Platelet-activating factor enhances urokinase-type plasminogen activator gene expression in corneal epithelium.
OBJECTIVE
To determine whether platelet-activating factor (PAF), a lipid mediator that is accumulated in the cornea after alkali burn, induces the gene expression of urokinase-type plasminogen activator (uPA) in the corneal epithelium. Possible signaling mechanisms of uPA gene induction by PAF also were examined.


METHODS
Rabbit corneas were cultured with or without PAF. One hour before stimulation, PAF antagonists or other modulators were added to PAF. In some experiments, the corneas were permeabilized to introduce guanosine triphosphate analogs into the corneal epithelial cells. Corneal epithelia were then harvested for Northern blot analysis, nuclear runoff transcription assay, and zymography.


RESULTS
Platelet-activating factor induced uPA mRNA expression in the corneal epithelium. New protein synthesis was not required for the induction of uPA mRNA. The induction was at the level of transcription as shown by nuclear runoff assays. Additionally, both actinomycin D and alpha-amanitin inhibited the increase in uPA mRNA by PAF. The message was translated into protein, which was secreted into the conditioned medium. An antagonist with high affinity for intracellular PAF binding sites (BN 50730) inhibited uPA gene expression and cellular secretion of the protein. The effect of PAF was not mediated by G proteins and was independent of protein kinase C- and cyclic adenosine monophosphate-dependent signal transduction pathways. Okadaic acid increased the expression of uPA and, at longer times, augmented the effect of PAF, suggesting that a signaling pathway that requires phosphorylation is involved in activated uPA mRNA synthesis.


CONCLUSIONS
After corneal injury and inflammation, PAF may be an important initiator of the proteolytic cascade, leading to epithelial defects and corneal ulceration. Antagonists of PAF could be useful in the prevention of these diseases.
Tao
Y
Y
LSU Eye Center, School of Medicine, Louisiana State University Medical Center, New Orleans 70112, USA.
Bazan
H E
HE
Bazan
N G
NG
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D015152:Blotting, Northern; D003315:Cornea / Q000187:drug effects / Q000201:enzymology*; D000242:Cyclic AMP / Q000502:physiology; D003609:Dactinomycin / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D004988:Ethers, Cyclic / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D019319:Okadaic Acid; D009924:Organ Culture Techniques; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits; D058924:Thienopyridines; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014230:Triazoles / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics*
D000546:Amanitins; D001381:Azepines; D004988:Ethers, Cyclic; D010972:Platelet Activating Factor; D010975:Platelet Aggregation Inhibitors; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D058924:Thienopyridines; D014230:Triazoles; C066686:BN 50730; D003609:Dactinomycin; D019319:Okadaic Acid; D000242:Cyclic AMP; D014568:Urokinase-Type Plasminogen Activator; D019204:GTP-Binding Proteins