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.
Immunobiology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
Chaitali
C
Banerjee
Immunobiology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
Preeti
P
Khatri
Gut Biology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
Rajagopal
R
Raman
Institute of Genomics and Integrative Biology, CSIR, Delhi, India.
Himanshi
H
Bhatia
Institute of Genomics and Integrative Biology, CSIR, Delhi, India.
Malabika
M
Datta
Immunobiology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
Shibnath
S
Mazumder
D016980:Aeromonas hydrophila / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002397:Catfishes; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005393:Fish Diseases / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D029941:Fish Proteins / Q000378:metabolism*; D016905:Gram-Negative Bacterial Infections / Q000378:metabolism / Q000473:pathology / Q000662:veterinary; D007668:Kidney / Q000378:metabolism* / Q000473:pathology; D020935:MAP Kinase Signaling System; D008264:Macrophages / Q000378:metabolism* / Q000473:pathology; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*
D029941:Fish Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048052:Mitogen-Activated Protein Kinase 3