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 1

LARP6 is upregulated in activated HSCs from human fibrotic livers.

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LARP6 is upregulated in activated HSCs from human fibrotic livers. (A) S...
(A) Schematic illustration of the human livers selected for snRNA-seq and snATAC-seq. (B) Fibrosis scores were graded by a pathologist in a double-blinded manner. (C) Livers were stained for Sirius Red, and the positive area was calculated as a percentage. Data are presented as mean ± SD. **P < 0.01, ***P < 0.001, by 1-way ANOVA followed by Tukey’s test. (D) Heatmap representing relative expression of LARP6 and selected genes specific for MASH and MetALD HSCs. LARP6 expression is highlighted in red. (E) SnRNA-seq uniform manifold approximation and projection (UMAP) plot (GSE244832) of the integrated dataset of liver cells from all donors, showing identified cell types (left) and color-coded UMAP for LARP6 expression (right). (F) HSCs were clustered according to gene expression profile using Seurat, version 4.0. snRNA-seq UMAP plot of the integrated dataset of HSCs from all donors, showing 4 HSC clusters (left), and the UMAP color-coded for LARP6 smoothed expression (right). (G) Heatmap representing relative expression of LARP6 and fibrogenic genes across HSC clusters. (H) SnATAC-seq normalized accessibility peaks of LARP6. Chromatin accessibility between the peak upstream of LARP6 (peak = chr15:70799442-70800783) was calculated in activated HSCs from fibrotic livers (MASH, MetALD) and quiescent HSCs from nonfibrotic livers (normal, MASL).