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 3

Knockdown of LARP6 inhibits HSC activation.

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Knockdown of LARP6 inhibits HSC activation. (A) Human liver tissues were...
(A) Human liver tissues were stained with an anti-LARP6 antibody. (B) The LARP6-positive area was calculated as a percentage. (C) LARP6-positive cells stained positive for activated HSC marker α-SMA on human fibrotic liver (scale bars: 100 or 250 μm). (D) Cultured human HSCs (donor D19) were stimulated with human TGF-β1 (5 ng/mL) for 24 hours. (E) We performed bulk RNA-seq of human HSCs (donor D22) transfected with either control or LARP6 targeting dsiRNA and present a heatmap showing the expression (red: upregulated; blue: downregulated) of HSC activation-related genes. Rep, repetition. (F) dsiRNA-transfected HSCs (donor D19) with or without TGF-β1 were stained with an anti–collagen type I (COL1) antibody, and the COL1-positive area was calculated and normalized by the cell number counted using DAPI (scale bar: 250 μm) (G) Collagen type I levels in the supernatant of human HSCs were measured with ELISA. (B, D, and F) Data are presented as mean ± SD (n = 3 or 4). *P < 0.05, **P < 0.01, ***P < 0.001, by 1-way ANOVA followed by Tukey’s test.