Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish
© Chou et al. 2015
Received: 21 April 2015
Accepted: 16 October 2015
Published: 17 November 2015
Mutations in mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) that occur during cell proliferation and tumor formation are well described. Information on the roles of MEK2 in these effects is still limited. We established a constitutive MEK2 transgenic zebrafish, Tg(krt14:MEK2S219D-GFP), to elucidate the role of MEK2 in skin tumor formation.
We found that both constitutive MEK2 and MEK1 are able to phosphorylate the extracellular signal-regulated kinase 1 (ERK1) protein. Transient expression of constitutive MEK2 and MEK1 in the zebrafish epidermis induced papillary formation at 48 h post-fertilization, but no effects were observed due to the expression of MEK1, MEK2, or the dominant negative form of MEK2. The transgenic zebrafish, Tg(krt14:MEK2S219D-GFP), developed skin papillomas in the epidermis within 6 days post-fertilization (dpf). The phospho-ERK signal was detected in section of skin papillomas in an immunohistochemical experiment. Treatment with 50 μM of the MEK inhibitor, U0126, had significantly decreased the skin papilloma formation in Tg(krt14:MEK2S219D-GFP) zebrafish by 6 dpf. In vitro and in vivo proliferation assay in COS-1 cells and in Tg(krt14:MEK2S219D-GFP) transgenic fish show significantly increased cell number and Ki-67 signaling.
Our data indicate that MEK2 is sufficient to induce epidermal papilloma formation through MAPK signaling in zebrafish, and this transgenic model can be used as a new platform for drug screening.
The Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is the downstream pathway of Ras which transforms its effects on its targets . When Ras is activated by a mutation, it was hypothesized that downstream components will be constitutively activated and corresponding abnormal gene expressions will occur that drive proliferation. Several model systems of skin tumors in mice have been established [1–5, 7, 8, 12, 20]. Zebrafish are a very useful and convenient model organism for studying human diseases. So far, no squamous skin cell tumor model has been established in zebrafish [9, 10, 16, 25, 29].
MEK2 was reported to be a factor related to invasion and metastasis of pancreatic tumors . The expression of Raf22w, v-Ha-Ras, or v-Src resulted in the constitutive activation of MEK1 and MEK2 . A mutation of p21ras in hepatocellular carcinoma (HCC) was associated with marked changes in expression of the function of MAPK cascades mainly through MEK1 . MEK1 is important for skin tumor development, and MEK2 could not compensate for this process with the loss of MEK1 function in a mouse model . The MEK1 and MEK2 signaling pathways were not redundant or interchangeable for cell proliferation in SK-MEL-28 melanoma cells .
However, it is unclear if MEK2 constitutively activated in zebrafish is sufficient to induce epidermal neoplasia. Herein, we established a stable transgenic zebrafish model with consistent expression of MEK2S219D under an epidermal-specific Krt14 promoter using the Tol2-transposone system to investigate the effects of MEK2S219D on inducing epidermal neoplasias in vivo. Further treatment with U0126 in this transgenic zebrafish tumor model was used to evaluate the cause of skin papilloma. We suggest that this model can serve as a useful platform for cancer drug screening.
Zebrafish (Danio rerio) were maintained at 28 °C on a 14-h light/10-h dark cycle. Embryos were incubated at 28 °C, and different developmental stages were determined according to the description in the Zebrafish Book .
Total RNA isolation
Total RNA was isolated from fertilized eggs (a pool of embryos at 12, 24, 36, 48, 72, 96, 120, and 144 h post-fertilization; hpf), using the RNAzol reagent (Tel-Test, Friendswood, TX, USA) according to instructions of the manufacturer.
Isolation of the full-length mek1, mek2, and erk1 cDNAs of zebrafish
cDNAs encoding the complete open reading frame (ORF) of zebrafish mek1/2 and erk1 were obtained by polymerase chain reaction (PCR) amplification using gene-specific primers according to the NCBI’s zebrafish EST database. PCR amplification was performed in a 50-μl reaction mixture containing 2 μl of first-strand cDNA, 0.5 μg of a forward primer (MEK1-F: 5′-CGG GAT CCA TGC AGA AAA GGA GGA AG-3′; MEK2-F: 5′-TAT TGG ATG TCA GTA GAG ACA ACC TGG-3′; and ERK1-F: 5′-CGG ACA GAA ACG ATG GCG GAA TCG-3′) and reverse primer (MEK1-R: 5′-GGG AAT TCC ATT CCC ACA CTG TGA GT-3′; MEK2-R: 5′-TTC AGC GCT ATG AGT GGG TGT GCT AGG-3′; and ERK1-R: 5′-CGT GAT GAC TGT CCC TCT CAG GAG-3′), 1.5 mM MgCl2, 0.2 mM dNTP, and 2.5 units ExTaq (Takara Shuzo, Shiga, Japan). Samples were incubated in a thermal cycler (SensoQuest, Göttingen, Germany). PCR amplification was performed; the DNA fragment of MEK1 and MEK2 was cloned into a pGEM-T Easy vector (Promega, Madison, WI, USA) as pGEMT-MEK1 and pGMET-MEK2 plasmids, and the resultant clones were subjected to a sequencing analysis.
Site-directed mutagenesis of zebrafish MEK1 and MEK2 cDNA
Site-directed mutagenesis was performed to generate plasmids encoding MEK1/2 mutants (S219D and S223A) using the pGEMT-MEK1 and pGEMT-MEK2 plasmid as a template for PCR amplification using specific oligonucleotide primers. The corresponding oligonucleotides used for PCR reaction were as follows with the altered bases underlined: MEK1S219D-F: 5′-GGA CAA CTC ATT GAC GAC ATG GCC-3′, MEK1S219D-R: 5′-GGC CAT GTC GTC AAT GAG TTG TCC-3′; MEK2S219D-F: 5′-CAG CTC ATC GAC GAT ATG GCC AAC TCC-3′, MEK2S219D-R: 5′-GGA GTT GGC CAT ATC GTC GAT GAG CTG-3′; and MEK2S223A-F: 5′-ATG GCC AAC GCC TTC GTT GGA ACA CGG-3′, MEK2S223A-R: 5′-CCG TGT TCC AAC GAA GGC GTT GGC CAT-3′. Sequences of the resultant plasmids were verified using DNA sequencing.
Construction of the expression plasmids
The 4-kb upstream region of the zebrafish krt14 gene (krt14 promoter) was amplified from a BAC clone, DKEYP-113D7 (Source Biosicence, Nottingham, UK), and inserted into the pGEM-T Easy cloning vector (Promega, Madison, WI, USA) as pGMET-krt14-pro plasmid. The krt14 promoter was then inserted into the pTol2-GFP vector  at appropriate sites to generate the pTol2-krt14-GFP plasmid. MEK1, MEK1S219D, MEK2, MEK2S219D, MEK2S223A, and ERK1 were inserted into pcDNA3-mCherry, pcDNA3-GFP, and pcDNA3-HA from PCR products as pcDNA3-MEK1-mCherry, pcDNA3-MEK1S219D-GFP, pcDNA3-MEK2-GFP, pcDNA3-MEK2S219D-GFP, pcDNA3-MEK2S223A-GFP, pcDNA3-ERK1-HA and then inserted into the pTol2-krt14-GFP vector to generate pTol2-krt14-MEK1-mCherry, pTol2-krt14-MEK1S219D-GFP, pTol2-krt14-MEK2-GFP, pTol2-MEK2S223A-GFP, and pTol2-krt14-MEK2S219D-GFP plasmids. The pcDNA3-GFP and pcDNA3-mCherry plasmids were constructed by inserting cDNA corresponding to the green fluorescence protein (GFP) and mCherry coding-region into pCMV-HA-YUN, which was a gift from Dr. H.J. Kung (Davis Cancer Center, University of California, Sacramento, CA, USA).
Microinjection of expression plasmid into zebrafish embryo
The expression plasmid was adjusted to a final concentration of 100 μg/ml in 1× Danieau solution (5 mM HEPES at pH 7.6, 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, and 0.6 mM Ca(NO3)2) containing 0.5 % phenol red and injected into a zebrafish embryo in the one-cell stage using the Narishige IM 300 microinjector system (Narishigi Scientific Instrument, Tokyo, Japan).
Cell culture and plasmid transfection
Monkey kidney fibroblast COS-1 cells (ATCC CRL-1650; VA, USA) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, UT, USA), supplemented with 10 % fetal bovine serum (FBS; Hyclone, UT, USA) in a humidified atmosphere of 5 % CO2 at 37 °C. COS-1 cells were transfected with pcDNA3, pcDNA3-MEK1-mCherry, pcDNA3-MEK1S219D-GFP, pcDNA3-MEK2-GFP, pcDNA3-MEK2S219D-GFP, pcDNA3-ERK1-HA plasmids, using Lipofectamine Plus reagent (Life technologies, Grand Island, NY, USA) according to manufacturer’s manner. Culture cells were harvested at 48 h post-transfection and lysed with lysis buffer. The protein level was analyzed by western blotting.
Total cell lysates from transfected cells were harvested and separated on 10 % sodium dodecylsulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5 % skim milk in phosphate-buffered saline (PBS) for 1 h at room temperature and then incubated at 4 °C with an anti-HA monoclonal antibody, anti-Actin polyclonal antibody (Santa Cruz, Dallas, TX, USA), and anti-mCherry polyclonal antibody (GeneTex, Hsinchu, Taiwan). Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).
Subcellular localization and image analysis
COS-1 cells transfected with pcDNA3-ERK1-HA alone or with pcDNA3-MEK1-mCherry, pcDNA3-MEK1S219D-GFP, pcDNA3-MEK2-GFP, pcDNA3-MEK2S219D-GFP, and pcDNA3-MEK2S223A-GFP for 48 h were fixed with 4 % paraformaldehyde and permeabilized in PBS with 0.1 % Triton X-100. Immunostaining was performed using an anti-HA antibody at 4 °C overnight, followed by incubation with a Cy2-conjugated goat anti-mouse antibody or Cy3-conjugated goat anti-mouse antibody for 30 min and DAPI (1 μg/ml) for 5 min at room temperature. Photoimages were prepared using an Olympus IX70-FLA inverted fluorescence microscope (Olympus, Tokyo, Japan) equipped with the SPOT system (Diagnostic Instruments, Sterling Heights, MI, USA).
Histology and Immunohistochemistry
Larvae in which a skin papilloma had formed from Tg(krt14-MEK2S219D-GFP) at 6 days post-fertilization (dpf) were fixed with 4 % paraformaldehyde for 1 day and dehydrated with 100 % methanol. Specimens were embedded in paraffin, sliced and stained with hematoxylin and eosin (H&E) or incubated with 3 % serum at room temperature for 1 h before incubation with an anti-pERK polyclonal antibody (Santa Cruz, Dallas, TX, USA) or anti-Ki-67 polyclonal antibody (GeneTex, Hsinchu, Taiwan) containing 5 % serum and 2 mg/ml bovine serum albumin (BSA) at 4 °C overnight. After washing with PBST, embryos were incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody at room temperature for 3 h. HRP labeling was visualized using DAB (Merck, Darmstadt, Germany) as a substrate. Nuclei were counterstained with hematoxylin (Merck, Darmstadt, Germany).
Chemical treatment and statistical analysis
Tg(krt14-GFP) and Tg(krt14-MEK2S219D-GFP) embryos were dechorionated at 1 dpf. Ten embryos with high GFP fluorescent signaling were transferred to each well in 1 ml water containing 50 μM U0126 (Sigma, MO, USA) or 0.01 % DMSO (Sigma, MO, USA) and incubated from 1 to 6 days at 28 °C incubator. The number of zebrafish forms any papilloma was assessed at 6 dpf under an Olympus IX70-FLA inverted fluorescence microscope. Results from three independent experiments are expressed as the mean ± standard deviation (SD). Statistical significance between groups was determined by an unpaired Student’s t-test. A probability of p < 0.001 was indicated as ***.
Cell proliferation assay
Cell proliferation was performed by trypan blue assay. Briefly, 3x104 COS-1 cells were seeded in 24-well plate overnight. COS-1 cells were transfected with 1 μg pcDNA3-GPF or pcDNA3-MEK2S219D-GFP plasmids using Lipofectamine Plus reagent (Life technologies, Grand Island, NY, USA) according to manufacturer’s manner. Culture cells were trypsinized and the number of viable cells was virtually counted with a hemocytometer at day 1, 2, 3 and 4 post-transfection. Three counts per well were performed on three different experiments. Statistical significance between groups was determined by an unpaired Student’s t-test. A probability of p < 0.05 was indicated as *.
Cloning and characterization of the zebrafish mek1 and mek2 (mek2a) cDNAs
The mek1 and mek2 cDNAs were isolated from mixed cDNA pools prepared from different developmental stages of zebrafish embryos by PCR amplification. Full-length cDNA of zebrafish mek1 and mek2 were 1191 and 1194 base-pair long, encoding two proteins of 396 and 397 amino acid residues, respectively. The overall amino acid sequence of zebrafish MEK1 showed 76 % identity and 85 % similarity to that of MEK2. The zebrafish MEK1 respectively showed 88 and 87 % identities to human and mouse MEK1 and shown 78 % identities to human and mouse MEK2 at the level of amino acid sequences (Fig. 1b). The encoded amino acid sequence of zebrafish MEK1 and MEK2 was highly homologous to human and mouse proteins. The phosphorylated site of MEK1 was also conserved (Fig. 1b, arrow). These data suggest that zebrafish MEK1 and MEK2 are highly conserved with mammalian MEK1 and MEK2.
Both MEK1 and MEK2 phosphorylated the ERK1 protein
It has been shown recently that overexpression of the ERK protein without active signaling caused its accumulation in the nuclei and the cytoplasm. In the presence of the active MEK protein, the ERK protein was translocate from nuclei into the cytoplasm . To further characterize the functions of MEK1 and MEK2, we examined the cellular localization of ERK1. In GFP, MEK1 or MEK2 expressed COS-1 cells, the ERK1 protein was accumulated in the nuclei and cytoplasm (Fig. 2b. A1-A4, B1-B4, D1-D4; arrow). In the presence of active MEK1S219D-GFP and MEK2S219D-GFP, the ERK1 protein was located in the cytoplasm (Fig. 2b, C1-C4, E1-E4). These results indicate that active zebrafish MEK1 and MEK2 both are able to induce ERK1 protein translocate from nuclei to the cytoplasm.
Transient expression of MEK1S219D and MEK2S219D induced papillary formation in the zebrafish epidermis
MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish
To further examine whether skin papilloma formation induced by MEK2 is through downstream signaling activation, we tested ERK phosphorylation of skin papilloma by an IHC experiment. Phosphorylated ERK is well known as a proliferation marker during tumor formation. Phosphorylation of ERK in skin papilloma of Homozygous Tg(krt14:MEK2S219D-GFP) larvae was detected by IHC experiment (Fig. 4f-F’). These results suggested that zebrafish constitutive activated MEK2S219D induced skin papilloma formation through phosphorylation of downstream ERK signaling.
U0126 inhibits skin papilloma formation induced by MEK2S219D
In vitro and in vivo cell proliferation assay in COS-1 cells and Tg(krt14:MEK2S219D-GFP) transgenic zebrafish
In this study, we established a transgenic zebrafish model to explore the role of MEK2 in skin tumor formation. Constitutive MEK1 and MEK2 phosphorylated ERK1 and induced downstream MAPK signaling activation. Transient expression of constitutive MEK2 and MEK1 in the epidermis had induced skin papillary formation at 3 dpf. Sporadic skin papilloma formation was revealed in the epidermis of Tg(krt14-MEK2S219D-GFP) transgenic zebrafish and had developed into skin tumors at 6 dpf. Treatment with U0126 could reduce skin tumor formation in Tg(krt14-MEK2S219D-GFP) transgenic zebrafish.
The functions of MEK1 and MEK2 in the Ras/Raf/Mek/Erk signaling pathway were well documented. During epidermal neoplasia formation, MEK2 and MEK1 showed mostly similar functions, but each had several differences. In zebrafish, the functions of MEK1 and MEK2 have not yet been characterized. From a phylogenic analysis, we identified and isolated zebrafish mek1 and mek2 cDNAs. We also isolated the downstream erk1 cDNA to characterize the function of these two MEK proteins. An in vitro experiment showed that both MEK1 and MEK2 were able to phosphorylate downstream ERK1 by causing it to mutate to the constitutive active form. The ERK1 protein is then translocate from nuclei to the cytoplasm. We conclude that zebrafish MEK1 and MEK2 could activate downstream gene expression through Erk1 phosphorylation.
The role of MEK2 in skin tumor development is still being debated. MEK2 functions in mouse melanoma formation but does not contribute to skin tumor formation [17, 26, 27]. Those transgenic mice that contained chemically induced carcinomas or knockout mice were used to characterize the functions of MEK1 and MEK2. The krt14-MEK1 transgenic mice exhibited moderate hyperplasia with spontaneous skin tumor formation at 5 weeks. Downstream signaling is activated by ornithine decarboxylase (ODC) expression . Surprisingly, skin tumors was sporadically formed just within 6 days in our Tg(krt14-MEK2S219D-GFP) transgenic zebrafish. The zebrafish MEK2 was sufficient to induce skin tumor formation through ERK1 activation.
In previous reports, the function of MEK1 was important in skin tumor formation. In our results, transient expression of activated MEK1 was produced papillary formation in the epidermis. The skin papilloma was also revealed in activated MEK2 expression. Although we did not establish transgenic zebrafish by expression of activated MEK1, we suggest that MEK1 also potentially to induce skin papilloma formation. Moreover, transient expression of constitutive MEK2 in the epidermis induced papillary formation. These papillae were then excised from the epidermis and placed in the medium. Collection of this skin cells and cultured in L15 medium could be continuously maintained for more than 1 week. Analysis of these skin cells showed intact nuclei, tubulin structure, and cell shape (data not shown). We suggest that these skin cells were experiencing hyper-proliferation due to MEK2 activation. This observation may be relevant to psoriasis but needs further investigation.
Ras proto-oncogenes are central regulators activating intracellular Ras-Raf-MEK-ERK signaling pathway in tumorigenesis. The zebrafish is increasingly used as a vertebrate model for studying tumorigenesis and drug screening. A transgenic approach is generally used for establishing a transgenic zebrafish. The Tg(fabp10:EGFP-krasv12) trangenic zebrafish specifically overexpressed high level of krasv12 in liver and progressed to hyperplasia and malignant tumor [22, 23]. Furthermore, the transgenic zebrafish overexpressed the BRAFV600E-mutated gene activated under microphthalmia-associated transcription factor (mitf) promoter induced melanocyte proliferation . The transgenic zebrafish also overexpressed krasG12V gene by using a pancreas specific transcription factor 1a promoter expressed in the pancreases, which caused pancreatic neoplasia . The overexpression of the krasG12V gene driven by cytokeratin 5 (krt5) or glial fibrillary acidic protein promoter induced brain tumor . Mutagenesis induced by a random insertion into the genome through a Tol2 transposon-mediated transgenic approach was used as new method for studying gene function during development [14, 21, 24]. A Tol2 transposon-mediated enhancer trap contains GFP without a promoter was used for establishing transgenic lines, but no phenotype was observed in 37 transgenic lines . The aforementioned method driven by the mini keratin8 promoter in the Tol2 transposon-mediated approach increased the transgenic efficiency to 16 %, but only 3.57 % of genes were inserted into the coding region . The Tol2 transposon-mediated enhancer-trap using an hsp70 promoter-driven GFP was used to screen gene mutagenesis. The transgenic efficiency was increased to 70 % but only 2.73 % recessive mutations were identified . To date, no gene mutation induced by Tol2 transposon-mediated enhancer trap has induced tumorigenesis. In our study, we established a Tol2 transposon-mediated Tg(Ka14:MEK2S219D-GFP) transgenic zebrafish by using a Ka14 promoter to constitutively activate MEK2S219D-GFP expression. Skip papilloma was observed at 3–6 dpf, and no other tumor type was observed in this transgenic line. We also established a Tg(Tol2:GFP) transgenic line by using Tol2 transposon-mediated approach, which did not display skin papilloma. Although we could not complicatedly exclude the possibility of a random insertion to the oncogene upstream of MEK and induction of the skin papilloma formation, we suggested that the frequency was extremely rare.
In summary, we showed that MEK2 is sufficient to induce skin papilloma formation. In vitro and in vivo experiments showed that activated MEK2 in MAPK pathway promoted downstream signaling through ERK1 phosphorylation. This activation could be inhibited by U0126 and reduced skin papilloma formation in transgenic zebrafish. This Tg(krt14-MEK2S219D-GFP) is the first established skin tumor model in zebrafish and could provide for anticancer drug screening to target the MAPK cascade.
Most cancer was associated with constitutive activation of ERK signaling, which was activated ERK1/2 by MEK1 and MEK2 upon activation of receptor tyrosine kinase. Our data supported that the constitutive activation of MEK2 was sufficiently induced tumor formation in Tg(krt14:MEK2S219D-GFP) transgenic zebrafish. This transgenic zebrafish might be provided for anticancer drug screening.
Taipei Medical University New Faculty Grant (TMU101-AE1-B22) and MOST (104-2311-B-038-001) supported this study. We thank the Zebrafish Core Laboratory of Taipei Medical University for help with the zebrafish experiments. We also thank the Department of Pathology of TMU for preparing H&E and IHC stains.
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