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• W2078072567 abstract "Epidermal growth factor (EGF) plays an important role in corneal epithelial migration and proliferation to improve the wound healing process. This study aimed to understand the role of NFκB in EGF-induced corneal epithelial wound healing through regulation of CTCF activity, which plays important roles in cell motility and migration to promote wound healing. The effect of NFκB p50 on corneal epithelial wound healing was investigated by comparing the eyes of wild-type and p50 knockout mice. We found that there was a significant retardation in corneal epithelial wound healing in the corneas of p50 knockout mice. Wound closure rates were measured in human corneal epithelial cells transfected with an NFκB activation-sensitive CTCF expression construct to demonstrate the effect of human CTCF expression under the control of EGF-induced NFκB activation on wound healing. EGF stimulation activated NFκB, which directly triggered the expression of the exogenous human CTCF in transfected cells and, subsequently, promoted human corneal epithelial cell motility, migration, and wound healing. Overexpression of CTCF in corneal epithelial cells and mouse corneas significantly enhanced the wound healing process. Furthermore, the effect of overexpressing NFκB p50 in corneal epithelial cells on the promotion of wound healing was abolished by knockdown of CTCF with CTCF-specific shRNA. Thus, a direct regulatory relationship between EGF-induced NFκB p50 and CTCF activation affecting corneal epithelial wound healing has been established, indicating that CTCF is, indeed, a NFκB p50-targeted and effective gene product in the core transcriptional network downstream from the growth factor-induced NFκB signaling pathway.Background: We study NF-κB-activated CTCF in EGF-induced wound healing.Results: Corneal epithelial wound healing was significantly impaired because of the lack of CTCF activity in NFκB-p50−/− mice. EGF-induced NFκB activation regulated CTCF by interacting with the CTCF promoter to increases motility, migration, and wound healing.Conclusion: CTCF is an NFκB-p50-interactive target in EGF-induced corneal epithelial wound healing.Significance: NF-κB-controlled CTCF activation plays important roles in EGF-regulated wound healing. Epidermal growth factor (EGF) plays an important role in corneal epithelial migration and proliferation to improve the wound healing process. This study aimed to understand the role of NFκB in EGF-induced corneal epithelial wound healing through regulation of CTCF activity, which plays important roles in cell motility and migration to promote wound healing. The effect of NFκB p50 on corneal epithelial wound healing was investigated by comparing the eyes of wild-type and p50 knockout mice. We found that there was a significant retardation in corneal epithelial wound healing in the corneas of p50 knockout mice. Wound closure rates were measured in human corneal epithelial cells transfected with an NFκB activation-sensitive CTCF expression construct to demonstrate the effect of human CTCF expression under the control of EGF-induced NFκB activation on wound healing. EGF stimulation activated NFκB, which directly triggered the expression of the exogenous human CTCF in transfected cells and, subsequently, promoted human corneal epithelial cell motility, migration, and wound healing. Overexpression of CTCF in corneal epithelial cells and mouse corneas significantly enhanced the wound healing process. Furthermore, the effect of overexpressing NFκB p50 in corneal epithelial cells on the promotion of wound healing was abolished by knockdown of CTCF with CTCF-specific shRNA. Thus, a direct regulatory relationship between EGF-induced NFκB p50 and CTCF activation affecting corneal epithelial wound healing has been established, indicating that CTCF is, indeed, a NFκB p50-targeted and effective gene product in the core transcriptional network downstream from the growth factor-induced NFκB signaling pathway. Background: We study NF-κB-activated CTCF in EGF-induced wound healing. Results: Corneal epithelial wound healing was significantly impaired because of the lack of CTCF activity in NFκB-p50−/− mice. EGF-induced NFκB activation regulated CTCF by interacting with the CTCF promoter to increases motility, migration, and wound healing. Conclusion: CTCF is an NFκB-p50-interactive target in EGF-induced corneal epithelial wound healing. Significance: NF-κB-controlled CTCF activation plays important roles in EGF-regulated wound healing. The corneal epithelial layer protects the eye structures behind it from environmental insults and infections to maintain the intact function of the vision system (1.Lu L. Reinach P.S. Kao W.W. Corneal epithelial wound healing.Exp. Biol. Med. 2001; 226: 653-664Crossref PubMed Scopus (333) Google Scholar). Corneal epithelial cells undergo a self-renewal process to replace the surface layer cells and repair corneal surface wounds dependent on the stimulation of growth factors. EGF is one of the growth factors that play important roles in corneal epithelial self-renewal and wound healing (2.Zhang Y. Akhtar R.A. Effect of epidermal growth factor on phosphatidylinositol 3-kinase activity in rabbit corneal epithelial cells.Exp. Eye Res. 1996; 63: 265-275Crossref PubMed Scopus (23) Google Scholar, 3.Zhang Y. Akhtar R.A. Epidermal growth factor stimulation of phosphatidylinositol 3-kinase during wound closure in rabbit corneal epithelial cells.Invest. Ophthalmol. Vis. Sci. 1997; 38: 1139-1148PubMed Google Scholar, 4.Zhang Y. Akhtar R.A. Epidermal growth factor stimulates phospholipase D independent of phospholipase C, protein kinase C or phosphatidylinositol-3 kinase activation in immortalized rabbit corneal epithelial cells.Curr. Eye Res. 1998; 17: 294-300Crossref PubMed Scopus (29) Google Scholar, 5.Zhang Y. Liou G.I. Gulati A.K. Akhtar R.A. Expression of phosphatidylinositol 3-kinase during EGF-stimulated wound repair in rabbit corneal epithelium.Invest. Ophthalmol. Vis. Sci. 1999; 40: 2819-2826PubMed Google Scholar, 6.Islam M. Akhtar R.A. Epidermal growth factor stimulates phospholipase Cγ1 in cultured rabbit corneal epithelial cells.Exp. Eye Res. 2000; 70: 261-269Crossref PubMed Scopus (24) Google Scholar, 7.Islam M. Akhtar R.A. Upregulation of phospholipase Cγ1 activity during EGF-induced proliferation of corneal epithelial cells. Effect of phosphoinositide-3 kinase.Invest. Ophthalmol. Vis. Sci. 2001; 42: 1472-1478PubMed Google Scholar, 8.Kang S.S. Li T. Xu D. Reinach P.S. Lu L. Inhibitory effect of PGE2 on EGF-induced MAP kinase activity and rabbit corneal epithelial proliferation.Invest. Ophthalmol. Vis. Sci. 2000; 41: 2164-2169PubMed Google Scholar, 9.Kang S.S. Wang L. Kao W.W. Reinach P.S. Lu L. Control of SV-40 transformed RCE cell proliferation by growth-factor-induced cell cycle progression.Curr. Eye Res. 2001; 23: 397-405Crossref PubMed Scopus (28) Google Scholar). EGF stimulates intracellular signaling pathways, including the NF-κB, PI3K/AKT, and MAPK/Erk cascades, to regulate cell cycle progression and to activate transcription factors that control the genetic responses (10.Madhani H.D. Fink G.R. The riddle of MAP kinase signaling specificity.Trends Genet. 1998; 14: 151-155Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 11.Hanahan D. Weinberg R.A. The hallmarks of cancer.Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22271) Google Scholar, 12.Li T. Lu L. Functional role of CCCTC binding factor (CTCF) in stress-induced apoptosis.Exp. Cell Res. 2007; 313: 3057-3065Crossref PubMed Scopus (23) Google Scholar, 13.Zhou H. Gao J. Lu Z.Y. Lu L. Dai W. Xu M. Role of c-Fos/JunD in protecting stress-induced cell death.Cell Prolif. 2007; 40: 431-444Crossref PubMed Scopus (38) Google Scholar, 14.Lu L. Wang L. Li T. Wang J. NF-κB subtypes regulate CCCTC binding factor affecting corneal epithelial cell fate.J. Biol. Chem. 2010; 285: 9373-9382Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Earlier studies demonstrate that the mitogenic effect of EGF on the proliferation of corneal epithelial cells requires suppression of the eye-specific Pax6 expression (15.Li T. Lu L. Epidermal growth factor-induced proliferation requires down-regulation of Pax6 in corneal epithelial cells.J. Biol. Chem. 2005; 280: 12988-91295Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The effect of EGF on suppressing Pax6 expression is through activation of CTCF, 2The abbreviations used are: CTCFCCCTC binding factorHTCEhuman telomerase-immortalized corneal epithelialHCEhuman SV-40 large T-transformed corneal epithelialTREtranscription response element. an epigenetic CCCTC binding factor and zinc finger protein (16.Li T. Lu Z. Lu L. Regulation of eye development by transcription control of CCCTC binding factor (CTCF).J. Biol. Chem. 2004; 279: 27575-27583Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In corneal epithelial cells, we found that EGF induces NF-κB subtype-specific signaling cascades to regulate CTCF activity and to promote cell proliferation (14.Lu L. Wang L. Li T. Wang J. NF-κB subtypes regulate CCCTC binding factor affecting corneal epithelial cell fate.J. Biol. Chem. 2010; 285: 9373-9382Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Our recent study demonstrates that CTCF is required for the EGF-induced alteration of focal adhesion and increases in cell motility and migration (17.Wang L. Deng S.X. Lu L. Role of CTCF in EGF-induced migration of immortalized human corneal epithelial cells.Invest. Ophthalmol. Vis. Sci. 2012; 53: 946-951Crossref PubMed Scopus (8) Google Scholar). However, the mechanisms of how EGF-induced activation of NF-κB and its subtypes regulate transcription activity of CTCF to effect corneal epithelial wound healing in the eye are still under investigation. CCCTC binding factor human telomerase-immortalized corneal epithelial human SV-40 large T-transformed corneal epithelial transcription response element. EGF activates transcription factors, such as NF-κB, CTCF, and other immediate early genes, upon exposure of mammalian cells to the growth factor (12.Li T. Lu L. Functional role of CCCTC binding factor (CTCF) in stress-induced apoptosis.Exp. Cell Res. 2007; 313: 3057-3065Crossref PubMed Scopus (23) Google Scholar, 18.Herrlich P. Ponta H. Rahmsdorf H.J. DNA damage-induced gene expression. Signal transduction and relation to growth factor signaling.Rev. Physiol. Biochem. Pharmacol. 1992; 119: 187-223Crossref PubMed Scopus (183) Google Scholar, 19.Holbrook N.J. Liu Y. Fornace Jr., A.J. Signaling events controlling the molecular response to genotoxic stress.EXS. 1996; 77: 273-288PubMed Google Scholar, 20.Belandia B. Latasa M.J. Villa A. Pascual A. Thyroid hormone negatively regulates the transcriptional activity of the β-amyloid precursor protein gene.J. Biol. Chem. 1998; 273: 30366-30371Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 21.Büscher M. Rahmsdorf H.J. Litfin M. Karin M. Herrlich P. Activation of the c-fos gene by UV and phorbol ester. Different signal transduction pathways converge to the same enhancer element.Oncogene. 1988; 3: 301-311PubMed Google Scholar, 22.Devary Y. Rosette C. DiDonato J.A. Karin M. NF-κB activation by ultraviolet light not dependent on a nuclear signal.Science. 1993; 261: 1442-1445Crossref PubMed Scopus (576) Google Scholar, 23.Li T. Dai W. Lu L. Ultraviolet-induced junD activation and apoptosis in myeloblastic leukemia ML-1 cells.J. Biol. Chem. 2002; 277: 32668-32676Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). NF-κB is an important gene regulator in the Rel transcription factor family involving inflammatory responses, developmental processes, cellular growth, and apoptosis (24.Courtois G. Gilmore T.D. Mutations in the NF-κB signaling pathway. Implications for human disease.Oncogene. 2006; 25: 6831-6843Crossref PubMed Scopus (411) Google Scholar, 25.Dutta J. Fan Y. Gupta N. Fan G. Gélinas C. Current insights into the regulation of programmed cell death by NF-κB.Oncogene. 2006; 25: 6800-6816Crossref PubMed Scopus (356) Google Scholar). CTCF is another gene regulator that plays important roles in the epigenetic regulation of genes. It functions as an insulator sensitive to DNA methylation to epigenetically control DNA imprinting and X chromosome inactivation during development (26.Baniahmad A. Steiner C. Köhne A.C. Renkawitz R. Modular structure of a chicken lysozyme silencer. Involvement of an unusual thyroid hormone receptor binding site.Cell. 1990; 61: 505-514Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 27.Bell K.D. Campbell R.J. Bourne W.M. Pathology of late endothelial failure. Late endothelial failure of penetrating keratoplasty. Study with light and electron microscopy.Cornea. 2000; 19: 40-46Crossref PubMed Scopus (61) Google Scholar, 28.Hark A.T. Schoenherr C.J. Katz D.J. Ingram R.S. Levorse J.M. Tilghman S.M. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus.Nature. 2000; 405: 486-489Crossref PubMed Scopus (1222) Google Scholar). Many studies demonstrate that CTCF also play a role as a transcription activator and repressor. Recent studies indicate that CTCF is involved in the regulation of cell migration in cancer cell proliferation, tumor suppression, and apoptosis (29.Qi C.F. Martensson A. Mattioli M. Dalla-Favera R. Lobanenkov V.V. Morse 3rd., H.C. CTCF functions as a critical regulator of cell-cycle arrest and death after ligation of the B cell receptor on immature B cells.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 633-638Crossref PubMed Scopus (56) Google Scholar, 30.Rasko J.E. Klenova E.M. Leon J. Filippova G.N. Loukinov D.I. Vatolin S. Robinson A.F. Hu Y.J. Ulmer J. Ward M.D. Pugacheva E.M. Neiman P.E. Morse 3rd, H.C. Collins S.J. Lobanenkov V.V. Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF.Cancer Res. 2001; 61: 6002-6007PubMed Google Scholar, 31.Docquier F. Farrar D. D'Arcy V. Chernukhin I. Robinson A.F. Loukinov D. Vatolin S. Pack S. Mackay A. Harris R.A. Dorricott H. O'Hare M.J. Lobanenkov V. Klenova E. Heightened expression of CTCF in breast cancer cells is associated with resistance to apoptosis.Cancer Res. 2005; 65: 5112-5122Crossref PubMed Scopus (80) Google Scholar). In corneal epithelial cells, EGF-induced activation of the NF-κB pathway regulates cell fate in a subtype-specific fashion through interactions with CTCF that function as a downstream component in the core transcriptional network (14.Lu L. Wang L. Li T. Wang J. NF-κB subtypes regulate CCCTC binding factor affecting corneal epithelial cell fate.J. Biol. Chem. 2010; 285: 9373-9382Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 32.Wang Y. Lu L. Activation of oxidative stress-regulated Bcl-3 suppresses CTCF in corneal epithelial cells.PLoS ONE. 2011; 6: e23984Crossref PubMed Scopus (3) Google Scholar). We found that, in corneal epithelial cells, CTCF is a targeted gene of the growth factor-induced pathways, including the Erk, AKT, and NF-κB signaling cascades. Activation of these signaling pathways by stimulation of EGF, insulin, and other stresses subsequently regulates the expression levels of CTCF to determine the corneal epithelial fate in the process of wound healing (12.Li T. Lu L. Functional role of CCCTC binding factor (CTCF) in stress-induced apoptosis.Exp. Cell Res. 2007; 313: 3057-3065Crossref PubMed Scopus (23) Google Scholar, 13.Zhou H. Gao J. Lu Z.Y. Lu L. Dai W. Xu M. Role of c-Fos/JunD in protecting stress-induced cell death.Cell Prolif. 2007; 40: 431-444Crossref PubMed Scopus (38) Google Scholar, 14.Lu L. Wang L. Li T. Wang J. NF-κB subtypes regulate CCCTC binding factor affecting corneal epithelial cell fate.J. Biol. Chem. 2010; 285: 9373-9382Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 32.Wang Y. Lu L. Activation of oxidative stress-regulated Bcl-3 suppresses CTCF in corneal epithelial cells.PLoS ONE. 2011; 6: e23984Crossref PubMed Scopus (3) Google Scholar). As previously described, EGF is an important growth factor in corneal epithelial wound healing. It facilitates corneal epithelial wound repair by promoting migration and proliferation in both in vivo and in vitro model systems (1.Lu L. Reinach P.S. Kao W.W. Corneal epithelial wound healing.Exp. Biol. Med. 2001; 226: 653-664Crossref PubMed Scopus (333) Google Scholar, 15.Li T. Lu L. Epidermal growth factor-induced proliferation requires down-regulation of Pax6 in corneal epithelial cells.J. Biol. Chem. 2005; 280: 12988-91295Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 33.Wang J. Lin A. Lu L. Effect of EGF-induced HDAC6 activation on corneal epithelial wound healing.Invest. Ophthalmol. Vis. Sci. 2010; 51: 2943-2948Crossref PubMed Scopus (13) Google Scholar, 34.Xu K.P. Ding Y. Ling J. Dong Z. Yu F.S. Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells.Invest. Ophthalmol. Vis. Sci. 2004; 45: 813-820Crossref PubMed Scopus (125) Google Scholar, 35.Yin J. Yu F.S. ERK1/2 mediate wounding- and G-protein-coupled receptor ligands-induced EGFR activation via regulating ADAM17 and HB-EGF shedding.Invest. Ophthalmol. Vis. Sci. 2009; 50: 132-139Crossref PubMed Scopus (49) Google Scholar). The question that remains to be answered is whether CTCF is one of the key factors that directly switch EGF-induced activation of NF-κB signaling to genetic responses that subsequently change corneal epithelial cell stages, resulting in the acceleration of wound healing. On the corneal surface, corneal epithelial wound healing requires proper activities of cell migration that are essential for successful re-epithelialization in the process of corneal epithelial self-renewal (1.Lu L. Reinach P.S. Kao W.W. Corneal epithelial wound healing.Exp. Biol. Med. 2001; 226: 653-664Crossref PubMed Scopus (333) Google Scholar). We demonstrate that EGF-induced CTCF activation accelerates corneal epithelial cell migration, which is favorable for wound healing and tissue repair in the cornea (15.Li T. Lu L. Epidermal growth factor-induced proliferation requires down-regulation of Pax6 in corneal epithelial cells.J. Biol. Chem. 2005; 280: 12988-91295Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16.Li T. Lu Z. Lu L. Regulation of eye development by transcription control of CCCTC binding factor (CTCF).J. Biol. Chem. 2004; 279: 27575-27583Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). However, the results obtained for EGF-induced NFκB subtype activation are sometimes contradictory, and the role of CTCF in corneal epithelial wound healing remains unclear. This study aimed to advance our understanding of how the EGF-induced NFκB subtype p50 directly activates CTCF to increase cell motility and migration in human corneal epithelial cells to promote corneal epithelial wound healing. We further revealed an EGF-induced activation of the NFκB p50 subtype that interacts with CTCF in the promoter region, resulting in the activation of CTCF and facilitating corneal epithelial wound healing. NF-κB p50 knockout transgenic mice (NF-κB 1−/−) and wild-type mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and genotypes of these mice were confirmed by PCR analysis from prepared tail DNA. All animal experiments were conducted in accordance with the institutional guidelines of the Animal Care and Use Committee according to National Institutes of Health guidelines. Human telomerase-immortalized corneal epithelial (HTCE) cells were cultured in a keratinocyte serum-free medium containing 120 μm calcium and supplemented with 0.4% bovine pituitary extract and 0.2 ng/ml EGF (Invitrogen). Human SV-40 large T-transformed corneal epithelial (HCE) cells were grown in Dulbecco's modified Eagle's medium/F-12 (1:1) containing 10% fetal bovine serum and 5 μg/ml insulin. Cells were cultured in an incubator supplied with 95% air and 5% CO2 at 37 °C. Culture media were replaced every 2 days, and cells were subcultured by treatment with 0.05% trypsin-EDTA. For EGF-induced experiments, cells were synchronized in growth factor-deprived medium for 24–48 h before EGF stimulation. Full-length cDNA encoding human p50 was cloned into the pcDNA4-to-A vector (Invitrogen), named pcDNA4-p50. Both the pcDNA4-p50 construct and the pcDNA4-to-A vector (control) were transfected into HTCE cells by FuGENE HD transfection reagent (Roche) for wound healing assays and Western blot analysis. For the experiments knocking down cellular NF-κB p50, p50-specific siRNA (GGGGCUAUAAUCCUGG-ACU (sense) and AGUCCAGGAUUAU-AGCCCC (antisense)) and control siRNA (Santa Cruz Biotechnology) were transfected into HCE cells that were subject to wound healing assays and Western blot analysis. Lentiviral particles containing shRNA of CTCF tagged with a variant of green fluorescent protein (Turbo-GFP, Sigma-Aldrich, St. Louis, MO) were packaged in HEK-293T cells (17.Wang L. Deng S.X. Lu L. Role of CTCF in EGF-induced migration of immortalized human corneal epithelial cells.Invest. Ophthalmol. Vis. Sci. 2012; 53: 946-951Crossref PubMed Scopus (8) Google Scholar). The viral concentrations in the culture medium were titrated by PCR after cotransfection of HTCE cells with pCMV-VSV-G, psPAX2, and pGIPZshRNA-CTCF fused to the GFP for 72 h (Open Biosystems, Huntsville, AL). The culture medium containing the lentiviral particles secreted from HEK-293T cells was added to HTCE cells, and infected clones stably expressing shRNAs were selected in selective culture with puromycin (2 μg/ml). HTCE cells infected with a pGIPZ-shRNA-control vector packed in the lentivirus served as controls. In addition, expression of GFP from the pGIPZ-TurboGFP vector allowed measuring of the efficiency of the viral infection and distinguishing green from non-green cells. The green cells integrated with shRNA were visualized by fluorescence microscope (Nikon). For corneal wound healing studies, the cDNA encoding the full-length CTCF gene were introduced into a linearized Adeno-x-vector using the In-Fusion HD cloning system (Clontech). The recombinant adenovirus was packaged in HEK293 cells and amplified by transfecting PacI-digested vectors. The viral titer of 109 plaque-forming units was obtained from crude viral lysates. The recombinant adenoviruses Adeno-x-vector (for controls) or Adeno-x-CTCF were added to the culture medium at 2.5 × 108 plaque-forming units/ml. After 5 days of incubation, the eyeballs were transferred to a new dish containing normal defined keratinocyte serum-free (KSF) medium without virus and incubated for additional 10–15 days. The medium was changed every other day and photographed with a Nikon fluorescent microscope. The wound area was calculated with Nikon Tis NIS-Elements software. NF-κB transcription response element (TREs) sties were inserted in five repeats upstream of the mini-CMV promoter followed by GFP or GFP plus full-length human CTCF cDNAs. The constructs were termed TRE-Control and TRE-CTCF, respectively. GFP served as a reporter of NF-κB activity. Lentiviral particles that contained NF-κB TRE-Control or TRE-CTCF were cotransfected with pCMV-VSV-G and psPAX2 into HEK-293T cells for packaging. The culture medium containing a high titer of lentivirus secreted from HEK-293T cells was collected and added to HTCE cells. All clones that were integrated with TRE-Control or TRE-CTCF were selected for 4 weeks by adding puromycin (2 μg/ml) to the culture medium to establish stable expression cell lines. Two wound healing assays were performed, including corneal wound healing in cultured whole-eye organ and a scratch-induced directional wound-healing assay. The corneas in cultured whole-eye organs were used for experiments of corneal epithelial wound healing. Under a dissecting microscope, the surface layer of the mouse corneas was debrided without damaging the basement membrane of the corneal epithelia using a corneal rust ring remover with a 0.5-mm burr (Algerbrush, The Alger Company, Inc., Lago Vista, TX). The whole eyeball was dissected and placed in culture wells (the corneas facing up) with the medium containing 10% fetal bovine serum and 1% antibiotic/antimycotic solution at 37 °C and 5% CO2 in a humidified incubator. The rate of epithelial healing in whole-eye organ culture was measured immediately after wounding. Eyeballs were taken from wild-type or NF-κB p50 knockout mice and allowed to heal under culture conditions. Lesions of the corneas were stained topically with fluorescein (fluorescein sodium 1.0% w/v) and photographed with an inverted microscope (Nikon). The corneal epithelial layer was removed in an area of 1.5-mm diameter (or 2-mm diameter for Ad-x-CTCF transduction assays) near the central cornea. Wounded corneas were incubated for 1–2 days under normal and EGF-induced conditions and up to 15 days in the absence of FBS and EGF. All animals used in our experiments were treated in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research using protocols approved and monitored by the LABioMed Animal Care Committee at the University of California Los Angeles School of Medicine. Corneal epithelial cells were seeded at 3 × 105 cells/well in 12-well plates and grown to 100% confluence. A cross-stripe scratch wound was made on the cell surface with a yellow micropipette tip. The wound area was measured by calculating the average values at multiple points (at least 10 points/wound along the edges) using commercial software (NIS-Element, Nikon, Tokyo, Japan) and photographed with an inverted microscope (Eclipse Ti, Nikon) during the healing period. The microscope was able to record exactly the same area at each time point by memorizing the x-y directions through a computer-controlled and motorized head stage. The width of the wounded area was measured, and the rate of wound closure was calculated using the units of micrometers/hour. The cell migration assay was performed following the instructions of the manufacturer (Transwell, Corning Inc., Corning, NY). The migration chamber culture insert contained a polyethylene terephthalate membrane 6.5 mm in diameter with an 8-μm pore size. HTCE cells expressing NF-κB TRE-CTCF or TRE-Control (5 × 104) were seeded in the culture insert (upper chamber) with plain medium and incubated for 24 h. EGF (20 ng/ml) or the sham was added to the culture insert, and the cells were incubated for 48 h. Migrated cells that grew on the culture well (bottom chamber) were counted and photographed with an inverted fluorescence microscope (Nikon). The cells were fixed in 4% paraformaldehyde, stained with 0.3% crystal violet, and photographed. The dye in the cells was then dissolved in 10% acetic acid, and the absorbance of the dissolved dye was measured at a wavelength of 600 nm. The Motility of HTCE cells expressing NF-κB TRE-CTCF and TRE-Control was measured using an inverted microscope (Eclipse Ti, Nikon) with the following functions: time-lapse videos of the phase contrast/fluorescent live images, built-in total internal reflection fluorescence and FRET, perfect focus system, and a digital charge-coupled device (CCD) camera at a time interval of 2 min for each photo. The system was equipped with a heated chamber at 37 °C and flushed with mixed 5% CO2 that kept the cells under normal culture conditions. Live cells were recorded for a period of 0.5–3 h. Cell motility was examined by tracking cell movements and distances (millimeters/hour) using an inverted microscope with a motorized head stage and software (Tis NIS-Elements, Nikon). Immunocytochemistry experiments were performed following a protocol as described previously (36.Wang L. Payton R. Dai W. Lu L. Hyperosmotic stress-induced ATF-2 activation through Polo-like kinase 3 in human corneal epithelial cells.J. Biol. Chem. 2011; 286: 1951-1958Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Briefly, mouse eyeballs were fixed with 4% paraformaldehyde and sectioned into 8-μm sections. The tissue section was perforated with 0.3% Triton X-100 in PBS (PBS-T). After being blocked with 2% BSA and 5% normal serum in PBS-T, the sections were incubated with primary antibody against CTCF (Millipore) in 1% BSA-0.1% Triton X-100-PBS for 16 h at 4 °C. Cy3-conjugated secondary antibody was applied in 1% BSA- 0.1% Triton X-100-PBS for 1 h at room temperature. Stained tissues were mounted with shield mounting medium (Vector Laboratories Inc.) and photographed using the Nikon Eclipse Ti inverted microscope with a ×60 oil total internal reflection fluorescence lens. Western blot analyses were performed by lysing corneal epithelial cells (2 × 105) in SDS sample buffer that contained 62.5 mm Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% glycerol, 50 mm DTT, and 0.01% (w/v) bromphenol blue. Proteins in the lysates were denatured by boiling for 5 min and being size-fractionated in 8–10% PAGE gels. Proteins in PAGE gels were electrotransferred to PVDF membranes (Millipore) by using a semidry gel transferring apparatus (Bio-Rad, CA). The PVDF membranes were exposed to the blocking buffer containing 5% nonfat milk in TBS and 0.1% Tween 20 (TBS-T) for 1 h at 22 °C and then incubated with the respective primary antibodies at 4 °C overnight. HRP-conjugated secondary antibody was applied in TBS-T buffer for 1 h at 22 °C. Western blot analyses were developed by an ECL Plus system (Santa Cruz Biotechnology) and visualized by exposure to x-ray films. Recent reports demonstrate that corneal epithelial wound healing is affected by a deficiency of IκB (in the NF-κB pathway) in the eyes of IκB knockout mice (37.Chen L. Meng Q. Kao W. Xia Y. IκB kinase β regulates epithelium migration during corneal wound healing.PLoS ONE. 2011; 6: e16132Crossref PubMed Scopus (20) Googl" @default.
• W2078072567 created "2016-06-24" @default.
• W2078072567 creator A5050487837 @default.
• W2078072567 creator A5061226816 @default.
• W2078072567 creator A5077852653 @default.
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• W2078072567 date "2013-08-01" @default.
• W2078072567 modified "2023-10-10" @default.
• W2078072567 title "Epidermal Growth Factor (EGF)-induced Corneal Epithelial Wound Healing through Nuclear Factor κB Subtype-regulated CCCTC Binding Factor (CTCF) Activation" @default.
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• W2078072567 doi "https://doi.org/10.1074/jbc.m113.458141" @default.
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