Hepatocyte nuclear factor 4α negatively regulates connective tissue growth factor during liver regeneration

Abstract

Liver regeneration after injury requires fine-tune regulation of connective tissue growth factor (Ctgf). It also involves dynamic expression of hepatocyte nuclear factor (Hnf)4α, Yes-associated protein (Yap), and transforming growth factor (Tgf)-β. The upstream inducers of Ctgf, such as Yap, etc, are well-known. However, the negative regulator of Ctgf remains unclear. Here, we investigated the Hnf4α regulation of Ctgf post-various types of liver injury. Both wild-type animals and animals contained siRNA-mediated Hnf4α knockdown and Cre-mediated Ctgf conditional deletion were used. We observed that Ctgf induction was associated with Hnf4α decline, nuclear Yap accumulation, and Tgf-β upregulation during early stage of liver regeneration. The Ctgf promoter contained an Hnf4α binding sequence that overlapped with the cis-regulatory element for Yap and Tgf-β. Ctgf loss attenuated inflammation, hepatocyte proliferation, and collagen synthesis, whereas Hnf4α knockdown enhanced Ctgf induction and liver fibrogenesis. These findings provided a new mechanism about fine-tuned regulation of Ctgf through Hnf4α antagonism of Yap and Tgf-β activities to balance regenerative and fibrotic signals.

Keywords: connective tissue growth factor (Ctgf); hepatocyte nuclear factor 4α (Hnf4α); liver injury; liver regeneration.

Figure 1

Ctgf deficiency is associated with reduced inflammation, hepatocyte proliferation, and collagen production after liver damage caused by ethanol/CCl₄ co‐treatment. Ctgf^k/k and Ctgf^f/f mice were subjected to ethanol/CCl₄ co‐treatment and were sacrificed at day 1 after CCl₄ administration. (A‐C) Ctgf loss was confirmed by qRT‐PCR analysis (A), Western blotting (B) and IHC staining (C). Downregulated Tnfα and IL1α expression in the damaged Ctgf^k/k livers were examined by qRT‐PCR analysis (D) and ELISA assays (E). (F) IHC staining showed that Ctgf deficiency reduced recruitment of CD11b⁺ macrophages and number of Ki67⁺ proliferating hepatocytes. Scale bar: 10 μm. Quantification was calculated from 10 random fields at 200× magnification based on staining of three different livers per group. Data are means ± SD (n = 3 per group). *P < .05 and **P < .005 (Student's t‐test)

Figure 2

Ctgf upregulation is associated with loss of Hnf4α as well as activation of Yap and Smad3 after EtOH/CCl₄‐induced liver injury. A, Western blotting determined expression patterns of tested genes in mouse livers after EtOH‐fed/CCl₄ co‐treatment. B, IHC labeled Hnf4α, Yap, Ctgf and GS on EtOH‐fed livers that received oil or CCl₄ administration. C and D, Dual staining showed periportal co‐localization of Ctgf and Hnf4α protein after liver injury. Arrows indicate the same periportal areas in each set of images. Scale bar: 100 μm

Figure 3

Ctgf upregulation is associated with transient decline of Hnf4α during early stage of liver regeneration following PH. Ctgf and key regulators for transcriptional reprogramming were examined by qRT‐PCR analysis (A) and Western blotting (B and C) in PH‐treated livers. Results in (A) were means ± SEM (n = 5 per group). *P < .05 and **P < .005 relative to tested genes of control group (0 hr). (D) IHC labeled Hnf4α, Yap, Ctgf, and the pericentral hepatocyte marker GS on PH treated livers. (E) Two sets of images are low and high magnifications of consecutive sections showing Ctgf in Hnf4α⁺ periportal hepatocytes. (F) The immunofluorescent staining confirmed Ctgf localization in Hnf4α⁺ periportal areas. Image were taken in the same areas for Ctgf (red) and Hnf4α (green). DAPI was stained for nucleus. Scale bar: 100 μm. hr, hour; PT, portal tract; CV, central vein

Figure 4

Hnf4α knockdown enhances Ctgf upregulation and liver fibrogenesis after ethanol/CCl₄ treatment. A and B, Out of four Hnf4α siRNAs (siHnf4α), #3 in piLentivirus vector had the highest efficiency to inhibit Hnf4α mRNA (A) and protein (B) in Hep1‐6 cells. Values in (A) were average ± SD from triplicated experiments. *P < .05 and **P < .005 relative to scramble controls. C‐E, Hnf4α knockdown in ethanol/CCl₄ treated livers carrying piLentivirus‐siHnf4α#3 caused increased expression of Ctgf, αSMA, and Collagen type I genes as determined by RT‐PCR (C), Western blotting (D), and IHC (E). Stained areas were quantified based on image analysis of at least 10 random fields (200× magnification). Data were expressed as means ± SEM (n = 3 animals per group). *P < .05 (Student's t test)

Figure 5

Hnf4α binds to CTGF promoter and exhibits antagonistic effects on Yap and Tgf‐β/Smad3 activities in vitro. A, A diagram shows cis‐elements in wild‐type human CTGF promoter (WT‐CTGFp) and mutant CTGFp. B, Myc‐tagged regulators were expressed in HEK293 cells and their binding to WT‐CTGFp was verified in ChIP assays. Fold enrichment data were expressed as means ± SEM from triplicated experiments. **P < .005 (Student's t test). C, Hnf4α activated WT‐CTGFp but not mutant CTGFp in HEK293 cells. D, Gel shift assays detected specific complexes of Hnf4α:Myc with a biotinylated WT probe, which could form super‐shifted bands with Hnf4α antibody (Ab) or combined treatment with Myc Ab. Excessive cold WT or labeled mutant probes competed with or disrupted the probe DNA/protein complex formation. E and F, Downregulation of HNF4α enhanced WT‐CTGFp activities in HepG2 cells, whereas HEK293 cells that overexpressed Hnf4α gene exhibited decrease in WT‐CTGFp activities. Data in (C, E and F) were expressed as means ± SD from triplicate tests. *P < .05 (Student’s t test)

Figure 6

A model about Ctgf regulation by Hnf4α, Yap, and Tgf‐β/Smad3 signaling during liver regeneration. Hepatocytes exit quiescence after injury and reset transcriptional programs regulated by Hnf4α and Yap for regeneration. Failure to regenerate causes scarring that overproduces profibrotic factors such as Ctgf and Tgf‐β. We propose that Ctgf production is a result of transient reprogramming in regenerating livers characterized by Hnf4α decline in conjunction with activation of Yap and Tgf‐β/Smad3 signaling. Hnf4α antagonism of Yap and Tgf‐β/Smad3 activities can downregulate Ctgf after the completion of liver regeneration. Otherwise, profibrotic signals are sustained leading to overproduction of Ctgf protein that may potentiate Tgf‐β actions and promote hepatic stellate cell activation during liver fibrosis