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. 2018 Aug 2;13(8):e0201744.
doi: 10.1371/journal.pone.0201744. eCollection 2018.

Accumulation of Citrullinated Glial Fibrillary Acidic Protein in a Mouse Model of Bile Duct Ligation-Induced Hepatic Fibrosis

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Accumulation of Citrullinated Glial Fibrillary Acidic Protein in a Mouse Model of Bile Duct Ligation-Induced Hepatic Fibrosis

Sung-Eun Kim et al. PLoS One. .
Free PMC article

Abstract

Hepatic stellate cells (HSCs) play pivotal roles in hepatic fibrosis as they synthesize glial fibrillary acidic protein (GFAP), which is increased in activated HSCs. GFAP-expressing HSCs and myofibroblasts accumulate in and around hepatic fibrosis lesions. Peptidylarginine deiminase 2 (PAD2) is responsible for the citrullination of GFAP (cit-GFAP). However, the involvement of PAD2 and cit-GFAP in hepatic fibrosis remains unclear. To determine the expression of PAD2 and cit-GFAP in hepatic fibrosis, C57BL/6 mice underwent bile duct ligation (BDL) or a sham operation. In BDL livers, the expression of PAD2 and its enzyme activity were significantly increased compared with controls. In addition, PAD2-postitive cells were rarely observed in only the portal vein and the small bile duct in sham-operated livers, whereas an increased number of PAD2-positive cells were detected in the bile duct and Glisson's sheath in BDL livers. Interestingly, PAD2 was colocalized with α-SMA-positive cells and CK19-positive cells in BDL livers, indicating upregulated PAD2 in activated HSCs and portal fibroblasts of the livers of BDL mice. We also found that citrullinated proteins were highly accumulated in the livers of BDL mice compared with controls. Moreover, the expression level of GFAP and the amount of cit-GFAP were higher in BDL livers than in control livers. In correlation with PAD2 localization, cit-GFAP was observed in α-SMA-positive and CK19-positive cells in the livers of BDL mice. These results suggest that the increased expression and activation of PAD2 along with increased citrullinated proteins, specifically cit-GFAP, may play important roles in the pathogenesis of hepatic fibrosis.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Development of hepatic fibrosis after BDL at week 3.
(A) Liver tissues from sham-operated controls did not show any hepatic inflammation or bile duct injury by hematoxylin and eosin staining. (B) Hepatic inflammation and expansion of portal tract tracts with bile duct proliferation and periportal fibrosis was noted in the livers of BDL mice. (C) Collagen deposition was not observed in liver tissues from sham-operated controls by Sirius red staining. (D) Collagen deposition was observed around the bile duct and fibrous septa in the livers of BDL mice. (E) mRNA levels of α-SMA and Col1a1 were analyzed by qPCR. (n = 9). ***P <0.001.
Fig 2
Fig 2. Comparison of PAD2 expression levels in livers from sham-operated control and BDL mice.
(A) The expression level of PAD2 protein in the liver tissues of control and BDL mice was analyzed by western blot using monoclonal anti-PAD2 antibody. (B) Densitometric analysis of PAD2 activity demonstrated that the liver tissue of BDL mice (n = 9) showed increased PAD2 activity compared with controls (n = 9). *P <0.05. (C) Immunohistochemical staining of PAD2 in liver tissues from controls and BDL mice. In BDL liver, an increased number of PAD2-postive cells was located in the bile duct and Glisson’s sheath.
Fig 3
Fig 3. Double immunofluorescence of PAD2 and α-SMA or CK19 in livers from sham-operated control and BDL mice.
Cryosections were examined under a confocal microscope. (A) PAD2 immunoreactivity was more intense in the livers of BDL mice than in the livers of sham-operated controls. Alpha-SMA immunoreactivity was also more intense in the livers of BDL mice than in the livers of controls. PAD2 was mainly colocalized with α-SMA-positive cells in and around the bile duct area (B) CK19 immunoreactivity was more intense in the livers of BDL mice than in the livers of controls. PAD2 was also colocalized with CK19-positive cells in and around the bile duct area.
Fig 4
Fig 4. Detection of citrullinated proteins in the livers of sham-operated control and BDL mice.
(A) The expression level of citrullinated protein in the liver tissues of control and BDL mice was analyzed by western blot using F95 antibody. Note that citrullinated proteins were increased in the liver tissues of BDL mice compared with controls. (B) Immunohistochemical staining of citrullinated proteins in liver tissues from controls and BDL mice. Citrullinated proteins were more prominent in the livers of BDL mice.
Fig 5
Fig 5. Detection of GFAP and cit-GFAP in livers from sham-operated control and BDL mice.
The expression levels of GFAP and cit-GFAP in the liver tissues of control and BDL mice were analyzed by western blot using rabbit polyclonal anti-GFAP (Abcam, Cambridge UK) and citrullinated GFAP (CTGF-122R and CTGF-1221) antibodies. Note that GFAP and cit-GFAP were increased in the liver tissues of BDL mice compared with controls.
Fig 6
Fig 6
Double immunofluorescence of cit-GFAP (CTGF-122R) with GFAP (A), α-SMA (B), F4/80 (C), CK19 (D) or SE-1 (E) in the livers of sham-operated control and BDL mice. Paraffin-embedded liver sections were examined under a confocal microscope. Scale bar, 20 μm.

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Sung Eun Kim was supported by a grant of the basic science research programme through the National Research Foundation (NRF) of Korea funded by the Ministry of Science and ICT (grant number: 2017R1C1B1005996) and Myoung Kuk Jang and Sung Eun Kim were supported by a Hallym University Research Fund (HURF-2015-56).
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