p63 controls metabolic activation of hepatic stellate cells and fibrosis via an HER2-ACC1 pathway

Abstract

The p63 protein has pleiotropic functions and, in the liver, participates in the progression of nonalcoholic fatty liver disease (NAFLD). However, its functions in hepatic stellate cells (HSCs) have not yet been explored. TAp63 is induced in HSCs from animal models and patients with liver fibrosis and its levels positively correlate with NAFLD activity score and fibrosis stage. In mice, genetic depletion of TAp63 in HSCs reduces the diet-induced liver fibrosis. In vitro silencing of p63 blunts TGF-β1-induced HSCs activation by reducing mitochondrial respiration and glycolysis, as well as decreasing acetyl CoA carboxylase 1 (ACC1). Ectopic expression of TAp63 induces the activation of HSCs and increases the expression and activity of ACC1 by promoting the transcriptional activity of HER2. Genetic inhibition of both HER2 and ACC1 blunt TAp63-induced activation of HSCs. Thus, TAp63 induces HSC activation by stimulating the HER2-ACC1 axis and participates in the development of liver fibrosis.

Keywords: ACC1; HER2; NASH; fibrosis; hepatic stellate cell; lipid metabolism; p63.

Graphical abstract

Figure 1

TAp63 protein is increased in activated hepatic stellate cells (HSCs) of livers with fibrosis (A) Representative dual immunofluorescence for α-SMA (red) and TAp63 (green) in control subjects (n = 3) and NAFLD patients with different stages of fibrosis (n = 16). Nuclei were stained with DAPI (blue). (B) Correlations of TAp63-stained area in α-SMA⁺ cells with fibrosis stage, NAS score, and serum AST and ALT levels (Spearman correlation test). (C) Representative dual immunofluorescence for α-SMA and TAp63 in liver sections from mice fed an MCDD for 6 weeks (n = 4), a CDHFD for 52 weeks (n = 4), or treated with carbon tetrachloride (0.6 mL/kg intraperitoneally [i.p.]) once per week for 6 weeks (n = 4). Untreated mice fed a standard chow diet were used as control group (n = 4).

Figure 2

p63 is upregulated in activated primary cultures of stellate cells from murine models or human patients (A) Levels of p63 mRNA in PmHSCs isolated from mice fed an MCDD for 6 weeks (n = 3) or (as a control) a standard diet (SD) for 6 weeks (n = 3). (B and C) Mice fed a CDHFD (n = 4) or SD for 52 weeks (n = 4) (B), and (C) treated with carbon tetrachloride (0.6 mL/kg i.p.) (n = 3) or (as a control) with vehicle (n = 4) once per week for 6 weeks. (D) mRNA expression of p63 and fibrogenesis markers in PrHSCs activated in culture (n = 4–8). (E) Expression of fibrotic markers and p63 in PhHSCs from a donor following the administration of 8 ng/mL TGF-β1 for 24 h (n = 8). (F) mRNA expression of fibrotic markers, total p63, TAp63, ΔNp63, and TAp63 protein levels in human LX-2 (n = 5–6) treated with TGF-β1 for 0, 12, or 24 h (n = 5–6). Hypoxanthine phosphoribosyltransferase (HPRT) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used to normalize mRNA and protein levels. Data are mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 3

Deficiency of TAp63 in HSCs protects against liver fibrosis in mice fed a CDHFD HSC-TAp63-KO or WT mice were fed a CDHFD for 20 weeks (n = 7). (A) Serum levels of AST. (B) Hepatic hydroxyproline levels. (C) Liver sections stained with H&E (top), oil red O (center), and Sirius red (bottom). Staining areas were quantified. (D) Expression of fibrosis markers in the liver. HSC-TAp63-KO or WT mice were fed an MCDD for 6 weeks (n = 6–7). (E) Serum levels of AST. (F) Hepatic hydroxyproline levels. (G) Liver sections stained with H&E (top), oil red O (center), and Sirius red (bottom). Staining areas were quantified. (H) Expression of fibrosis markers in the liver. HPRT was used to normalize mRNA levels. Data are mean ± SEM. ∗p < 0.05; ∗∗p < 0.01. See also Figure S1.

Figure 4

Genetic inhibition of p63 attenuates the metabolic and fibrogenic activation induced by TGF-β1 in PhHSCs and human LX-2 cell line (A and B) OCR (A) and (B) ECAR in PhHSCs silencing p63 and treated with TGF-β1 for 24 h. Arrows indicate the time point at which metabolic modulators (oligomycin [Oligo], phenylhydrazone [FCCP], rotenone/antimycin A [Rot/AA] and 2-deoxyglucose [2-DG]) were added to the assay. Parameters of mitochondrial and glycolytic function were calculated (n = 2–4). (C) Graph depicting the effect of TGF-β1 or sip63 treatments on quiescent or energetic metabolic states, based on quantification of ECAR and OCR during basal metabolism. (D) Expression of fibrotic markers (n = 4). (E and F) OCR I and (F) ECAR in LX-2 cells silencing p63 and treated with TGF-β1 for 24 h. (G) Basal energetic metabolic states, based on quantification of ECAR and OCR during basal metabolism (n = 4–6). (H) Expression of fibrotic markers (n = 6). (I) Expression of markers of lipid metabolism (n = 6). (J and K) ACC activity (n = 4) (J), and (K) protein levels of ACC and p-ACC (n = 5) in LX-2 cells silencing p63 and treated with TGF-β1. HPRT and GAPDH were used to normalize mRNA and protein levels. Data are mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S2 and S3.

Figure 5

Overexpression of TAp63 activates human LX-2 cells (A) mRNA expression of TAp63 and ΔNp63 (n = 4–6), and protein levels of TAp63 (n = 5–6) in LX-2 cell after overexpressing TAp63. (B) OCR. (C and D) ECAR (C) and (D) basal metabolism in LX-2 cells overexpressing TAp63 (n = 3). Arrows indicate the time point at which metabolic modulators (Oligo, FCCP, Rot/AA, and 2-DG) were added to the assay. (E) Expression of markers of lipid metabolism (n = 4–6). (F and G) Protein levels of ACC and p-ACC (n = 4) (F), and (G) mRNA levels of fibrogenic markers (n = 4). HPRT and GAPDH were used to normalize mRNA and protein levels. Data are mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S4.

Figure 6

Pharmacological or genetic inhibition of ACC1 blunts the metabolic and fibrogenic activation induced by TAp63 in human HSCs (A) ACC1 mRNA levels (n = 6) and ACC activity (n = 4) in LX-2 cells overexpressing TAp63 and treated with 0.5 μM FIR. (B) OCR (n = 3). (C) ECAR (n = 4). Arrows indicate the time point at which metabolic modulators (Oligo, FCCP, Rot/AA, and 2-DG) were added to the assay. (D) Graph depicting the effect of overexpression of TAp63 and administration of FIR on quiescent or energetic metabolic states, based on quantification of ECAR and OCR during basal metabolism. (E) Expression of fibrogenic markers (n = 6). (F) ACC1 mRNA levels (n = 6) and ACC activity (n = 4) in LX-2 cells overexpressing TAp63 and silencing ACC1. (G) OCR (n = 2–3). (H) ECAR (n = 4). (I) Basal metabolism. (J) Expression of fibrogenic markers (n = 6). HPRT was used to normalize mRNA levels. Data are mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S5.

Figure 7

Genetic inhibition of HER2 blocks the fibrogenic activation induced by TAp63 in human HSCs (A and B) mRNA levels of different transcription factors known to regulate ACC1 expression in LX-2 cells (A) treated with TGF-β1 and silencing p63 (n = 6) and (B) overexpressing TAp63 (n = 4). (C) Protein levels of HER2 in LX-2 cells treated with TGF-β1 for 24 h and overexpressing TAp63 are also shown (n = 5–6) (see also A). (D) mRNA expression of HER2. (E and F) ACC1 (E) and (F) fibrogenic markers in LX-2 cells overexpressing TAp63 following the silencing of HER2 (n = 6). (G) Diagram depicting putative binding sites for TAp63 in the HER2 gene promoter. (H) Levels of HER2 promoter activity in LX-2 cells transfected with TAp63 (n = 6). (I) Nebulosa plots of the HER2 density in the uniform manifold approximation and projection visualization of cirrhotic HSCs (n = 4) and normal HSCs (n = 4). HER2 expressions in both conditions are also shown. (J) Expression of HER2 in primary human HSCs treated with TGF-β1 (n = 8). HPRT and GAPDH were used to normalize mRNA and protein levels. Data are mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S6 and S7.