bg-CAT is a naturally existing 72-kDa complex of non-lens bg-crystallin (a-subunit, CAT-a) and trefoil factor (b-subunit, CAT-b), with a non-covalently linked form of ab2, identified from frog Bombina maxima skin secretions. The N-terminal part (CAT-aN, 1-170 residues) of CAT-a are two bg-crystallin domains, while the rest C-terminal part (CAT-aC) shows sequence homology to membrane insertion domain of Clostridium perfringens epsilon toxin. It was previously found that bg-CAT is able to upregulate many cell-stress related and proinflammatory proteins in Human umbilical vein endothelial cells. And bg-CAT can also strongly activate caspase-1 in Human umbilical vein endothelial cells and human colon cancer cell line HCT116 cells. Based on these findings, we hypothesized that bg-CAT may be involved in innate immune system. And we can further suppose that immune regulation may be the physiological function of bg-CAT in Bombina maxima skin. The term inflammasome was coined to describe a high molecular molecular weight complex that activates inflammatory caspases ultimately caspase-1 and induces the maturation and secretion of proinflammatory cytokines such as IL1b and IL18. Both pathogen associated molecular patterns (PAMPs) and Danger associated molecular patterns (DAMPs) activate inflammasomes. In the present study, bg-CAT was found to activate NLRP3 inflammasomes in THP-1 cells, which subsequently induced the maturation and secretion of proinflammatory cytokines IL1b and IL18. After exposure to 1nM concentration of bg-CAT, THP-1 cells showed the phenomena of potassium efflux, calcium influx and ROS generation. Extracellular high concentration of potassium and calcium chelator BAPTA-AM strongly inhibited the activation of NLRP3 inflammasome and IL1b secretion in THP-1 cells. These results suggested that potassium efflux and increase of the calcium concentration inside the cell were essential for the activation of NLRP3 inflammaosomes by bg-CAT. Pyropotosis is a more recently recognized form of regulated cell death dependent of the activation of caspase-1. Caspase-1 dependent pores are formed on the plasma membrane of pyroptotic cells. Subsequently, cell contents are released out of the cells through these pores. The released cell contents would activate inflammatory responses. bg-CAT was able to induce cell death of human dermal fibroblasts and cell death effect could be inhibited with caspase-1 inhibitor. Thus, we deduced that the bg-CAT induced cell death type in fibroblasts was pyroptosis. The cell death effect could also be strongly inhibited with both extracellular calcium chelator EGTA and intracellular calcium chelator BAPTA-AM. These findings strengthened the importance of calcium in the process of pyroptosis induced by bg-CAT. Lysosomes inside the fibroblast cells were disrupted and the endocytosis inhibitor M-beta-CD and cytochalasin D could inhibit the pyroptosis, which suggested that endocytosis of bg-CAT may be essential for it to exert its function. The findings that bg-CAT could activate NLRP3 inflammasome and induce pyroptosis strongly supported the hypothesis that bg-CAT may act as immune modulator. Thus, we proposed a new concept, Regional innate danger signal (RIDS). RIDS locates in limited organs or tissues without biological function in its resident location. But when the body is on pathological condition, the RIDS may reach other tissues or organs, and trigger immune responses. bg-CAT might be a typical example. To further prove our hypothesis, fibroblast cells originated from Bombina maxima skin were cultured. In the present study, we proposed the concept Regional innate danger signal (RIDS) for the first time. And also, our present study may be beneficial to understand the basic mechanisms of inflammasome activation and pyroptosis. The methods of amphibian cell culture generated may be useful for amphibian physiological study.
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