Role for WNT16B in human epidermal keratinocyte proliferation and differentiation

The secreted glycoprotein WNT family proteins have been shown to regulate a number of basic cell functions such as cell proliferation, cell fate, polarity, differentiation and migration (Giles et al., 2003; Moon et al., 2002). There are currently at least 19 human WNT genes identified, some of which have different isoforms (Miller, 2002). These WNTs are classified into two groups according to their ability to stabilise β-catenin, leading to the activation of T-cell factor/lymphoid enhancer factor (TCF/LEF) promoters, which in turn leads to transcriptional induction of various target genes such as Myc and cyclin D1 (canonical pathway). The non-canonical or planar cell polarity (PCP) pathway is characterised by an inability to stabilise β-catenin and/or signalling through Ca²⁺ flux and activation of the Jun N-terminal kinases (JNKs) (Kohn and Moon, 2005; Veeman et al., 2003). The canonical WNT pathway has been implicated in embryonic development (Nelson and Nusse, 2004), tumourigenesis (Giles et al., 2003) and stem cell maintenance (Sato et al., 2004; Willert et al., 2003). Although the non-canonical pathway is largely implicated in the cell polarity that is important during development (Fanto and McNeill, 2004; Kohn and Moon, 2005; Veeman et al., 2003), emerging evidence has shown that this pathway also plays an important role in tumourigenesis (Caldwell et al., 2004; Liang et al., 2003; Nateri et al., 2005; Pukrop et al., 2006; You et al., 2004).

WNT16 expression has been linked to lineage specification in B-cell development (Muschen et al., 2002) and a previous study has shown that WNT16 was activated in the bone marrow of patients with pre-B acute lymphoblastoid leukaemia containing a t(1;19) chromosomal translocation (McWhirter et al., 1999). We have previously identified two closely related human WNT16 isoforms showing differential expression in adult human tissues with evidence that the two WNT16 isoforms are likely to be products of two independent promoters rather than splice variants from a single promoter (Fear et al., 2000). However, little is known about the signalling mechanisms of the two isoforms and their functional significance in cell functions. Our finding that WNT16 protein was upregulated in basal cell carcinoma (BCC) prompted the investigation into the function of the two WNT16 isoforms in both primary normal human epidermal keratinocytes (NHEKs) and the immortalised keratinocyte cell line (RTS3b). We provide the first evidence that WNT16B has a role in mediating keratinocyte proliferation, possibly by activating a non-canonical WNT pathway.

We have previously reported differential mRNA expression of two WNT16 isoforms in various adult human tissues (Fear et al., 2000). The two WNT16 isoforms have different 5′ untranslated regions (UTR) and first exons, which appear to be independently controlled by two putative promoters (Fear et al., 2000) (Fig. 1A). Protein sequence alignment of the first exon of WNT16A and WNT16B isoforms showed no significant sequence homology (Fig. 1B). However, both isoforms share exons 2-4. Overall protein sequence similarity between WNT16A and WNT16B is ∼92%.

The mRNA expression profile of the two WNT16 isoforms in normal human skin (NS), primary epidermal keratinocytes (K), hair bearing scalp skin (HS), primary, hair follicle derived, dermal papilla (DP) cells and basal cell carcinomas (BCCs) was performed by semi-quantitative RT-PCR using WNT16 isoform-specific primers. Endogenous WNT16A was not detectable by RT-PCR in any of the investigated tissues (data not shown) or cells (see Fig. 2B). By contrast, WNT16B was detected in all samples at varying expression levels as determined by semi-quantitative RT-PCR (Fig. 1C). Densitometry of data in Fig. 1C indicates a significant ∼threefold (Student's t-test, P<0.001) increased WNT16B expression in primary cultured keratinocytes over NS and HS cells, whereas DP cells have ∼1.8-fold (P<0.05) increased WNT16B expression compared with HS cells (Fig. 1D). Interestingly, all three independent nodular BCCs were found to have increased levels (∼3.2-fold, P<0.001) of WNT16B expression compared to NS and HS cells.

WNT16 protein expression pattern was investigated by immunohistochemistry using a WNT16-specific antibody on paraffin-embedded sections of normal human skin and BCC. WNT16 was expressed throughout the suprabasal layers (stratum spinosum and stratum granulosum) but discretely excluded the basal cell layer (stratum basalae) of normal skin (Fig. 1E,F). By contrast WNT16 protein expression was found to be cytoplasmic within the BCC tumour mass with increased WNT16 expression at the peripheral palisading advancing edge of the tumour (Fig. 1G,H). WNT16 protein expression was also detected in normal skin within the bulge region or stem-cell compartment (Fig. 1J) and DP cells of the catagen hair follicle (Fig. 1K). Faint WNT16 protein expression was detected within the sebaceous glands, but the regressing epithelial strand of a catagen hair follicle was predominantly negative for WNT16 (Fig. 1K).

To study the role of each isoform in primary and immortalised keratinocytes, we generated retrovirus bearing either CMV-WNT16A or CMV-WNT16B isoforms with EGFP as a marker for simultaneous protein expression under the IRES promoter. Using the WNT16-specific antibody we performed immunocytochemistry on retrovirally transduced keratinocytes to detect protein expression of both isoforms of WNT16. We consistently detected high levels of WNT16B but not WNT16A protein expression (Fig. 2A). RT-PCR was used to determine whether WNT16A mRNA was being expressed. These experiments confirmed that WNT16A mRNA was expressed in both primary NHEK and RTS3b keratinocytes transduced with the WNT16A isoforms (Fig. 2B). Although, endogenous WNT16B mRNA was readily detectable in both cell lines, no endogenous WNT16A mRNA was detected by PCR. Protein analysis by western immunoblotting of WNT16A-transduced NHEKs and RTS3b did not demonstrate WNT16A protein expression; EGFP expression was detected by fluorescence microscopy (Fig. 2C). Interestingly, a lower molecular mass (∼30-35 kDa; Fig. 2C) protein was detected in WNT16A-transduced cells. With this observation of a smaller-than-predicted WNT16A protein, we investigated whether the expression plasmids were capable of expressing a full-length WNT16A (and B) protein using in vitro translation. We found that the WNT16A and WNT16B plasmids were indeed translated into a full-length protein with a predicted band size at ∼40 kDa (Fig. 2D). To verify the identity of these bands, a western blot was carried out on the protein lysates generated by in vitro translation and we were able to show that the WNT16 antibody was able to detect both isoforms (Fig. 2E). The fact that we failed to detect WNT16A protein expression in cells could be because the WNT16A mRNA is not translated into protein in vivo or the protein undergoes a rapid post-translational modification or degradation. This is supported by our observations that WNT16A mRNA was detectable in cells transduced with WNT16A and that WNT16A mRNA could be translated into protein. In further support of this notion, the lower molecular mass band (∼30-35 kDa; Fig. 2C) consistently detected in WNT16A-expressing cell lysates might represent a degradation product of WNT16A protein. As we were unable to detect either endogenous WNT16A mRNA or protein in cells or skin tissues, we decided to restrict this study to further characterisation of the role of WNT16B, which we have shown is upregulated in BCCs. Further studies are required to delineate the mechanism of WNT16A protein processing in cells, which are beyond the scope of the present study.

We first investigated the ability of WNT16B to stabilise β-catenin using western immunoblotting and immunocytochemistry. In transduced primary NHEKs, WNT16B failed to induce nuclear β-catenin translocation (Fig. 2F). Control cells treated with lithium chloride (LiCl, 5 mM, 24 hours) showed nuclear localisation of β-catenin (Rao et al., 2005) indicating that the canonical WNT pathway remained intact in the mock-infected keratinocytes. We further showed that WNT16B is a β-catenin-independent non-canonical WNT using the TCF/LEF luciferase assay. Primary NHEK cells overexpressing WNT16B (by retroviral co-transduction with pSIN-OT) did not activate the TCF/LEF reporter in NHEKs whereas LiCl (as a positive control for β-catenin activation) significantly activated TCF/LEF reporter (3.1-fold; Fig. 2G). Similarly, in 293T cells, co-transfection of WNT16B with the active (pOT) TCF/LEF reporter did not activate luciferase activity, whereas, the known canonical WNT1 significantly (P<0.001) activated pOT luciferase activity (∼13-fold; Fig. 2H). Both the negative control EGFP-expressing cells and background control pOF-expressing cells showed minimal luciferase activity. These results suggest that WNT16B does not activate canonical WNT signalling via β-catenin in both primary human keratinocytes and a non-keratinocyte epithelial cell line (293T), indicating that this mechanism is conserved in two diverse epithelial cell types. Further experiments showed that WNT16B overexpression in NHEKs and a keratinocyte cell line RTS3b showed no increase in total β-catenin protein levels when compared with control cells expressing EGFP (Fig. 2I, right panel). To investigate whether WNT16B can activate a non-canonical pathway, we performed immunoblotting to detect the activated/phosphorylated forms of Jun and JNK protein levels in cells (Kohn and Moon, 2005; Veeman et al., 2003). In primary NHEKs, WNT16B expression clearly showed increased protein levels of phospho-Jun and phospho-JNK compared with control cells expressing EGFP alone (Fig. 2I, right panel). Collectively these data suggest that in primary keratinocytes WNT 16B may signal via the non-canonical Jun-N-terminal kinase cascade involving phosphorylation of Jun and JNK (Kohn and Moon, 2005; Veeman et al., 2003) and not via the classical canonical WNT pathway.

We performed keratinocyte proliferation assays to determine the growth rates of primary NHEKs transduced with EGFP, WNT16B, β-catenin or S33Y (constitutively active β-catenin) (Morin et al., 1997). Transduced cells were plated at low density and left to grow for 9 days and the number of live cells was determined by either measuring total adherent cell protein concentration (Fig. 3A) or by measuring ATP concentrations in adherent cells (Fig. 3B). Cells expressing WNT16B, β-catenin and S33Y had slightly elevated growth rates comparable with EGFP-expressing cells. There was no difference in the growth rate between EGFP and untransduced cells (data not shown). At day 9, these cells were harvested for FACS (propidium iodide) analysis to determine the cell-cycle profile (Fig. 3C). Cell-cycle profiles of WNT16B, β-catenin and S33Y were similar to control EGFP-expressing cells.

Normal primary NHEK usually survive less than 3-4 passages in serum-free keratinocyte culture system (data not shown). To test whether WNT16B was prolonging the clonogenicity of primary keratinocytes, EGFP or WNT16B transduced cells were allowed to grow and were passaged three times before fluorescence and phase-contrast images were captured. We found that WNT16B-expressing cells significantly prolonged cell survival compared with control cells expressing EGFP alone (Fig. 3D). In another experiment, EGFP or WNT16B-transduced cells were seeded at high density (∼2-3 million cells/35 mm²) and left to grow for 1 week with a daily medium change. The number of viable adherent cells was measured after 1 and 7 days in culture (Fig. 3E). WNT16B-expressing cells showed significant (P<0.001) cell survival compared with EGFP-expressing cells.

To verify the role of WNT16 in cell survival, we used the DNA-based short hairpin (sh)RNA interference technique to achieve isoform-specific gene silencing. Co-transfection of each WNT16 isoform with either shRNA against WNT16A (shA), against WNT16B (shB) or both (shAB) showed significant (P<0.001) isoform-specific knockdown 48 hours post transfection in RTS3b keratinocytes. WNT16B was knocked down by shB and shAB but not by shA (Fig. 4A,B). The effect of RNAi could be prolonged by growing shRNA-transfected cells in puromycin selection culture medium (data not shown). Using the WNT16-specific antibody for western immunoblotting, we showed that in both NHEKs and RTS3b, the WNT16B protein levels were significantly reduced in shB and shAB but not in shA-transfected cells (Fig. 4C). This confirmed the specificity and efficiency of shRNA interference used in this study to achieve WNT16B isoform-specific gene silencing. Using this shRNA gene silencing technique, we investigated the role of endogenous WNT16B in keratinocyte proliferation. In primary NHEKs, silencing endogenous WNT16B by shB significantly reduced cell survival (P<0.01; Fig. 4D) within a 7-day culture period. Transfection with control shRNA had little effect on keratinocyte cell proliferation.

To investigate the possibility of an involvement of β-catenin in WNT16B-induced cell proliferation and to further investigate non-canonical signalling of WNT16B, endogenous β-catenin was silenced in 293T cells using a commercially available siRNA specific to human β-catenin (siβCat, cat. no. 42816, Ambion). This siβCat has been used by other researchers to achieve significant β-catenin knockdown (Castellone et al., 2005; Yang et al., 2005). To validate this specificity, we co-transfected siβCat (10 nM; 48 hours) with or without EGFP, Wnt1 or β-catenin expression plasmids in the presence of the TCF/LEF luciferase reporter (pGL3-OT) to quantify the activity of β-catenin in 293T cells (Fig. 4E). siβCat specifically and significantly inhibited Wnt1 (2.7-fold; P<0.01) and β-catenin (3.6-fold; P<0.001) induced activity. Having confirmed the specificity of siβCat in our system, we proceeded to investigate its effect on Wnt16B-induced proliferation advantage in 293T cells. Cells were co-transfected (FuGENE 6, Roche) with or without siβCat in the absence or presence of EGFP or Wnt16B expression plasmids. These cells were left to grow for 48 hours before replating them in 96-well plates (n=6 for each treatment) at low starting cell density. Cells were left for 7 days and the density of live cells was measured using the CellTitre-Glo luminescent assay (Promega) to quantify ATP levels of metabolically active cells. This experiment was repeated with similar results. siβCat significantly reduced cell proliferation in both control cells and also in cells transfected with EGFP or Wnt16B (Fig. 4F). This result indicates that attenuation of β-catenin is detrimental to cell survival, which is in accordance with previous reports (Fodde et al., 2001; Hadjihannas et al., 2006; Kaplan et al., 2004; Olmeda et al., 2003).

To investigate the biological functions of WNT16B in keratinocyte proliferation and differentiation, we have used the three-dimensional organotypical keratinocyte co-culture system as described previously (Ojeh et al., 2001). Primary NHEK and RTS3b cells transduced with either EGFP or WNT16B were used in the organotypical co-cultures for assessing epidermal keratinocyte proliferation and differentiation (Fig. 4E,F). Transduced cells were verified by fluorescence microscopy and western blotting to ensure the expression of each WNT16 isoform before use in organotypical cultures. In both NHEK and RTS3b organotypical culture systems, WNT16B expression, but not EGFP, significantly induced (2.3-fold, P<0.01 in NHEK and 5.6-fold, P<0.001 in RTS3b; Fig. 4F) a phenotype equivalent to epidermal hyperproliferation, confirming the role of WNT16B in promoting keratinocyte proliferation as seen in experiments above using monolayer cultures.

We have previously shown that WNT16A and WNT16B isoforms were differentially expressed in various adult human tissues (Fear et al., 2000) but little is known about their biological functions in human skin and keratinocytes. This study investigated the roles of WNT16A and WNT16B isoforms in human epidermal keratinocytes using a range of tissue, cell and molecular techniques to characterise their biological roles in human skin. We first investigated and compared the WNT16 isoform mRNA expression levels using semi-quantitative RT-PCR. We found that WNT16B mRNA expression levels were ∼threefold higher in both cultured primary NHEKs and BCCs than in normal human skin. Immunohistochemistry showed that WNT16 protein was expressed throughout the suprabasal layers but not in the basal cells in normal human skin. In the hair follicle, WNT16 protein was detected in the `stem-cell' bulge compartment and also discrete expression was found within the dermal papilla. Although the antibody recognises both WNT16 isoforms, the lack of detectable WNT16A mRNA suggests the antibody is detecting WNT16B expression. These patterns of WNT16 protein localisation suggest that WNT16B may be involved in epidermal keratinocyte proliferation and also differentiation, hair follicle development and/or lineage specification or stem cell maintenance within the hair follicle. In the BCCs, WNT16 immunoreactivity was detected within the BCC tumour islands and especially at the peripheral palisading edge of the tumour. Using WNT16 isoform-specific RT-PCR, WNT16B was upregulated in the BCCs compared with normal skin. WNT16B is also upregulated in both chronic lymphocytic leukaemia (Lu et al., 2004) and acute lymphoblastoid leukaemia (Mazieres et al., 2005), which indicates that WNT16B might play a key role in tumourigenesis.

To further dissect the role of each of the WNT16 isoforms, retrovirus bearing either WNT16A or WNT16B isoforms was used to constitutively express each isoform in primary and immortalised human keratinocytes. Using RT-PCR, in vitro translation and immunoblotting, we confirmed that retrovirally transduced cells constitutively expressed each WNT16 isoform. However, we had difficulty detecting WNT16A protein in transduced cells, despite the presence of high levels of mRNA, suggesting that WNT16A may undergo rapid protein degradation or post-translational modification. The mechanism of WNT16A protein processing is beyond the scope of this study and further studies are required to delineate its mechanism.

To investigate whether WNT16B acts through the canonical WNT signalling pathway by inducing the accumulation of nuclear β-catenin and subsequent activation of TCF/LEF factors, an antibody against β-catenin was used to detect its protein levels and intracellular localisation within primary NHEKs transduced with either EGFP or WNT16B. Overexpression of WNT16B in keratinocytes neither increased β-catenin protein level nor induced nuclear β-catenin accumulation, suggesting that WNT16B does not activate through the canonical WNT signalling. This is further confirmed by our TCF/LEF (TOPFLASH) reporter assay showing no activation by WNT16B in both primary NHEK and 293T cells. We have provided the first evidence to suggest that in keratinocytes, WNT16B activates a non-canonical WNT pathway through the N-Jun-terminal kinase cascade by activating Jun and JNK (Kohn and Moon, 2005; Veeman et al., 2003). These results are in agreement with a previous study showing the lack of canonical WNT activation by WNT16B even in the presence of canonical WNT receptors Frizzled (Fzd5) and low-density-lipoprotein receptor-related proteins (LRP6) (Lu et al., 2004). Overexpression of WNT16B enhanced cell proliferation to confirm whether this was mediated via canonical or non-canonical WNT pathways we used a specific siRNA against β-catenin in 293T cells to silence endogenous β-catenin. We observed that WNT16B-induced proliferation was antagonised by β-catenin knockdown. However, in control experiments, silencing endogenous β-catenin also caused a significant reduction in cell proliferation compared with control cells (untransfected). Because siRNA against β-catenin inhibited cell proliferation in control cells we were unable to confirm whether the WNT16B effect on cell proliferation was specifically mediated via a non-canonical WNT pathway. This is consistent with previous studies showing that β-catenin has a role in a number of WNT-signalling-independent events such as cell-cycle G2-M phase progression (Olmeda et al., 2003), mitotic spindle assembly (Hadjihannas et al., 2006; Kaplan et al., 2004; Ridanpaa et al., 2001) and apoptosis (Olmeda et al., 2003). A recent study has shown that inhibition of WNT16B expression by RNA interference correlated with decreased β-catenin, dishevelled-2 and survivin protein levels in leukaemia cells with t(1;19) chromosomal translocation but not in control cells (Mazieres et al., 2005), indicating that WNT16B may also activate canonical WNT signalling in some cell types. Nevertheless, the role of canonical WNT signalling in BCCs remains debatable. One study using small number of BCC tumours found that 14 of the 20 BCCs were positive for nuclear β-catenin (Yamazaki et al., 2001) whereas another study using a larger number (46) of tumours found elevated cytoplasmic β-catenin levels in BCCs (Lo Muzio et al., 2002). Recently, a study using 86 BCCs, nuclear β-catenin was found in only a small subset (23%) of BCCs (Saldanha et al., 2004), indicating that canonical WNT signalling may be an effect rather than causal in BCC tumourigenesis.

To further characterise the cellular functions of WNT16B in keratinocytes, we compared the rate of cell proliferation and cell-cycle profiles in primary NHEKs. Proliferation rate was significantly increased by overexpression of WNT16B, indicating a positive role in cell growth and proliferation. FACS analysis showed insignificant alteration of the cell-cycle profile in WNT16B-expressing cells compared with control cells, indicating that WNT16B may not be directly involved in the cell cycle to enhance cell proliferation. Further experiments showed that overexpression of WNT16B in primary NHEK prolonged survival and clonogenicity. This suggests that upregulation of WNT16B might delay keratinocyte terminal differentiation thereby enhancing cell proliferation and prolonging clonogenicity. This might also explain the increased WNT16B expression in BCCs (especially in the peripheral palisading advancing edge of the tumour, Fig. 1G,H) and also in proliferating cultures of primary NHEKs (Fig. 1C,D) but not in normal skin. To further investigate the role of WNT16B in antagonising keratinocyte differentiation, a stratification-induced differentiation/cell death assay showed that WNT16B-transduced RTS3b keratinocytes were protected from terminal differentiation and cell death. This is consistent with our notion that WNT16B provided a survival advantage and prolonged proliferation, possibly through inhibition of keratinocyte differentiation. The acquisition of enhanced proliferation and survival advantage in cells overexpressing WNT16B could be attenuated by isoform-specific RNAi knockdown of WNT16B confirming its role in cell growth and proliferation. These results agree well with a previous study showing that isoform-specific RNAi knockdown of WNT16B increased apoptosis in leukaemia cells (Mazieres et al., 2005). We have presented several lines of evidence that WNT16B enhances cell proliferation and prolonged clonogenicity of primary keratinocytes in monolayer cultures. The effect of WNT16B on cell proliferation was also reproduced in a three-dimensional organotypical culture system using both primary and immortalised keratinocytes. WNT16B was found to induce a phenotype equivalent to hyperproliferation of epidermal keratinocytes (Fig. 4G,H), confirming its role in promoting cell proliferation.

In summary, this study investigated and compared the cellular functions of the two isoforms of WNT16 in human keratinocytes. We have presented the first evidence that WNT16B is upregulated in BCCs and its overexpression in keratinocytes led to a cellular hyperproliferation phenotype in a three-dimensional organotypical culture system. Using a variety of cellular and molecular approaches, we confirmed that WNT16B expression enhances keratinocyte proliferation in both primary and immortalised human keratinocytes possibly through a non-canonical WNT signalling pathway via the N-Jun-terminal kinase cascade. We hypothesise that upregulation of WNT16B plays an important role in tumourigenesis by conferring cell growth/proliferation, prolonged clonogenicity and cell survival advantage by antagonising or delaying keratinocyte differentiation.

The cloning of human WNT16A and WNT16B isoforms and WNT16 isoform-specific PCR primers was as described previously (Fear et al., 2000). Primers used for WNT16A were W16aF, 5′-CAGAAAGATGGAAAGGCACC-3′ and W16aR, 5′-ATCATGCAGTTCCATCTCTC-3′ (276 bp product). Primers used for WNT16B were W16bF, 5′-TGCTCGTGCTGTTCCCCTAC-3′ and W16bR, 5′-ATCATGCAGTTCCATCTCTC-3′ (226 bp product). GapF, 5′-CCCATCACCATCTTCCAGGAGC-3′ and GapR, 5′-CCAGTGAGCTTCCCGTTCAGC-3′ (based on the human glyceraldehyde-3-phosphate dehydrogenase, GAPDH: GenBank Accession no. NM_002046) primers were used to detect GAPDH (473bp) as a reference housekeeping gene in cDNA samples used for PCR. Semi-quantitative PCR conditions were 3 minutes at 95°C, 24 cycles of 30 seconds at 94°C, 30 seconds at 60°C, 60 seconds at 72°C, then 5 minutes at 72°C final extension. After agarose gel electrophoresis, images were captured using an AutoChemi imaging system (UVP, Upland, CA) and PCR products were densitometrically quantified using the LabWorks image acquisition and analysis software (UVP).

pEGFP/IRES-WNT16A/B were obtained by high fidelity PCR (pfu-Turbo DNA polymerase, Stratagene) cloning using the following primers: sense Eco16A-F, 5′-GCGGAATTCATGGAAAGGCACCCA-3′ (for WNT16A) or sense Eco16B-F, 5′-GCGGAATTCATGGACAGGGCGGCG-3′ (for WNT16B) and antisense Kpn16AB-R, 5′-GCGGTTACCTTACTTGCAAGTGTG-3′, into EcoRI-KpnI sites of pEGFP/IRES vector (BD Biosciences). The CMV-WNT16A/B-IRES-EGFP fragment was subsequently subcloned into the pSIN (Deng et al., 1997) retroviral vector kindly provided by Paul Khavari (Stanford University School of Medicine, CA). All plasmid constructs were verified by both restriction enzyme digest and sequencing analyses (BigDye Terminator Cycle Sequencing Kit, Applied Biosystems). pGL3-OT/OF luciferase reporter, pCL-β-Catenin (wild type) and pCL-β-Catenin-S33Y (constitutively active mutant) (Morin et al., 1997) were kindly provided by Bert Volgelstein (The John Hopkins Oncology Centre, Baltimore, MD). The pGL3-OT reporter was later subcloned into the pSIN retroviral vector (pSIN-OT) to perform luciferase reporter assays in primary human epidermal keratinocytes.

NHEKs were cultured in a low-Ca²⁺ (0.06 mM) EpiLife® keratinocyte growth medium (M-EPI-500-CA; Cascade Biologics, TCS CellWorks Ltd., Buckinghamshire, UK) supplemented with bovine insulin, hydrocortisone, bovine transferrin, human epidermal growth factor and bovine pituitary extract (ZHS-8943; Cascade Biologics). Spontaneously immortalised human epidermal keratinocytes (RTS3b) (Purdie et al., 1993) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 25% Ham's F12 medium, 10% foetal calf serum (FCS), and various mitogens (0.4 μg/ml hydrocortisone, 0.1 nM cholera toxin, 5 μg/ml transferrin, 20 pM lyothyronine, 0.18 mM adenine, 5 μg/ml insulin and 10 ng/ml epidermal growth factor). The human embryonic epithelial 293T and Phoenix (293T-derived) cell lines were cultured in DMEM supplemented with 10% FCS. All cells were grown at 37°C in a humidified atmosphere of either 5% (for EpiLife) or 10% (for DMEM) CO₂.

Retroviral supernatant was produced as previously described (Deng et al., 1997; Teh et al., 2002). Briefly, cells to be retrovirally transduced were plated at 10⁵ cells/well into six-well plates 24 hours before transduction. Each well was pre-incubated for 5-10 minutes in 1 ml of culture medium containing 5 μg/ml polybrene (hexadimethrine bromide; Sigma) before replacing with 2 ml of retroviral supernatant containing the same concentration of polybrene to facilitate infection. The six-well plate was then centrifuged (350 g) at 32°C for 1 hour before retroviral supernatant was replaced with normal growth medium and kept in normal culture condition.

PolyA⁺ mRNA was extracted directly from tissues or cells in culture using the Dynabeads® mRNA Direct kit (Dynal ASA, Oslo, Norway) and purified mRNA was then reverse transcribed (RT) into cDNA using the Reverse Transcription System kit (Promega). Control untranscribed mRNA was used as a control for genomic DNA contamination in subsequent PCR analysis. Primers were designed to amplify products with exons encompassing multiple introns, hence any genomic DNA contamination in PCR would give rise to significantly larger sized products. None of these mRNA controls produced any PCR products eliminating genomic DNA contamination.

The translation of WNT16A and WNT16B protein isoforms from the pcDNA-WNT16A/B-expression vectors were verified using the T7-TNT Quick Coupled Translation System (Promega) according to manufacturer's instructions. Briefly, translated lysates containing [³⁵S]methionine-incorporated proteins separated in 10% SDS-polyacrylamide gel was fixed (10% acetic acid, 10% ethanol, 1% glycerol) and vacuum dried (80°C, 1 hour) on Whatman filter paper before exposure to Phospho-Screen (24 hours) and subsequent image scanning using a PhosphorImager (STORM 840, Molecular Dynamics, Amersham).

WNT16 was immunostained with a rabbit polyclonal antibody (H-96; Santa Cruz Biotechnology) on paraffin sections as described (Ghali et al., 1999). The reaction product was visualised using diaminobenzidine as a chromogenic substrate. Protein samples were separated on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membrane (Hybon-C Extra, Amersham Pharmacia) according to standard protocols. Protein loading was normalised to total protein levels determined using the Bradford protein assay (Sigma). Antibodies used were rabbit polyclonal anti-WNT16 (1:1000; H-96 Santa Cruz Biotechnology), mouse monoclonal pan-anti-β-catenin (1:1000 dilution; Transduction Laboratories), rabbit polyclonal anti-phospho-c-Jun (Ser73) (1:1000; Cell Signalling Technology), rabbit polyclonal anti-phospho-JNK (Thr183/Tyr185) (1:1000; Cell Signalling) and mouse monoclonal anti-GAPDH (1:10,000; Abcam). Secondary horseradish peroxidase-linked antibodies were obtained commercially (Dako) and immunodetection was performed with enhanced chemiluminescence reagent (Amersham Pharmacia).

For canonical WNT reporter assay, pGL3-OT (containing a wild-type binding sequence) or pGL3-OF (mutant binding sequence) (Shih et al., 2000) and pcDNA3.1-LacZ (Invitrogen, San Diego, CA) were co-transfected with control (pEGFP-C3; BD Biosciences) or various WNT expression vectors into 293T cells using FuGENE 6 (Roche Biochemicals). To perform reporter assay in NHEKs, cells were co-transduced with pSIN-OT and EGFP or WNT16B viral supernatants. After 24 hours (or 72 hours for NHEK transduced cells), transfected cells were harvested for luciferase assays as previously described (Teh et al., 2002). Statistical analysis was performed using the GraphPad InStat software (V2.04a, GraphPad Software, San Diego, CA) for Student's t-test analysis.

For FACS-propidium iodide analysis (performed by Derek Davis at FACS Lab, Cancer Research UK, Lincoln Inn Fields, London, UK), culture medium was centrifuged together with trypsinised cells to collect all cells including detached cells. Each cell pellet was resuspended in 100 μl PBS before addition of 1 ml 70% ethanol and subsequently stored at –20°C. For the cell viability assay, at indicated time points, cells were harvested for measuring cell viability by using either the Bradford protein assay (Sigma) to determine the number of adherent cells or the CellTiter-Glo™ luminescent assay (Promega) to quantify ATP levels of metabolically active cells.

We used a H1 promoter-driven hairpin siRNA vector (pSilencer 3.1-H1 puro; Ambion, Cambridgeshire, UK) containing the following siRNA target sequences: human WNT16A (shA, 5′-AAGACCTCCCTATGGTGGTTG-3′ based on AF152584/gi:5020353), WNT16B (shB, 5′-AAACTGGATGTGGTTGGGCAT-3′ based on NM_057168/gi:17402815), WNT16 (shAB, 5′-AAGCTGGGCTGCGCCAATTTG-3′ for silencing both WNT16A and WNT16B isoforms) and control (shCtrl, containing a random sequence with no biological activity; Ambion). Cells were transfected with shRNA using Transfast™ Reagent (Promega) according to manufacturer's protocol. Transfected cells were grown in culture medium containing puromycin (2 μg/ml) to select for constitutive shRNA-expressing cells. siRNA specific to human β-catenin (siβCat, cat. no. 42816, Ambion) were used to knock down β-catenin expression in 293T cells by transient transfection (FuGENE 6, Roche Biochemicals).

The organotypic keratinocyte cultures were performed as previously described (Ojeh et al., 2001). Briefly, glycerol-preserved skin (Euro Skin Bank, Beverwijk, Holland) was washed three to five times in PBS and incubated in PBS containing antibiotic mix (600 units/ml penicillin-G, 600 μg/ml streptomycin sulphate, 250 μg/ml gentamicin sulphate and 2.5 μg/ml fungizone) at 37°C for up to 10 days. Epidermis was then mechanically removed using forceps, and de-epidermalised dermis (DED) was cut into 2×2 cm squares and placed in culture plates with the papillary dermal surface on the underside. Stainless steel rings were placed on top of the dermis, and normal human dermal fibroblasts (5×10⁵ cells) were seeded into the rings on the reticular dermal surface. After a 24-hour incubation, the DED was inverted to orient the papillary dermal surface on top before the rings were replaced and keratinocytes (3×10⁵ cells) were seeded inside the rings. After 2 days, the DED composite was raised to the air-liquid interface in the same orientation, by placing on stainless steel grids for 14 days. The medium was refreshed every 3 days. After 14 days, the composites were removed from the grids, fixed in 10% formalin and embedded in paraffin. Deparaffinised sections were stained with haematoxylin and eosin for histological examination.

Cultured cells expressing EGFP (enhanced green fluorescence protein) were visualised using a light microscope (Leica DM IRB) fitted with a krypton/argon mixed-gas laser for emitting excitation wavelength at 488 nm. Images were captured with a digital imaging system (Leica DC200 imager with a DC Viewer software V3.2; Leica Microsystems Ltd., Heerbrugg, Germany) attached to the microscope. Live-cell EGFP fluorescence expression levels (Fig. 4A,B) or organotypic epidermal area thickness (Fig. 4E,F) were quantified using Adobe Photoshop CS (Adobe Systems Inc., USA) utilising the Colour Range tool for EGFP quantification or Lasso tool for manual selection of epidermal area and subsequent pixel density readout from the Histogram data chart.

This work was supported by funding from the Cancer Research UK and the Association for International Cancer Research. We thank Derek Davis for FACS analysis.