Hepatocyte Transforming Growth Factor-β-Oxysterol-Binding Protein-Related Protein 3 Signaling Axis Promotes Hepatic...

Prolonged activation of hepatocyte transforming growth factor β (TGFβ) signaling has been identified as a key driver of hepatic stellate cell activation, leading to liver fibrosis in chronic liver diseases. However, the mechanisms by which overactivation of TGFβ signaling within hepatocytes promotes hepatic stellate cell activation and liver fibrosis remain poorly understood. The expression of OSBPL3, enconding oxysterol-binding protein-related protein 3 (ORP3), is markedly induced in human fatty liver samples. However, the regulation and function of hepatic ORP3 in the pathogenesis of liver fibrosis are currently unknown.

Primary mouse hepatocytes were used to examine the effects of TGFβ onOrp3 mRNA and ORP3 protein expression. Adenovirus-mediated overexpression or knockdown of Orp3 was used to manipulate hepatocyte -ORP3 expression, and the effects on TGFβ signaling, TGFβ-induced profibrogenic factor expression, and in vivo liver fibrosis development were assessed. Crosstalk between hepatocyte ORP3 and hepatic stellate cells was examined using a conditioned medium transfer system.

TGFβ induces Orp3 mRNA and ORP3 protein expression in mouse hepatocytes primarily via the noncanonical TGFβ pathway that involves p38/c-Jun N-terminal kinase and E2F transcription factor 1. Elevated hepatic Orp3 and OSBPL3 expression was detected in mouse models of diet-induced liver fibrosis and in livers from human patients with alcoholic steatohepatitis. Data mining of a single-nucleus RNA sequencing dataset on human metabolic dysfunction-associated steatohepatitis samples revealed that OSBPL3 transcripts are enriched in hepatocytes, endothelial cells, and hepatic stellate cells. In vitro analysis showed that ORP3 is both necessary and sufficient for TGFβ-induced expression of profibrogenic factors, notably thrombospondin-1 and connective tissue growth factor, in hepatocytes. Overexpression of Orp3 in hepatocytes enhances hepatic stellate cell activation in an in vitro conditioned medium-transfer experiment, in part via connective tissue growth factor. Furthermore, acute hepatic Orp3 depletion protects mice from hepatotoxin carbon tetrachloride-induced liver fibrosis. Mechanistically, the profibrogenic action of hepatocyte ORP3 depends on stabilization of Yes-associated protein, a key driver of liver fibrosis.

Orp3 is a novel target gene induced by TGFβ signaling within hepatocytes. ORP3 then promotes TGFβ's fibrogenic actions by stimulating hepatic stellate cell activation and liver fibrosis in a feed-forward loop.