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

Abstract

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.

Intracellular Ca2+ homeostasis and JAK1/STAT3 pathway are involved in the protective effect of propofol on BV2 microglia against hypoxia-induced inflammation and apoptosis

“…Besides, intracellular Ca 2+ overload could upregulate the expression of several down-stream proteins. In the situation of ischemic myocardial stress response, CAMKIIα could activate ERK and NF-κB, thus regulating inflammation and injury[12]. In our study, as shown in Fig 3, CoCl 2 treatment disrupted the intracellular Ca 2+ hemostasis and inducing the phosphorylation of CAMKIIα, ERK and NF-κB.…”

Lactobacillus acidophilus attenuates Aeromonas hydrophila induced cytotoxicity in catla thymus macrophages by modulating oxidative stress and inflammation

“…Therefore, to examine whether increased ROS and RNS generation observed earlier is a prerequisite to apoptosis production in CTM cells, the apoptotic population by flow cytometry using Annexin-V/PI assay was ascertained. A. hydrophila mediated apoptosis has been earlier reported in murine macrophages and head kidney macrophages of fish (Mazumdar et al, 2004;Banerjee et al, 2012;Banerjee et al, 2014a). However, there are no existing reports of the apoptosis induction in the thymic macrophages in response to A. hydrophila challenge.…”

“…Besides, it has been fairly well reported that the ROS accumulation plays a crucial role in A. hydrophila induced macrophage apoptosis (Fierro-Castro et al, 2012;Chakrabarti et al, 2014). ROS generation in macrophages initiates a series of reactions including iNOS mediated NO production leading to biomolecular damage, apoptosis and release of cytokine cascade (Banerjee et al, 2014a). In-vitro piscine macrophage models can thus serve as an excellent platform for exploring several distinct mechanisms of disease progression (Hightower and Renfro, 1988;Fierro-Castro et al, 2013).…”

Ameliorating ER-stress attenuates Aeromonas hydrophila-induced mitochondrial dysfunctioning and caspase mediated HKM apoptosis in Clarias batrachus

“…Altered Ca 2+ -homeostasis is primal to ER-stress. After observing Ca 2+ and its dependent molecules critical for A. hydrophila -induced HKM apoptosis46 we questioned the involvement of ER-stress in terms of CHOP-BiP-eIF2α-spree and caspase-12 activation.…”

“…A. hydrophila -induced macrophage apoptosis is executed by an intracellular proteolytic cascade of caspases, where initiator caspases are triggered by a range of intracellular signalling molecules to accomplish programmed demolition of cell through caspase-3 activation346. As an early initiator of the process, the role of calcium (Ca 2+ ) is foremost7.…”