TLR4-mediated pyroptosis in human hepatoma-derived HuH-7 cells induced by a branched-chain polyunsaturated fatty acid, geranylgeranoic acid

Abstract

A branched-chain polyunsaturated fatty acid, geranylgeranoic acid (GGA; C20:4), which is an endogenous metabolite derived from the mevalonate pathway in mammals, has been reported to induce cell death in human hepatoma cells. We have previously shown that the lipid-induced unfolded protein response (UPR) is an upstream cellular process for an incomplete autophagic response that might be involved in GGA-induced cell death. Here, we found that Toll-like receptor 4 (TLR4)-mediated pyroptosis in HuH-7 cells occurred by GGA treatment. The TLR4-specific inhibitor VIPER prevented both GGA-induced cell death and UPR. Knockdown of the TLR4 gene attenuated GGA-induced cell death significantly. Upon GGA-induced UPR, caspase (CASP) 4 (CASP4) was activated immediately and gasdermin D (GSDMD) was translocated concomitantly to the plasma membrane after production of the N-terminal fragment of GSDMD. Then, cellular CASP1 activation occurred following a second gradual up-regulation of the intracellular Ca2+ concentration, suggesting that GGA activated the inflammasome. Indeed, the mRNA levels of NOD-like receptor family pyrin domain containing 3 (NLRP3) and interleukin-1 β (IL1B) genes were up-regulated dramatically with translocation of cytoplasmic nuclear factor-κB (NF-κB) to nuclei after GGA treatment, indicating that GGA induced priming of the NLRP3 inflammasome through NF-κB activation. GGA-induced up-regulation of CASP1 activity was blocked by either oleic acid, VIPER, MCC950 (a selective inhibitor of the NLRP3 inflammasome), or CASP4-specific inhibitor peptide cotreatment. Pyroptotic cell death was also confirmed morphologically by bleb formation in time-series live cell imaging of GGA-treated cells. Taken together, the present results strongly indicate that GGA causes pyroptotic cell death in human hepatoma-derived HuH-7 via TLR4 signalling.

Pyroptosis, metabolism, and tumor immune microenvironment

“…In fact, GGA is a branched‐chain polyunsaturated fatty acid, an endogenous metabolite derived from the mevalonate pathway in mammals. 212 …”

“…It has been reported that geranylgeranoic acid (GGA) (C20:4) could induce pyroptotic cell death in human hepatoma‐derived HuH‐7 via toll‐like receptor 4 (TLR4) signaling pathway. In fact, GGA is a branched‐chain polyunsaturated fatty acid, an endogenous metabolite derived from the mevalonate pathway in mammals 212 …”

Age-Dependent Decrease in Hepatic Geranylgeranoic Acid Content in C3H/HeN Mice and Its Oral Supplementation Prevents Spontaneous Hepatoma

“…In these studies, GGA was reported to induce cell death in human hepatoma cells via endoplasmic reticulum stress response and incomplete autophagic response [ 7 , 8 ]. Most recently, we reported that GGA induces Toll-like receptor 4 (TLR4)-mediated pyroptosis in human hepatoma-derived cells [ 9 ].…”

A rapid increase in lysophospholipids after geranylgeranoic acid treatment in human hepatoma-derived HuH-7 cells revealed by metabolomics analysis

“…We have reported that GGA induces cell death in human hepatoma cells via the unfolded protein response (UPR ER ) and an incomplete response of autophagy [ 10 , 11 ]. Most recently, we concluded that GGA induces Toll-like receptor 4 (TLR4)-signaled pyroptosis in human hepatoma cells [ 12 ].…”

“…GGA causes activation of caspase-4 via TLR4-mediated UPR ER , resulting in cleavage of gasdermin D (GSDMD) and the translocation of its N-terminal fragment to the plasma membrane leading to the formation of the GSDMD pore [ 12 ]. Another TLR4 signaling causes increased reactive oxygen species production in the mitochondria, leading to the transfer of NF-κB to the nucleus and priming the NLRP3 inflammasome.…”