Increased platelet-activating factor receptor gene expression by corneal epithelial wound healing.
OBJECTIVE
Platelet activating factor (PAF) is a potent inflammatory mediator the synthesis of which increases in the cornea after injury. The effects of PAF are mediated by receptors (PAF-R), which are present in target cells. This study was undertaken to investigate the effects of wound healing, PAF, and growth factors on modulating PAF-R mRNA levels in corneal epithelial cells.


METHODS
Cultures of rabbit corneal epithelial (RCE), rabbit limbal epithelial (RLE), rabbit corneal fibroblast (RCF), and rabbit corneal endothelial (RCEn) cells, as well as rabbit corneal keratocytes (RCKs) were used. For the in vivo wound-healing experiments, a 7-mm central corneal deepithelialization was performed in anesthetized rabbits. For the in vitro experiments, wounded rabbit corneas were maintained in organ culture. Corneas were stimulated with 120 nM PAF or preincubated with PAF antagonists, cyclohexamide (CHX) or actinomycin D (AcD) before adding PAF. RCE cells were stimulated with transforming growth factor (TGF)-beta1, -beta2, and, -beta3, basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF); and hepatocyte growth factor (HGF). Total RNA was isolated and PAF-R expression evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis, and quantitative RT-PCR.


RESULTS
PAF-R mRNA was expressed in RCE, RLE, and RCEn cells and RCKs, but not in RCFs. After epithelial injury, PAF-R expression increased from 2.5 to 4 times, both in vitro and in vivo. Addition of cPAF further stimulated PAF-R gene expression in epithelium, which was abolished by PAF antagonists. Quantitative RT-PCR revealed that PAF stimulated PAF-R mRNA threefold after injury. The induction of PAF-R by its agonist required previous injury and was inhibited by AcD but not by CHX. Treatment of RCE cells with TGF-beta1, -beta2, or -beta3, HGF, and KGF increased mRNA in PAF-R; however, bFGF had no effect.


CONCLUSIONS
Corneal injury produces changes in PAF-R mRNA expression. Whereas stroma fibroblastic cells lost the PAF-R gene expression found in keratocytes, corneal epithelial injury upregulated PAF-R mRNA. These results suggest that activation of selective growth factors and increases in PAF synthesis after injury stimulate PAF-R gene transcription and constitute important feedback mechanisms needed to maintain the inflammatory process and regulate epithelial wound healing.
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Department of Ophthalmology and Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, USA.
Bazan
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D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects; D065306:Corneal Injuries; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D051526:Fibroblast Growth Factor 10; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D006133:Growth Substances / Q000494:pharmacology; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D009924:Organ Culture Techniques; D010972:Platelet Activating Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis*; D011817:Rabbits; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta; D015854:Up-Regulation; D014945:Wound Healing
D051526:Fibroblast Growth Factor 10; D006133:Growth Substances; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D016212:Transforming Growth Factor beta; C039351:platelet activating factor receptor; D016222:Fibroblast Growth Factor 2; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D017228:Hepatocyte Growth Factor