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. 2024 Apr 1;134(7):e166731.
doi: 10.1172/JCI166731.

Hepatocyte-specific CCAAT/enhancer binding protein α restricts liver fibrosis progression

Tingting Yan  1   2 , Nana Yan  1   2 , Yangliu Xia  1 , Vorthon Sawaswong  1 , Xinxin Zhu  3 , Henrique Bregolin Dias  1 , Daisuke Aibara  1 , Shogo Takahashi  1 , Keisuke Hamada  1 , Yoshifumi Saito  1 , Guangming Li  4 , Hui Liu  5 , Hualong Yan  6 , Thomas J Velenosi  1 , Kristopher W Krausz  1 , Jing Huang  6 , Shioko Kimura  1 , Yaron Rotman  7 , Aijuan Qu  3 , Haiping Hao  2 , Frank J Gonzalez  1
Affiliations

Hepatocyte-specific CCAAT/enhancer binding protein α restricts liver fibrosis progression

Tingting Yan et al. J Clin Invest. .

Abstract

Metabolic dysfunction-associated steatohepatitis (MASH) - previously described as nonalcoholic steatohepatitis (NASH) - is a major driver of liver fibrosis in humans, while liver fibrosis is a key determinant of all-cause mortality in liver disease independent of MASH occurrence. CCAAT/enhancer binding protein α (CEBPA), as a versatile ligand-independent transcriptional factor, has an important function in myeloid cells, and is under clinical evaluation for cancer therapy. CEBPA is also expressed in hepatocytes and regulates glucolipid homeostasis; however, the role of hepatocyte-specific CEBPA in modulating liver fibrosis progression is largely unknown. Here, hepatic CEBPA expression was found to be decreased during MASH progression both in humans and mice, and hepatic CEBPA mRNA was negatively correlated with MASH fibrosis in the human liver. CebpaΔHep mice had markedly enhanced liver fibrosis induced by a high-fat, high-cholesterol, high-fructose diet or carbon tetrachloride. Temporal and spatial hepatocyte-specific CEBPA loss at the progressive stage of MASH in CebpaΔHep,ERT2 mice functionally promoted liver fibrosis. Mechanistically, hepatocyte CEBPA directly repressed Spp1 transactivation to reduce the secretion of osteopontin, a fibrogenesis inducer of hepatic stellate cells. Forced hepatocyte-specific CEBPA expression reduced MASH-associated liver fibrosis. These results demonstrate an important role for hepatocyte-specific CEBPA in liver fibrosis progression, and may help guide the therapeutic discoveries targeting hepatocyte CEBPA for the treatment of liver fibrosis.

Keywords: Fibrosis; Hepatology; Macrophages; Transcription.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Hepatic CEBPA expression is decreased by MASH and tracks with human liver fibrosis.
(A) CEBPA mRNA in human livers. n = 13 for normal group, and n = 28 for MASH group. (B–D) Correlation of CEBPA mRNA with fibrosis gene mRNAs in human livers by nonparametric Pearson’s test. (E–G) Representative Western blot of CEBPA p42 and p30 protein in human livers (E) and quantitation (F–G, n = 12). (H and I) Representative images of CEBPA (red), hepatocyte marker HepPar1 (green), and DAPI (blue) immunofluorescence in liver biopsies from patients with histologically normal livers, F0-4 MASLD livers and quantitation of the percentage of CEBPA positive cells among HepPar1 positive cells (pink indicates red nuclear CEBPA merged with blue DAPI; n = 3 for control normal livers, n = 9 for F0, n = 9 for F1, n = 7 for F2, n = 6 for F3 and n = 7 for F4 MASLD livers). (J–N) Cebpa mRNA in liver (J, n = 5) and primary hepatocytes (K, n = 4), representative liver CEBPA p42 and p30 protein (L), representative CEBPA IHC staining (M) and quantitation (N, n = 3) of C57BL/6N mice fed HFCFD for 13 or 26 weeks. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed unpaired student’s t test for A, F, and G, while 1-way ANOVA followed by Dunnett’s multiple-comparisons test for I–K and N, compared with control group. Scale bar: 50 μm.
Figure 2
Figure 2. Constitutive hepatocyte CEBPA loss enhances MASH-associated liver fibrosis.
(A) Representative histological staining. (B–E) Liver mRNAs in fibrosis and inflammation for mice fed a HFCFD for 16 weeks (B, n = 8), and the quantitation of liver Sirius red staining (C, n = 8), CD45 staining (D, n = 3) and PAS staining (E, n = 3) for mice. (F–I) Liver mRNAs in fibrosis and inflammation for mice fed a HFCFD for 9 months (F, n = 12 for Cebpafl/fl mice and n = 11 for CebpaΔHep mice) and the quantitation of liver Sirius red staining (G, n = 8), CD45 staining (H, n = 3) and PAS staining (I, n = 4). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed unpaired student’s t test. Scale bars: 50 μm for Oil red O staining; 100 μm for others.
Figure 3
Figure 3. Hepatocyte CEBPA loss at the progressive stage of MASH exacerbates liver fibrosis.
(A–D) Time scheme (A and C), liver mRNAs in fibrosis and inflammation of livers from 22-week HFCFD-fed mice dosed with tamoxifen for the last 10 weeks (12W+10W; B, n = 12 for Cebpafl/fl mice and n = 11 for CebpaΔHep mice) or 26-week HFCFD-fed mice dosed with tamoxifen for the last 10 weeks (16W+10W; D, n = 8 for Cebpafl/fl mice and n = 6 for CebpaΔHep,ERT2 mice). (E) Liver pictures for mice treated as schemed in C. (F) Representative histological staining, quantitation of liver Sirius red staining (G, n = 6 for 12W+10W and I, n = 5 for 16W+10W) and CD45 staining (H and J, n = 3). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by 2-tailed unpaired student’s t test. Scale bar: 50 μm for Oil red O staining; 100 μm for others.
Figure 4
Figure 4. Hepatocyte CEBPA represses Spp1 expression and OPN release in mice.
(A) Venn diagram showing genes upregulated (left) or downregulated (right) by hepatocyte CEBPA knockout. (B) Volcano plots for RNA-Seq analyses of livers (HFCFD2W and HFCFD12W+T10W). Log2FC, FC > 2. P, Padj < 0.05. P and log2FC, Padj < 0.05 and FC > 2. (C–F) liver Spp1 mRNA and Serum OPN in CebpaΔHep,ERT2 mice fed a HFCFD and dosed with tamoxifen as indicated (C and D, n = 6–12) and CebpaΔHep mice fed a HFCFD for 2 weeks (n = 5 or 6), 16 weeks (n = 8), and 9 months (n = 11 or 12). T10D or T10W, tamoxifen for the last 10 days or 10 weeks. (G) Spp1 mRNA in the livers (n = 5), primary hepatocytes (n = 4) from C57BL/6N mice fed a 13-week HFCFD or 26-week HFCFD with statistics calculated by 1-way ANOVA with Dunnett’s multiple-comparisons test and human livers (n = 13 or 28). (H) Representative OPN protein in human livers and quantitation (n = 12). (I) Correlation analyses of fibrosis gene mRNAs with SPP1 mRNA in human livers by nonparametric Pearson’s test. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed unpaired student’s t test unless otherwise stated.
Figure 5
Figure 5. CEBPA represses Spp1 expression and OPN release in primary hepatocytes in vitro.
(A–D) Cebpa and Spp1 mRNAs (A, n = 4), OPN protein (B and C, n = 3), and supernatant OPN (D, n = 5) of primary hepatocytes from chow-fed mice. (E–H) Cebpa and Spp1 mRNAs (E, n = 4), OPN protein (F–G, n = 4), and supernatant OPN (H, n = 4) in 48 hour Ad-GFP or Ad-CEBPA-treated primary WT hepatocytes. (I and J) Schematic diagram of the mouse Spp1 promoter illustrating the CEBPREs (I) and luciferase reporter assays (J, n = 4). (K and L) ChIP assay, relative CEBPA enrichment on CEBPRE1-4 (P1–P4) (K, n = 3) or H3K4Me3 enrichment on RLP30/HOXD10 (L, n = 3). (M) ChIP assay, relative H3K27Ac enrichment on CEBPRE1, n = 3. Data represent mean ± SEM. *P <0.05, **P < 0.01, ***P < 0.001, and ###P < 0.001 compared with each control group or as indicated by 2-way ANOVA with Šidák’s multiple-comparisons test for J and M or by 2-tailed unpaired student’s t test for others.
Figure 6
Figure 6. Hepatocyte CEBPA deficiency–induced Spp1 expression and OPN release activates HSCs to promote MASH fibrosis.
(A) Fibrosis gene mRNAs of primary HSCs treated with supernatants from hepatocytes or with mouse OPN antibody (OPN Ab), n = 4. shCtrl, control scrambled shRNA; shSpp1, Spp1 shRNA. #P < 0.05, ##P < 0.01, ###P < 0.001 compared with WT+shCtrl, while *P < 0.05, **P < 0.01, ***P < 0.001 compared with KO+shCtrl. (B–F) Liver Cebpa and Spp1 mRNAs (B, n = 9), serum OPN (C, n = 9), representative histological staining (D), quantitation of Sirius red staining (E, n = 9), and liver mRNAs in fibrosis and inflammation (F, n = 9) in 16-week HFCFD-fed CebpaΔHep mice treated with AAV8-control scrambled shRNA (control) or AAV8-Spp1 shRNA (shSpp1). (G–K) Liver Cebpa and Spp1 mRNAs (G, n = 6), serum OPN (H, n = 6), representative histological staining (I), quantitation of Sirius red staining (J, n = 6) and liver mRNAs in fibrosis and inflammation (K, n = 6) in CebpaΔHep,ERT2 mice fed a HFCFD for 24 weeks and treated with tamoxifen for the last 12 weeks with AAV8 dosing at 1 week prior to tamoxifen dosing. Data represent mean ± SEM. Cebpafl/fl Control or shSpp1, Cebpafl/fl mice treated with AAV8-control scrambled shRNA or AAV8-shSpp1. CebpaΔHep or CebpaΔHep,ERT2 Control or shSpp1, CebpaΔHep or CebpaΔHep,ERT2 mice treated with AAV8-control scrambled shRNA or AAV8-shSpp1. #P < 0.05, ##P < 0.01, ###P < 0.001 compared with Cebpafl/fl Control, while *P < 0.05, **P < 0.01, ***P < 0.001 compared with CebpaΔHep Control or CebpaΔHep,ERT2 Control by 2-way ANOVA with Šidák’s multiple-comparisons test. Scale bars: 100 μm.
Figure 7
Figure 7. Hepatocyte-specific CEBPA knockout enhances CCl4-induced liver fibrosis via Spp1 induction.
(A) Liver Cebpa and Spp1 mRNAs (n = 6), serum OPN (n = 6), liver mRNAs in fibrosis and inflammation (n = 6), and quantitation of Sirius red staining (n = 6) and CD45 staining (n = 3) for 4-week CCl4-treated mice. (B) Liver Cebpa and Spp1 mRNAs (n = 9-13), serum OPN (n = 9-13), liver mRNAs in fibrosis and inflammation (n = 9-13), and quantitation of Sirius red staining (n = 8) and CD45 staining (n = 3) for 8-week CCl4-treated mice. (C) Representative histological staining for the livers from 4-week CCl4-treated mice and 8-week CCl4-treated mice. (D–E) Liver Cebpa and Spp1 mRNAs, serum OPN, liver mRNAs in fibrosis and inflammation (D, n = 6), representative histological staining and quantitation of Sirius red staining (E, n = 5) in 4-week CCl4-treated mice dosed with AAV8-control scrambled shRNA (control) or AAV8-Spp1 shRNA (shSpp1). Data represent mean ± SEM. #P < 0.05, ##P < 0.01, ###P < 0.001 compared with Cebpafl/fl group, while *P < 0.05, **P < 0.01, ***P < 0.001 compared with the CebpaΔHep group by 2-tailed unpaired student’s t test for A–C or by 2-way ANOVA with Šidák’s multiple-comparisons test for D–E. Scale bars: 100 μm.
Figure 8
Figure 8. AAV8-TBG-Cebpa reduces liver fibrosis in HFCFD-fed mice.
(A–E) Preventive dosing scheme (A), liver mRNAs in fibrosis and inflammation (B), serum OPN (C), quantitation of Sirius red staining (D) and representative histological staining (E), scale bar: 100 μm for H&E and Sirius red staining; 50 μm for Oil red O staining; n = 5. (F–J) Therapeutic dosing scheme (F), liver mRNAs in fibrosis and inflammation (G), serum OPN (H), quantitation of Sirius red staining (I), and representative histological staining (J). Scale bar: 100 μm; n = 6. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by 2-tailed unpaired student’s t test.
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Comment in

  • Protective hepatocyte signals restrain liver fibrosis in metabolic dysfunction-associated steatohepatitis

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