Hepatocyte Stress Increases Expression of Yes-Associated Protein and Transcriptional Coactivator With PDZ-Binding Motif in Hepatocytes to Promote Parenchymal Inflammation and Fibrosis

Abstract

Background and aims: Activated hepatocytes are hypothesized to be a major source of signals that drive cirrhosis, but the biochemical pathways that convert hepatocytes into such a state are unclear. We examined the role of the Hippo pathway transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in hepatocytes to facilitate cell-cell interactions that stimulate liver inflammation and fibrosis.

Approach and results: Using a variety of genetic, metabolic, and liver injury models in mice, we manipulated Hippo signaling in hepatocytes and examined its effects in nonparenchymal cells to promote liver inflammation and fibrosis. YAP-expressing hepatocytes rapidly and potently activate the expression of proteins that promote fibrosis (collagen type I alpha 1 chain, tissue inhibitor of metalloproteinase 1, platelet-derived growth factor c, transforming growth factor β2) and inflammation (tumor necrosis factor, interleukin 1β). They stimulate expansion of myofibroblasts and immune cells, followed by aggressive liver fibrosis. In contrast, hepatocyte-specific YAP and YAP/TAZ knockouts exhibit limited myofibroblast expansion, less inflammation, and decreased fibrosis after CCl₄ injury despite a similar degree of necrosis as controls. We identified cellular communication network factor 1 (CYR61) as a chemokine that is up-regulated by hepatocytes during liver injury but is expressed at significantly lower levels in mice with hepatocyte-specific deletion of YAP or TAZ. Gain-of-function and loss-of-function experiments with CYR61 in vivo point to it being a key chemokine controlling liver fibrosis and inflammation in the context of YAP/TAZ. There is a direct correlation between levels of YAP/TAZ and CYR61 in liver tissues of patients with high-grade nonalcoholic steatohepatitis.

Conclusions: Liver injury in mice and humans increases levels of YAP/TAZ/CYR61 in hepatocytes, thus attracting macrophages to the liver to promote inflammation and fibrosis.

FIGURE 1.. Hepatocyte-specific YAP expression activates αSMA⁺ myofibroblasts and recruits immune cells resulting in liver fibrosis

A. Timeline of AAV-Cre delivery to adult TetOYAP mice, induction of YAP-Tg expression and subsequent sample collection. Representative α-smooth muscle actin (αSMA) and collagen staining from livers of TetOYAP mice at the indicated timepoints after doxycycline administration. B. Confocal immunofluorescent liver staining of YAP-Tg mice after 2 weeks for the indicated proteins. Boxed area in upper picture is enlarged in the image below. C. Immunofluorescent F4/80 liver staining of Control and YAP-Tg mice after 2 weeks. D. Representative flow cytometry of liver neutrophils (CD45⁺CD11b⁺Ly6G⁺) and macrophages (CD45⁺CD11b⁺Ly6G⁻F4/80⁺) one week after YAP-Tg induction. Percentages are of the gated population. Right, dot plots of all flow cytometry experiments (n=8, control; n=9, YAP-Tg). E. Dot plots of whole liver RT-qPCR of the noted genes after YAP-Tg or YAP-Tg mice treated with clodronate (YAP-Tg/Clod) two weeks after induction. Horizontal line represents the mean, each dot represents an experiment. Error bars indicate standard error. *p<0.05, **p<0.01, ***p<0.001. F. Immunoblots of the indicated proteins in YAP-Tg or YAP-Tg/Clod two weeks after induction. G. Representative αSMA and collagen stain of YAP-Tg and YAP-Tg/Clod two weeks after YAP-Tg induction.

FIGURE 2.. Clonal YAP-Tg Cells Intimately Interact with Myofibroblasts and Immune Cells

A. Immunofluorescent staining of either αSMA, Sox9, or F4/80 in clonal YAP-Tg populations after 4 and 8 weeks. EYFP labels YAP-Tg cells if doxycycline is administered. B. Clonal analysis of control versus YAP-Tg cells after various time points for Ctgf and Cyr61 using RISH. EYFP labels YAP-Tg cells if doxycycline is administered. C. Diagram/representative flow cytometry plots of strategy to isolate hepatocytes by tdTomato⁺ gene expression D. qPCR dot plots of Hippo pathway target genes from sorted tdTomato⁺ hepatocytes. E. qPCR dot plots of cyto/chemokines from control (Ctl) and YAP-Tg hepatocytes after 2 weeks of YAP-Tg expression. **p<0.01 ***p<0.001. Horizontal line represents the mean, each dot represents an experiment.

FIGURE 3.. Multiple Forms of Toxic/Metabolic Liver Injury Lead to Increases in YAP/TAZ

A. Chronic CCl₄ treatment diagram and IHC for YAP expression after chronic CCl₄ treatment. B. Whole liver immunoblot of YAP and TAZ after chronic CCl₄ treatment. C. CDE diet administration diagram and IHC for YAP expression after one week treatment. D. Whole liver immunoblot of YAP, and TAZ after CDE treatment for 7 and 14 days. E. IHC for YAP expression in Adiponectin-Cre IR/IGFR mice and littermate controls at 12 months of age. Associated picrosirius red staining is seen below each IHC staining. F. Whole liver YAP/TAZ immunoblots of IR/IGFR mice. G. Dot plots of whole liver qRT-PCR of littermate control and IR/IGFR mice at 12 months (n=3, each). For immunoblots, Ratio indicates relative enrichment of indicated protein after normalization to GAPDH. *p<0.05, **p<0.01. Error bars indicate standard error of the mean.

FIGURE 4.. YAP and TAZ are Predominantly Found within Hepatocytes of the Liver

A. Experimental design of Yap fl/fl(YKO) or Yap/Taz fl/fl(DKO) hepatocyte knockout and chronic CCl₄ treatment. Controls-YKO mice given AAV-LacZ. Representative YAP IHC of the indicated treatments. Box indicates chronic CCl₄ treatment. B. Whole liver immunoblot of YAP, TAZ, and GAPDH after the indicated chronic CCl₄ treatments. C. Representative tdTomato (Red) and EdU (Green) staining in Control and YKO mice subjected to a single dose of CCl₄. Dot plot to the right indicates the relative number of EdU⁺ cells per tdTomato⁺ population for each treatment (n=4, each). **p<0.01. Error bars indicate standard error of the mean.

FIGURE 5.. Loss of Hepatocyte YAP or YAP/TAZ attenuates Liver Fibrosis after CCl₄ Injury

A. Representative Picro-Sirius Red staining of mice from the indicated genotypes after chronic CCl₄ treatment. Dot plot to the right indicates quantification of collagen staining per unit area. B. Representative αSMA staining of mice from the indicated genotypes after chronic CCl₄ treatment. C. Representative F4/80 staining of mice from the indicated genotypes after chronic CCl₄ treatment. D. Representative flow cytometry of liver macrophages one day after chronic CCl₄ treatment. Percentages in the graph are of the gated population. Dot plot to the right of all performed experiments (n=5, each). Horizontal line represents the mean, each dot represents an experiment. Bars above each graph indicate standard error of the mean. **p<0.01, ***p<0.001.

FIGURE 6.. After Injury, YKO Hepatocytes Express More Metabolic and Fewer Immune Cell Migratory Programs than Controls

A. Treatment strategy diagram to analyze hepatocytes by RNA-seq. B. Principal component analysis (PCA) of the indicated treatments. C. Heat map of 1749 differentially expressed genes from the indicated treatments. D. Top and bottom differentially expressed gene programs identified by GSEA for Ctl vs DKO. E. Top and bottom differentially expressed gene programs identified by GSEA for Ctl/CCl₄ vs DKO/CCl₄

FIGURE 7.. Cyr61 is the Primary Macrophage Chemoattractant Target of the Hippo Pathway

A. Cyr61 mRNA hepatocyte expression under the indicated conditions. Transcripts per million base pairs (TPM). B. Representative EGFP staining of control and Cyr61-EGFP reporter mouse after chronic CCl₄ treatment. Asterisk marks central vein. Dotted line indicates area of EGFP⁺ cells. C. Strategy for either EGFP or Cyr61 expression in hepatocytes using AAV-TBG-EGFP or AAV-TBG-Cyr61. Below are whole-liver immunoblots of Cyr61, αSMA, F4/80, and β-actin for the indicated treatments one week after infection. D. Strategy for Cyr61 deletion utilizing AAV-TBG-saCas9/Cyr61 sgRNAs delivered to TetOYAP mice with AAV-Cre. Middle: Gross pictures of Control, YAP-Tg, and YAP-Tg/sgCyr61 livers two weeks after YAP induction. Lower: Graph of relative weight between YAP-Tg and YAP-Tg/sgCyr61 livers at the indicated times (1 week, n=6/group; 2 week, n=5/group). E. Immunoblots of control, YAP-Tg, YAP-Tg/sgCyr61 whole livers for Cyr61, αSMA, F4/80, YAP, and β-actin after 2 weeks of TetOYAP induction. F. Representative immunostaining of YAP-Tg and YAP-Tg/sgCyr61 mice for F4/80 and αSMA after 2 weeks of TetOYAP induction. G. Representative flow cytometry of liver macrophages in YAP-Tg and YAP-Tg/sgCyr61 liver after 2 weeks TetOYAP induction. Percentages shown are of gated population. Dot plot to the right of all performed experiments (n= 5, each). Horizontal line represents the mean, each dot represents and experiment. Bars above each graph indicate standard error of the mean. H. Dot plots of Col1A1 and Timp1 expression by RT-qPCR from YAP-Tg and Yap-Tg/sgCyr61 whole livers at 2 weeks after TetOYAP induction. *p<0.05, **p<0.01, ***p<0.001

FIGURE 8.. High Clinical NASH Activity Scores are Directly Associated with YAP/TAZ/CYR61 Expression

A. Immunostaining of representative human livers with low and high NASH activity for YAP and HNF4α. Right, dot plot quantification of each patient for YAP⁺ nuclei/HNF4α⁺. Arrowheads indicate double positive cells. B. Immunostaining (YAP, TAZ) or RISH (CYR61) of a cohort of human livers with low and high NASH activity (n=10, each) showing representative staining for each group. Graphs to the right show the relationship of YAP or TAZ immunostaining of each subject with respect to CYR61 expression. ***p<0.001