFP, WNT16A or WNT16B. (B) RT-PCR gel electrophoresis detection of WNT16A or WNT16B mRNA expression using isoform-specific primers in NHEKs and RTS3b keratinocytes transduced with either WNT16A (16A), WNT16B (16B) or EGFP (C). GAPDH was amplified as a loading control. WNT16A/B expression plasmids were used as positive controls for each isoform. (C) Western blot detection of WNT16 protein in NHEKs and RTS3b keratinocytes transduced with either WNT16A or WNT16B. A lower molecular mass product (30-35 kDa; X) was detected in WNT16A-tranduced cell lysates. Corresponding fluorescence micrographs show the expression level of WNT16A or B in respective samples before harvesting for immunoblotting. (D) In vitro protein translation of pcDNA-WNT16A or B expression plasmids produced expected size proteins (40 kDa for both isoforms). pGL3 (luciferase gene) and pcDNA3.1 were used as positive (+ve) and negative (–ve) controls, respectively. (E) The in vitro translated protein lysates (from D) were subsequently used for immunoblotting with the WNT16 antibody, showing positive immunoreactivity in both WNT16A and B isoforms. (F-H) WNT16 isoforms do not activate through a canonical WNT signalling pathway in human keratinocytes. (F) Immunocytochemistry of endogenous -catenin proteins (using a pan--catenin antibody) in primary NHEKs transduced with either EGFP or WNT16B or control cells treated with LiCl (5 mM, 24 hours). (G) TCF/LEF luciferase reporter assay on primary NHEKs co-transduced with pSIN-OT and either EGFP or WNT16B. CTRL cells were mock transduced with retrovirus bearing no expression gene. Positive control cells (for activation of canonical WNT signalling) were treated with LiCl (5 mM) to activate LEF/TCF promoter activity. (H) TCF/LEF luciferase reporter assay on 293T cells co-transfected with EGFP, WNT1 or WNT16B. pOT (grey bar) or pOF (black bar) indicate cells co-expressing luciferase reporter bearing an active or mutant TCF/LEF binding sites, respectively. ***P<0.001 indicates significant activation of TCF/LEF promoter activity. (I) Immunoblotting of WNT16B, -catenin, phospho-Jun, phospho-JNK, and GAPDH in the indicated cell types expressing either WNT16B or EGFP.

View larger version (52K): [in this window] [in a new window]   Fig. 3. WNT16B but not WNT16A enhanced keratinocyte cell proliferation. Growth curves of primary NHEKs retrovirally transduced with EGFP, WNT16A, WNT16B, -catenin or S33Y (constitutively active -catenin) determined by measuring total protein concentration of adherent cells (A) or ATP concentration (B) at the indicated time points. Insets show the relative growth rate at 50% confluency of each curve. (C) Cell-cycle profile determined by FACS (propidium iodide) analysis at the end of the cell proliferation assay (day 9) in A and B, with corresponding phase-contrast micrographs. (D) Primary NHEK transduced with either EGFP, WNT16A or WNT16B were cultured in low Ca2+ (0.06 mM) medium without irradiated 3T3 feeders and subcultured regularly up to passage 3. Phase-contrast and fluorescence micrographs were taken at post-transduction passage 1 and 3. (E) RTS3b keratinocytes were cultured at high cell density (2-3x106 cells/35 mm2) for 7 days to induce contact inhibition and stratification-induced cell death. The number of viable adherent cells was quantified using CellTitre-Glo to measure the ATP concentrations of surviving cells at day 1 and 7. Each bar represents a mean ± s.e.m. of triplicate samples. *P<0.01 and ***P<0.001 indicates statistically significant reduction in cell viability at day 7 compared with day 1.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Silencing endogenous WNT16B gene expression decreased keratinocyte proliferation and cell survival. (A) Fluorescence micrographs showing isoform-specific siRNA gene silencing in WNT16A or WNT16B-transduced RTS3b keratinocytes transfected with either shCtrl (control shRNA with random sequence), shA (against WNT16A), shB (against WNT16B) or shAB (against both WNT16 isoforms). All cells were maintained in culture medium containing puromycin (2 µg/ml) to select for shRNA-expressing cells. (B) Pixel densitometry of fluorescence images in A. Each bar represents % mean ± s.e.m. (n=5 images) of green fluorescence. ***P<0.001 indicates statistically significant gene knockdown by respective shRNA expression. (C) Western immunoblotting of WNT16B protein (arrows) from cell lysates collected from WNT16-expressing NHEK or RTS3b cells transfected with each shRNA, as indicated, confirming the specific knockdown of WNT16 protein expression by RNAi. (D) RTS3b keratinocytes transduced with either EGFP or WNT16B or mock transduced (empty virus, grey bar) were transfected with each of the indicated shRNAs and cultured at high density for 7 days to promote stratification-induced cell death. Cells transfected with shRNA were grown in culture medium containing puromycin (2 µg/ml) to select for shRNA-expressing cells. Viable cells were quantified at post shRNA transfection day 1 and 7. Each bar represents mean ± s.e.m. of three samples. *P<0.05 and **P<0.01 indicate statistically significant effects induced by shRNA expression. This experiment has been reproduced twice on separate occasions with similar results. (E,F) Silencing -catenin attenuated 293T cell proliferation. (E) Transient co-transfection of siCat (10 nM, 48 hours) with EGFP, Wnt1 or -catenin in the presence of pGL3-OT luciferase reporter in 293T cells. Each bar represents a mean ± s.e.m. of triplicate samples. **P<0.01 and ***P<0.001 indicate statistically significant inhibition of TCF/LEF promoter activity by siCat. (F) Transient co-transfection of siCat in the absence (CTRL) or presence of either EGFP or WNT16B expression vectors in 293T cells. Cell density at day 7 post-transfection was determined using CellTitre-Glo to measure ATP concentrations of surviving cells. Each bar represents mean ± s.e.m. n=6. *P<0.05 and **P<0.01 indicate statistically significant inhibition of cell proliferation by siCat. (G,H) Constitutive expression of WNT16B induces hyperproliferation in organotypical culture system. (G) Representative haematoxylin and eosin sections of organotypic cultures using EGFP, WNT16A or B-transduced NHEK or RTS3b cells seeded on de-epidermalised dermis (DED). (H) Area quantification of epidermal thickness in organotypical cultures performed in A. Each bar represents a mean epidermal area above the DED of four independent organotypical cultures performed on separate occasions. **P<0.01 and ***P<0.001 indicate statistically significant thickening of epidermal layer above the DED.