Aeromonas hydrophila-induced alterations in cytosolic calcium activate pro-apoptotic cPKC-MEK1/2-TNFα axis in infected headkidney macrophages of Clarias gariepinus.

ABSTRACT

Alterations in intracellular-calcium (Ca2+)i homeostasis is critical to Aeromonas hydrophila-induced headkidney macrophages (HKM) apoptosis of Clarias gariepinus, though the implications are poorly understood. Here, we describe the role of intermediate molecules of Ca2+-signaling pathway that are involved in HKM apoptosis. We observed phosphoinositide-3-kinase/phospholipase C is critical for (Ca2+)i release in infected HKM. Heightened protein kinase-C (PKC) activity and phosphorylation of MEK1/2-ERK1/2 was noted which declined in presence of 2-APB, Go6976 and PD98059, inhibitors to IP3-receptor, conventional PKC isoforms (cPKC) and MEK1/2 respectively implicating Ca2+/cPKC/MEK-ERK1/2 axis imperative in A. hydrophila-induced HKM apoptosis. Significant tumor necrosis factor-α (TNFα) production and its subsequent reduction in presence of MEK-ERK1/2 inhibitor U0126 suggested TNFα production downstream to cPKC-mediated signaling via MEK1/2-ERK1/2 pathway. RNAi and inhibitor studies established the role of TNFα in inducing caspase-8-mediated apoptosis of infected HKM. We conclude, alterations in A. hydrophila-induced (Ca2+)i alterations activate cPKC-MEK1/2-ERK1/2-TNFα signaling cascade triggering HKM apoptosis.