TWEAK induces liver progenitor cell proliferation

Abstract

Progenitor ("oval") cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors.

Figure 1

TWEAK transgene is expressed primarily in the livers of 2 independent transgenic lines. (A) AAT promoter directs expression of TWEAK transgene to the liver and at low levels to the kidney. Quantitative PCR using transgene-specific primers was done with cDNA pooled from 3 adult transgenic animals. Data was normalized to the levels of the GAPDH gene. (B) Time course of TWEAK and (C) Fn14 mRNA expression in TWEAK-Tg vs. Ntg mice was measured by quantitative PCR. Each time point represents the combined RNA from 3 different animals. (D) TWEAK protein expression in 2 independent transgenic founder lines as compared with that in a control littermate. TWEAK protein was detected by staining frozen liver sections with a hamster anti-TWEAK mAb.

Figure 2

TWEAK overexpression promotes oval cell hyperplasia. (A–D) H&E staining of 8- to 10-week-old (A and B) Ntg or (C and D) TWEAK-Tg livers. Magnification, ×320 (A and C); ×500 (B and D). (E–G) A6 (red) and CD34 (green) expression in Ntg livers. (E) A6 expression on bile duct epithelium. (F) CD34 expression on bile duct epithelium. (G) Coexpression of A6 and CD34. (H–J) TWEAK-Tg livers showing increased expression of (H) A6 and (I) CD34 in oval cells and bile ducts and (J) increased coexpression of A6 and CD34. Original magnification, ×200 (E–J).

Figure 3

TWEAK overexpression in transgenic mice promotes early proliferation and increased turnover of oval cells with no detectable damage in the parenchyma. (A and B) Proliferation, as measured by PCNA staining of TWEAK-Tg and Ntg livers at the indicated ages. (A) PCNA-positive periportal oval cells and BECs show increased proliferation in TWEAK-Tg vs. Ntg livers. (B) PCNA-positive hepatocytes. (C and D) Apoptosis detected by TUNEL staining of TWEAK-Tg and Ntg livers at the indicated ages. (C) Apoptotic periportal oval cells and BECs show increased proliferation in TWEAK-Tg vs. Ntg livers. (D) Apoptotic hepatocytes. PCNA- or TUNEL-positive cells were counted in 20 different fields (magnification, ×200). *P < 0.05 by 2-tailed Student’s t test.

Figure 4

Increased Fn14 mRNA expression in the hyperplastic regions of adult TWEAK-Tg relative to Ntg livers. Fn14 mRNA expression was detected by in situ hybridization using a murine Fn14 riboprobe template. (A–D) Ntg liver in (A) E18.5 embryo, (B) newborn, and (C) 8-week-old mouse. Darkfield (top) and brightfield (bottom) views are shown. Magnification, ×250. (D) Ntg 8-week-old liver. Magnification, ×640. (E–H) TWEAK-Tg liver in (E) E18.5 embryo, (F) newborn, and (G) 8-week-old mouse. Darkfield (top) and brightfield (bottom) views are shown. Magnification, ×250. (H) TWEAK-Tg 8-week-old liver. Magnification, ×640.

Figure 5

Ad-TWEAK administration in adult mice induces oval cell and biliary duct hyperplasia in the absence of significant liver damage. Mice were dosed with either Ad-TWEAK or Ad-control, as described in Methods, and livers were obtained 3 or 7 days later for TUNEL staining or 7 days later for H&E, A6, and CD34 staining. Serial frozen sections were stained with A6 or CD34 in order to assess marker coexpression. (A–C) Ad-TWEAK treated mice. (A) H&E staining showing a marked oval cell and ductal hyperplasia. (B) Extensive A6 and (C) CD34 coexpression on oval cells and duct-like cells and on BECs. (D–F) Ad-control–treated mice. (D) H&E staining showing an inflammatory response induced by Ad-control vector. (E) A6 and (F) CD34 coexpressed on bile ducts in the portal tract region and CD34 additionally expressed on vessels. Magnification, ×200. (G) Serum ALT measured in IU/l 3 days after adenoviral administration, showing no signs of liver damage. (H) TUNEL staining at days 3 and 7 after Ad-control or Ad-TWEAK administration. Magnification, ×400.

Figure 6

Fn14 and TWEAK are upregulated in the reactive portal area (including oval cells and bile ducts) in the DDC-fed mice but not in control mice fed regular diet with no DDC. Fn14 expression was detected by in situ hybridization. Dark- and brightfield pictures of the same field are displayed in (A) a control mouse on regular diet and (B) a DDC-fed mice on day 7. TWEAK expression in (C) control mouse on regular diet and (D) DDC-fed mouse on day 7. Magnification, ×250.

Figure 7

TWEAK pathway blockade in DDC-fed mice inhibits the proliferation of A6-expressing oval cells. DDC-fed mice were dosed with either hamster anti-TWEAK mAb, hamster control mAb, or PBS and sacrificed 7 days later. Frozen liver sections were stained for A6 expression. The amount of positive staining was then quantitated using MetaMorph software. (A) Mouse fed regular diet with no DDC. (B) DDC-fed mouse. (C) Control Ig-treated DDC-fed mouse. (D) Hamster anti-TWEAK mAb–treated DDC-fed mouse. Magnification, ×200. (E) Quantitation of A6 staining using MetaMorph software. *P < 0.05 by 2-tailed Student’s t test.

Figure 8

Mitogenic effect of TWEAK on oval cells is Fn14 dependent. Fn14-KO or wild-type mice were dosed with Ad-TWEAK or Ad-control as described in Methods, and frozen livers were obtained 7 days later and stained for A6 expression. The amount of positive staining was then quantitated using MetaMorph software. Ad-TWEAK–treated (A) wild-type mice and (B) Fn14-KO mice. Ad-control–treated (C) wild-type mice and (D) Fn14-KO mice. Magnification, ×100. (E) Quantitation of A6 staining using MetaMorph software. *P < 0.02 by 2-tailed Student’s t test.

Figure 9

DDC-fed Fn14-KO mice have reduced proliferation of A6-expressing oval cells. Fn14-KO mice or age-matched control littermates were fed with the DDC diet or with regular chow. Mice were sacrificed 14 days later, and frozen liver sections were stained for A6 expression, which was then quantified using MetaMorph software. Wild-type mice fed (A) regular diet or (B) DDC-containing diet. Fn14-KO mice fed (C) regular diet or (D) DDC-containing diet. Magnification, ×200. (E) Quantitation of A6 staining using MetaMorph software. *P < 0.02 by 2-tailed Student’s t test.

Figure 10

TWEAK induces proliferation of NRC-1 cells. NRC-1 cells were cultured with a range of TWEAK concentrations in serum-free medium for 48 hours. Then TWEAK-containing medium was renewed, with the addition of BrdU, and incubated a further 24 hours. Positive cells and total cells were counted in 10 randomly chosen fields. Results are expressed as percentage of BrdU-positive cells. Graph shows the combined results of 3 independent experiments.

Figure 11

Fn14 expression in human liver. Serial frozen human liver sections were stained with either anti-Fn14 mAb or H&E. (A) Normal liver. (B) NASH cirrhosis. (C) Alcoholic cirrhosis (end stage). (D) HCV with HCC. Magnification, ×210.