Galectin-3 modulates phagocytosis-induced stellate cell activation and liver fibrosis in vivo

Abstract

Hepatic stellate cells (HSC), the key fibrogenic cells of the liver, transdifferentiate into myofibroblasts upon phagocytosis of apoptotic hepatocytes. Galectin-3, a β-galactoside-binding lectin, is a regulator of the phagocytic process. In this study, our aim was to study the mechanism by which extracellular galectin-3 modulates HSC phagocytosis and activation. The role of galectin-3 in engulfment was evaluated by phagocytosis and integrin binding assays in primary HSC. Galectin-3 expression was studied by real-time PCR and enzyme-linked immunosorbent assay, and in vivo studies were done in wild-type and galectin-3(-/-) mice. We found that HSC from galectin-3(-/-) mice displayed decreased phagocytic activity, expression of transforming growth factor-β1, and procollagen α1(I). Recombinant galectin-3 reversed this defect, suggesting that extracellular galectin-3 is required for HSC activation. Galectin-3 facilitated the α(v)β(3) heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin α(v)β(3) resulted in decreased phagocytosis. Galectin-3 expression and release were induced in active HSC engulfing apoptotic cells, and this was mediated by the nuclear factor-κB signaling. The upregulation of galectin-3 in active HSC was further confirmed in vivo in bile duct-ligated (BDL) rats. Galectin-3(-/-) mice displayed significantly decreased fibrosis, with reduced expression of α-smooth muscle actin and procollagen α1(I) following BDL. In summary, extracellular galectin-3 plays a key role in liver fibrosis by mediating HSC phagocytosis, activation, and subsequent autocrine and paracrine signaling by a feedforward mechanism.

Fig. 1.

Galectin-3 is required for the phagocytosis of apoptotic bodies (AB) by hepatic stellate cells (HSC) by facilitating the integrin α_vβ₃-mediated tethering. Wild-type (wt) and galectin-3^−/− HSC were incubated with AB in serum-free conditions in the presence or absence of recombinant galectin-3 (rGal3) (1 μM) for 16 h (A). Lactose (Lac, 50 mM) was applied before the AB and rGal3. Phagocytosis of AB was significantly decreased in galectin-3^−/− HSC. This was reversed by incubating the cells with rGal3. The competitive inhibitor of galectin binding, lactose, reduced the phagocytic rate in rGal3-treated cells, suggesting that extracellular galectin-3 is necessary for phagocytosis to occur. Data are means ± SE; n = 5. *P < 0.05, **P < 0.01, and ##P < 0.0001. Primary HSC were treated with anti-integrin α_v and β₃ antibodies (10 μg/ml) or the same amount of IgG isotype, followed by AB for 16 h (B). The HSC phagocytic activity was significantly suppressed by the integrin antibodies. Data are means ± SE, n = 4, *P < 0.01. Binding assay was performed on primary HSC transfected with either pBJ1integrin β₃ (Intβ3^wt) or a binding-deficient mutant (Intβ3^D119A) to study galectin-3 binding to integrins (C). Galectin-3 binding was diminished in Intβ3^D119A -transfected cells, indicating that this pair is important in the galectin-3-mediated phagocytic activity; n = 3, **P < 0.05. Immunocytochemistry and confocal microscopy were performed to analyze the galectin-3 (red) and integrin β₃ (green) signals in the above experiment (D). Colocalization of the signals was seen in the Intβ3^wt-transfected HSC (a, arrows), whereas in the Intβ3^D119A -transfected cells no colocalization was seen (b). In nontransfected (NT) cells, no significant colocalization was seen. (c). Bar = 25 μM.

Fig. 2.

Galectin-3 is necessary for phagocytosis-induced HSC activation. The wt and galectin-3^−/− HSC were treated with AB in serum-free medium. In wt HSC, the expression of procollagen α1(I) and transforming growth factor (TGF)-β1 has increased significantly, whereas incubation with rGal3 only did not induce activation. There was no increase in the profibrogenic transcripts in galectin-3^−/− cells. However, after the cells were incubated with rGal3 and AB, the expression of TGF-β and procollagen α1(I) was induced. βACT, β-actin. Data are means ± SE, n = 4. *P < 0.05 and #P < 0.001.

Fig. 3.

Engulfment of AB results in galectin-3 induction via nuclear factor (NF)-κB. Primary HSC were treated with AB or AB plus caffeic acid phenethyl ester (CAPE, A) or transfected by control vector or dominant-negative IκB expression vector (DN-IκB, B). Real-time PCR showed a significant induction of galectin-3 mRNA by phagocytosis, and this was abolished by CAPE (A, #P < 0.005) or DN-IκB (B, mean ± SE, n = 3, **P < 0.01 and #P < 0.005). Electrophoretic mobility shift assay (EMSA) studies confirmed that phagocytosis induced NF-κB activation, which was significantly blunted by CAPE (C and D, densitometry data, control denotes no AB exposure). Arbp, acidic ribosomal phosphoprotein P0. Data are means ± SE, n = 3. **P < 0.01.

Fig. 4.

Phagocytosis of AB promotes galectin-3 secretion by HSC (A) and Kupffer cells (B). Primary rat HSC or Kupffer cells were incubated with AB in serum-free medium for 48 h. The medium was collected, and enzyme-linked immunosorbent assay (ELISA) was performed to quantify the galectin-3 released by the cells. The galectin-3 level in the medium was significantly higher in AB-treated HSC (mean ± SE, n = 3, ##P < 0.0001) and Kupffer cells (*P < 0.05) compared with the nontreated controls.

Fig. 5.

Galectin-3 is upregulated in livers from bile duct-ligated (BDL) animals. HSC were isolated from BDL and sham-operated rats (A). The expression of galectin-3 was significantly increased in HSC from BDL animals (*P < 0.05). Immunofluorescence and confocal microscopy studies using anti-galectin-3 (B, a and d) and anti-α-smooth muscle actin (α-SMA) antibodies (B, b and e) indicated colocalization of the α-SMA and galectin-3 signals (B, c and f, overlay images).

Fig. 6.

Liver fibrosis is decreased in galectin-3^−/− mice. Picrosirius staining demonstrates decreased fibrosis following BDL in galectin-3^−/− mice (A). The areas of fibrosis were significantly lower in the galectin-3^−/− mice following BDL (mean ± SE, 5 different areas counted in 5 mice, P < 0.05, B). The collagen level in the liver was assessed by hydroxyproline assay (C). In wt mice, increased levels of hydroxyproline were seen, whereas in the galectin-3^−/− mice this was significantly decreased (P < 0.05). The expression of procollagen α1(I) and α-SMA was examined by real-time PCR (D). In wt mice following BDL, the expression of both transcripts has significantly increased, whereas in the galectin-3^−/− mice the increase was significantly blunted (mean ± SE, n = 3, *P < 0.05 and **P < 0.01). The expression of TGF-β1 and tissue inhibitor of matrix metalloproteinase-1 was also significantly reduced in the galectin-3 ^−/− mice. Data are means ± SE, N = 3 (*P < 0.05, E). The inflammatory markers tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, interleukin (IL)-1β, and IL-6 have decreased in galectin-3^−/− BDL mice; however, only the reduction of MCP-1 was significant. Data are means ± SE, n = 3 (*P < 0.05, F).

Fig. 7.

The role of galectin-3 in HSC activation. Galectin-3 induces integrin-mediated uptake of AB, NF-κB activation, and an increase in galectin-3 production and secretion. Thus galectin-3 may regulate fibrosis by a feedforward mechanism.