Signal transduction of the prophenoloxidase activating system of prawn haemocytes triggered by CpG oligodeoxynucleotides

Abstract

Intracellular phenoloxidase (PO) activity in haemocyte lysate supernatant (HLS) of giant freshwater prawn ( Macrobrachium rosenbergii) was shown to be enhanced by CpG oligodeoxynucleotide (ODN) 2006, but not by so-ODN13. When haemocytes were treated in vitro with 50 μg/ml of ODN2006 for 30 min, the increases in both intra- and extracellular stimulated PO activity (PO S) and extracellular total PO activity (PO T) and the reduction of PO T suggest that the PO activity of haemocytes is enhanced by ODN2006 stimulation, but new prophenoloxidase (proPO) is not synthesised. In an attempt to determine which signal transduction pathway is involved in the activation of the proPO system, haemocytes were separately treated with activators or inhibitors of specific signalling components. The results show that there was an increase in both intra- and extracellular PO T of haemocytes treated with sodium fluoride (a G-protein activator); the addition of phosphokinase A (PKA)-activating 8-bromo-cAMP to haemocytes only increased intracellular PO T, and the addition of either phorbol-12-myristate-13-acetate (PMA; a phosphokinase C (PKC) activator) or caffeine (a phosphodiesterase inhibitor) only increased extracellular PO T. When PMA-stimulated haemocytes were treated with chelerythrine (a PKC inhibitor), the induced extracellular PO T was significantly reduced. Furthermore, the study of ODN2006-stimulated haemocytes treated with chelerythrine or palmitoyl- dl-carnitine (a PKC inhibitor) showed that the enhancement effects of ODN2006 on the intra- and extracellular PO S and extracellular PO T were significantly decreased. ODN-stimulated haemocytes treated with genistein (an inhibitor of protein tyrosine kinase) showed a further increase in extracellular PO T, but the other PO activities remained the same as those of the ODN-stimulated group. These results suggest that the activation of the proPO system of prawn haemocytes, including degranulation and PO activity, is induced by ODN2006 via a PKC-activating signalling pathway, but negatively regulated via the tyrosine kinase pathway.