Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM), an interleukin-6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes. Interestingly, OSM is expressed in CD45 ϩ hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo.
Interleukin‐3 (IL3) was shown recently to utilize the transcription factor Stat5, but the genes regulated by this pathway and the biological consequence of Stat5 activation remained to be determined. In order to study the role of Stat5 in IL3 signalling, we constructed a dominant‐negative Stat5 protein by C‐terminal truncation, and inducibly expressed it in an IL3‐dependent cell line. The effect of dominant‐negative Stat5 induction on expression of IL3 early response genes was examined, and expression of several genes, including cis, osm and pim‐1 was inhibited profoundly. The expression of c‐fos was also reduced, but to a lesser extent. While activated Ras alone (though not Stat5 alone) could induce c‐fos, maximal expression required the action of both Ras and Stat5. Interestingly, although the membrane‐proximal region of the IL3 receptor beta‐chain is responsible for both Jak2‐Stat5 activation and c‐myc induction, c‐myc levels were not affected by the dominant‐negative Stat5. Thus, the signals directed by this membrane‐proximal domain, which is essential for transducing a DNA synthesis signal, can be separated further into Stat5 or c‐myc pathways. The net effect of dominant‐negative Stat5 expression was partial inhibition of IL3‐dependent growth. This provides the first direct evidence that Stat5 is involved in regulation of cell proliferation.
Interleukin 3 (IL‐3) and granulocyte‐macrophage colony stimulating factor (GM‐CSF) exert their biological functions through acting on a specific receptor which consists of a ligand‐specific alpha subunit and the shared common beta subunit. Inhibition by genistein of a subset of IL‐3/GM‐CSF‐mediated signals, including c‐myc induction, resulted in the abrogation of DNA synthesis, however, IL‐3 still protected cells from apoptotic cell death. Conversely, a C‐terminal truncated form of the GM‐CSF receptor, which is missing a critical cytoplasmic region required for activation of the Ras/Raf‐1/MAP kinase pathway, induced DNA synthesis, but failed to prevent cell death in response to GM‐CSF. Consequently, cells died by apoptosis in the presence of GM‐CSF, despite displaying a transient mitogenic response. However, expression of activated Ras protein complemented defective signalling through the mutant receptor and supported long‐term proliferation in concert with GM‐CSF. These results indicate that IL‐3 and GM‐CSF prevent apoptosis of hematopoietic cells by activating a signalling pathway distinct from the induction of DNA synthesis and that long‐term cell proliferation requires the activation of both pathways.
Two distinct signaling pathways regulate the survival of interleukin-3 (IL-3)-dependent hematopoietic progenitors. One originates from the membrane-proximal portion of the cytoplasmic domain of the IL-3 receptor (c chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor and is involved in the regulation of Bcl-x L through activation of STAT5. The other pathway emanates from the distal region of the c chain and overlaps with downstream signals from constitutively active Ras proteins. Although the latter pathway is indispensable for cell survival, its downstream targets remain largely undefined. Here we show that the expression of Bim, a member of the BH3-only subfamily of cell death activators, is downregulated by IL-3 signaling through either of two major Ras pathways: Raf/mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/mammalian target of rapamycin. Akt/phosphokinase B does not appear to play a significant role in this regulatory cascade. Bim downregulation has important implications for cell survival, since enforced expression of this death activator at levels equivalent to those induced by cytokine withdrawal led to apoptosis even in the presence of IL-3. We conclude that Bim is a pivotal molecule in cytokine regulation of hematopoietic cell survival.
Liver development is regulated by soluble factors as well as cell^cell contacts. We previously reported that oncostatin M (OSM) induced hepatic maturation in a primary culture of embryonic day 14 liver cells. While OSM expression in the liver starts in mid gestation and decreases in postnatal stages, hepatocyte growth factor (HGF) is mainly expressed in the liver in the first few days after birth. In this study, we compared the effect of OSM and HGF on the differentiation of fetal hepatic cells in vitro. Like OSM, HGF in the presence of dexamethasone induced expression of glucose-6-phosphatase, tyrosine amino transferase and carbamoyl-phosphate synthase, and accumulation of glycogen in fetal hepatic cells, although to a lesser extent than OSM. Interestingly, while both OSM and HGF upregulated production of albumin, secretion of albumin occurred only in response to OSM. In addition, although hepatic maturation induced by OSM depends on STAT3, HGF failed to activate STAT3 and HGF-induced differentiation was independent of STAT3. These results indicate that OSM and HGF induce hepatic maturation through different signaling pathways. ß
Liver development is a sequential array of distinct biological events. Each step of differentiation is regulated by intrinsically programmed mechanisms as well as by extracellular signals. The establishment of cell culture systems that recapitulate each stage of liver development has led to the identification of several extracellular signals that affect hepatocytic differentiation. Furthermore, studies on genetically engineered animals, especially knockout and transgenic mice, have highlighted a number of molecules essential for liver development. By applying primary culture techniques to analyses of mutant mice, it is now possible to link extracellular signals to intracellular pathways that provoke cellular responses of differentiation. Improvement in gene transfer technology utilizing viral vectors has further expanded the molecular analysis of liver development. In this review article, we summarize recent advances and attempt to describe the molecular basis of liver development from beginning to end as a sequential event.
Embryonic liver is a transient site for definitive hematopoiesis. Along with maturation of the bone marrow and spleen, hematopoietic cells relocate from the liver to their final destinations while the liver starts organizing its own structure and develops numerous metabolic functions toward adult. Recently, it was demonstrated that the signal exerted by oncostatin M (OSM) through gp130 plays a pivotal role in the maturation process of the liver both in vitro and in vivo. However, the molecular basis underlying the termination of embryonic hematopoiesis remains unknown. In this study, we report that primary culture of fetal hepatic cells from embryonic day 14.5 murine embryos supported expansion of blood cells from Lin ؊ Sca-1 ؉ c-Kit ؉ cells, giving rise to myeloid, lymphoid, and erythroid lineages. Of interest, promotion of hepatic development by OSM and glucocorticoid strongly suppressed in vitro hematopoiesis. Consistent with these results, hepatic culture from the embryonic day 18.5 liver no longer supported hematopoiesis. These data together with the previous observations suggest that the signals exerted by OSM and glucocorticoid induce hepatic differentiation, which in turn terminate embryonic hematopoiesis and promote relocation of hematopoietic cells.
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