Epithelial Phenotype in Human Keratinocytes

In the current study, we investigated the effects of p63 modulation in epithelial plasticity in human keratinocytes. p63 isoforms ΔNp63α, ΔNp63β, and ΔNp63γ were ectopically expressed in normal human epidermal keratinocytes (NHEK). Epithelial or mesenchymal state was determined by morphological changes and altered expression of various markers, e.g., fibronectin (FN), ECadherin (E-Cad), and keratin 14 (K14). Overexpression of ΔNp63α and ΔNp63β , but not ΔNp63γ isoforms, led to morphological changes consistent with epithelial-mesenchymal transition (EMT). However, only ΔNp63α overexpression was able to maintain the morphological changes and molecular phenotype consistent with EMT. Interestingly, knockdown of all p63 isoforms by transfection of p63 siRNA also led to EMT phenotype, further confirming the role of p63 in regulating epithelial phenotype in NHEK. EMT in NHK accompanied loss of GrainyheadLike 2 (GHRL2) and miR-200 family gene expression, both of which play crucial roles in determining epithelial phenotype. Modulation of GRHL2 in NHK also led to congruent changes in p63 expression. Chromatin immunoprecipitation (ChIP) revealed direct GRHL2 binding to p63 promoter; GRHL2 knockdown in NHK led to impaired binding of GRHL2 and changes in the histone marks consistent with p63 gene silencing. These data indicate the presence of a reciprocal feedback regulation between p63 and GRHL2 in NHEK to regulate epithelial plasticity. p63 is a transcription factor that regulates epithelial phenotype and keratinocyte proliferation and is an important marker of epidermal and stratified keratinocyte stem cells (KSCs) (4,5). p63 is expressed as six isoforms. Three of these isoforms contain the N-terminal transactivation domain (TAp63α, TAp63β, TAp63γ), whereas http://www.jbc.org/cgi/doi/10.1074/jbc.M115.659144 The latest version is at JBC Papers in Press. Published on June 17, 2015 as Manuscript M115.659144 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from p63 and GRHL2 determine epithelial plasticity in human keratinocytes 2 the remainder (ΔNp63α, ΔNp63β, ΔNp63γ) do not (6). Our recent study demonstrated that transduction of the ΔNp63α isoform and intact TGF-β signaling is necessary to induce epithelialmesenchyme transition (EMT) in normal human epidermal keratinocytes (NHEK) (4). EMT is a process by which epithelial cells exhibit morphologic and molecular changes consistent with mesenchymal phenotype and is involved in tissue and organ development and repair (7). Cells with EMT phenotype exhibit loss of epithelial morphology and development of elongated, spindled, mesenchymal morphology (8). EMT is facilitated by the loss of cell-cell adhesion mediated by E-Cadherin (E-Cad), and loss of ECad expression serves as a marker for EMT (9). Also, loss of keratin 14 (K14) and enhanced expression of fibronectin (FN) and vimentin serve as mesenchymal markers (9-11). GRHL2 is a novel transcription factor involved with epithelial morphogenesis, cell proliferation, and differentiation (12). Our recent studies showed that GRHL2 transcriptionally regulates broad spectrum of target genes including the human telomerase (hTERT) gene, proliferating cell nuclear antigen (PCNA), and epidermal differentiation complex (EDC) genes (12,13). Phenotypically, GRHL2 promotes epithelial cell proliferation and inhibits keratinocyte differentiation in NHEK (12). Other studies suggest its inhibitory role in EMT and apoptosis in breast cancer cell lines, demonstrating its involvement in cancer progression (14). Mechanistically, GRHL2 downregulates zinc finger E-box binding protein 1 (ZEB1), which triggers EMT through suppressing E-Cad expression (15). Thus, GRHL2 is a determinant of epithelial phenotype through transcriptional network of its target genes. Our previous study demonstrated that ectopic ΔNp63α expression in NHEK yielded EMT phenotype in a TGF-β dependent manner (4). In this report, we show that induced EMT phenotype is specific to ectopic overexpression of ΔNp63α among the p63 isoforms. Alternatively, knockdown of all isoforms of p63 also led to EMT phenotype through loss of GRHL2 and miR-200 family genes. Lastly, we found that GRHL2 directly binds to the p63 promoter, and that p63 and GRHL2 regulate their mutual expression. This reciprocal feedback loop between p63 and GRHL2 maybe critical in determining epithelial plasticity. EXPERIMENTAL PROCEDURES Cells and cell culture Primary cultured single cell-suspension of NHEK were isolated from discarded foreskin epidermal tissues and grown in EpiLife medium supplemented with growth factors (Invitrogen, Carlsbad, CA), as described elsewhere (16). Normal human oral keratinocytes (NHOK) isolated from separated keratinizing oral epithelial tissues were prepared similar to NHEK. NHEK were serially subcultured and passed at 70% confluence until they reached replicative senescence. Replication kinetics were recorded as described previously (16). Normal human oral fibroblasts (NHOF) were obtained from discarded gingival connective tissue and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). Bone marrow mesenchymal stromal cells (BM-MSCs) and dental pulp stem cells (DPSCs) were cultured in α-MEM medium (Invitrogen) supplemented with 10% FBS (Invitrogen), and 5 mg/mL gentamicin sulfate (Gemini Bio-Products, West Sacramento, CA). SCC4 and SCC9 cancer cell lines were cultured in DMEM/F12 (Invitrogen) and supplemented with 10% FBS and 0.4 pg/ml hydrocortisone, as described elsewhere (17). Immortalized keratinocyte cell line HaCaT cells were grown in EpiLife medium supplemented with growth factors (Invitrogen). Cells were maintained at 37°C at 5% CO2 in a humidified chamber. Retroviral vector construction and transduction of cells – We constructed retroviral vectors expressing human wild-type ΔNp63α, ΔNp63β, and ΔNp63γ. Full-length cDNAs of ΔNp63 were cloned from the cDNA library obtained from primary human keratinocytes by PCR amplification. ΔNp63α, ΔNp63β, and ΔNp63γ cDNAs were subcloned into pLXSN retroviral expression vector (Clontech, Mountainview, CA) at XhoI/BamHI restriction sites, and isoforms were confirmed by sequencing. Retroviral construction and infection were performed as described elsewhere (18), and the infected cells were selected with 200 μg/ml G418 (Sigma, St. Louis, MO). G418-resistant cells were continually by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from p63 and GRHL2 determine epithelial plasticity in human keratinocytes 3 maintained by serial subculture as outlined above. Transwell Migration Assay – Transwell chambers with polycarbonate membranes were used to measure cell migration (Corning Inc., Corning, NY), according to previously described methods (19). NHEK/LXSN, NHEK/ΔNp63α, NHEK/ΔNp63β, and NHEK/ΔNp63γ cells were cultured in EpiLife (Invitrogen) and seeded in the upper chamber of the transwell. Cells were allowed to migrate for 24 hours, and the transwell was washed with 1x phosphate-buffered saline (PBS) and fixed in 10% formalin for 10 min. Cells were then stained with 1% crystal violet dissolved in 10% formalin for a duration of 1 hour. Nonmigratory cells were removed from the chamber and transwells were photographed. Immunofluorescence Staining – Cells were fixed for 15 minutes in 3.7% formaldehyde prior to permeabilization with 0.25% Triton X-100 for 10 minutes. Cells were then blocked in 10% normal goat serum for 1 hour. Antibodies against p63 (A4A), E-Cad, β-catenin, ZEB1 (Santa Cruz Biotech, Santa Cruz, CA), GRHL2 (Abnova, Taipei City, Taiwan), and anti-FN (Sigma) were used as primary antibodies, and Alex Fluor 488 IgG (Invitrogen) was used as secondary antibody. Cells were counterstained with DAPI, and Olympus BH2-RFCA fluorescence microscope was used to obtain images. siRNA Transient Transfection NHEK were transfected with non-specific, scrambled siRNA (Si-SCR) or siRNA targeting human p63 (Si-p63) at a concentration of 10 nM using Lipofectamine reagents (Invitrogen) according to the manufacture’s instructions. NHEK were transfected with Si-p63 once every 3 days over the course of 9 days. NHEK growth medium was replaced with mesenchymal growth medium (αMEM medium/10% FBS, 5 mg/mL gentamicin sulfate) upon morphological transition to mesenchymal phenotype. Western Blotting Whole cell extracts were isolated from the cultured cells, fractionated by sodium dodecylsulfate polyacrylamide gel electrophoresis, and transferred to Immobilon membrane (Millipore, Billerica, MA). Antibodies against p63 (A4A), p63 (H129), E-Cad, K14, ZEB1, N-Cad, GAPDH (Santa Cruz Biotech), GRHL2 (Abnova), FN and Snail (Sigma) were used. Chemiluminescence signal was detected by using the HyGLO Chemiluminescent HRP antibody detection reagent (Denville Scientific, South Plainfield, NJ). Real-time qRT-PCR Total RNA was extracted from cultured cells by using RNeasy Plus Mini kit (Qiagen, Valencia, CA). Reverse transcription (RT) was performed with 5 μg RNA by using the method described elsewhere (20). qRT-PCR was performed for the relative mRNA expression of TAp63, ΔNp63, and GRHL2, relative microRNA expression of miR-200a, miR-200b, miR-200c, miR-141, miR-429 and enrichment of GRHL2, H3K4Me3 and H3K27Me3 in triplicates for each sample with LC480 SYBR Green I master (Roche, Basel, Switzerland) using LightCycler 480 (Roche). Second derivative Cq value was calculated after running a total of 50 cycles in order to compare relative change in mRNA and microRNA expression. The primer sequences and the PCR conditions will be available on request. F-Actin Staining / Stress Fiber Formation– Cells were fixed for 10 minutes in 3.7% formaldehyde prior to permeabilization with 0.1% Triton X-100 for 5 minutes. Cells were stained with Alexa Fluor 594 Phalloidin (Invitrogen) for 20 minutes at room temperature. Cells were counterstained with DAPI, and Olympus BH2RFCA fluorescence microscope was used to obtain images. Lentiviral vector construction and knockdown of endogenous GRHL2 – Lentiviral vector expressing short hairpin RNA (shRNA) against GRHL2 (Sh-GRHL2) and control vector expressing enhanced green fluorescence protein (EGFP) were constructed as described earlier (21). Endogenous GRHL2 was knocked down with ShGRHL2. All lentiviral vectors used allow identification of infected cells by GFP fluorescent signal under epifluorescence microscope. Dual-Luciferase Reporter Assay – Promoter regions of GRHL2 and p63 were cloned into pGL3B-Luc reporter plasmid (Promega, Madison, WI) expressing firefly luciferase. The promoterluciferase constructs were transfected into SCC4 or SCC9 using Lipofectin Reagent (Invitrogen), along with pRL-SV40 containing Renilla luciferase cDNA under the control of SV40 enhancer/promoter. After 48 hours posttransfection, cells were collected and the lysates were prepared using Dual Luciferase Reporter Assay System (Promega). Firefly and Renilla by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from p63 and GRHL2 determine epithelial plasticity in human keratinocytes 4 luciferase activities were measured using a luminometer (Turner Designs, Sunnyvale, CA). Renilla luciferase activity was used to control for the varied transfected efficiency. The gene promoter activity, reflected by the firefly luciferase activity, was determined as the mean of at least triplicates per experiment. Chromatin Immunoprecipitation (ChIP) / ChIP-qPCR Assay ChIP assay was performed as described previously (12). Nuclear proteins were cross-linked to DNA by adding 10% formaldehyde for 10 minutes, and 0.15 M glycine was used to stop cross-linking. The cells were then collected in ice-cold phosphate-buffered saline (PBS) supplemented with a protease inhibitor mixture and lysed in buffer (1% SDS, 10mM EDTA, protease inhibitors, 50mM Tris-HCl, pH 8.1). Genomic DNA was sonicated to produce DNA fragments 300–1000 bp in length. Cellular lysates were diluted 1:10 in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM NaCl, protease inhibitors, 16.7mM Tris-HCl, pH 8.1). Chromatin solutions were incubated with 5 μl p63 or GRHL2 primary antibody (or 5 μl of mouse IgG) overnight at 4°C with rotation. Immunocomplexes were precipitated with 30 μl of protein G-agarose slurry (Upstate Biotechnology, Lake Placid, NY, USA) and eluted in 500 μl of buffer (1% SDS, 100mM NaHCO3). Precipitated DNA was recovered by phenol extraction and used for PCR amplification to check the enrichment of p63, GRHL2, H3K4Me3 or H3K27Me3 in gene promoter regions. qPCR was performed with the purified DNA using LightCycler 480 (Roche). Samples pulled down with IgG were included as the negative control. qPCR readout was normalized relative to the amount of amplification from input. The promoter regions analyzed in this study for p63 or GRHL2 enrichment is near transcription start sites (TSS) (available upon request), which contribute to core promoter activity and our previous data shown that GRHL2 binds to this region to regulate gene expression (12).