Regulators of fetal liver differentiation in vitro (original) (raw)
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Regulation of rat liver maturation in vitro by glucocorticoids
Molecular and Cellular Biology, 1988
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 kil...
European Journal of Biochemistry, 1997
Tyrosine aminotransferase (TyrAT) is one of several gluconeogenic enzymes which appear postnatally in humans and rodents in response to increased glucocorticoid and glucagon levels and decreased insulin. Primary cultured fetal rat hepatocytes older than day 15 of gestation (>E15) transcribe the TyrAT gene in response to the synergistic effect of dexamethasone and Nh,2'-O-dibutyryl-adenosine 3',5'-monophosphate (Bt,cAMP), whereas less mature hepatocytes (<E15) do not [Shelly, L. L. & Yeoh, G. C. T. (1991) Eur: J. Biochem. 199, 475-4811. Therefore, we consider >El5 hepatocytes, and not <El5 hepatocytes, to be determined. This study reports that 11.1 kb of sequences upstream of the TyrAT transcription start site, which include a CAMP-responsive element (CRE) and a glucocorticoid-responsive element (GRE), are required for correct developmental regulation of gene expression in determined fetal hepatocytes. In contrast, the TyrAT CRE alone does not have this capability. Dexamethasone augments basal and Bt,cAMP-stimulated activity of the TyrAT CRE alone, suggesting that synergism may be due to interaction between the glucocorticoid and CAMP-signaling pathways. However, Bt,cAMP does not further increase dexamethasone-induced activity of the 11.1 kb 5' sequences when the TyrAT CRE is removed, thus excluding interaction of Bt,cAMP with the glucocorticoid pathway. Finally, insulin inhibition of dexamethasone-induced gene transcription is shown to be conferred by TyrAT 5' sequences. This study shows that cellular components, other than those which mediate hormonal regulation of genes, are required for determination of hepatocytes with respect to TyrAT. Since this phenomenon is observed with transient transfections, it is unlikely to involve higher-order chromatin structure.
The Journal of Cell Biology, 1989
A fetal rat hepatocyte culture system has been used to study the molecular mechanisms of tyrosine aminotransferase (TAT) gene expression during development. It has previously been shown that TAT activity can be detected in 19-d, but not 15-d, gestation hepatocytes on the first day of culture (Yeoh, G. C. T., E A. Bennett, and I. T. Oliver. 1979. Biochem. J. 180:153-160). In this study enzyme activity, synthesis, and mRNA levels were determined in hepatocytes isolated from 13-, 15-, and 19-d gestation rats maintained in culture for 1, 2, or 3 d and exposed to dexamethasone. TAT expression is barely detectable in 13-d gestation hepatocytes even after 3 d in culture. Hepatocytes isolated from 15-d gestation fetuses have undetectable levels of enzyme activity and synthesis on the first day of culture; both can be assayed by days 2 1. Abbreviations used in this paper: GAPDH, glyceraldehyde-3-phosphatedehydrogenase; TAT, tyrosine aminotransferase.
The Biochemical journal, 1981
The glucocorticoid receptor activity that can be detected in the liver from 15-day foetal rats would appear to be associated with the haemopoietic cells. In hepatocytes, purified by culture for 1-2 days from 15-day foetal rats, the glucocorticoid receptor activity is low and dexamethasone does not induce the enzyme tyrosine aminotransferase. If culture is continued both receptor activity and steroid responsiveness are acquired. Cultured hepatocytes from 19-day foetal liver contain receptor from the first day of culture and, furthermore, the subsequent level of response to glucocorticoids is directly correlated with the actual receptor concentration. It would appear that the glucocorticoid receptor is not acquired by hepatocytes until after 18 days of gestation. Nevertheless, the fact that bromodeoxyuridine has no effect on the rate of accumulation of receptor in hepatocytes suggests that the differentiative event leading to the subsequent appearance of the receptor has already occur...
Glucocorticoids are Insufficient for Neonatal Gene Induction in the Liver
Proceedings of The National Academy of Sciences, 1998
Glucocorticoids and their receptor (GR) play a key role in perinatal gene induction. In the liver, the GR is essential for the neonatal induction of a number of genes, including that coding for tyrosine aminotransferase (TAT). To assess the function of the GR in the perinatal period, we have compared the activity of two types of glucocorticoid responsive elements in transgenic mice; one is the Tat gene glucocorticoid-responsive unit (GRU), an assembly of numerous binding sites for transcription factors, including the GR; the other is a simple dimer of high-affinity GR binding sites (GREs). Both elements confer strong glucocorticoid response in the adult liver. However, only the Tat GRUs are able to promote neonatal induction; the GRE dimer is unresponsive. Because this dimer is responsive to glucocorticoid administration in the neonate, the absence of neonatal induction is not due to the inactivity of the GR at this stage. At birth, the neonate has to withstand a brief period of starvation and hypoglycemia, a nutritional and hormonal situation that resembles fasting in the adult. In transgenic mice, the responses at birth and after fasting in the adult are similar: the Tat GRUs but not the dimeric GREs are activated. Our results show that, in rodents, glucocorticoids are not sufficient for neonatal gene induction in the liver and support the conclusion that the hypoglycemia at birth is the main trigger for expression.
European Journal of Biochemistry, 1991
Fetal hepatocyte cultures were used to investigate tyrosine aminotransferase (TyrAT) expression during development. Previous studies showed that TyrAT is synthesized by hepatocytes isolated from 15-day-gestation fetuses maintained in culture for two or more days, then exposed to dexamethasone. TyrAT expression was essentially undetectable on the first day of culture of hepatocytes derived from 15-day-gestation, or less mature, fetuses. Dexamethasone and cAMP are potent inducers of TyrAT and they synergistically induce TyrAT to extremely high levels when added simultaneously to cultured fetal hepatocytes.
Journal of Medical Primatology, 2013
Background-The objective of this study was to develop a cell culture system for fetal baboon hepatocytes and to test the hypotheses that (1) expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase-1 (PEPCK-1) is upregulated in hepatocytes isolated from fetuses of nutrient-restricted mothers (MNR) compared to ad libitum-fed controls (CTR) and (2) glucocorticoids stimulate PEPCK-1 expression. Methods-Hepatocytes from 0.9G CTR and MNR fetuses were isolated and cultured. PEPCK-1 protein and mRNA levels in hepatocytes were determined by western blot and quantitative PCR, respectively. Results-Fetuses of MNR mothers were intrauterine growth restricted (IUGR). Feasibility of culturing 0.9G fetal baboon hepatocytes was demonstrated. PEPCK-1 protein levels were increased in hepatocytes isolated from IUGR fetuses and PEPCK-1 mRNA expression was stimulated by glucocorticoids in fetal hepatocytes. Conclusions-Cultured fetal baboon hepatocytes that retain their in vivo phenotype provide powerful in vitro tools to investigate mechanisms that regulate normal and programmed hepatic function.
European Journal of Biochemistry, 1981
Whereas dexamethasone is unable to induce tbe premature formation of hepatic tyrosine aminotransferase whcn administered to foetal rats in utero, the stcroid can induce the enzyme in foetal rat liver if the liver is first removed from the environment in utero and grown in culture. Dexamethasone produced a significant induction of the enzyme at a concentration of 0.1 nM in cultured foetal hepatocytes, but for optimal induction the cells werc exposed to 10 nM for 15 h. Growing the hepatocytes in the presence of physiological concentrations of insulin had no effect on the enzyme activity in control cells. However, the induction of the enzyme by dexamethasone was markedly diminished in the presence of insulin. This effect of insulin is both time-dependent and dose-dependent with significant inhibition being obtained with 1 n M insulin. Growing foetal hepatocytes in the presence of insulin has no effect on either the cellular level of glucocorticoid receptor or on the ability of dcxamethasonereccptor complexes to undergo nuclear translocation suggesting that insulin inhibits some event subsequent to translocation.