Origin of corticosteroid-binding globulin in fetal rat. Comparative dynamics of corticosteroid-binding globulin, alpha-fetoprotein, and albumin secretion in primary cultures of fetal rat hepatocytes

The origin of corticosteroid-binding globulin (CBG) and its evolution in comparison with alpha-fetoprotein (AFP) and albumin synthesis, during early development of rat liver (days 13 and 15 of fetal life), have been investigated using cultured fetal hepatocytes. Synthesis and secretion of CBG, AFP, and albumin is evidence by cycloheximide-sensitive [14C]leucine incorporation into immunoprecipitable polypeptides secreted by cultured hepatocytes into the medium, two-dimensional immunoelectrophoretic and autoradiographic identification of newly synthesized labeled proteins, corticosterone and estradiol-17 beta binding to CBG and AFP, respectively, and indirect immunofluorescence localization of AFP, albumin, and CBG in cultured fetal hepatocytes. CBG, albumin, and AFP accounted for 6, 11, and 25% (in 13-day-old rat fetuses) and 5, 15, and 28% (15-day-old rat fetuses), respectively, of the total secreted proteins in the culture medium. The rates of CBG, AFP, and albumin (counts/minute of secretion [14C]leucine incorporated per milligram of cell protein/hour of culture) in the hepatocytes of 15-day-old rat fetuses were 1.48-, 2.1-, and 2.57-fold higher, respectively, than in the 13-day-old rat fetuses. These results indicate that fetal liver is also active in CBG synthesis, along with AFP and albumin, as early as day 13 of fetal life and that the synthetic rates of these secretory proteins depend upon the developmental stage of the fetal liver. This developmental related change in the rate of synthesis of CBG by the fetal hepatocytes may regulate the level of free (active) glucocorticoid in the fetal circulation and thereby the initiation and regulation of glucocorticoid-dependent processes during the crucial stages of the differentiation of fetal liver and other developing tissues.     

Kinetics of albumin- and alpha-fetoprotein-production during rat liver development

Synthesis of most of the plasma proteins is one of the main functions of the hepatocytes. Albumin synthesis is quantitatively the most abundant. In the present study we investigated albumin- and alpha-fetoprotein-gene-expression, and the function of the secretory apparatus during rat liver development. To this purpose we used the method of radioactive biosynthetic labeling of newly synthesized albumin and alpha-fetoprotein (AFP) to monitor the secretory capacity of endodermal cells derived from ventral foregut region (embryonic day 10, E10), and of embryonic and fetal hepatoblasts. Synthesis and secretion of albumin and AFP were already detected in the low numbered ventral foregut endodermal cells; fibrinogen synthesis was detectable in the E12 hepatoblasts, which were in higher number. The whole secretory machinery was functional from the earliest stages of liver development, and the speed of secretion was comparable with that of the adult hepatocytes. There was almost 4-fold increase of hepatoblasts cell volume in fetal stage compared with embryonic stage. The model used suggests that the hepatocyte secretory apparatus is already functional before the emergence of the liver bud. This is the first comparative report to analyze the hepatocyte secretory function, cell proliferation and cell volume during liver development.     

Regulation of rat liver maturation in vitro by glucocorticoids.

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.     

Chimeric molecule IL-6/soluble IL-6 receptor is a potent mitogen for fetal hepatocytes

A novel recombinant molecule, termed IL-6c and consisting of a chimera of interleukin 6 (IL-6) and its soluble receptor is extremely potent in stimulating proliferation of hematopoietic progenitors. We investigated the effect of the IL-6c on the proliferation and differentiation of E14 fetal hepatocytes. IL-6c, in a dose-dependent manner, stimulated proliferation of E14 fetal rat hepatocytes. Adult hepatocyte mitogens together with IL-6c showed no further effect on proliferation. Hematopoietic stem cells mitogens SCF and flt3 ligand (FL) were also mitogenic for fetal hepatocytes, but did not further enhance the effect of IL-6c on cell proliferation. IL-6c decreased expression of fetal markers alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase, and induced expression of adult enzyme glucose-6-phosphatase (Gluc-6-P) in E14 hepatocytes. On the other hand, IL-6c strongly reduced, in a dose-dependant manner, expression of albumin and tyrosine aminotransferase (TAT). However, when the cells were grown for 3 days with IL-6c, and IL-6c was removed for the next 5 days, expression of albumin and TAT returned to levels found in control cultures. In conclusion, IL-6c stimulated proliferation and affected gene expression in fetal hepatocytes in culture.     

New primary culture systems to study the differentiation and proliferation of mouse fetal hepatoblasts

Hepatoblasts have the potential to differentiate into both hepatocytes and biliary epithelial cells through a differentiation program that has not been fully elucidated. With the aim to better define the mechanism of differentiation of hepatoblasts, we isolated hepatoblasts and established new culture systems. We isolated hepatoblasts from E12.5 fetal mouse liver by using E-cadherin. The E-cadherin+ cells expressed alpha-fetoprotein (AFP) and albumin (Alb) but not cytokeratin 19 (CK19). Transplantation of the E-cadherin+ cells into mice that had been subjected to liver injury or biliary epithelial injury led to differentiation of the cells into hepatocytes or biliary epithelial cells, respectively. In a low-cell-density culture system in the absence of additional growth factors, E-cadherin+ cells formed colonies of various sizes, largely comprising Alb-positive cells. Supplementation of the culture medium with hepatocyte growth factor and epidermal growth factor promoted proliferation of the cells. Thus the low-cell-density culture system should be useful to identify inductive factors that regulate the proliferation and differentiation of hepatoblasts. In a high-cell-density system in the presence of oncostatin M+dexamethasone, E14.5, but not E12.5, E-cadherin+ cells differentiated into mature hepatocytes, suggesting that unidentified factors are involved in hepatic maturation. Culture of E-cadherin+ cells derived from E12.5 or E14.5 liver under high-cell-density conditions should allow elucidation of the mechanism of hepatic differentiation in greater detail. These new culture systems should be of use to identify growth factors that induce hepatoblasts to proliferate or differentiate into hepatocytes and biliary epithelial cells.     

Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

alpha 1-Fetoprotein production during the hepatocyte growth cycle of developing rat liver.

The relation of AFP production to DNA synthesis was investigated in newborn rat liver and in primary cultures of fetal rat hepatocytes, by combining immunoperoxidase AFP localization and autoradiography after ³H-thymidine labelling. The vast majority of AFP-positive hepatocytes did not incorporate ³H-thymidine after ≤4-h isotope pulses, suggesting that in the developing liver, essentially no production of AFP occurs in S, G₂ or M phases of the hepatocyte cell life cycle. Serial or continuous thymidine labelling experiments further indicated that post-mitotic hepatocytes constitute a sizable fraction of AFP-producing cells.     

Regulators of fetal liver differentiation in vitro

Seventeen-day-old fetal rat hepatocytes were employed to examine factors required to promote differentiation in vitro. In the absence of effectors, primary fetal hepatocytes dedifferentiated, as characterized by the rapid decline in synthesis of fetal alpha-fetoprotein (AFP), albumin, and transferrin. On the other hand, cells maintained in the presence of glucocorticoid hormone produced high levels of albumin and transferrin. Glucocorticoid could not prevent the decline in fetal AFP synthesis, but induced synthesis of the 65K variant AFP--the major AFP species produced by adult rat liver. Fetal hepatocytes maintained in the presence of 8-bromo-cAMP (8-BrcAMP), or methyl isobutyl xanthine (MIX), an agent that increases intracellular cAMP levels, synthesized high levels of fetal AFP and albumin but reduced levels of transferrin. Both glucocorticoid and 8-BrcAMP or MIX induced expression of adult liver-specific genes such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), suggesting that these fetal hepatocytes have matured. Cells maintained in the presence of glucocorticoid hormone and MIX (or 8-BrcAMP) contained more albumin, TAT, and PEPCK mRNAs and synthesized increased amounts of the 65K variant AFP than those with either agent alone. However, the glucocorticoid/MIX cells produced intermediate levels of the fetal AFP and transferrin. Our data indicate that both glucocorticoid hormone and cAMP are necessary for optimal differentiation of fetal hepatocytes in vitro.     

Identification of candidate growth-regulating genes that are overexpressed in late gestation fetal liver in the rat

We have shown previously that hepatocyte proliferation in the late gestation fetal rat is mediated by growth factor-independent mechanisms that are distinct from the signaling pathways that promote proliferation of adult rat hepatocytes. In the present studies, we identified six candidate growth-regulating genes that are overexpressed in fetal rat liver (embryonic day 19, 2 days pre-term) relative to adult rat liver using suppressive subtractive hybridization. These included the following: Grb10, a growth factor receptor binding protein; eps15, a growth factor receptor substrate; nuc2+, a retinoblastoma protein binding protein; cdc25B, a cell cycle tyrosine phosphatase; the peroxisome proliferator-activated receptor PPAR alpha; and a deoxyuridine triphosphatase that functions as a PPAR alpha binding partner. In every case, the ontogeny of the expression of these genes declined postnatally in a manner consistent with the transition from a fetal to an adult hepatocyte phenotype. None were found to be cell cycle-dependent, in that they did not show expression that followed perinatal changes in hepatocyte cell cycle activity. Based on our identification of these genes and previous work characterizing their role in growth regulation, we conclude that they may contribute to the mitogenic signaling phenotype of fetal rat hepatocytes.     

Long-term maintenance of hepatocyte functional activity in co-culture: requirements for sinusoidal endothelial cells and dexamethasone

Sinusoidal cells isolated from adult rat liver have been established in primary culture and in cell line. The presence of factor VIII R:Ag and peroxidatic/phagocytosis activities were the criteria used to distinguish in freshly isolated cells the endothelial cells from the Kupffer cells and suggested the endothelial origin of the cell line. Using a co-culture system, the effect of sinusoidal liver cells on hepatocyte functional activity was characterized. A plateau in which the state of differentiation was stabilized could be generated for co-cultured hepatocytes isolated from adult rat and a disappearance of the initial expression of alpha 1-fetoprotein (AFP) and the increase and/or maintenance of albumin secretion were measured with co-cultured hepatocytes isolated from suckling rat. The presence of dexamethasone was required for such beneficial effect. The hepatocyte-stabilizing activity was also produced by a pulmonary endothelial cell line.     

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.