RNA-binding protein LARP6 coordinates hepatic stellate cell activation and liver fibrosis
Hyun Young Kim, Orel Mizrahi, Wonseok Lee, Sara B. Rosenthal, Cuijuan Han, Brian A. Yee, Steven M. Blue, Jesiel Diaz, Jyotiprakash P. Jonnalagadda, Lena A. Street, Kanani Hokutan, Haeum Jang, Charlene Miciano, Chen-Ting Ma, Andrey A. Bobkov, Eduard Sergienko, Michael R. Jackson, Marko Jovanovic, Branko Stefanovic, Tatiana Kisseleva, Gene W. Yeo, David A. Brenner
Hyun Young Kim, Orel Mizrahi, Wonseok Lee, Sara B. Rosenthal, Cuijuan Han, Brian A. Yee, Steven M. Blue, Jesiel Diaz, Jyotiprakash P. Jonnalagadda, Lena A. Street, Kanani Hokutan, Haeum Jang, Charlene Miciano, Chen-Ting Ma, Andrey A. Bobkov, Eduard Sergienko, Michael R. Jackson, Marko Jovanovic, Branko Stefanovic, Tatiana Kisseleva, Gene W. Yeo, David A. Brenner
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Research Article Cell biology Hepatology

RNA-binding protein LARP6 coordinates hepatic stellate cell activation and liver fibrosis

Abstract

Metabolic syndrome and excessive alcohol consumption (MetALD) result in liver injury and fibrosis, which are driven by increased collagen production by activated hepatic stellate cells (HSCs). Our previous studies demonstrated that LARP6, an RNA-binding protein, may facilitate collagen production. However, the expression and function of LARP6 as a regulator of fibrosis development in a disease-relevant model remain poorly understood. We demonstrated that LARP6 was upregulated in human activated HSCs in metabolic dysfunction–associated steatohepatitis (MASH) and MetALD. By using single-nucleus RNA-seq and assay for transposase-accessible chromatin sequencing, we showed that JUNB upregulated LARP6 expression in activated HSCs. Moreover, LARP6 knockdown in human HSCs suppressed fibrogenic gene expression. By integrating enhanced crosslinking and IP analysis and ribosome profiling in HSCs, we showed that LARP6 interacted with mature mRNAs comprising more than 300 genes, including RNA structural elements within COL1A1, COL1A2, and COL3A1 to regulate mRNA expression and translation. IP–mass spectrometry analysis demonstrated LARP6 protein–protein interactions with mRNA translation components and the actin cytoskeleton. Furthermore, Dicer substrate siRNA-based HSC-specific gene knockdown or pharmacological inhibition of LARP6 attenuated fibrosis development in human MASH and MetALD liver spheroids. Our results suggest LARP6 plays a key role in fibrogenic gene regulation and that targeting LARP6 in human HSCs may represent a therapeutic approach for liver fibrosis.

Authors

Hyun Young Kim, Orel Mizrahi, Wonseok Lee, Sara B. Rosenthal, Cuijuan Han, Brian A. Yee, Steven M. Blue, Jesiel Diaz, Jyotiprakash P. Jonnalagadda, Lena A. Street, Kanani Hokutan, Haeum Jang, Charlene Miciano, Chen-Ting Ma, Andrey A. Bobkov, Eduard Sergienko, Michael R. Jackson, Marko Jovanovic, Branko Stefanovic, Tatiana Kisseleva, Gene W. Yeo, David A. Brenner

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Figure 2

JUNB binds near the LARP6 promoter region and regulates its expression.

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JUNB binds near the LARP6 promoter region and regulates its expression. ...
(A) Dot plot showing TFs with significant motifs in LARP6 promoter-linked peaks (links defined through Cicero). Dot size is scaled to the FIMO score, and dot color is scaled to the FIMO P value. The peak near the LARP6 promoter region is highlighted red. (B) Cultured human HSCs (donor D19) were transfected with JUNB-targeting dsiRNA (dsiJUNB) or dsi-negative control (dsiControl), and the efficiency of gene knockdown was measured. (C) Expression of the LARP6 gene was assessed in dsiJUNB.1-transfected (vs. vehicle-transfected) HSCs ± TGF-β1 after 24 hours of stimulation. (D) Cultured human HSCs were transfected with LARP6 overexpression vector, and (E) LARP6 gene expression was assessed in JUNB-overexpressing vector-transfected (vs. dsiControl-transfected HSCs ± TGF-β1 after 24 hours of stimulation. (F) LARP6 locus-specific ChIP analysis was performed using human HSCs ± TGF-β1. (B–E) Data are reported as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, by 1-way ANOVA followed by Tukey’s test.