Divergent roles of three cytochrome c in CTSB-modulating coelomocyte apoptosis in Apostichopus japonicus.

Cytochrome c plays crucial roles in apoptosis and the immune response. We previously demonstrated that cathepsin B from Apostichopus japonicus (AjCTSB) induces coelomocyte apoptosis. However, the mechanistic explanation and the regulation of this process have not been investigated. In the present study, we identified three cytochrome c cDNAs from A. japonicus (designated Ajcytc1, Ajcytc-1, and Ajcytc-2) using expressed sequence tag- (EST) and RACE-based approaches. The deduced amino acid sequences of the three cytochrome isoforms contained conserved CXXCH motifs, which are involved in binding heme and maintaining proteolytic activity. Time course expression analysis in vitro and in vivo revealed that the three cytochrome isoforms were induced upon pathogen challenge and LPS exposure. More importantly, AjCTSB knockdown by siRNA dramatically increased mitochondrial membrane potential (ΔΨm) in a time-dependent manner based on JC-1 fluorescent probe staining. AjCTSB knockdown also resulted in decreased expression of these three cytochromes 24 h after siAjCTSB transfection. Functional analysis using isoform-specific siRNAs revealed that Ajcytc-1, but not Ajcytc1 or Ajcytc-2, is involved in coelomocyte apoptosis. Moreover, the transcript level of Ajcaspase-3, an apoptosis executioner, was also consistently down-regulated upon silencing of Ajcytc-1 but not Ajcytc1 or Ajcytc-2. Collectively, these results indicate that Ajcytc1, Ajcytc-1, and Ajcytc-2 play distinct roles in mediating the immune response to bacteria according to AjCTSB expression. Moreover, Ajcytc-1 could be released upon dissipation of the ΔΨm, which could further trigger coelomocyte apoptosis through the activation of Ajcaspase-3.