Lipocalin‐2 Stimulates Proliferation and a Profibrotic Cholangiocyte Phenotype during Cholestasis

Lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL), is a secreted glycoprotein that has been shown to regulate cell proliferation, innate immunity, metabolism, and tumor metastasis in several cell types. LCN2 expression is elevated in hepatic tissues in experimental liver injury models and patients with alcoholic hepatitis. LCN2 has been shown to stimulate proliferation and metastasis of cholangiocarcinoma. However, the role of cholangiocyte LCN2 and LCN2 receptors (NGALR/24p3-R) during cholestasis and primary sclerosing cholangitis (PSC) has not been evaluated. Therefore, we aimed to assess the role of LCN2 and its receptor, 24p3-R, in acute and chronic cholestatic models.LCN2 and 24p3-R were evaluated in liver sections, total liver, and isolated cholangiocytes from wild-type (WT; C57) normal and BDL (7 days) mice and WT (FVB/NJ) and Mdr2-/- mice (model of primary sclerosing cholangitis, PSC) by immunohistochemistry and qPCR, respectively. LCN2 levels were evaluated by ELISA in cholangiocyte supernatants (6 hrs) collected from animal models and serum from BDL and Mdr2-/- and WT controls and late-stage PSC patients and healthy human controls. Serum bile acid (BA) levels were measured using a commercial BA assay kit in BDL, Mdr2-/- , and control mice. In vitro, both human (H69) and mouse pooled (MPC) cholangiocytes were treated with either 50 ng/ml LCN2 or 100 mM taurocholic acid (TCA) for 24 hrs after which the expression of proliferation, fibrosis, LCN2, and 24p3-R genes was determined via qPCR.There were significant increases in LCN2 and 24p3-R immunostaining in BDL mice compared to controls. By qPCR significant increases were observed for both LCN2 and 24p3-R in total liver and isolated cholangiocytes from BDL and Mdr2-/- mice compared to WT. Increased levels of LCN2 were found in serum from PSC patients and serum and supernatants from isolated cholangiocytes from BDL and Mdr2-/- mice compared to control. In vitro, MPC cells treated with LCN2 had increases in fibrotic marker Col1a1, proliferation marker PCNA, and inflammatory markers IL-1b and IL-6. Also, significant increases were seen in fibrotic markers (aSMA, FN-1, and TGF-b1) and LCN2 receptor 24p3-R in H69 cells treated with LCN2 compared to untreated cells. As expected, increased serum BA levels were observed in both BDL and Mdr2-/- mice compared to controls. H69 cells treated with TCA also had significant increases in FN-1, TGF-b1, and the LCN2 receptor 24p3-R, indicating that BA may regulate 24p3-R expression.LCN2 and 24p3-R expression is elevated in cholestatic models and stimulates a profibrotic cholangiocyte phenotype. These findings suggest that LCN2 may play a crucial role in the pathogenesis of cholestatic liver injury.