Chronic human liver diseases are characterized by continuous liver damage and hepatocyte loss, with subsequent activation of the progenitor cell compartment . Despite the capacity of the adult human and rodent liver to reply to loss of its cellular mass, it is widely acknowledged that impairment of the regenerative capacity of hepatocytes may result in proliferation and migration of cells forming ductular structures, initially close to the biliary tree. The capacity of these ductular structures to proliferate and differentiate into hepatocytes, in association with impaired liver regeneration, has led to the conclusion that they may represent progeny of facultative liver stem cells [31, 32]. In recent years, considerable interest has been devoted to the hepatic progenitor cells, as tumors showing hepatic progenitor cell features have a worse prognosis and a higher recurrence rate compared to tumors lacking these characteristics [5, 21].
The present study has provided evidence that in the human progenitor cell compartment SB3 is detectable in the same ductular structures that express stem/progenitor cell markers, with multiple differentiating capabilities [18, 19, 33]. Within the ductular reaction areas, a high proportion of CK-7 and CK-19 positive cells presented indeed a positive staining for SB3. This SB3 positive cell population was also positive for EpCAM and CD90, markers of mesenchymal and foetal liver stem cells. This is the first report, at the best of our knowledge, indicating that SB3 is present in human stem/progenitor cells of hepatic origin. The lack of the hematopoietic markers CD34 and CD117 in the same areas may exclude that this population derives from circulating hematopoietic cells, rather supporting its belonging to a distinct liver stem cell-like population [34, 35].
In addition, to evaluate the expression of SB3 in the liver stem/progenitor cell compartment, EpCAM + cells were isolated by immunomagnetic sorting from foetal and adult human livers. A bulk of recent literature deals with the role of EpCAM + cells as hepatic resident stem/progenitor cells [36, 37]. Moreover, microarray expression data from a panel of human tissue generated using GNF Expression Atlas 2 Data from U133A chip (publicy available through the UCSC Genome Browser website http://genome.ucsc.edu/), further confirms that EpCAM mRNA is robustly expressed in foetal liver. The detection of SB3 RNA in the EpCAM + cell fractions of human foetal and adult livers supports strongly the presence of this serpin in hepatic progenitor cells .
An animal model of stem/progenitor cell induction after acute liver injury determined by LPS/D-GalN  has also been studied. In this model hepatic injury is mediated by macrophages  and proinflammatory cytokines, including IL-1, IL-6, IL-12 and TNF-α  lead to hepatocyte necrosis and to massive hepatocyte apoptosis . Members of the TNF family are the best characterized stimuli for stem/progenitor cell proliferation, an event that shares an unusual reciprocal relationship with the proliferation of hepatocytes . In our experimental conditions, using total cell lysates, a progressive increase of mouse SB3 and a parallel decrease of activated caspase 3 was detectable in the early phase of acute liver injury. We cannot exclude the possibility that the observed increase in mouse SB3 may be contributed by other cell types, rather than the progenitors, since activated B lymphocytes have been also reported to express SB3 . Transcription analysis confirmed the presence of SB3-homologous only in the liver of injured mice and sequence analysis proved its belonging to mouse Serpinb3b. This serpin, one of the four isoforms of the Serpinb3 mouse gene, is the counterpart of the human SB3 and shares 73% aminoacid similarity. Both are dual cross-class inhibitors of cysteine and serine proteinases [16, 44], supporting the hypothesis that they retained these activities from an ancestral SCCA-like gene.
SB3 is a multifunctional protein that, beyond its antiprotease activity, makes cells more resistant to several killing mechanisms by inhibition of apoptosis . The results obtained in LPS/D-GalN treated mice confirmed that overexpression of Serpinb3b in the liver was associated with a marked decrease of the activated caspase 3, which represents a well-defined hallmark of apoptosis.
SB3 has been detected recently in hepatoblastoma, the embryonal tumor of the liver, and a direct correlation was observed between its gene expression, the up-regulation of Myc oncogene and tumor extension . In addition, in vitro experiments documented the primary involvement of SB3 in the up-regulation of Myc transcription . This serpin has been also shown to be able to increase TGF-β expression in hepatic cell lines and in primary hepatocytes . Chronic stimulation of hepatic progenitor cells by TGF-β has been shown recently to induce their transformation into cancer stem cell/tumor-initiating cells , suggesting the involvement of triggering autocrine and paracrine mechanisms in hepatocarcinogenesis. These observations, together with the fact that SB3 was found to promote epithelial to mesenchymal transition and to increase cell proliferation and invasiveness , may suggest a carcinogenic potential of hepatic progenitor cells expressing this serpin.