Ectodysplasin regulates corneal epithelial cell proliferation
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In the cornea, healing of the wounded avascular surface is an intricate process comprising the involvement of epithelial, stromal and neuronal cell interactions. These interactions result to the release of various growth factors that play prominent roles during corneal wound healing response. Bone morphogenetic proteins (BMPs) are unique multi-functional potent growth factors of the transforming growth factor-beta (TGF-β) superfamily. Treatment of corneal epithelial cells with substance P and nerve growth factor resulted to an increase in the expression of BMP7 mRNA. Since BMP7 is known to modulate the process of corneal wound healing, in this present study, we investigated the influence of exogenous rhBMP7 on human corneal epithelial cell and stromal cell (SFs) function. To obtain a high-fidelity expression profiling of activated biomarkers and pathways, transcriptome-wide gene-level expression profiling of epithelial cells in the presence of BMP7 was performed. Gene ontology analysis shows BMP7 stimulation activated TGF-β signaling and cell cycle pathways, whereas biological processes related to cell cycle, microtubule and intermediate filament cytoskeleton organization were significantly impacted in corneal epithelial cells. Scratch wound healing assay showed increased motility and migration of BMP7 treated epithelial cells. BMP7 stimulation studies show activation of MAPK cascade proteins in epithelial cells and SFs. Similarly, a difference in the expression of claudin, Zink finger E-box-binding homeobox 1 was observed along with phosphorylation levels of cofilin in epithelial cells. Stimulation of SFs with BMP7 activated them with increased expression of α-smooth muscle actin. In addition, an elevated phosphorylation of epidermal growth factor receptor following BMP7 stimulation was also observed both in corneal epithelial cells and SFs. Based on our transcriptome analysis data on epithelial cells and the results obtained in SFs, we conclude that BMP7 contributes to epithelial-to-mesenchymal transition-like responses and plays a role equivalent to TGF-β in the course of corneal wound healing.
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EGF-induced activation of the epigenetic CCCTC binding factor (CTCF) plays an important role in corneal epithelial cell proliferation by suppressing the Pax6 gene. The present study focused on further understanding the role of CTCF in mediating EGF-induced migration of immortalized human corneal epithelial cells.CTCF activities in human corneal epithelial cells immortalized by telomerase (HTCE cells) and SV-40 (HCE cells) transformation were suppressed and enhanced by CTCF mRNA knockdown and by overexpressing CTCF cDNA, respectively. EGF-induced cell migration was evaluated by linear scratch wound healing, a cell migration assay, and live cell motility GFP-tracking with a fluorescence microscope. Immunochemical analysis was performed for detecting focal adhesion changes in EGF-induced and CTCF activity-altered cells.EGF-induced wound closure and cell migration rates of human corneal epithelial cells were significantly suppressed and enhanced by CTCF mRNA knockdown and by overexpression of CTCF, respectively. CTCF mRNA knockdown also markedly suppressed cell motility, determined by using a live-cell-tracking system in GFP-tag-expressed HTCE cells. Finally, alterations of EGF-stimulated focal adhesion were observed in CTCF knockdown HTCE cells by immunostaining of F-actin and vinculin in cytoskeleton reorganization.CTCF, an epigenetic regulator and transcription factor, involves EGF-induced increases in cell motility and migration. CTCF plays an essential role in growth factor-regulated human corneal epithelial cell wound healing.
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Injury-mediated corneal epithelial wound healing in vivo is mediated through different cell signaling pathways depending on whether or not the basement membrane is removed. Given this dependence, we ascertained if c-jun N-terminal kinase (JNK/SAPK) mitogen-activated protein kinase (MAPK) cell signaling mediates this response in vivo and in vitro, irrespective of the presence or absence of the basement membrane. Furthermore, in vitro the relative contribution was determined by the JNK/SAPK pathway to that of its p38 and ERK MAPK counterparts in mediating injury-induced increases in cell migration.Corneal epithelial debridement was performed in C57BL/6 mice and their organ-cultured eyes without removal of the basement membrane. In rabbits, following basement membrane removal by keratectomy, fluorescein-staining monitored reepithelialization was performed as in the mice. Immunohistochemistry evaluated changes in JNK phosphorylation status and localization. JNK inhibitor I and its inactive analogue determined if JNK signaling activation contributes to wound healing. BrdU staining assessed cell proliferation. A scratch wound assay of healing rates in SV40-immortalized human corneal epithelial cell line (HCEC) evaluated the relative contributions by p38 and ERK and JNK MAPK signaling activation to wound healing. A TUNEL assay probed for apoptosis after wound closure of HCEC. MTT assay evaluated corneal epithelial viability.Two hours following mice corneal epithelial debridement, phospho-JNK was transiently upregulated in the nucleus, whereas total JNK was constitutively expressed. JNK inhibitor I suppressed epithelial spreading in organ-cultured mouse eyes and rabbit corneal blocks, irrespective of the presence or absence of basement membrane. No proliferation was detected at the wound edges. In HCEC, a p38 (SB203580) and a JNK pathway inhibitor (JNK inhibitor I) inhibited migration rates more than U0126-induced ERK, whereas the JNK inhibitor I inactive analogue had no effect. JNK pathway inhibition wound closure in this region was not associated with either any TUNEL or BrdU-positive cells. Cell viability was unaffected by any of these MAPK inhibitors.JNK/SAPK pathway activation stimulates wound healing in vitro and in vivo, irrespective of the presence or absence of the basement membrane. Therefore, studies on how wound closure is elicited in HCEC are relevant for identifying how MAPK signaling mediates this response in vivo and in organ-cultured eyes. This realization suggests that the JNK signaling system has a role in vivo that is intermediate to those of ERK and p38 in mediating increases in cell migration.
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