RESUMO
Cellular Factor XIII (cFXIII) mRNA is rapidly upregulated in the fish retina after optic nerve injury (ONI). Here, we investigated the molecular mechanism of cFXIII gene activation using genetic information from the A-subunit of cFXIII (cFXIII-A). Real-time PCR that amplified the active site (exons 7-8) of cFXIII-A showed increased cFXIII-A mRNA in the retina after ONI, whereas the PCR that amplified the activation peptide (exons 1-2) showed no change. RT-PCR analysis that amplified exons 1-8 showed two bands, a faint long band in the control retina and a dense short band in the injured retina. Therefore, we conclude that activated cFXIII-A mRNA after ONI is shorter than that of the control retina. Western blot analysis also confirmed an active form of 65â¯kDa cFXIII-A protein in the injured retina compared to the control 84â¯kDa protein. 5'-RACE analysis using injured retina revealed that the short cFXIII-A mRNA lacked exons 1, 2 and part of exon 3. Exon 3 has two sites of heat shock factor 1 (HSF-1) binding consensus sequence. Intraocular injection of HSF inhibitor suppressed the expression of cFXIII-A mRNA in the retina 1 day after ONI to 40% of levels normally seen after ONI. Chromatin immunoprecipitation provides direct evidence of enrichment of cFXIII-A genomic DNA bound with HSF-1. The present data indicate that rapid HSF-1 binding to the cFXIII-A gene results in cleavage of activation peptide and an active form of short cFXIII-A mRNA and protein in the zebrafish retina after ONI without thrombin.