Differential sensitivity of HTC and Fu5-5 cells for induction of tyrosine aminotransferase by 3',5'-cyclic adenosine monophosphate

The two independently derived hepatoma cell lines (HTC and Fu5-5) have previously been shown to display different sensitivities for the induction of tyrosine aminotransferase (TAT) enzyme activity and mRNA levels by glucocorticoids with the enzyme being half-maximally induced at approximately 7-fold higher concentrations of dexamethasone in HTC cells than in Fu5-5 cells. In the present study we investigated the induction of TAT activity by cAMP in order to see whether the difference is limited to the steroidal induction. Using the stable cAMP derivative (8-(4-chlorophenylthio)-cAMP) as an inducer, we found that a 6-fold higher cAMP concentration was needed in HTC cells to achieve the same extent of enzyme induction as in Fu5-5 cells. The induction of TAT enzyme activity could be accounted for by an increased amount of TAT mRNA. Further experiments involving sequential addition of both inducers in general showed a synergism of steroids and cAMP for TAT induction in HTC cells only at submaximal concentrations of steroid; in Fu5-5 cells, the occurrence of synergism depended on the order of addition of inducers. The maximal response in HTC cells was limited to the value that could be achieved by induction with steroid alone. In Fu5-5 cells, however, the steroid response could be augmented when cAMP was added to cells already maximally induced by steroid. This demonstrates that the effect of a combination of cAMP and steroids depends on their concentration, the sequence of their addition, and the rat hepatoma cell line used. Collectively the data suggest that a common pretranslational event determines the differential sensitivity of TAT induction by glucocorticoids and by cAMP in HTC and Fu5-5 cells. Furthermore a second, or possibly the same, common event also regulates the maximum level of TAT induction that is obtainable under most conditions with glucocorticoids and/or cAMP.     

Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescence-activated cell sorting

We have used a mouse mammary tumor virus (MMTV)-infected rat hepatoma cell line as a model system for studying glucocorticoid action. These cells induce tyrosine aminotransferase and MMTV in response to the synthetic glucocorticoid, dexamethasone. The major viral antigen, a glycoprotein of 52,000 daltons (gp52), appears on the surface of infected cells in amounts which reflect the cytoplasmic content of viral RNA. Using an anti-gp52 antiserum and a fluorescence-activated cell sorter (FACS), we have selected variants which display low levels of pg52 in the presence of the hormone. Multiple cycles of enrichment for cells that fluoresce weakly in the presence of hormone have generated a population which fails to produce a detectable increase in cell surface gp52 in response to dexamethasone. This population of nonresponders and a number of independent clones derived from this population were analyzed for their ability to induce gp52 and TAT and for these presence of glucocorticoid receptors. All nonresponder clones exhibited little or no induction of either glucocorticoid-inducible marker. Two of the clones contained reduced levels of glucocrticoid receptor, while the remainder of the clones showed no detectable specific hormone binding. These results provide genetic evidence that a single class of glucocorticoid receptors is involved in the induction of both MMTV and TAT in HTC cells.     

Antiglucocorticoid steroids have increased agonist activity in those hepatoma cell lines that are more sensitive to glucocorticoids

FU5-5 rat hepatoma (Reuber H35) cells are hypersensitive in that the same percentages of full induction of tyrosine aminotransferase (TAT) occur at much lower concentrations of glucocorticoids than in the related HTC rat hepatoma (Morris) cells. Unexpectedly, these hypersensitive FU5-5 cells also exhibited more agonist activity with the affinity labeling antiglucocorticoids cortisol 21-mesylate and dexamethasone 21-mesylate than did HTC cells (Mercier et al., Endocrinology 112, 601-609 [1983]). In the present study, several other antiglucocorticoids (11-desoxycortisone, progesterone, dexamethasone oxetanone, and RU 486 in addition to dexamethasone 21-mesylate) and the antiandrogen cyproterone acetate were examined to see if chemically unreactive, reversible antisteroids also would exhibit an altered activity (i.e. increased agonist activity) in FU5-5 cells. Each antiglucocorticoid examined did display a 2-fold increased amount of agonist activity in FU5-5 cells, as compared to HTC cells; only RU 486 was predominantly an antagonist in FU5-5 cells but the potency of RU 486 was about 9-fold less than in HTC cells. Dexamethasone, and especially progesterone, was metabolized in FU5-5 and HTC cells. However, differential metabolism in FU5-5 vs HTC cells cannot account for the increased induction of TAT in FU5-5 cells since the amount of agonist activity seen for dexamethasone mesylate (or its metabolites) depended not on the cell type used but rather on the glucocorticoid inducible enzyme monitored, i.e. TAT or glutamine synthetase. The combined data suggest that the hypersensitivity of FU5-5 cells towards glucocorticoid induction of TAT may be linked with the ability of both reversible and irreversible antiglucocorticoids to display increased TAT agonist activity in FU5-5 cells. This behavior was somewhat steroid specific since the antiandrogen cyproterone acetate did not display increased TAT agonist activity in FU5-5 cells compared to HTC cells and was only 2-fold less effective as an antiglucocorticoid in FU5-5.     

Dual regulation of protein maturation in viral infected rat HTC hepatoma cells by glucocorticoids and progesterone

Dexamethasone, a synthetic glucocorticoid, is required for full posttranslational maturation of mouse mammary tumor virus (MMTV) phosphoproteins and glycoproteins in M1.54 cells, a viral infected rat hepatoma (HTC) cell line. Pulse-chase radiolabeling with [35S]methionine revealed that steroids with known glucocorticoid activity (such as dexamethasone and hydrocortisone) regulated the maturation of both MMTV polyproteins in a manner proportional to their occupancy for glucocorticoid receptors and their biological potency. In contrast, progesterone selectively induced the proteolytic processing of MMTV phosphoproteins but simultaneously antagonized the dexamethasone-regulated maturation of MMTV glycoproteins and all other tested glucocorticoid responses. Exposure to suboptimal concentrations of both progesterone and dexamethasone fully stimulated the processing of MMTV phosphoproteins, suggesting that steroid receptors occupied with combinations of either steroid functionally interact at the putative maturation gene. Moreover, treatment with either actinomycin D, a potent inhibitor of de novo RNA synthesis, or RU38486, a synthetic antagonist of glucocorticoid and progesterone action, prevented both the dexamethasone and progesterone-regulated induction of MMTV phosphoprotein maturation. Sedimentation velocity and saturation binding analysis revealed that the sizes and concentrations of hepatoma cell progesterone and dexamethasone binding activities are similar while specific binding of the active progestin R5020 was not detected in either M1.54 cells or the glucocorticoid receptor deficient HTC cell line MSN6.10.2. Taken together, our results demonstrate that two distinct classes of steroid hormones can uniquely alter the posttranslational maturation of a specific subset of phosphoprotein substrates by a common glucocorticoid receptor-dependent process.     

Genetic evidence that the steroid-regulated trafficking of cell surface glycoproteins in rat hepatoma cells is mediated by glucocorticoid-inducible cellular components

The biological control of posttranslational maturation and compartmentalization reactions that operate upon proteins during transport to their final cellular destinations is crucial for normal cellular function. Using the expression of mouse mammary tumor virus (MMTV) glycoproteins as sensitive probes in the viral-infected rat hepatoma cell line M1.54, we have discovered and documented a novel glucocorticoid-regulated trafficking pathway that controls the cell surface localization of MMTV glycoproteins. One complement-selected derivative of M1.54 cells, CR4, failed to compartmentalize cell surface MMTV glycoproteins in the presence of dexamethasone. To test genetically if this glycoprotein trafficking pathway is mediated by cellular or viral gene products, CR4 cells were fused with uninfected Fu5 rat hepatoma cells. Indirect immunofluorescence of CR4 X Fu5 heterokaryons revealed that Fu5 complemented the defect in CR4 only after exposure to 1 microM dexamethasone. The glucocorticoid inhibition of Fu5 proliferation was exploited to recover the receptor-deficient uninfected derivative EDR3 that expressed a 100-fold lower level of [3H]dexamethasone binding activity. Analysis of CR4 X EDR3 cell fusions by indirect immunofluorescence revealed that EDR4 cells complemented CR4 in a dexamethasone-dependent manner, suggesting that EDR3 supplied a functinal trafficking component while CR4 provided a functional glucocorticoid receptor to the heterokaryons. Taken together, our results demonstrate that cellular-encoded glucocorticoid-inducible components mediate the regulated trafficking of cell surface MMTV glycoproteins.     

Sphingosine inhibition of tyrosine aminotransferase and tryptophan oxygenase induction by dexamethasone in primary culture of rat hepatocytes

The effect of sphingosine, a known selective inhibitor of protein kinase C, on the induction of tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) by dexamethasone was studied in the primary culture of rat hepatocytes to determine the possible involvement of protein kinase C in the expression of glucocorticoid action. Sphingosine inhibits the induction of TAT by dexamethasone in a concentration- and time-dependent manner in primary culture of rat hepatocytes. It does not inhibit the induction of TAT by Bt2cAMP. Sphingosine inhibits also the induction of TO by dexamethasone in a manner similar to TAT inhibition. It has no effect on the activity of lactate dehydrogenase, a cytosolic marker enzyme and on the protein content of the cultured hepatocytes. These findings indicate that endogenous modulator of protein kinase C, such as sphingosine, may influence the expression of glucocorticoid action in rat hepatocytes.     

Dexamethasone-mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorticoid action.

We have investigated the mechanisms by which dexamethasone (a synthetic glucocorticoid) stimulates the production of mouse mammary tumor virus (MMTV) by cell cultures derived from mammary carcinomas of GR mice. Treatment of these cells with dexamethasone stimulates a rapid accumulation of intracellular virus-specific RNA which is dependent upon RNA synthesis but not upon DNA or protein synthesis. The effect of dexamethasone is probably mediated by a specific and saturable glucocorticoid receptor. We conclude that the accumulation of MMTV RNA is a primary response to dexamethasone and that the rate of synthesis of MMTV RNA is probably accelerated by treatment with dexamethasone.     

Description and analysis of differential sensitivity to glucocorticoids in Fao cells

This study shows that the derived hepatoma cell line Fao displays different sensitivities for glucocorticoid induction of tyrosine aminotransferase (TAT), alanine aminotransferase (AAT) and gamma-glutamyltransferase (GGT). This was seen in the different behaviors of nine steroids with respect to these three effects: (1) in the presence of full agonists (dexamethasone or deacylcortivazol), half-maximal induction of GGT occurred at approx 5- to 6-fold higher agonist concentrations than those required for half-maximal induction of AAT and TAT; (2) in the presence of full antagonists (RU 486, R5020, or progesterone) the GGT response induced by an equal agonist concentration was inhibited at concentrations approx 4- to 5-fold lower than those required for an equivalent inhibition of TAT response; (3) in the presence of cortexolone, deoxycorticosterone, 11 beta-hydroxyprogesterone and dexamethasone-3'-oxetanone, there was a partial agonistic effect (30-50%) on TAT and AAT responses, whereas there was a mainly antagonistic effect (very weak agonistic effect: 0-10%) on GGT response; (4) regardless of the steroid or its full or partial agonist activity, a given TAT induction level (50%, for example) always corresponded to the same AAT and GGT induction levels (50 and 10% respectively). We provide evidence showing that the three above-mentioned biological responses are mediated via the same type of glucocorticoid receptor binding site. Consequently, this differential behavior probably originates from a phenomenon occurring after the common steps (activation, translocation) that follow the formation of the steroid-receptor complex. This leads us to propose a model in which this phenomenon is assumed to originate from a difference in the affinities of the activated receptor for the nuclear acceptor sites of the TAT and GGT genes.     

Glucocorticoid requirement for tyrosine aminotransferase induction by adenosine 3':5'-cyclic phosphate analogs in somatic cell hybrids.

The ability of adenosine 3′:5′-cyclic phosphate (cyclic AMP) analogs to induce l-tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5; TAT) in a rat hepatoma (H35)-rat liver cell (BRL) hybrid (BF5) and a subclone which has lost 29 chromosomes (BF5-1-1) has been analyzed. Cyclic AMP analogs alone were unable to increase TAT activity in either hybrid cell line or in the “normal” liver cells despite three- to fivefold induction of this enzyme in the hepatoma parental cells. In contrast, dexamethasone by itself reproducibly increased TAT activity both in BF5-1-1 cells and in the parental H35 hepatoma cells. Pretreatment of the hybrid cells with dexamethasone revealed a synergistic increase in TAT activity when a cyclic AMP analog was added. From studies of the thermal stability and immunological inhibition of TAT activity, it is concluded that the low basal activity in BRL, BF5, and BF5-1-1 cells represents tyrosine transamination catalyzed by a different aminotransferase, whereas all the induced activity does represent bona fide TAT. The results suggest that functional TAT mRNA may not be present in significant quantities in the hybrid cells in the absence of adrenal steroids and that this could account for the inability of cyclic AMP analogs to exert their presumably translational effect on TAT synthesis.     

The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction.

Sequences within the long terminal repeat region (LTR) of mouse mammary tumour virus (MMTV) confer progestin inducibility to either the tk-promoter or the MMTV-promoter in T47D cells, a human mammary tumour cell line which possesses high constitutive levels of progesterone receptor. In a clone of MCF7 cells, another human mammary tumour cell line with a low level of progesterone receptor, as well as in rat fibroblasts, glucocorticoid but not progestin induction is observed. The effect of the progesterone analogue R5020 is much more pronounced than the effect of dexamethasone, and at the concentrations required for maximal induction, R5020 does not significantly compete with binding of dexamethasone to the glucocorticoid receptor. In conjunction with previous results on the DNA binding of the glucocorticoid and progesterone receptors, these data show that two different steroid hormones, acting through their respective receptors, can mediate the induction of gene expression by interacting with the same DNA sequences. Our results suggest that the hormone regulatory element of MMTV may primarily be a progesterone-responsive element in mammary cells.     

Pretranslational regulation of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (GTP) synthesis by glucagon and dexamethasone in adult rat hepatocytes.

The regulation of synthesis of the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and of tyrosine aminotransferase (TAT) by glucagon and glucocorticoid hormones was studied in hepatocytes maintained in suspension culture for 7 h. Specific antibodies were used to measure relative rates of enzyme synthesis after pulse-labelling of the cells with [3H]leucine or [35S]methionine. Concomitantly, amounts of mRNA were quantified after translation in vitro in a reticulocyte lysate and specific immunoprecipitation of the proteins. Glucagon stimulated the rate of synthesis of PEPCK by 4-6-fold and that of TAT by 6-8-fold in 2h. In contrast, dexamethasone had little effect on PEPCK synthesis, whereas it increased TAT synthesis by 5-9-fold. When used in combination, the two hormones displayed additive effects on TAT synthesis, whereas the glucocorticoid hormone strongly potentiated stimulation of PEPCK synthesis by glucagon. In every instance, changes in rates of synthesis of the two enzymes were totally accounted for by increases in amounts of the corresponding functional mRNA, suggesting a pretranslational site of action for both glucagon and dexamethasone.     

Effect of dibutyryl cyclic AMP and dexamethasone on glutamine synthetase gene expression in rat astrocytes in culture

Astrocytes are the primary site of glutamate conversion to glutamine in the brain. We examined the effects of treatment with either dibutyryl cyclic AMP and/or the synthetic glucocorticoid dexamethasone on glutamine synthetase enzyme activity and steady-state mRNA levels in cultured neonatal rat astrocytes. Treatment of cultures with dibutyryl cyclic AMP alone (0.25 mM–1.0 mM) increased glutamine synthetase activity and steady state mRNA levels in a dose-dependent manner. Similarly, treatment with dexamethasone alone (10^–7–10^–5 M) increased glutamine synthetase mRNA levels and enzyme activity. When astrocytes were treated with both effectors, additive increases in glutamine synthetase activity and mRNA were obtained. However, the additive effects were observed only when the effect of dibutyryl cyclic AMP alone was not maximal. These findings suggest that the actions of these effectors are mediated at the level of mRNA accumulation. The induction of glutamine synthetase mRNA by dibutyryl cyclic AMP was dependent on protein synthesis while the dexamethasone effect was not. Glucocorticoids and cyclic AMP are known to exert their effects on gene expression by different molecular mechanisms. Possible crosstalk between these effector pathways may occur in regulation of astrocyte glutamine synthetase expression.Abbreviations used GS glutamine synthetase - dbcAMP dibutyryl cyclic AMP - MEM minimal essential medium - cyx cycloheximide - GRE glucocorticoid response element - CRE cyclic AMP response element     

High frequency variation in mammary tumor virus expression in cell culture.

Clonal derivatives of C3HMT murine mammary cell lines in culture demonstrate conversion of mammary tumor virus (MMTV) expression at a rate of appriximately 6 per 100 clones. This alteration is largely unidirectional from a relatively high level (MMTV(H)) to a 10 fold lower level (MMTV(L)). This high rate of MMTV(L) variant conversion is in apparent contrast to the presumably mutational rate (approximately 3 per million cells) that governs development of resistance to 6-thioguanine (TG) in the same mammary cells. In somatic cell hybrids between different MMTV TGr clones and mouse or hamster TK- cells, expression of constitutive levels of MMTV and responsiveness to dexamethasone induction is dominant. Thus MMTV expression is regulated by at least two levels of positive control, constitutive expression and glucocorticoid stimulation, but the former is subject to a high rate of variant formation.     

Glucocorticoid regulation of phenylethanolamine N-methyltransferase in vivo.

In vivo, supraphysiological doses of glucocorticoids are required to restore adrenal medullary phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) activity after hypophysectomy. However, in vitro, phenylethanolamine N-methyltransferase gene expression appears normally glucocorticoid-responsive. To explore this paradox, rats were given dexamethasone or the type II-specific glucocorticoid RU28362 (1-1000 micrograms/day), and adrenal phenylethanolamine N-methyltransferase activity and mRNA levels were determined. At low doses (1-30 micrograms/day), neither steroid altered mRNA whereas at higher doses (100-1000 micrograms/day), mRNA rose 10- to 20-fold, with dexamethasone approximately 3 times as potent as RU28362. In contrast, enzyme activity fell with low doses of either steroid, consistent with suppression of ACTH and endogenous steroidogenesis. At higher doses of RU28362, enzyme activity remained low and unchanged despite increased mRNA expression, whereas higher doses of dexamethasone progressively restored the enzyme to normal. These findings suggest 1) that glucocorticoid regulation of phenylethanolamine N-methyltransferase activity occurs largely independent of gene expression; 2) that glucocorticoid effects on enzyme activity are primarily indirect, probably through cosubstrate regulation and/or enzyme stabilization; and 3) that these effects are not mediated via a classical (type II) glucocorticoid receptor mechanism, given the high doses of dexamethasone and corticosterone required and the inability of RU28362 to mimic the effects of these less selective steroids.