Hepatic Prominin-1 expression is associated with biliary fibrosis

Abstract

Background: Intrahepatic biliary fibrosis, as seen with cholestatic liver injuries such as biliary atresia, is mechanistically distinct from fibrosis caused by hepatocyte toxicity. We previously demonstrated the expansion of cells expressing the stem/progenitor cell marker Prominin-1, within regions of developing fibrosis in biliary atresia. Thus, we hypothesized that Prominin-1 expression is biliary fibrosis-specific.

Methods: Gene expression of Prominin-1 was analyzed in adult mice undergoing either cholestatic bile duct ligation or hepatotoxic carbon tetrachloride administration by quantitative polymerase chair reaction. Lineage tracing of Prominin-1-expressing cells and Collagen-1α-expressing cells was performed after bile duct ligation in Prominin-1^{cre-ert2-lacz};Gfp^lsl and Collagen-1α^Gfp transgenic mice, respectively.

Results: Prominin-1 expression increased significantly after bile duct ligation compared with sham (6.6 ± 0.9-fold change at 2 weeks, P < .05) but not with carbon tetrachloride (-0.7 ± 0.5-fold change, not significant). Upregulation of Prominin-1 was observed histologically throughout the liver as early as 5 days after bile duct ligation in Prominin-1^{cre-ert2-lacz} mice by LacZ staining in nonhepatocyte cells. Lineage tracing of Prominin-1-expressing cells labeled prior to bile duct ligation in Prominin-1^{cre-ert2-lacz};Gfp^lsl mice, demonstrated increasing colocalization of GREEN FLUORESCENT PROTEIN with biliary marker CYTOKERATIN-19 within ductular reactions up to 5 weeks after bile duct ligation consistent with biliary transdifferentiation. In contrast, rare colocalization of GREEN FLUORESCENT PROTEIN with mesenchymal marker α-SMOOTH MUSCLE ACTIN in Prominin-1^{cre-ert2-lacz};Gfp^lsl mice and some colocalization of GREEN FLUORESCENT PROTEIN with PROMININ-1 in Collagen-1α^Gfp mice, indicate minimal contribution of Prominin-1 progenitor cells to the pool of collagen-producing myofibroblasts.

Conclusion: During biliary fibrosis Prominin-1-expressing progenitor cells transdifferentiate into cells within ductular reactions. This transdifferentiation may promote fibrosis.

Figure 1

(A) Collagen deposition demonstrated by sirius red staining with BDL and CCl₄ liver injuries (counterstained with eosin only). (B) Gene expression early and later after BDL and CCL₄. Scale bar = 50 µm. * denote p < 0.05.

Figure 2

LacZ staining (counterstained with eosin only) in Prom1^{cre-ert2-lacz} mice 5 days after undergoing (A) sham operation or (B) BDL. Note the greater blue staining, which mark sites of Prom1 expression, after BDL (B, and inset B’) compared to sham. Scale bar = 50 µm.

Figure 3

Lineage tracing of Prom1-expressing cells labeled with Green Fluorescent Protein, via tamoxifen injection 3 weeks prior to BDL, 3 and 5 weeks after BDL in Prom1^{cre-ert2-lacz};GFP^lsl mice. CK19 (red). Arrowheads mark GFP^posCK19^pos (yellow) cells. Scale bar = 25 µm.

Figure 4

Lineage tracing of Prom1-expressing cells labeled with Green Fluorescent Protein, via tamoxifen injection 3 weeks prior to BDL, 3 and 5 weeks after BDL in Prom1^{cre-ert2-lacz};GFP^lsl mice. (A) NTPdase II (red), (B) αSMA (red). Arrowheads mark yellow co-positive cells. Note the presence of red single positive cells. Scale bar = 25 µm.

Figure 5

Immunofluorescence co-localization of GFP, CK19, and PROM1 in Col1α^Gfp mice 3 weeks after BDL. Arrowheads mark yellow cells co-positive for PROM1 and GFP (Collagen). Scale bar = 75 µm.