Role of Calmodulin-Calmodulin Kinase II,cAMP/Protein Kinase A and ERK 1/2 on Aeromonas hydrophila-Induced Apoptosis of Head Kidney Macrophages

The role of calcium (ca2+) and its dependent protease calpain in aeromonas hydrophila-induced head kidney macrophage (hkm) apoptosis has been reported. here,we report the pro-apoptotic involvement of calmodulin (cam) and calmodulin kinase ii gamma (camkiig) in the process. we observed significant increase in cam levels in a. hydrophila-infected hkm and the inhibitory role of bapta/am,egta,nifedipine and verapamil suggested cam elevation to be ca2+-dependent. our studies with cam-specific sirna and the cam inhibitor calmidazolium chloride demonstrated cam to be pro-apoptotic that initiated the downstream expression of camkiig. using the camkiig-targeted sirna,specific inhibitor kn-93 and its inactive structural analogue kn-92 we report cam-camkiig signalling to be critical for apoptosis of a. hydrophila-infected hkm. inhibitor studies further suggested the role of calpain-2 in camkiig expression. camk kinase (camkk),the other cam dependent kinase exhibited no role in a. hydrophila-induced hkm apoptosis. we report increased production of intracellular camp in infected hkm and our results with kn-93 or kn-92 implicate the role of camkiig in camp production. using sirna to pkaca,the catalytic subunit of pka,anti-pkaca antibody and h-89,the specific inhibitor for pka we prove the pro-apoptotic involvement of camp/pka pathway in the pathogenicity of a. hydrophila. our inhibitor studies coupled with sirna approach further implicated the role of camp/pka in activation of extracellular signal-regulated kinase 1 and 2 (erk 1/2). we conclude that the alteration in intracellular ca2+ levels initiated by a. hydrophila activates cam and calpain-2; both pathways converge on camkiig which in turn induces camp/pka mediated erk 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected hkm. © 2014 banerjee et al.