Interaction of glucocorticoid hormones and cyclic nucleotides in induction of tyrosine aminotransferase in cultured hepatoma cells.

Reproducible induction of the enzyme tyrosine aminotransferase by dibutyryl cAMP (Bt2cAMP) in a line of HTC hepatoma cells in suspension culture requires that the cells be preinduced with dexamethasone, a synthetic glucocorticoid which itself induces tyrosine aminotransferase. Concentrations of dexamethasone that do not induce tyrosine aminotransferase fail to support Bt2cAMP induction, removal of the steroid from the medium leads to a loss of the Bt2cAMP effect, and an HTC cell line whose aminotransferase is not steroid-inducible does not respond to the cyclic nucleotide. We show that the further induction of tyrosine aminotransferase by Bt2cAMP in dexamethasone-treated cells is due to an increased rate of enzyme synthesis. The cyclic nucleotide has no effect on aminotransferase synthesis in cells grown in the absence of steroid. Several lines of evidence suggest that dexamethasone acts at a step beyond the activation of protein kinase by cAMP: (a) basal levels of cAMP are not altered by growth of HTC cells in dexamethasone; (b) accumulation of cAMP from the medium is not enhanced; (c) the glucocorticoid does not induce cAMP-dependent protein kinase in HTC cells; and (d) there is no augmentation of cAMP binding to the regulatory protein, nor is there any change in cAMP activation of protein kinase caused by growth in dexamethasone. These results help define a system that should be useful in studying the interaction of cyclic nucleotides and steroid hormones.     

The effect of hormones on glucocorticoid binding capacity of rat liver cytosol.

The relationship between the glucocorticoid binding capacity of rat liver cytosol and the activity of tyrosine aminotransferase has been studied in adrenalectomized male rats. Bilateral adrenalectomy of male rats caused an increase within 3 days in the level of specific dexamethasone binding of liver cytosol accompanied by a rapid decrease in tyrosine aminotransferase activity. Known inducers of tyrosine aminotransferase were administered in vivo to test their effect on dexamethasone binding capacity, in order to determine whether the induction was by an indirect mechanism involving an increase in glucocorticoid binding capacity. Insulin, adrenalin, glucagon, dibutyryl cyclic AMP and oestradiol caused a significant increase in the activity of the enzyme, with no change in the specific dexamethasone binding. Tetracosactrin, a synthetic analogue of ACTH, had no effect on either parameter. It was concluded that the induction of tyrosine aminotransferase by the compounds tested was not mediated by an increase in glucocorticoid receptor activity.     

Hormonal regulation of plasminogen activator in rat hepatoma cells

Plasminogen activators are membrane-associated, arginine-specific serine proteases which convert the inactive plasma zymogen plasminogen to plasmin, an active, broad-spectrum serine protease. Plasmin, the major fibrinolytic enzyme in blood, also participates in a number of physiologic functions involving protein processing and tissue remodelling, and may play an important role in tumor invasion and metastasis. In HTC rat hepatoma cells in tissue culture, glucocorticoids rapidly decrease plasminogen activator (PA) activity. We have shown that this decrease is mediated by induction of a soluble inhibitor of PA activity rather than modulation of the amount of PA. The hormonally-induced inhibitor is a cellular product which specifically inhibits PA but not plasmin. We have isolated variant lines of HTC cells which are selectively resistant to the glucocorticoid inhibition of PA but retain other glucocorticoid responses. These variants lack the hormonally-induced inhibitor; PA from these variants is fully sensitive to inhibition by inhibitor from steroid-treated wild-type cells. Cyclic nucleotides dramatically stimulate PA activity in HTC cells in a time- and concentration-dependent manner. Paradoxically, glucocorticoids further enhance this stimulation. Thus glucocorticoids exert two separate and opposite effects on PA activity. The availability of glucocorticoid-resistant variant cell lines, together with the unique regulatory interactions of steroids and cyclic nucleotides, make HTC cells a useful experimental system in which to study the multihormonal regulation of plasminogen activator.     

Hormonal deinduction of tyrosine aminotransferase

The effects of several antagonists of glucocorticoid action on a line of hepatoma cells (HTC strain) have been studied in order to determine their mechanisms of action. The induction of tyrosine aminotransferase by dexamethasone can be partially or totally inhibited if an antagonist is added simultaneously with dexamethasone or some time later. Antagonists, even if they have as much affinity for the cytoplasmic receptor as dexamethasone, must be administered at a 100-fold excess as compared to dexamethasone. Their receptor binding kinetics are not identical to those of inducer steroids: moreover there is no correlation between relative binding affinities and anti-inducing capacities. A short contact between the cells and the antagonist is sufficient to obtain a full antagonistic effect, but the antagonist is inactive if administered and removed from the cells before induction. An interpretation if suggested, considering these results which do not find a satisfactory explanation in the classical theory of receptor action.     

Enhancement by streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine of the tyrosine aminotransferase activity in cultured rat liver cells: role of dexamethasone and NAD

The activity of tyrosine aminotransferase (TAT) (EC 2.6.1.5) was enhanced 3-fold after a 5-h exposure of cultured rat liver cells (RLC) to streptozotocin (SZ) at concentrations higher than 100 microgram/ml (0.38 mM) in the presence of 10 nM dexamethasone, a potent glucocorticoid inducer for the enzyme. The structurally related carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also enhanced the aminotransferase in the presence of the glucocorticoid, but its optimal concentration was at 100 ng/ml (0.68 microM). While the cellular NAD (NAD+ + NADH) concentration was reduced to 60% of the control levels, the rate of poly(ADP-ribose) formation in the isolated cell nuclei was unaffected by treating the cells with SZ. The enhancement of tyrosine aminotransferase by SZ and MNNG was effectively prevented by nicotinamide. Using nicotinamide and its derivatives such as 1-methyl-, N'-methyl- or 6-amino-derivatives it was found that the degree of enzyme induction is almost inversely proportional to the cellular NAD content, though the activity of nuclear poly(ADP-ribose)polymerase remains unchanged. The results indicate that SZ or MNNG, in combination with dexamethasone, stimulate the induction of tyrosine aminotransferase through their NAD lowering action.     

Binding of glucocorticoid antagonists to androgen and glucocorticoid hormone receptors in rat skeletal muscle

The binding of ten steroids possessing antiglucocorticoid activity has been studied in rat skeletal muscle cytosol. The affinity of these steroids for both the androgen and the glucocorticoid receptors was determined by competition with radioactive R1881 (methyltrienolone, metribolone) and dexamethasone, respectively. The antiglucocorticoid activity of these compounds was assessed in rat hepatoma (HTC) cells by measuring their inhibitory effect on the glucocorticoid-induced tyrosine aminotransferase activity. This led to identification of five novel in vitro glucocorticoid antagonists. All the steroids tested bound to both the glucocorticoid and the androgen receptors in muscle. Four steroids had an affinity for the glucocorticoid receptor higher than for the androgen receptor. The assumption is made that the steroids tested also behave as antagonists when binding to the glucocorticoid receptor in muscle and behave as agonists when binding to the androgen receptor. On this basis, the data allow one to compute a potential anticatabolic (PAG) and a potential anabolic (PAA) index for each compound. These indices might be of predictive value to determine whether these steroids exert their anabolic action in muscle through the glucocorticoid receptor or through the androgen receptor. The data also make it unlikely that satellite cells are a preferential target for anabolic steroids in muscle.     

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.     

The regulation of hepatic tyrosine aminotransferase

Tyrosine aminotransferase induction has been studied in hepatocytes from untreated, partially and fully glucocorticoid-induced rats: enzyme activities were initially 12.9 +/- 1.7 (n = 16), 41.4 +/- 3.2 (n = 6) and 117.9 +/- 10.5 (n = 7) munits/mg protein, respectively. Untreated or fully induced hepatocytes maintain initial levels, whereas partially induced hepatocytes increase their tyrosine aminotransferase activity even in the presence of actinomycin D. Fully induced hepatocytes possess a normal protein synthetizing machinery and the mechanisms to degrade selectively tyrosine aminotransferase. The effect of progesterone treatment is consistent with these cells retaining a high dexamethasone level. Glucagon induces tyrosine aminotransferase via its second messenger, cyclic AMP. This induction decreases dramatically with in vivo glucocorticoid treatment. Time courses and effects of inhibitors are consistent with these in vivo and in vitro treatments being alternative methods of inducing tyrosine aminotransferase by the same basic pretranslational step.     

Glucocorticoid regulation of hepatic cytosolic glucocorticoid receptors in vivo and its relationship to induction of tyrosine aminotransferase

Regulation of rat hepatic cytosolic glucocorticoid receptors was studied using our newly developed exchange assay. Injecting 1 mg of dexamethasone or corticosterone into 150-250 g adrenalectomized rats caused a rapid decline in glucocorticoid receptor binding. Glucocorticoid receptor levels were depressed 80-90% in less than 15 min after hormone treatment, and remained low for about 24-48 h after glucocorticoid administration. 80-90% of glucocorticoid receptor binding was regenerated by 48 h, and complete binding was recovered by 72 h. Regenerated glucocorticoid receptor binding (48-72 h after first hormone injection) could be re-depressed by a second injection of the hormone. Similar results were obtained using normal (intact) rats. Optimum induction of tyrosine aminotransferase activity was obtained within 2 h following the first hormonal injection. Induction of tyrosine aminotransferase activity (measured 2 h after a second injection of the glucocorticoid) correlated with glucocorticoid receptor levels. Thus, 1 mg of dexamethasone or corticosterone greatly enhanced the liver tyrosine aminotransferase activity in the adrenalectomized rats (not previously hormone treated) and in adrenalectomized rats previously injected (48-72 h) with 1 mg of the glucocorticoid hormone. Enhancement of tyrosine aminotransferase activity was lowest 16-24 h after the first hormone injection (when receptor levels were extremely low). These results indicate that the induction of liver tyrosine aminotransferase activity by glucocorticoid hormones is correlated with cytosolic glucocorticoid receptor levels.     

Regulation of cytosolic aspartate aminotransferase mRNAs in the Fao rat hepatoma cell line by dexamethasone, insulin and cyclic AMP

Glucocorticoid hormones increase the activity of cytosolic aspartate aminotransferase (cAspAT) in the Fao rat hepatoma cell line. Maximal increase (6-10-fold) was observed 48 h following the addition of the glucocorticoid agonist dexamethasone at a concentration of 0.1 microM. The effect of dexamethasone was specific since it was not mimicked by sex steroids and was inhibited by the glucocorticoid antagonist RU 486. Insulin (0.1 microM) inhibited by more than 50% the induction of cAspAT by glucocorticoids. The cAMP analog, 8-bromoadenosine 3',5'-monophosphate (Br8cAMP, 0.5 mM), potentiated the effect of dexamethasone (2-3-fold) and partially relieved the inhibitory effect of insulin on the induction by dexamethasone. Both insulin and Br8-cAMP had no significant effect on basal activity. The mitochondrial isoenzyme was insensitive to the various hormonal treatments. Northern blot analysis revealed the presence of two major (2.1-kb and 1.8-kb) and one minor (4-kb) mRNA species hybridizing with a rat cAspAT probe. The regulation of these mRNAs by glucocorticoids, insulin and cAMP correlated with the variation of the cAspAT activity, suggesting that these hormones act at the pretranslational level. We compared the regulation of cAspAT mRNAs with those of tyrosine aminotransferase mRNA. Both were similarly increased by dexamethasone but the latter was also increased by cAMP even in the absence of the glucocorticoid agonist. In addition, the increase in tyrosine aminotransferase mRNA was inhibited by cycloheximide whereas the increase in cAspAT mRNAs was not. These results show that there are significant differences in the regulation of cAspAT and tyrosine aminotransferase by glucocorticoids and other hormones, although both enzymes probably contribute to the same metabolic pathway.     

Glucocorticoid hormone receptor mediated induction of metallothionein synthesis in HeLa cells

HeLa cells grown in chemically defined medium lacking glucocorticoids synthesize metallothioneins, low molecular-weight heavy-metal binding proteins. Dexamethasone and hydrocortisone increase the rate of metal-lothionein synthesis five- to ten-fold. Maximal induction is achieved with 10^–8M dexamethasone and 10^–7M hydrocortisone. Half-maximal induction is achieved at 5 ± 10^–9M dexamethasone and 5 ± 10^–8M hydrocortisone. Although carried for many generations in the absence of any glucocorticoids, HeLa cells (clone S) contain 25,000 specific ³H-dexamethasone receptors that translocate into the nucleus after one hour of incubation. ³H-dexamethasone binds to a single class of receptors with an apparent Kd = 18.8 nM. A variety of steroids can be classified into three classes, based on their effect on metallothionein synthesis: (a) full agonists (optimal inducers), (b) intermediate effectors which have either partial agonist or antagonist activities, and (c) inactive steroids. There is a correlation between the effects on metallothionein synthesis of different steroids and their ability to compete with ³H-dexamethasone binding. We conclude that metallothionein is induced in HeLa cells by a glucocorticoid receptor mediated mechanism.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

Metyrapone modulation of tyrosine aminotransferase induction by dexamethasone in cultured hepatocytes

Metyrapone, an inhibitor of cytochrome P-450-dependent monooxygenases, enhanced the induction of tyrosine aminotransferase by dexamethasone in primary cultures of hepatocytes, while it had no effect on the basal level of the enzyme activity in the absence of the hormone. The amplification of the hormonal induction of tyrosine aminotransferase activity was strictly correlated with the concentration and with the inhibitory action of the compound on cytochrome P-450. The phenomenon occurred even at the maximally effective concentrations of dexamethasone, thus showing that metyrapone is a 'Glucocorticoid Potency Amplifier'. The dexamethasone activity amplification by metyrapone could be the consequence of a modulation of the glucocorticoid biotransformations due to the cytochrome P-450 inhibitor.     

Glucocorticoid induction of tyrosine aminotransferase in kidney cortex

It has recently been reported that the glucocorticoid receptors present in kidney occur as two distinct forms which are segregated in the cortex and the medulla. We were interested in determining if glucocorticoid induction of the enzyme tyrosine aminotransferase (L-tyrosine: 2 oxoglutarate aminotransferase, E.C.2.6.1.5) also differed in these two areas of the kidney. Administration of the synthetic glycocorticoid, dexamethasone, resulted in a 2-fold induction of tyrosine aminotransferase in kidney cortex of adrenalectomized rats and no induction of the enzyme in kidney medulla. Examination of this response in rat brain revealed no induction of the enzyme by dexamethasone in this tissue.     

The influence of culture conditions on the induction of tyrosine aminotransferase by cyclic nucleotides in rat hepatoma cells.

The increase in tyrosine aminotransferase activity which occurs in rat hepatoma tissue culture (HTC) cells in response to cyclic AMP analogs has been shown to be an enzyme induction, similar to the larger response observed in certain other hepatoma cells and in liver. A specific antibody to tyrosine aminotransferase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by N6,O2'-dibutyryl cyclic AMP in HTC cells. The effect on tyrosine aminotransferase is also produced by various 8-substituted derivatives of cyclic AMP and occurs whether or not the enzyme has been preinduced with a glucocorticoid. The response of the enzyme is greater when HTC cells are maintained in monolayer than in suspension cultures. Neither cell growth nor serum is required for the response.     

Dexamethasone induction of an inhibitor of plasminogen activator in HTC hepatoma cells

Incubation of HTC rat hepatoma cells with dexamethasone causes a rapid decrease in cellular plasminogen activator (PA) activity. Mixing experiments show the presence of an inhibitor of PA in dexamethasone-treated cells. This study investigates whether the decrease in PA activity is secondary to the induction of an inhibitor by glucocorticoids, to a decrease in the amount of PA, or to a combination of both mechanisms. PA and its inhibitor are dissociated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, and both activities are then recovered and quantitated. HTC cells have two major forms of PA with Mr values of 110,000 and 64,000. Although PA activity in the unfractionated extracts from dexamethasone-treated cells is inhibited by 90% relative to control, there is no decrease in the total activity of sodium dodecyl sulfate-dissociated PA activity, suggesting that dexamethasone causes no decrease in the amount of the enzyme. PA inhibitor activity migrates as a single band of Mr = 50,000. The total activity of inhibitor increases in a time-dependent fashion, reaching a maximum of greater than 10 times control after a 4-6-h incubation with 0.1 microM dexamethasone. The induction of inhibitor requires both RNA and protein synthesis and shows a dependence on dexamethasone concentration identical to that for responses known to be mediated by glucocorticoid receptors. We conclude that dexamethasone inhibits PA activity by inducing the synthesis of an inhibitor rather than by decreasing the amount of PA.     

Lack of glucose effect on the induction of 5-aminolevulinate synthetase and tyrosine aminotransferase in the isolated perfused rat liver.

In the isolated perfused rat liver, both 5-aminolevulinate synthetase and tyrosine aminotransferase were induced by the addition of 3.5 mmol/l allylisopropylacetamide and 58 mumol/l dexamethasone to the perfusion medium. Glucose (40 mmol/l) did not affect either the induction of these enzymes or the intrahepatic level of cyclic AMP. The results suggest that the glucose effect on the induction of 5-aminolevulinate synthetase and tyrosine aminotransferase in vivo is mediated by extrahepatic factors.     

Tyrosine kinase inhibitors block the glucocorticoid stimulation of prostaglandin endoperoxide H synthase expression in amnion cells.

Human amnion cells in primary culture respond to glucocorticoids in a characteristic fashion by the increased expression of the inducible prostaglandin endoperoxide H synthase isoenzyme, PGHS-2. Since PGHS-2 induction by agonists generally involves tyrosine kinases, we examined the possibility that the glucocorticoid stimulation of PGHS-2 in the amnion cells is tyrosine kinase dependent. PGHS-2 expression was stimulated in confluent, serum-starved amnion cells with dexamethasone, and the effect of the tyrosine kinase inhibitors herbimycin A and tyrphostins AG126, AG1288, and A1 on enzyme activity induction was determined. All four inhibitors blocked the increase of PGHS activity in a concentration-dependent manner with IC50 values of 0.077 +/- 0.05, 15.38 +/- 5.14, 20.91 +/- 3.1, and 29.77 +/- 8.21 microM, respectively (mean +/- SE, n = 4). Dexamethasone increased (approximately twofold) the tyrosine phosphorylation of 120-, 110-, and 77-kDa proteins in cell extracts, and herbimycin A selectively blocked the phosphorylation of the 110-kDa phosphoprotein. The stimulation of the steady-state level of PGHS-2 mRNA by dexamethasone was also inhibited by herbimycin A. These results suggest that glucocorticoids induce PGHS-2 expression in amnion cells with the involvement of tyrosine kinase(s). The role of tyrosine kinase dependent mechanisms in the control of amnion cell responsiveness to corticosteroids remains to be established.     

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.