Effects of epiplakin-knockdown in cultured corneal epithelial cells
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To investigate effects of knockdown of epiplakin gene expression on the homeostasis of cultured corneal epithelial cell line. We previously reported acceleration of corneal epithelial wound healing in an epiplakin-null mouse.
Gene expression of epiplakin was knockdowned by employing siRNA transfection in SV40-immortalized human corneal epithelial cell line. Protein expression of E-cadherin, keratin 6 and vimentin was examined by western blotting. Cell migration and proliferation were examined by using scratch assay and Alamar blue assay, respectively.
Scratch assay and Alamar blue assay showed migration and proliferation of the cells was accelerated by epiplakin knockdown. siRNA-knockdown of epiplakin suppressed protein expression of E-cadherin, keratin 6 and vimentin.
Decreased expression of E-cadherin, keratin 6 and vimentin might be included in the mechanisms of cell migration acceleration in the absence of epiplakin. The mechanism of cell proliferation stimulation by epiplakin knockdown is to be investigated.
KeywordsEpiplakin Knockdown Corneal epithelium Culture Migration Proliferation E-cadherin
Epiplakin was one of intermediate filament-related components and was originally identified as an autoantigen that reacted with serum from a patient with subepidermal blistering disease , . Epiplakin is homologous to plectin and other members of the plakin family , . Human epiplakin is a 552-kDa protein that is expressed in various epithelial tissues, i.e., epidermis, esophagus, outer root sheath of hair follicles and mucous epithelial cells , . Studies by using an epiplakin-null mouse line showed that lacking epiplakin accelerates migration of epidermal keratinocytes in mice in vivo and also showed that this is also the case in outgrowth of keratinocytes from explanted skin tissue in vitro, although the exact mechanism of the phenomena is to be revealed . We reported that corneal epithelium, a non-keratinizing stratified squamous epithelium, also express epiplakin and its loss accelerates cell migration-dependent healing of the corneal epithelium in mice. The in vivo study showed reduced E-cadherin protein and mRNA in epiplakin -null corneal epithelium, although E-cadherin expression level is not altered in epidermis by the loss of epiplakin . In the present study we investigated behaviors, i.e., cell migration and proliferation, of epiplakin-knockdowned cultured corneal epithelial cell line and also examined the expression level of E-cadherin, keratin 6 and vimentin, both members of intermediate filament involved in cell migration , in the cells, although decreased epiplakin expression does not solely explain the mechanism of cell migration acceleration in corneal epithelium.
Experimental protocols and the use of experimental mice were approved by the DNA Recombination Experiment Committee and the Animal Care and Use Committee of Wakayama Medical University and conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Araki-Sasaki SV40-immortalized human corneal epithelial cell line was obtained from RIKEN Laboratories, Tsukuba Japan. The phenotype of this cell line retains many of the properties identified in their primary cell counterpart . They were grown to confluence in DMEM/F12 medium containing 200 U/ml of penicillin and streptomycin, 5 % FBS, 0.1 μg/ml cholera toxin, 5 μg/ml insulin, and 10 ng/ml human epidermal growth factor (EGF).
Knockdown of epiplakin in a corneal epithelial cell line in vitro
Expression of epiplakin was knockdowned in Araki-Sasaki cell line by employing a commercially available siRNA for epiplakin. The cells (2 × 105) in each well of a six well plate in 2 ml antibiotic-free normal growth medium supplemented with 5 % fetal bovine serum. The cells were incubate at 37 °C under 5.0 % CO2 until the cells are 60–80 % confluent. Human epiplakin 1 siRNA (sc-77799, Santa Cruz Biotechnology, CA) or control siRNA (sc-37007, Santa Cruz Biotechnology) was transfected for 6.0 h according to the protocol prepared by the manufacturer using siRNA Transfection Medium (sc-36868, Santa Cruz Biotechnology). Cells without siRNA were also prepared (control). Normal growth medium (1.0 ml) containing 2 times the normal serum and antibiotics concentration (2× normal growth medium) was added to each well without removing the transfection mixture.
Cell migration by scratch assay
The cells were cultured for 72 h after siRNA transfection procedure. The medium was then replaced with fresh 1× normal growth medium and processed for scratch assay as previously reported. Two liner defects were produced by a silicone needle in monolayer of the cells in control culture and siRNA epiplakin knockdown culture. Six cultures were prepared for each condition. The closure of the defect was evaluated at two independent points in each defect under phase-contrast microscopy at every 3 h until 30 h post-wounding. The width of the remaining defect was measured at four independent points at each time point and statistically analysed by Mann–Whitney U-test.
Cell proliferation assay
Araki-Sasaki cells were treated with epiplakin-siRNA or control siRNA as described above. Cells were seeded into wells of 96-well plates at the concentration of 1.0 ×104 cells/100 l/well 72 h after siRNA treatment, and incubated for 24 h. Twenty-six wells were prepared for each condition. Cell proliferation was assayed by using Alamar blue (Trek Diagnostic Systems, West Sussex, UK) according to the manufacturer’s protocol . After a wash with phosphate-buffered saline (PBS), 40 μL Alamar blue was diluted in culture medium (1:2).Thirty and sixty min later, the optical absorbance at 570 nm was measured at 570 nm.
Western blotting for epiplakin, E-cadherin, keratin 6 and vimentin
The efficacy of knockdown of epiplakin protein expression was first evaluated by using western blotting with anti-epiplakin antibody (T-16, sc-87104, Santa Cruz:diluted 1:500) as previously reported . Then protein expression of E-cadherin, keratin 6 and vimentin was assayed by western blotting as previously reported . SDS-PAGE was done with the gel of Mini-PROTEAN® TGX™ Gel (Bio-Rad, #456-1096) under 200 V. The PVDF membrane was Immobilon-P Membrane, PVDF, 0.45 µm, 26 × 26 cm sheet.
(Merk Millipore Corporation, #IPVH304F0) and transfer the proteins under 5 V. Antibodies used were anti-E-cadherin antibody (G-10, sc-8426, Santa Cruz Biotechnology, diluted 1:1000) and anti-kerarin 6 antibody (LHK6, sc-53260, Santa Cruz Biotechnology, diluted 1:1000), and anti-vimentin antibody (C-20, sc-7557, Santa Cruz Biotechnology, diluted 1:1000).
Results and discussion
Further study is to be conducted to uncover the mechanism of cell migration acceleration in the absence of epiplakin in vivo and in vitro in order to understand the biological mechanism underlying corneal epithelial wound healing.
SiRNA knockdown of epiplakin gene expression accelerated migration and proliferation of corneal epithelial cell in cell culture. Decreased expression of E-cadherin, keratin 6 and vimentin might be included in the mechanisms of cell migration acceleration in the absence of epiplakin. The mechanism of cell proliferation stimulation by epiplakin knockdown is to be investigated.
SS conceived and designed the experiments. MK and YO performed the experiments. TM and OY participated in study design, data analysis. All authors read and approved the final manuscript.
The authors thank Prof. Sakuhei Fujiwara, Department of Dermatology, Ohita University School of Medicine, Ohita, Japan, for his helpful suggestion.
The authors declare that they have no competing interests.
This study was supported by Grant of Wakayama Medical Award for Young Researchers (to MK) and the Grants from the Ministry of Education, Science, Sports and Culture of Japan (C21592241 to YO, C19592036 to SS). The abstract of the manuscript was presented by Dr. Kokado at the ARVO Annual meeting (2009, 2012, Fort Lauderdale, FL).
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