Prospero-related homeobox 1 and liver receptor homolog 1 coordinately regulate long-term proliferation of murine fetal hepatoblasts

Abstract

During early to late-fetal liver development, bipotential hepatoblasts proliferate and differentiate into hepatocytes and cholangiocytes. The prospero-related homeobox 1 gene (Prox1) is expressed in hepatoblasts, and the inactivation of Prox1 causes defective early liver development, in particular, faulty migration of fetal hepatoblasts. Prox1 binds to another hepatocyte-enriched transcription factor, liver receptor homolog 1 (Lrh1), and suppresses its transcriptional activity. However, the molecular mechanism by which Prox1 and Lrh1 regulate the characteristics of fetal hepatic cells remains unknown. We investigated the contribution of Prox1 and Lrh1 in early liver development. Embryonic day 13 liver-derived CD45-Ter119-Dlk+ cells were purified as fetal hepatic stem/progenitor cells, and formation of colonies derived from single cells was detected under low-density culture conditions. We found that overexpression of Prox1 using retrovirus infection induced migration and proliferation of fetal hepatic stem/progenitor cells. In contrast, overexpression of Lrh1 suppressed colony formation. Prox1 induced the long-term proliferation of fetal hepatic stem/progenitor cells, which exhibited both high proliferative activity and bipotency for differentiation. Prox1 up-regulated expression of cyclins D2, E1, and E2, whereas it suppressed expression of p16(ink4a), the cdk inhibitor. In addition, overexpression of Prox1 significantly inhibited the proximal promoter activity of p16(ink4a). These results suggested that Prox1 and Lrh1 coordinately regulate development of hepatic stem/progenitor cells and that Prox1 induces fetal hepatocytic proliferation through the suppression of the promoter activity of p16(ink4a).