Abstract:
In this study, by specifically focusing on the role of ASC in melanoma and NLRP3 activation in response to double stranded RNA, we try to provide answers to more general but basic questions: “How and why would cancer cells alter the function of inflammatory proteins?” and “How can NLRP3 inflammasome recognize varying PAMPs and DAMPs with totally different natures and molecular structures?”, respectively. Although having mRNA expression for all necessary NLRP3 inflammasome components, healthy primary melanocytes showed no response to NLRP3 activating signals as measured by IL-1 beta secretion via ELISA. Whereas ASC was silenced at the transcription level, the protein was present and functional in certain melanoma cell lines. Moreover, knock-down of Asc in these cell lines by shRNA resulted in diminished NLRP3 activation. Overall, our observations support the idea that cancer cells may activate the nonfunctional inflammasome machinery, supposedly to increase cytokine secretion which may help angiogenesis and subsequently metastasis. In the second part of our study, we investigate dsRNA recognition mechanism and pathway leading to the NLRP3 inflammasome activation. Treating LPS primed Ips-1/Trif DKO macrophages we observed that Ips-1 is required for intracellular pIC recognition. Inflammasome activity analysis of Bax/Bak DKO macrophages and immunofluorescence assay for active Bax showed that, Ips-1 acts on apoptotic Bcl2 family members Bax/Bak triggering programmed cell death mechanism. Resulting membrane permeabilization causes loss of cytosolic K+ ion which is sensed by NLRP3 leading to NLRP3 inflammasome activation. Our data provides the lacking unifying model of NLRP3 activation mechanism: Any pathogenic organism or danger signal causes loss of membrane integrity. Resulting damage disturbs the active regulation of cytosolic ion balance, causing loss of K+ from cytosol, which is sensed by NLRP3 inflammasome resulting in NLRP3 inflammasome activation.
