Physiological ranges of matrix rigidity modulate primary mouse hepatocyte function in part through hepatocyte nuclear factor 4 alpha

Abstract

Matrix rigidity has important effects on cell behavior and is increased during liver fibrosis; however, its effect on primary hepatocyte function is unknown. We hypothesized that increased matrix rigidity in fibrotic livers would activate mechanotransduction in hepatocytes and lead to inhibition of liver-specific functions. To determine the physiologically relevant ranges of matrix stiffness at the cellular level, we performed detailed atomic force microscopy analysis across liver lobules from normal and fibrotic livers. We determined that normal liver matrix stiffness was around 150 Pa and increased to 1-6 kPa in areas near fibrillar collagen deposition in fibrotic livers. In vitro culture of primary hepatocytes on collagen matrix of tunable rigidity demonstrated that fibrotic levels of matrix stiffness had profound effects on cytoskeletal tension and significantly inhibited hepatocyte-specific functions. Normal liver stiffness maintained functional gene regulation by hepatocyte nuclear factor 4 alpha (HNF4α), whereas fibrotic matrix stiffness inhibited the HNF4α transcriptional network. Fibrotic levels of matrix stiffness activated mechanotransduction in primary hepatocytes through focal adhesion kinase. In addition, blockade of the Rho/Rho-associated protein kinase pathway rescued HNF4α expression from hepatocytes cultured on stiff matrix.

Conclusion: Fibrotic levels of matrix stiffness significantly inhibit hepatocyte-specific functions in part by inhibiting the HNF4α transcriptional network mediated through the Rho/Rho-associated protein kinase pathway. Increased appreciation of the role of matrix rigidity in modulating hepatocyte function will advance our understanding of the mechanisms of hepatocyte dysfunction in liver cirrhosis and spur development of novel treatments for chronic liver disease. (Hepatology 2016;64:261-275).

Figure 1

Increased pathological severity grade of liver fibrosis corresponds to increased quantified fibrillar collagen deposition. (A) Representative images of H&E and Sirius Red staining (brightfield & polarized light) demonstrate distinct patterns of collagen deposition depending on the model and severity of experimental liver fibrosis. Scale bars represent 200μm. (B) Quantification of fibrillar collagen deposition in Sirius Red-stained liver sections. *p<0.05 and **p<0.01 by one-way ANOVA and Tukey’s post-hoc test for pairwise comparison. There were 3 mice per group and 3 sections were quantified per mouse, n=3. Error bars represent SEM.

Figure 2

Liver fibrosis results in treatment and region specific increases in tissue matrix stiffness as determined by AFM. (A) Diagram depicting the directionality of AFM measurement acquisition either along fibrotic tracts or orthogonal to fibrotic tracts. (B) Quantitative AFM analysis of tissue matrix stiffness of a liver lobule from untreated normal liver. Measurements were taken either from central (location ratio 0) to portal zones (location ratio 1) or from portal to portal zones. Tissue matrix rigidity did not vary significantly within normal liver lobules and averaged around 150Pa. (C) AFM analysis orthogonal to fibrotic tracts demonstrates that tissue stiffening is region specific with the greatest increases in matrix rigidity in areas of greatest fibrosis. Measurements were carried out within a liver lobule starting remote from the fibrotic tract (location ratio 0) toward the fibrotic tract (location ratio 1). Graphs depict changes between all conditions (no treatment, black; CCl_4, blue; DDC, red), between mild (orange) vs. severe (blue) CCl₄-induced fibrosis, or between mild (purple) vs. severe (red) DDC-induced fibrosis. *p<0.01 between all conditions and **p<0.01 for the two treatment groups compared to non-treated mice within the region of comparison denoted by dotted line. (D) AFM analysis demonstrates significant increases in tissue stiffness along fibrotic tracts in both CCl₄-induced and DDC-induced disease compared to non-treated normal liver. Measurements were carried out either from central-central zones or portal-portal zones depending on disease model and severity. *p<0.01 between all conditions across the entire fibrotic tract. Sample size = 3 mice for each condition and 3 lobules were examined per mouse. For “All Conditions” plots, severe and mild forms of disease were combined so that n=6 for CCl₄ and DDC treatment. Statistical significance was calculated using one-way ANOVA and Tukey’s post-hoc test for pairwise comparison. Error bars represent SEM.

Figure 3

Primary hepatocyte cell area increases logarithmically with increasing matrix stiffness. (A) Primary hepatocytes were cultured on collagen-conjugated polyacrylamide gels at 8 different stiffnesses: 75, 140, 400, 1k, 2.7k, 6k, 22k, and 60k Pa. Images were taken after 24h of culture and the area of individual cells were determined by digital imaging analysis. Data represent the average of 2–5 independent experiments in which at least 20 cells were measured for each stiffness level per experiment. The best-fit curve follows a logarithmic function with an R² = 0.95 as demonstrated by the semi-log plot. The inset linear plot shows that the most dynamic range for increasing cell size was between 75 and 1k Pa. Error bars represent SEM. (B) Representative phase-contrast images of single-cell primary hepatocytes cultured at 140, 1k, 6k, and 60k Pa for 24h. Scale bar = 25μm. Total magnification 80x.

Figure 4

Matrix rigidity modulates various primary hepatocyte functions to differing degrees. (A) Albumin production, (B) glycogen storage, and (C) cytochrome P450 1A activity were measured quantitatively with correction for cell numbers after 24h of culture on matrices of varying stiffness. Data represent the average of 3 independent experiments. *p<0.05 by Student’s t-test. Error bars represent SEM.

Figure 5

HNF4α expression is decreased in fibrotic livers in vivo and in hepatocytes cultured on stiff matrix in vitro. (A) HNF4α (red) was co-stained with fibronectin (green) to depict areas of fibrosis in livers of untreated, CCl₄-, and DDC-treated mice. Images are representative of at least 3 mice per group and at least 3 sections evaluated per mouse. Scale bars represent 100μm. (B) The percentage of HNF4α⁺ cells per low-power field in the livers of untreated, CCl₄-, and DDC-treated mice were quantified by digital imaging analysis. Sample size = 3 mice for each group with at least 3 sections evaluated per mouse. *p<0.01 by one-way ANOVA and Tukey’s post-hoc test for pairwise comparison. Error bars represent SEM. (C) Expression levels of hepatocyte nuclear factor 4 alpha (Hnf4a), bile acid-CoA:amino acid N-acyltransferase (Baat), factor VII (F7), and glycogen synthase 2 (Gys2) mRNA were significantly inhibited on matrices stiffer than 140Pa in hepatocytes after 24h culture as determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Data represent the average of 3 independent experiments. *p<0.05 and **p<0.005 by Student’s t-test. Error bars represent SEM.

Figure 6

HNF4α transcriptional regulation is maintained in primary hepatocytes cultured on normal liver matrix stiffness in vitro. (A) Primary hepatocytes were isolated from the Hnf4a^fl/fl:Alb Cre⁺ mice and wildtype littermates (Hnf4a^fl/fl:Alb Cre⁻) and cultured on 140Pa collagen-PA gels for 24h. Expression of gene targets positively regulated by HNF4α (Baat, F7, and Gys2) was decreased whereas targets negatively regulated by HNF4α (Snai1 and Vim) were increased in Hnf4a^fl/fl:Alb Cre⁺ hepatocytes, indicating that maintained expression of those functional genes on 140Pa matrix was dependent on continued HNF4α expression. Data represent the average of 5 independent experiments. (B) Primary hepatocytes were cultured on 1kPa matrix and transfected with either control plasmid or plasmid over-expressing HNF4α. Gene expression as determined by qRT-PCR was determined 24h after transfection. HNF4α-plasmid transfection achieved high over-expression of Hnf4a mRNA (>85-fold compared to control plasmid transfection) and resulted in decreased expression of Snai1. Data represent the average of 3 independent biological samples. *p<0.05 and **p<0.005 by Student’s t-test. Error bars represent SEM.

Figure 7

Mechanotransduction is activated in primary hepatocytes by fibrotic levels of matrix stiffness in vivo and in vitro. (A) Activated phospho-FAK^Y397 (green) was expressed in hepatocytes near fibrotic tracts (highlighted by fibronectin staining in red) in the livers of CCl₄- and DDC-treated mice. Expression of phospho-FAK^Y397 and activated β₁-integrin (green) were localized to hepatocytes by HNF4α (red) co-staining to demonstrate activation of mechanotransduction within the hepatocytes of fibrotic livers. Images are representative of at least 3 mice per group and at least 3 sections evaluated per mouse. There was minimal background staining with secondary antibody-only controls (data not shown). (B) Isolated primary hepatocytes were cultured on 140, 1k, and 60k Pa matrices for 2h and cell lysates prepared for immunoblotting. Blot images are from one representative experiment. Densitometry graph shows the specific activation of phospho-FAK^Y397 relative to total FAK averaged over 5 independent experiments. *p<0.005 by Student’s t-test. Error bars represent SEM.

Figure 8

Inhibition of HNF4α expression in primary hepatocytes at fibrotic levels of matrix rigidity is mediated predominantly by the Rho/ROCK pathway. (A) Isolated primary hepatocytes were cultured on 1kPa matrices for 24h in the presence of control or inhibitors of FAK (FAKi-14), ROCK (Y-27632), myosin contractility (blebbistatin), or MEK (U-0126). Expression of Hnf4a and its target genes, Baat and Vim, were analyzed by qRT-PCR. Expression of Hnf4a and its positively regulated target, Baat, was significantly increased by blocking the Rho/ROCK pathway with Y-27632. Expression of Vim, a negatively regulated target of Hnf4a, was significantly inhibited by both FAK and ROCK inhibition. Data are representative of 3–5 independent experiments. *p<0.05 and **p<0.01 by Student’s t-test. Error bars represent SEM. (B) Primary hepatocytes were cultured on 140Pa matrix, 1kPa matrix, or 1kPa matrix in the presence of FAK or ROCK inhibitors for 12h, 24h, or 48h. HNF4α protein expression was determined by immunoblotting and quantified compared to the GAPDH housekeeping protein. Densitometry calculations were normalized to HNF4α expression in freshly isolated hepatocytes.