Extracellular matrix protein 1 binds to connective tissue growth factor against liver fibrosis and ductular reaction

Abstract

Background: Extracellular matrix protein 1 (ECM1) can inhibit TGFβ activation, but its antifibrotic action remains largely unknown. This study aims to investigate ECM1 function and its physical interaction with the profibrotic connective tissue growth factor (CTGF) in fibrosis and ductular reaction (DR).

Methods: Ecm1 knockouts or animals that ectopically expressed this gene were subjected to induction of liver fibrosis and DR by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or α-naphthyl-isothiocyanate (ANIT). ECM1 and CTGF were also examined in the livers of patients with alcohol-associated liver disease (ALD) or ethanol-exposed animals that were fed the western diet for 4 months in the WDA model with liver pathology resembing ALD in patients.

Results: ECM1 bound to CTGF in yeast two-hybrid systems, cultured liver cells, and cholestatic livers damaged by DDC or α-naphthyl-isothiocyanate. This interaction blocked integrin αvβ6-mediated TGFβ activation, thereby reducing fibrotic responses in vitro. ECM1 downregulation was associated with biliary CTGF induction during human ALD progression. In experimental models, Ecm1 loss enhanced susceptibility to DDC-induced cholestasis with upregulation of Ctgf, αvβ6, alpha-smooth muscle actin, procollagen type I, serum transaminase, and total bilirubin levels in germline knockouts, whereas forced expression of this gene significantly attenuated DR and biliary fibrosis after the feeding of DDC or α-naphthyl-isothiocyanate containing diets. Moreover, ectopic Ecm1 inhibited not only alcohol-associated fibrosis but also TGFβ-mediated deregulation of hepatocyte nuclear factor 4α, preventing the production of the fetal p2 promoter-driven isoforms in the WDA model.

Conclusions: We uncover a novel antifibrotic action by ECM1 that binds CTGF and inhibits integrin αvβ6-mediated TGFβ activation. Targeting its loss has therapeutic potential for the treatment of DR and liver fibrosis in chronic conditions, such as cholangiopathy and ALD.

Graphical abstract

FIGURE 1

Ecm1 binds to Ctgf and reduces αvβ6-mediated TGFβ activation leading to attenuated. fibrosis in vitro. (A) Yeast two-hybrid analyses showed that all co-transformants expressing AD and BD constructs grew on permissive media but only the ones containing interactors (Ecm1 with Ctgf or the CtgfIV mutant) could survive on selective media (with 3-AT but lacking leucine, tryptophan, and histidine). (B) The immunofluorescent staining showed colocalization between Ecm1:FLAG and Ctgf:V5 fusion proteins in transfected WB-F344 cells and primary human BDEC. (C) M2 antibody (Ab) that recognized the Ecm1:FLAG protein specifically pulled down Ctgf:V5 and CtgfIV:V5 proteins from conditioned media of the rat hepatic progenitor WB-F344 cells, whereas no immunoprecipitation signal was seen from CtgfI-III:V5 or vector control in immunoprecipitation assays (left). M2 Ab rather than mouse IgG controls could also pull down Ctgf:V5 from the human BDEC lysates (right). Equal input of tested proteins was shown. (D) Dose-dependent binding of MBP-Ctgf or CtgfIV fusion proteins to recombinant ECM1:FLAG protein was detected by M2 Ab-conjugated HRP in solid-phase assays. MBP protein only showed background signal. Values were represented means ± SD. **p<0.01; *p<0.05. Student t test. (E) Western blotting detected the expression of β6:HA, Ctgf:V5, and Ecm1:FLAG in established WB-F344 stable cell lines and the human BDEC. (F and G) Ecm1 reduced αvβ6-mediated and Ctgf-potentiated TGF-β1 activation in the WB-F344 stable cell lines and and the human BDEC based on plasminogen activator inhibitor 1-driven luciferase activity in co-cultured transfected mink lung epithelial cell (F) and ELISA from the conditioned media of these cells (G). (H) Presence of Ecm1:FLAG significantly reduced the expression of CTGF, αSMA, and COLLAGEN genes in LX-2 cells that were exposed to conditioned media containing β6:HA with or without Ctgf:V5. Values in (F–H) are means ± SD from triplicate experiments. ***p<0.001; **p<0.01; *p<0.05 in Student t test. Abbreviations: AD, activation domain; BD, binding domain; BDEC, bile ductular epithelial cell; CTGF, connective tissue growth factor; ECM1, extracellular matrix protein 1; HA, hemagglutinin; MBP, maltose binding protein; αSMA, α-smooth muscle actin.

FIGURE 2

ECM1 loss is associated with Ctgf induction in ductular reaction and liver fibrosis during human chronic liver disease progression. (A) immunohistochemical analysis detected extensive ductular reaction positive for CTGF in reactive ducts of human AH livers compared to normal (N) livers. Scale bar: 100 μm. (B) Upregulation of biliary and fibrosis markers including CTGF transcript (from the public data set GSE143318) was associated with loss of ECM1 in human AH livers (from the public data set GDS4389 and GSE143318), PSC and PBC livers (from GSE159676). **p<0.01; *p<0.05 in Student t test. (C) Western blotting and densitometric analyses indicated the loss of ECM1 and induction of CTGF proteins in livers of patients with AS, ASH, and AC compared to healthy livers (n=2 per group). Abbreviations: AC, alcohol-associated cirrhosis; AH, alcohol-associated hepatitis; AS, alcohol-associated steatosis; ASH, alcohol-associated steatohepatitis; CTGF, connective tissue growth factor; ECM1, extracellular matrix protein 1; PBC, primary biliary cholangitis; PSC, primary sclerosing cholangitis; αSMA, α-smooth muscle actin.

FIGURE 3

Ecm1 deficiency promotes biliary fibrosis and DR during DDC-induced liver damage. Ecm1 KO or WT littermates (7-week-old, n=4) were fed 0.01% DDC-containing diet for 5 days. quantitative reverse transcriptase-PCR analyses (A), Western blotting with densitometric analysis (B), and immunohistochemical analysis (C) detected elevated levels of biliary markers and fibrosis-related genes in KO compared to WT littermates in untreated conditions and after DDC feeding. αSMA was detected with Vector blue substrates. Relative expressions were calculated in comparison to WT livers (n=4–5/group). Data were represented as means ± SEM. **p<0.01; *p<0.05. Student t test. Graphs are quantification of stained areas based on image analysis of at least 10 random fields (×200 magnification) per liver. Scale bar: 100 μm. (D and E) The loss of Ecm1 was associated with elevated levels of hepatic hydroxyproline content (D) and liver injury markers (E) in KO (n=4) compared to WT (n=5). Data were represented as means ± SEM. *p<0.05. Student t test. Abbreviations: CTGF, connective tissue growth factor; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; ECM1, extracellular matrix protein 1; KO, knockout; αSMA, α-smooth muscle actin; WT, wild type.

FIGURE 4

Ecm1 binds to Ctgf and reduces biliary fibrosis and DR during DDC-induced liver damage. Ectopic expression of Ecm1 in AAV8 virus caused downregulation of biliary markers and fibrosis-related genes at 14 days after DDC feeding shown in quantitative reverse transcriptase-PCR analyses (A), Western blotting with densitometric analyses (B), and immunohistochemical analysis (C). Dual staining were carried out using rabbit anti-Ki67 and mouse anti-CK19 antibodies followed by detection with Vector blue and Vector red substrates respectively. Integrin αvβ6 and αSMA were also detected by Vector blue. Sirius red staining was performed to label collagen deposit. Relative expressions were calculated in relation to GFP livers. Data were represented as means ± SEM (n=4/group). **P<0.01; *P<0.05. Student t test. Scale bar: 200 μm. Graphs are quantification of stained areas based on image analysis of at least ten random fields (200x magnification) per animal. Ectopic Ecm1 also decreased hepatic hydroxyproline content (D) and serum levels of liver injury markers (E). Data were expressed as means ± SEM (n=4/group). *p<0.05. Student t test. (F) Immunoprecipitation assays using M2 antibody pulled down Ctgf in the Ecm1:FLAG expressing livers. Equal Ctgf was input in the assays. (G) Proximity ligation assay assay detected Ecm1:FLAG and Ctgf interaction (red signal) on liver sections from animals that received AAV8-Ecm1:FLAG or AAV8-GFP infection and 14-day 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding. Scale bar: 50 μm. Abbreviations: CTGF, connective tissue growth factor; ECM1, extracellular matrix protein 1; αSMA, α-smooth muscle actin.

FIGURE 5

Ecm1 binds to Ctgf and attenuates the ANIT-induced biliary fibrosis. Quantitative reverse transcriptase-PCR analyses (A), Western blotting with densitometric analyses (B), and immunohistochemical analysis (C) showed that ectopic expression of Ecm1 in AAV8 virus could downregulate expression of Itgβ6, Ctgf, αSMA, and procollagen type I genes at 1 month after the α-naphthyl-isothiocyanate feeding. Relative expressions were calculated in comparison to to AAV8-GFP-infected controls. Data were represented as means ± SEM (n=4/group). **p<0.01; *p<0.05. Student t test. Scale bar: 100 μm. Graphs are quantification of stained areas based on image analyses of at least 10 random fields (×200 magnification) per liver. Ectopic expression of Ecm1 gene also decreased levels of hepatic hydroxyproline content (D) and liver injury markers (E) compared to the GFP controls (n=4). Data are represented as means ± SEM. *p<0.05. Student t test. (F) Immunoprecipitation assays using M2 antibody pulled down Ctgf in the α-naphthyl-isothiocyanate damaged, Ecm1:FLAG expressing livers. Equal input of Ctgf was also shown. (G) Proximity ligation assay assays detected direct binding between Ecm1:FLAG and Ctgf proteins (red signal) on liver sections from mice that received AAV8-Ecm:FLAG or GFP infection before the one-month α-naphthyl-isothiocyanate feeding. Scale bar: 50 μm. Abbreviations: CTGF, connective tissue growth factor; ECM1, extracellular matrix protein 1; αSMA, α-smooth muscle actin.

FIGURE 6

Forced expression of Ecm1 reduces fibrosis during the WDA-induced liver injury. Q-RTPCR analyses (A), western blotting with densitometric analysis (B), and immunohistochemical analysis (C) showed lower induction of Ctgf, a Hnf4α-p2 promoter-derived transcript, αSMA, and procollagen type I in the Ecm1:FLAG-infected livers compared to GFP controls at 16 weeks post the feeding of western diet and alternate drinking of 10% to 20% ethanol. Data were represented as means ± SEM (n=4/group). **p<0.01; *p<0.05. Student t test. Scale bar: 200 μm. Graphs are quantification of stained areas based on image analysis of at least 10 random fields (×200 magnification) per liver. (D) Decreases in hepatic hydroxyproline content were also found in the Ecm1:FLAG-infected livers compared to GFP controls (n=4/group). Data are means ± SEM. *p<0.05. Student t test. (E) Western blotting indicated less deregulation of Hnf4α in the Ecm1:FLAG expressing livers (n=4) than the GFP controls (n=4) during the WDA-induced liver injury. Abbreviations: CTGF, connective tissue growth factor; ECM1, extracellular matrix protein 1; αSMA, α-smooth muscle actin.