Hepatic progenitor cells express SerpinB3

Abstract

Background: In the setting of liver injury hepatic progenitor cells are activated, counterbalancing the inhibited regenerative capacity of mature hepatocytes. Chronic activation of this compartment may give rise to a subset of liver tumours with poor prognosis. SerpinB3, a serpin over-expressed in injured liver and in primary liver cancer, has been shown to induce apoptosis resistance, epithelial to mesenchymal transition and to increase TGF-beta and Myc expression. Aim of the present study was to explore the presence of SerpinB3 in hepatic progenitor cells in human livers and in a mouse model of liver stem/progenitor cell activation.Hepatic progenitor cells were analysed in foetal and adult livers at protein and transcriptional levels. To induce experimental activation of the liver stem/progenitor compartment, C57BL/6J mice were injected with lipopolysaccharide plus D-galactosamine and were sacrificed at different time points. Liver cDNA was amplified using specific primers for mouse-homologous SerpinB3 isoforms and automatically sequenced.

Results: The presence of SerpinB3 in the progenitor cell compartment was detected in sorted human foetal and adult epithelial cell adhesion molecule (EpCAM) positive liver cells. By immunohistochemistry SerpinB3 was found in human cirrhotic livers in portal areas with progenitor cell activation showing ductular proliferation. CK-7, CK-19, EpCAM and CD-90 positive cell were also positive for SerpinB3. In the animal model, time course analysis in liver specimens revealed a progressive increase of SerpinB3 and a parallel decrease of activated caspase 3, which was barely detectable at 20 hours. Transcription analysis confirmed the presence of SerpinB3-homologous only in the liver of injured mice and sequence analysis proved its belonging to mouse Serpinb3b.

Conclusion: SerpinB3 is highly expressed in hepatic stem/progenitor cell compartment of both foetal and adult livers.

Figure 1

Immunohistochemistry in human liver cirrhosis. Immunohistochemistry on serial liver sections obtained from a cirrhotic patient with ductular proliferation. A) Representative images show negative control (C-) and immunostaining for cytokeratin 7 (CK-7), cytokeratin 19 (CK-19) and for SerpinB3. B) Serial liver sections immunostained for SerpinB3, EpCAM, CD-90, CD-34 and CD-117. Original magnification as indicated.

Figure 2

Stem/progenitor cells analysis. A: Flow cytometry analysis of stem/progenitor cells isolated from foetal or adult livers. Cells were isolated by magnetic immunoselection for EpCAM. Flow cytometry analysis, where cells were stained with anti-EpCAM and anti-NCAM antibodies, demonstrated the enrichment of EpCAM + cells. A significant number of EpCAM + cells were also positive for NCAM. B: RNA analysis for SerpinB3 in human liver stem/progenitor cells. In the real time amplification for human SerpinB3 data were normalized to GAPDH housekeeping gene and the y-axis represents the relative mean mRNA level of the SB3 gene expressed by 2^-ΔCt*10^-5, with the bars representing the standard error. In the lower panel agarose gel electrophoresis of individual corresponding samples is shown as example.

Figure 3

Western blot analysis. Upper Panel) Representative images of Western blot analysis for Serpinb3, activated Caspase 3 and PCNA in liver homogenates of two mice sacrificed at 5 hours (lanes 1,2) and of two mice sacrificed at 20 hours (lanes 3,4) after treatment with LPS/D-galactosamine. The liver of a control mouse has also been shown. Lower Panel) Densitometric analysis of the above results expressed as optical density (OD) of each molecule. The quantitative densitometric values are normalized to β-actin.

Figure 4

Sequence analysis of Serpinb3b cDNA. Sequence analysis of Serpinb3b cDNA confirmed the presence of SerpinB3-homologous only in the liver of injured mice, being not detectable in controls. Homology search analysis proved its belonging to mouse Serpinb3b.