Hepatocyte NAT10-mediated ac4C regulation contributes to TGFβ1-associated Kupffer cell -HSC communication in steatotic liver disease.

BACKGROUND AND OBJECTIVE: Obesity and metabolic syndrome impose chronic lipotoxic stress on hepatocytes, contributing to macrophage reprogramming and fibrosis progression in steatotic liver disease. However, how soluble hepatocyte-derived factors regulate Kupffer cell-hepatic stellate cell (HSC) crosstalk remains unclear. This study investigated the role of hepatocyte NAT10-mediated N4-acetylcytidine (ac4C) RNA modification and explored potential ac4C-dependent targets involved in this process. METHODS: Hepa1-6 cells were exposed to palmitic acid with or without NAT10 knockdown, and nd key findings were validated in primary mouse hepatocytes. Hepatocyte-conditioned media were applied to primary Kupffer cells and a Kupffer-HSC coculture system. ac4C-RIP-qPCR and mRNA stability assays were performed. Recombinant TGFβ1 was used for rescue analysis. In vivo, hepatocyte-specific NAT10 knockdown was achieved using AAV8-TBG-shNat10 in a high-fat diet plus fructose model. RESULTS: NAT10 knockdown reduced TGFβ1 secretion from lipotoxic hepatocytes without affecting cell viability. Conditioned media induced Spp1 and Lgals3 expression in Kupffer cells, which was attenuated by NAT10 silencing. In the Kupffer-HSC coculture system, Kupffer cells primed by lipotoxic hepatocyte-conditioned media markedly increased HSC activation, as indicated by elevated α-SMA and Collagen I expression. This effect was diminished when Kupffer cells were exposed to media from NAT10-deficient hepatocytes, and recombinant TGFβ1 partially restored HSC activation. Tgfb1 showed reduced ac4C enrichment and mRNA stability following Nat10 knockdown, suggesting that it is a NAT10-responsive ac4C-associated transcript under lipotoxic conditions. In vivo, hepatocyte-specific NAT10 knockdown reduced hepatic TGFβ1 levels, decreased CLEC4F+LGALS3+ pro-fibrotic Kupffer cells, and alleviated liver fibrosis. CONCLUSIONS: Hepatocyte NAT10-mediated ac4C regulation contributes to pro-fibrotic Kupffer-HSC crosstalk in steatotic liver disease, at least partly through TGFβ1-associated soluble hepatocyte-derived signaling.