Characteristics of an endogenous glucocorticoid receptor stabilizing factor.

A heat-stable preparation from rat liver cytosol increases the glucocorticoid binding capacity of rat thymocyte cytosol and stabilizes thymocyte binding capacity that has been activated by dithiothreitol. The liver preparation contains at least two heat-stable activities. The activity that increases glucocorticoid binding capacity may represent a rather short lived reducing activity that can be separated from a small M, factor (or factors) that, like molybdate, stabilizes reduced receptors. In unheated cytosol, the majority of the heat stable, receptor-stabilizing activity is present in association with macromolecules but this is released to a small molecular weight form on heating. The properties of this receptor-stabilizing factor have been examined in a rat liver cytosol assay where the lability of the receptor has been increased by washing the cytosol free of low Mr substances by filtration on an Amicon UMlO filter. The binding capacity of the washed cytosol preparation is stabilized by addition of boiled cytosol prepared from several rat tissues. The heat-stable factor activity is acid labile and is not affected by incubation with a variety of hydrolytic enzymes, including proteases, nucleases, glycohydrolases, phospholipase A, lipase, or alkaline phosphatase. The endogenous heat-stable factor(s) is not a nucleotide, a cyclic nucleotide, pyridoxal phosphate, or inorganic phosphate. Steroid-bound receptors in cytosol washed free of low molecular weight substances are more rapidly transformed to the DNA-binding state on incubation at 15 “C than those in normal cytosol. This rapid transformation is inhibited by addition of the heat-stable factor preparation. The two heat-stable activities, inhibition of receptor inactivation and inhibition of receptor transformation, coelute from Sephadex G-10 and from Dowex 1 columns. Both activities have been purified more than 200-fold with respect to Lowry-reactive material. The heat-stable factor appears to be widely distributed as the activity has been found in primitive eukaryotes, like the lobster and yeast, as well as in avians, amphibians, and mammals. This heat-stable factor(s) may play a role in maintaining the glucocorticoid receptor in the untransformed, steroid-binding form that is found in the cytoplasm.

A heat-stable preparation from rat liver cytosol increases the glucocorticoid binding capacity of rat thymocyte cytosol and stabilizes thymocyte binding capacity that has been activated by dithiothreitol. The liver preparation contains at least two heat-stable activities. The activity that increases glucocorticoid binding capacity may represent a rather short lived reducing activity that can be separated from a small M, factor (or factors) that, like molybdate, stabilizes reduced receptors. In unheated cytosol, the majority of the heat stable, receptor-stabilizing activity is present in association with macromolecules but this is released to a small molecular weight form on heating.
The properties of this receptor-stabilizing factor have been examined in a rat liver cytosol assay where the lability of the receptor has been increased by washing the cytosol free of low Mr substances by filtration on an Amicon U M l O filter. The binding capacity of the washed cytosol preparation is stabilized by addition of boiled cytosol prepared from several rat tissues. The heat-stable factor activity is acid labile and is not affected by incubation with a variety of hydrolytic enzymes, including proteases, nucleases, glycohydrolases, phospholipase A, lipase, or alkaline phosphatase. The endogenous heat-stable factor(s) is not a nucleotide, a cyclic nucleotide, pyridoxal phosphate, or inorganic phosphate.
Steroid-bound receptors in cytosol washed free of low molecular weight substances are more rapidly transformed to the DNA-binding state on incubation at 15 "C than those in normal cytosol. This rapid transformation is inhibited by addition of the heat-stable factor preparation. The two heat-stable activities, inhibition of receptor inactivation and inhibition of receptor transformation, coelute from Sephadex G-10 and from Dowex 1 columns. Both activities have been purified more than 200-fold with respect to Lowry-reactive material.
The heat-stable factor appears to be widely distributed as the activity has been found in primitive eukaryotes, like the lobster and yeast, as well as in avians, amphibians, and mammals. This heat-stable factor(s) may play a role in maintaining the glucocorticoid receptor in the untransformed, steroid-binding form that is found in the cytoplasm.
8 T o whom correspondence should be addressed.
When cytosols from a variety of tissues are incubated at 25 "C, there is a rapid loss of ability to hind glucocorticoids in a specific manner (1,2). We have shown that inactivated receptors in cytosols prepared from mouse L cells or rat thymic lymphocytes can be reactivated to the steroid binding form by a process that utilizes ATP and a heat-stable factor or factors (3-5). The activation' process in rat thymocyte cytosol also requires the presence of a reducing agent, like dithiothreitol, for maximum activation to occur (5). The requirement for a sulfhydryl-protecting agent in cytosols prepared from rat thymocytes (6), lung, and spleen (7) stands in marked contrast to many other well studied systems (e.g. Refs. 4, 7, and 8) where sulfhydryl-protecting agents have no effect on glucocorticoid binding activity.
We have shown that heated cytosol from mouse L cells increases the glucocorticoid binding capacity of rat thymocyte cytosol at 0 to 4 "C (3), and Granberg and Ballard (7) have shown that boiled rat liver cytosol increases the specific binding capacity of rat lung cytosol. Although some of this activating effect of heated cytosol preparations can be explained by the presence of sulfhydryl-reducing activity, neither the observations of Granberg and Ballard (7) nor those of our own laboratory ( 5 ) are completely explained on that basis alone. We have shown, for example, that a heated rat liver preparation both increases the specific binding capacity of thymocyte cytosol and stabilizes receptors that have been activated with dithiothreitol (5). In this paper we will show that the liver preparation cont.ains at least two heat-stable activities, one of which, like sulfhydryl-reducing agents, activates thymocyte glucocorticoid binding capacity and a second component which, like molybdate, stabilizes the unbound receptor to inactivation and inhibits transformation of the bound receptor to the DNA-binding state.

Stabilization and Activation of Receptors in Thymocyte
CytosoE-Addition of dithiothreitol to thymocyte cytosol in-' It should be noted that the term "activation" will he used throughout this paper to describe the process whereby the receptor is converted from a nonbinding form to a form that binds steroids. We use the term "transformation" to describe the process whereby the ste-

Glucocorticoid Receptor Stabilizing Factor
creases glucocorticoid binding capacity and, as shown in Fig.  1, the binding capacity is stabilized by heated liver cytosol or by molybdate. If the binding capacity is partially inactivated by incubating for 30 min a t 25 "C and the factor preparation or molybdate is added, stabilization still occurs (Fig. 1). There is no effect, however, if additional dithiothreitol is added. The heated liver preparation has no binding capacity of its own but it can activate thymocyte binding capacity in the absence of dithiothreitol as shown in Fig. 2. Although the activation provided by dithiothreitol is stabilized by molybdate, we have repeatedly observed that the activation provided by the factor preparation is not stabilized by molybdate. Unlike the factor preparation, molybdate alone does not activate or stabilize the binding capacity.
After fractionation of the boiled liver cytosol by Amicon filtration, the stabilizing activity is recovered in the fraction  Table I, if the fraction with M , < 1,000 is added to filtered cytosol at twice the concentration used in the experiment of Fig. 3, some stabilization is observed, suggesting that the M, of the stabilizing factor may be near 1,000 by this method, It is clear from Table I that  concentration of components greater than M, = 10,000 or mixing of the nonstabilizing fractions does not inhibit inactivation.
The heated factor preparation and molybdate both have an effect on the receptor in the absence of added reducing activity. This can be seen in the experiment of Fig. 4A where it is clear that in the presence of molybdate the receptor is maintained in a state such that addition of dithiothreitol at any time permits complete reactivation of the binding capacity. The effect of factor in the absence of dithiothreitol is like that of molybdate in that subsequent addition of dithiothreitol will activate the binding capacity well beyond the values observed in the presence of the reducing agent alone (Fig. 4B). It  to inactivation of the factor as that can be incubated alone or with thymocyte cytosol for many hours at 25 "C without loss of activity (Table 11).
It is clear that the stabilizing activity of the heated liver cytosol is different from the activating activity. As shown in Table 11, the activating activity (increase in binding activity in the absence of dithiothreitol) is lost when the factor is preincubated. The activating and stabilizing activity can also be separated by chromatography on Sephadex G-10 where the activating activity is eluted fEst (data not shown). Granberg and Ballard ( 7 ) proposed that the activation of rat lung receptors by heated rat liver cytosol was due to reduction of receptor sulfhydryl groups and it is likely that the activating activity we are observing represents a reducing action that is rather short lived in the system compared to that of dithiothreitol. This would explain why activation provided by the factor preparation is not maintained by molybdate.
In unheated cytosol, most of the stabilizing activity is found in association with components of M , > 30,000. As shown in  were incubated a t 20 "C, and at various times, rtliquots were removed and the specific binding capacity was assayed at 0 "C. The inset shows a similar experiment with normal and twice washed cytosol incubated at 0 "C. liver cytosol that has been washed by filtration through an Amicon UMlO filter is inactivated more rapidly than that of unfiltered cytosol a t both 0 and 20 "C (Fig. 5). When cytosol is washed more than once, the zero time binding value is lowered whereas a single washing results in little or no loss of initial binding capacity. If the filtrate from undiluted cytosol (data not shown) or the heat-stable factor preparation (Fig.  6A) is added to an equal volume of filtered liver cytosol, the rate of loss is inhibited in a concentration-dependent manner (Fig. 6B). Triamcinolone acetonide-bound receptors in filtered cytosol are transformed to the DNA-binding state more rapidly than those in normal cytosol (Fig. 7A) and the rate of transformation is inhibited by the heat-stable factor preparation (Fig. 7B). If steroid-bound receptors are transformed by heating and the factor is then added, subsequent binding to , and, at various times, aliquots were removed and the specific binding capacity was assayed. B, filtered cytosol was incubated at 20 "C for 1 h in the presence of various concentrations of heat-treated liver cytosol. After 1 hr, the specific binding capacity was determined in 0.5-ml aliquots at 0 "C as described under "Experimental Procedures." The results are presented as a percentage of the binding present in filtered cytosol at zero time. The concentration of factor preparation represents the milligrams of protein derived from the heat-treated liver cytosol which are present/ml of final incubation mixture. The arroul points to the value obtained with the 1:1 (v/v) ratio of fdtered liver cytosol and heat stable factor preparation utilized in many of the experiments in this paper. were prebound with [ 'HI triamcinolone acetonide for 2 h a t 0 "C and then incubated a t 15 "C. At various times, aliquots were removed and assayed for specific binding and for binding to DNA-cellulose. The binding to DNAcellulose is presented as the percentage of specific binding present in each 0.2-ml aliquot. B, filtered cytosol, prebound with steroid, was incubated a t 15 "C in the presence of an equal volume of buffer (0) or heated cytosol (0). In this experiment, 0.6-ml aliquots of each incubation were assayed for binding to DNA-cellulose, and the results are presented as counts/min bound/aliquot. f, heat-treated liver cytosol.

FRACTION NUMBER
r' " I 1 1.61 C FIG. 9. Sephadex G-10 chromatography of filtered heat-stable factor. Filtered boiled cytosol was chromatographed on a Sephadex G I 0 column as described under "Experimental Procedures." The eluates were monitored at 280 nm (. . . . j; 2-ml fractions were collected, combined, lyophilized, and redissolved in 2 ml of distilled water. A, each fraction was incubated with an equal volume of filtered cytosol for 45 min at 20 "C and the specific binding capacity (0) was assayed. Aliquots of each fraction were also incubated at 15 "C with an equal volume of filtered cytosol prebound with ["H]triamcinolone acetonide, and binding to DNA-cellulose (0) was assayed after 1 h, as described under "Experimental Procedures." Each fraction was assayed for ( B ) conductivity (X---Xj and inorganic phosphate (Hj and for (Cj Lowry (A) and fluorescamine (A---Aj reactive material.

FRACTION NUMBER FRACTION NUMBER
DNA is not inhibited. Sodium molybdate (10 mM) also blocks both receptor inactivation and transformation in filtered liver As shown in Table IV, incubation of heated cytosol with a variety of hydrolases does not substantially affect the stabilizing activity. Incubation at 37 "C for 120 h with 500 pg/ml of pronase or for 45 min with calf intestine alkaline phosphatase does not affect the factor activity. It is also unaffected by Nethylmaleimide.
We have previously reported that the binding capacity of rat liver cytosol is partially stabilized by several nucleotides (8). ATP inhibits inactivation of binding capacity in filtered cytosol with a maximum effect observed at 1 to 3 mM (Fig.  8A). The nonmetabolizable ATP analog AMP-PCP' produces a modest stabilizing effect and, as shown in Table V, it inhibits part of the stabilization provided by the heat-stahle factor preparation, as well as that provided by ATP by decreasing I The abbreviations used are: AMP-PCP, adenylyl (8,y-methylene)-diphosphonate; &pes, 4-(2-hydrox~~eth~~lj-I-piperazineethanesulfonic acid. cytosol.
the stabilizing activity of each to the level observed with AMP-PCP alone.
The stabilizing factor is not ATP. The boiled preparation contains only 2 p~ ATP, a concentration that is a t least two orders of magnitude too low to account for any stabilization. Extraction of heated liver cytosol with activated charcoal does not affect its stabilizing activity whiIe greater than 99% of added [''HIATP (10 mM) is removed. Other nucleotides and cyclic nucleotides should also be eliminated by the charcoal extraction procedure. Although CAMP does stabilize somewhat, it does so only a t concentrations that are at least two orders of magnitude higher than those that exist endogenously. Cyclic GMP has no stabilizing effect. The sum of these observations makes it very unlikely that the factor is a nucleotide.
Sodium phosphate produces a little stabilization with a maximum effect at about 10 mM being only about a third of that provided by the heat-stable factor preparation (Fig. 8B). The concentration of inorganic phosphate in the heated factor preparation is 7 to 8 mM. Inorganic phosphate does not inhibit transformation in filtered cytosol at any concentration and, as with other salts, at 50 mM sodium phosphate and above, transformation is stimulated. Thus, it is clear that neither the receptor-stabilizing nor the transformation-inhibiting activity can be due to inorganic phosphate. Litwack and co-workers (16,17) have suggested that pyridoxal 5"phosphate may act as an endogenous receptor "modulator." The factor cannot be pyridoxal 5"phosphate as this compound (0.01-10 mM) does not inhibit receptor inactivation in filtered cytosol. Molecular Sieve and Ion-Exchange Chromatography of the Heat-stable Factor-When boiled cytosol that has been filtered through an Amicon UMlO filter is chromatographed on Sephadex G-10 in 0.1 M Hepes buffer, both transformationinhibiting activity and receptor-stabilizing activity are recovered in a major peak that elutes after the excluded material (Fig. 9A). A small but consistent peak of stabilization is eluted with the major salt peak (determined by conductivity, Fig.  9B). The factor is separated from about 70% of the salt but it co-elutes with both inorganic phosphate and the major peak of Lowry-reactive material (Fig. 9, B and C). The major peak of stabilizing activity is recovered in the same region from longer Sephadex G-10 columns eluted with 200 mM triethylammonium bicarbonate buffer, suggesting that the elution pattern does not reflect ionic interactions with the gel matrix and that the M , is considerably less than 700 by this method.
Boiling the Sephadex G-10 peak material for 6 h in 6 N HC1 eliminates both receptor-stabilizing and transformation-inhibiting activities.
Using the receptor-stabilizing assay, we have determined that the factor in boiled cytosol is adsorbed to Dowex AG 1-X8 (HCOJ but not to Dowex 50W-H' using 0.1 mM Hepes, pH 7.35, for the column wash. In the experiment of Fig. 10, the fractions containing the major peak of receptor-stabilizing activity from Sephadex (2-10 were combined and chromatographed on Dowex 1. Both the receptor-stabilizing and transformation-inhibiting activities elute in a sharp peak at about 0.25 M triethylammonium bicarbonate. This peak contains all of the inorganic phosphate and a small amount of fluorescamine-reactive material. Although the activity is recovered in a single major peak from both Sephadex G-10 and Dowex 1, it is not yet known if the factor is a single entity or whether the activity represents the action of multiple small M , negatively charged compounds present in cytoplasm.
In the experiment of Table VI, both inhibition of receptor inactivation and transformation were assayed with multiple concentrations of the heat-st,able factor preparation at each step through Dowex 1. Inhibition was plotted as shown in Fig.  11 and units of inhibitory activity were calculated from the 50% inhibitory concentration as described under "Experimental Procedures." As can he seen from Table VI, the factor activity is separated from a considerable amount of the Lowryand fluorescamine-reactive material through the Sephadex G-10 step. Both the rate of receptor inactivation and the rate of transformation are increased by salt, and desalting with Sephadex yields some increase in activity in both assays, commensurate with elimination of the salt effect. Although the Dowex step is useful for defining the charge behavior of the factor, it is not of much value in purification as the apparent yield is low.
As the major peak of receptor-stabilizing activity and the transformation-inhibiting activity co-elute on Sephadex G-10 and Dowex 1 chromatography, it is possible that the same heat-stable component is responsible for both activities. We would speculate that the receptor-stabilizing factor that we have reported in this and previous papers (3, 5) may be identical with the small M,. heat-stable transformation inhibitor reported by others (18)(19)(20)(21). The heat-stable factor should not be considered to have a function that is unique to glucocorticoid receptor systems. Sato et al. (22) have recently reported that transformation of both androgen and estrogen receptors is also inhibited by a small M , (dialyzable) factor (or factors) present in cytosol. As the rates of receptor inactivation and transformation are both increased when the inhibitor is removed, it seems plausible to suggest that the factor plays some role in the regulation of these processes.
Although the work we have presented in this paper has focused on heat-stable factor in rat liver cytosol, we have found the same activity in boiled supernatants prepared from rat brain, heart, kidney, lung, muscle, spleen, and thymus. As shown in Table VII, receptor-stabilizing activity is present in heated liver cytosol from chicken and frog as well as mammals and it exists in primitive systems, like lobster and yeast, which are lower on the evolutionary scale than the point at which glucocorticoid receptors emerged. The boiled preparations from chicken liver, lobster, and yeast have also been tested for inhibition of transformation and found to be active in that assay as well. We have not found factor activity in calf serum or in boiled sonicates prepared from Esch,erichia coli and Salmonelfa typhimurium.