Pleiotrophin regulates the ductular reaction by controlling the migration of cells in liver progenitor niches

Abstract

Objective: The ductular reaction (DR) involves mobilisation of reactive-appearing duct-like cells (RDC) along canals of Hering, and myofibroblastic (MF) differentiation of hepatic stellate cells (HSC) in the space of Disse. Perivascular cells in stem cell niches produce pleiotrophin (PTN) to inactivate the PTN receptor, protein tyrosine phosphatase receptor zeta-1 (PTPRZ1), thereby augmenting phosphoprotein-dependent signalling. We hypothesised that the DR is regulated by PTN/PTPRZ1 signalling.

Design: PTN-GFP, PTN-knockout (KO), PTPRZ1-KO, and wild type (WT) mice were examined before and after bile duct ligation (BDL) for PTN, PTPRZ1 and the DR. RDC and HSC from WT, PTN-KO, and PTPRZ1-KO mice were also treated with PTN to determine effects on downstream signaling phosphoproteins, gene expression, growth, and migration. Liver biopsies from patients with DRs were also interrogated.

Results: Although quiescent HSC and RDC lines expressed PTN and PTPRZ1 mRNAs, neither PTN nor PTPRZ1 protein was demonstrated in healthy liver. BDL induced PTN in MF-HSC and increased PTPRZ1 in MF-HSC and RDC. In WT mice, BDL triggered a DR characterised by periportal accumulation of collagen, RDC and MF-HSC. All aspects of this DR were increased in PTN-KO mice and suppressed in PTPRZ1-KO mice. In vitro studies revealed PTN-dependent accumulation of phosphoproteins that control cell-cell adhesion and migration, with resultant inhibition of cell migration. PTPRZ1-positive cells were prominent in the DRs of patients with ductal plate defects and adult cholestatic diseases.

Conclusions: PTN, and its receptor, PTPRZ1, regulate the DR to liver injury by controlling the migration of resident cells in adult liver progenitor niches.

Keywords: CELL MIGRATION; CHOLESTATIC LIVER DISEASES; FIBROSIS; IMMUNOHISTOCHEMISTRY; STEM CELLS.

Figure 1

Adult liver stromal cells produce pleiotrophin (PTN). (A) PTN gene expression was assessed in primary hepatocytes (Hep), hepatic stellate cells (HSC), and liver sinusoidal endothelial cells (LSEC), as well as the ductular progenitor cell line, 603B by qRT-PCR. Results are normalised to S9 expression and expressed as fold-change over Hep (n=3–5 independent isolations). (B) Representative staining for PTN-driven GFP expression in PTN-GFP mice after sham operation or bile duct ligation (BDL) (magnification, ×10) and (C) Quantification of GFP(+) cells from 20 random fields. Results are expressed as fold-change over sham-operated controls. (D) Changes in PTN expression in during culture activation of primary HSC were analysed by qRT-PCR. Results are normalised to S9 and expressed as fold-change relative to day 0 (n=3–6 independent isolations). (E) PTN expression in whole liver from sham-operated or BDL mice was analysed by qRT-PCR. Results are normalised to S9 and expressed as fold-change over sham-operated control. (F) Double-staining demonstrating co-localisation of GFP (green) with either desmin (brown) or elastin (brown) in PTN-GFP mice after BDL (magnification: ×40; inset ×100). Double positive cells are indicated by black arrows. *p<0.05; **p<0.01; ***p<0.001.

Figure 2

Pleiotrophin (PTN) deficiency enhances the ductular reaction during cholestatic injury. (A) Representative H&E staining of liver sections from wild-type and PTN deficient mice (n=7–9 mice per group; magnification ×4). Differences in portal tract size were evaluated by morphometry and graphed as mean±SEM relative to sham-operated control. Serum bilirubin levels in sham-operated or bile duct ligation (BDL) wild type (WT) and PTN-knockout (KO) mice. Values are expressed as mean±SEM relative to WT sham-operated controls (mg/dL). (B) Representative immunohistochemistry (magnification, ×20), corresponding quantification of immunostained images and qRT-PCR analysis of Krt19 mRNA in whole liver RNA is shown in panels to the right. Results are expressed relative to sham-operated WT mice. (C) Representative sections of Sirius red stained liver (magnification, ×4) with Col1α1 qRT-PCR analysis and quantification of hepatic hydroxyproline in sham-operated or BDL WT and PTN-KO mice. Results are expressed relative to WT sham-operated mice. *p<0.05; ** p<0.01; ***p<0.001.

Figure 3

Pleiotrophin (PTN) inhibits liver cell migration and epithelial-to-mesenchymal transition (EMT). Cell migration was assessed by wound-healing assay in the murine ductular progenitor line 603B (A), rat hepatic stellate cells (HSC) line 8B (B), and primary HSC isolated from either wild type (WT) or PTN- knockout (KO) mice (C) in the presence of either PBS (untreated, UT) or PTN (PTN; 100 ng/mL) then quantified after 48 h (magnification, ×10). Leading edge of the repairing cell monolayers is indicated by yellow bars. Results are expressed as mean±SEM of three independent measurements. (D) SNAIL gene expression by qRT-PCR in 603B and 8B treated with either PBS (UT) or PTN (100 ng/mL) for 48 h. Results are normalised to S9 and expressed as fold-change relative to UT control for each cell type. (E) Changes in expression of either mesenchymal or epithelial (boxed) genes were assessed by qRT-PCR analysis in myofibroblastic-hepatic stellate cells (MF-HSC) treated with either PBS (UT) or PTN (100 ng/mL) for 48 h. Results are normalised to S9 and expressed as fold-change relative to UT control. *p<0.05; **p<0.01.

Figure 4

Protein tyrosine phosphatase receptor zeta-1 (PTPRZ1) signalling is required for pleiotrophin (PTN)-mediated inhibition of cell migration. (A) PTPRZ1 gene expression in different types of liver cells, and whole liver from either sham-operated or bile duct ligation (BDL) mice, was analysed by qRT-PCR. Results are normalised to S9 and expressed as fold-change relative to primary hepatocytes or sham-operated controls respectively. (B) Representative liver sections for PTPRZ1 immuno-staining (brown) from either sham-operated or BDL mice (magnification, ×10). Double immunostaining reveals co-localisation (red arrows) of PTPRZ1 (brown) and desmin (green) with a double-stained cell highlighted (inset, magnification ×100). (C) Changes in total tyrosine phosphorylation over time in 603B treated with PTN (100 ng/mL) were visualised by Western analysis using anti-PY20 antibody. Phosphoprotein expression (arrow) at decreased exposure is highlighted in boxed region. Tubulin was used as protein loading control. (D) Wound-healing assay in primary hepatic stellate cells ( isolated from either WT or PTPRZ1- knockout (KO) mice and treated either PBS (UT) or PTN (100 ng/mL). Leading edge of the repairing cell monolayers is indicated by yellow bars. Cellular migration was quantified and represented graphically with results expressed as mean±SEM of three independent measurements. *p<0.05; ***p<0.01.

Figure 5

Protein tyrosine phosphatase receptor zeta-1 (PTPRZ1) deficiency inhibits the ductular reaction during cholestatic injury. (A) Representative sections of H&E stained livers (magnification, ×10) from wild type (WT) or PTPRZ1-deficient mice after sham-operation or bile duct ligation (BDL) are shown. Portal tract diameter was evaluated by morphometry with results expressed as mean±SEM relative to WT sham-operated mice. (B) Representative immunohistochemistry (magnification, ×20), corresponding quantification of immunostained images and qRT-PCR analysis of Krt19 mRNA in whole liver RNA is shown in panels to the right. Results are expressed relative to sham-operated WT mice. (C) Representative Sirius red-stained sections in WT or PTPRZ1-deficient mice after sham-operation or BDL (magnification, ×20) with associated morphometric analysis and quantification of hepatic hydroxyproline content to assess fibrosis. Results are displayed as mean±SEM relative to sham-operated WT mice. *p<0.05 **p<0.01.

Figure 6

Protein tyrosine phosphatase receptor zeta-1 (PTPRZ1)(+) cells accumulate during the ductular reaction in humans. (A) PTPRZ1 is not detectable in normal adult human liver (NHL). Patients with ductal plate abnormalities, for example, adult polycystic liver disease (B) and von Meyenburg complexes (C), demonstrated a marked increase in PTPRZ1 expression, particularly in ductular cells. (D, E) PTPRZ1 (brown) localises in (D) Krt7(+) ductular cells (green) and (E) Desmin(+) stromal cells (green) in representative liver sections from patients with von Meyenburg complexes (magnification ×10; inset ×100). PTPRZ1-positive ductular cells were also evident in liver sections from representative patients with primary biliary cirrhosis (F; magnification ×100), primary sclerosing cholangitis (G; magnification ×100) with ductular reactions. Arrows indicate PTPRZ1 positive cells in atypical ductular structures. (H) Quantification of PTPRZ1-positive ductular cells in liver sections from PBC patients (n=2) and PSC patients (n=4) relative to NHL (n=3 individuals).