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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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 5

CCN1 promotes cholangiocyte proliferation through NF-κB–mediated Jag1 expression.

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CCN1 promotes cholangiocyte proliferation through NF-κB–mediated Jag1 ex...
(A) Expression of indicated genes was assessed by qRT-PCR in LMCCs transfected with siCcn1 or control siRNA. **P < 0.004, Student’s t test. (B) JAG1 protein was detected by immunoblotting in lysates of LMCCs incubated with WT CCN1, CCN1-DM, CCN1-D125A, or BSA (4 μg/ml each) for 2 days, and Jag1 mRNA was quantified by qRT-PCR. *P < 0.02, **P < 0.004, Student’s t test. (C) BrdU incorporation and total cell numbers were assessed in cholangiocytes 2 days after transfection with siJag1 or control siRNA. Knockdown of JAG1 was confirmed by immunoblotting. *P < 0.02, **P < 0.004, Student’s t test. (D) Jag1 mRNA was measured by qRT-PCR in cells treated with siRNAs targeting indicated integrins. *P < 0.02, **P < 0.004, Student’s t test. (E) Phosphorylation of NF-κB p65 was detected by immunoblotting in cholangiocytes incubated with CCN1 for 3 hours with or without pretreatment (30 minutes) with BAY11-7082 (5 μM). Levels of phosphorylated p65 were normalized to total p65. Pi, phosphorylated. (F) LMCCs were treated with siRNA targeting indicated integrins before CCN1 treatment and assayed for phosphorylation of NF-κB p65 by immunoblotting. (G) Jag1 mRNA was measured by qRT-PCR in LMCCs treated with CCN1 for 3 hours, with or without preincubation with BAY11-7082, or in cells transfected with siRNA targeting NF-κB p65. Knockdown of p65 was confirmed by immunoblotting. **P < 0.004, Student’s t test. (H) BrdU incorporation was assessed in LMCCs treated with BSA (4 μg/ml), CCN1 (4 μg/ml), NBD (25 μM), or control peptide (25 μM). *P < 0.02, **P < 0.004, Student’s t test. (I) Jag1 mRNA expression was analyzed with cells treated as in H. *P < 0.02, **P < 0.004, Student’s t test. All data are expressed as mean ± SD of triplicate determinations.