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.
Highlights
A 20-carbon branched-chain polyunsaturated fatty acid or acyclic diterpenoid acid of geranylgeranoic acid (GGA) was first recognised as an acyclic retinoid because the acid showed agonist activity as a ligand for retinoic acid receptor-β, retinoid-X receptor-α, and cellular retinoic acid-binding protein [1]
We demonstrated that GGA-induced unfolded protein response (UPR) and GGA-induced cell death are both sensitive to cotreatment with VIPER, a specific inhibitor of Toll-like receptor 4 (TLR4) signalling, GGA-induced cell death is preventable by knockdown of TLR4 gene expression, early transient activation of CASP4 is induced via GGA-induced UPR, and GGA stimulates NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome priming to activate CASP1 by NLRP3 inflammasome activation through TLR4 signalling
As expected, we found that both UPR and cell death induced by GGA treatment were blocked by VIPER, a specific inhibitor of TLR4
Summary
A 20-carbon branched-chain polyunsaturated fatty acid or acyclic diterpenoid acid of geranylgeranoic acid (GGA) was first recognised as an acyclic retinoid because the acid showed agonist activity as a ligand for retinoic acid receptor-β, retinoid-X receptor-α, and cellular retinoic acid-binding protein [1]. N-acetyl-aspartyl-glutamyl-valyl-aspartyl-aldehyde (Ac-DEVD-CHO), a specific inhibitor of caspase (CASP)-3/7, was unable to block GGA-induced cell death, indicating that GGA did not induce typical apoptosis, but rather caspase-3/7-independent cell death [2]. We investigated another form of programmed cell death, autophagic cell death, after GGA treatment. Α-tocopherol, a fat-soluble antioxidant vitamin, efficiently prevented ψm dissipation and GGA-induced cell death [2]. This suggested that mitochondrial superoxide hyperproduction might be indispensable for GGA-induced cell death
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