CCN1 induces hepatic ductular reaction through integrin αvβ5–mediated activation of NF-κB
Ki-Hyun Kim, … , Gianfranco Alpini, Lester F. Lau
Ki-Hyun Kim, … , Gianfranco Alpini, Lester F. Lau
Published March 30, 2015
Citation Information: J Clin Invest. 2015;125(5):1886-1900. https://doi.org/10.1172/JCI79327.
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Research Article Hepatology

CCN1 induces hepatic ductular reaction through integrin αvβ5–mediated activation of NF-κB

Abstract

Liver cholestatic diseases, which stem from diverse etiologies, result in liver toxicity and fibrosis and may progress to cirrhosis and liver failure. We show that CCN1 (also known as CYR61), a matricellular protein that dampens and resolves liver fibrosis, also mediates cholangiocyte proliferation and ductular reaction, which are repair responses to cholestatic injury. In cholangiocytes, CCN1 activated NF-κB through integrin αvβ5/αvβ3, leading to Jag1 expression, JAG1/NOTCH signaling, and cholangiocyte proliferation. CCN1 also induced Jag1 expression in hepatic stellate cells, whereupon they interacted with hepatic progenitor cells to promote their differentiation into cholangiocytes. Administration of CCN1 protein or soluble JAG1 induced cholangiocyte proliferation in mice, which was blocked by inhibitors of NF-κB or NOTCH signaling. Knock-in mice expressing a CCN1 mutant that is unable to bind αvβ5/αvβ3 were impaired in ductular reaction, leading to massive hepatic necrosis and mortality after bile duct ligation (BDL), whereas treatment of these mice with soluble JAG1 rescued ductular reaction and reduced hepatic necrosis and mortality. Blockade of integrin αvβ5/αvβ3, NF-κB, or NOTCH signaling in WT mice also resulted in defective ductular reaction after BDL. These findings demonstrate that CCN1 induces cholangiocyte proliferation and ductular reaction and identify CCN1/αvβ5/NF-κB/JAG1 as a critical axis for biliary injury repair.

Authors

Ki-Hyun Kim, Chih-Chiun Chen, Gianfranco Alpini, Lester F. Lau

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Figure 6

CCN1 promotes cholangiocyte proliferation through JAG1/NOTCH1 pathway.

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CCN1 promotes cholangiocyte proliferation through JAG1/NOTCH1 pathway. (...
(A) Protein lysates of LMCCs transfected with siRNAs targeting Ccn1 or Jag1 were analyzed for NICD of NOTCH1 and NOTCH2 by immunoblotting. (B) Proliferation of cholangiocytes transfected with siNotch1 was assessed by BrdU incorporation. **P < 0.01, Student’s t test. (C) NOTCH 1 NICD was detected by immunoblotting in cells treated with siCcn1, siJag1, or a nontargeting control and incubated with or without soluble JAG1 (2 μg/ml), CCN1 (4 μg/ml), or BSA for 2 days. (D) Proliferation of cells treated with siCcn1 or control siRNA and incubated with or without soluble JAG1 (2 μg/ml) was evaluated by cell numbers and BrdU incorporation. *P < 0.04, **P < 0.01, Student’s t test. (E) NOTCH1 NICD was detected by immunoblotting in cholangiocytes incubated with DAPT (10 μM) or vehicle (DMSO) for 24 hours. (F and G) Proliferation of cells was assessed by counting of cell numbers (F) and immunohistochemical detection of Ki67-positive cells (G). Percentages of Ki67-positive cells relative to total number of cells were counted in 5 randomly chosen high-power fields. **P < 0.01, Student’s t test. (H) Freshly isolated primary cholangiocytes (98.5% ± 0.4% IgG2a-positive) were cultured with BSA or CCN1 (4 μg/ml) for 2 days, and proliferation was assessed by cell numbers. **P < 0.01, Student’s t test. (I) BrdU incorporation was quantified in freshly isolated primary cholangiocytes treated with BSA (4 μg/ml), CCN1 (4 μg/ml), soluble JAG1 (2 μg/ml), DAPT (10 μM), cilengitide (1 μM), NBD (25 μM), or control peptide (25 μM). Where indicated, CCN1 (4 μg/ml) was added with other inhibitors. *P < 0.04, **P < 0.01, Student’s t test. All data are expressed as mean ± SD of triplicate determinations.