Vesicular nucleotide transporter (VNUT)-dependent ATP secretion by hepatic stellate cells promotes liver fibrosis

Although liver fibrosis presents a substantial global health challenge, therapeutic options that directly target liver fibrosis remain limited. Hepatic stellate cells (HSCs) are key contributors to fibrosis through extracellular matrix production. This study uncovers a previously unrecognized function of HSCs: ATP secretion. We found that HSCs express the vesicular nucleotide transporter (VNUT) on secretory vesicles and actively release ATP. In mouse HSCs, VNUT is localized to the cytosol and around lipid droplets. In a thioacetamide-induced liver fibrosis model, VNUT inhibition with clodronate suppressed HSC proliferation and fibrosis progression, while restoring AMPK phosphorylation. In human hepatic stellate LX-2 cells, VNUT colocalized with v-SNARE proteins VAMP3 and VAMP7 and the vesicular proton pump V-ATPase. ATP secretion from LX-2 cells was observed upon stimulation with the Ca²⁺ ionophore ionomycin and was inhibited by Ca²⁺ chelation or low temperature, supporting an exocytotic mechanism. Clodronate and VNUT-targeting siRNA significantly reduced ATP release. Thapsigargin, an inducer of endoplasmic reticulum Ca²⁺ release, upregulated VNUT expression, suggesting a transcriptional regulation of VNUT-dependent ATP release by Ca²⁺ signaling. TGF-β1 stimulation also upregulated VNUT expression, suggesting its involvement in TGF-β1-induced fibrogenesis pathway. Additionally, serotonin was identified as an ATP secretion stimulator in LX-2 cells, and this effect was blocked by clodronate. Platelets-a major peripheral serotonin source-were increased in TAA-treated liver and found adjacent to serotonin receptor 5-HT2B-positive HSCs. Clodronate treatment reduced CD41-positive platelets in liver tissue. These findings highlight VNUT-mediated ATP secretion as a key regulator of HSC function and a potential therapeutic target for liver fibrosis.