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Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible
J. steroid Biochem.
Vol.
20, No.
3, pp. 683-689,
1984
0022-4731184 $3.00 +O.OO
Printed in Great Britain. All rights reserved
Copyright 0 1984Pergamon Press Ltd
EVIDENCE THAT pH INDUCED ACTIVATION OF THE RAT HEPATIC GLUCOCORTICOID-RECEPTOR COMPLEX IS IRREVERSIBLE PETER V. BODINE*, THOMAS J. SCHMIDTQ and GERALD LtTwAck*t$ Department of Biochemistry* and The Fels Research Institute?, Temple University School of Medicine, Philadelphia, PA 19140, U.S.A. (Received 22 June 1983) Summary-The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15°C 30 min), and then returning it to pH 6.5 for a second incubation (15°C 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide @HITA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [rH]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [‘HITA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and been used to study pH induced activation of the DEAE-cellulose chromatography have glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.
INTRODUCTION
The formation of the cytoplasmic glucocorticoid-receptor complex is thought to initiate a 2-step process that ultimately leads to the translation of mRNA into specific proteins. The first step, activation [for review see 1 and 21, is believed to involve a conformational change of the steroid-receptor complex which results in the exposure of basic amino acid residues on the surface of the complex [3]. The second step, translocation, involves the movement of the activated complex from the cytoplasm to the nucleus where it then is believed to interact with acceptor sites within the chromatin. Activation has been shown to occur in viuo under physiological conditions [4, 51, and can be achieved in vitro by elevated temperature [3,6-91, increased ionic strength [3,8, lo], increased pH [l l-131, dilution [8, 141, or gel filtration [15-171. Bailly et a/.[ 181 previously presented evidence which suggested that in vitro pH induced activation of the rat hepatic glucocorticoid-receptor is reversible. They showed that incubating previously pH activated steroid-receptor complexes at a lower pH reduced the binding of those complexes to isolated nuclei; similar results were also obtained using activafPresent address: Department of Physiology and Biophysics, College of Medicine, University of Iowa, 5432 Bowen Science Building, Iowa City, Iowa 52242, U.S.A. §To whom s B 20,13-A
correspondence
should
tion induced by increased ionic strength. From this study the authors postulated that there exists an equilibrium between the unactivated and activated steroid-receptor complexes, and that one can shift this equilibrium by simply reversing the conditions. However, activation in this study was based solely on the binding of steroid-receptor complexes to isolated nuclei. We sought to further study the possible reversibility of pH induced activation of the glucocorticoid-receptor complex by using DNAcellulose and hydroxylapatite (HAP) binding to quantitate activation, and DEAE-cellulose chromatography to visualize both activated and unactivated steroid-receptor complexes. Although the combination of DNA-cellulose binding and chromatography on DEAE-cellulose has been used extensively to various aspects of glucostudy corticoid-receptor complex activation [ 11, this is the first known report of their combined usage to study possible reversibility of pH induced activation. By these criteria, we demonstrate that in vitro pH induced activation of the glucocorticoid-receptor complex is irreversible. MATERIALS
Animals
and preparations
AND METHODS
of cytosol
Adrenalectomized male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington,
be addressed. 683
684
PETER
V. BODINEet al
MA) were used 4-8 days after surgery. The 150 to 175 g rats were fed a normal chow diet and maintained on 0.9% NaCl. The animals were killed by decapitation and the livers were perfused in situ through the portal vein with cold 0.9% NaCl. The livers were removed and homogenized in an equal volume of TSM buffer (50 mM Tris-HCl, 0.25 M sucrose, 3 mM MgCl,, pH 8.0 at 0-4”C). The homogenate was centrifuged at 105,OOOg for 1 h at 04°C in a Beckman L5-50 ultracentrifuge (Beckman Instruments, Palo Alto, CA). The upper lipid layer was discarded and the cytosol was removed and stored under a liquid nitrogen gas phase until further use. Steroid binding to glucocorticoid receptors in the frozen cytosol was stable for at least 1 month. Cytosolic ([‘HITA)’
binding of [‘Hltriamcinolone and pH adjustments
acetonide
Aliquots of cytosol were diluted 2: 1 with cold TSM buffer (pH 7.2 at 0-4”C) and incubated for 2 h at 0-4”C with 60 nM [6,7-‘HITA (31.3 Ci/mmol; New England Nuclear Corp., Boston, MA) in the presence or absence of lOOO-fold excess of nonradioactive TA (Sigma Chemical Co., St. Louis, MO) to determine nonspecific binding to DNA-cellulose and hydroxylapatite. The initial pH of the cytosol was 7.5 & 0.2. pH adjustments of the cytosol were accomplished by adding 1 N HCl or 1 N NaOH (lo-15 pi/ml) at t&&C to bring the cytosol to pH 6.5 f 0.1 or 8.5 + 0.1, respectively. Hydroxylapatite
(HAP)
binding assay
The total amount of [‘HITA-receptor complexes present was measured by the hydroxylapatite technique [ 191. Aliquots (50 ~1) of the labeled cytosol were incubated in duplicate for 45 min at 04°C with 400~1 of 10% (w/v) HAP (Bio Rad Laboratories, Richmond, CA) in a KP buffer (50 mM potassium phosphate, pH 7.0 at 04C). After the incubation, 2 ml of the cold KP buffer were added to each tube. The suspensions were agitated and centrifuged for 5 min at 600g in a clinical centrifuge at 4°C. The HAP pellets obtained after three additional washes with the same buffer were resuspended in 5 ml of Liquiscint Scintillation Cocktail (National Diagnostics, Somerville, NJ). Radioactivity was determined with an average counting efficiency of 30% for tritium. DNA-cellulose
binding assay
LeFevre et aZ.[20] have previously shown that DNA-cellulose binding can be employed to differentiate between nonactivated and activated glucocorticoid-receptor complexes. This technique facilitates the measurement maximally of only 5&70x of the activated complexes. However, in a given experiment using the same batch of DNA‘Triamcinolone acetonide is the trivial name for 9-fluoro118,21 -dihydroxy- 16a,17- 1-[-methylethylidenebis(oxy)] pregna- 1,4-diene-3,20-dione.
cellulose, this maximal binding remains relatively constant, and therefore this batch assay may be employed for comparative measurements. In the present study, the binding of previously activated [‘HITA-receptor complexes to DNA-cellulose was determined by the procedure of Kalimi er a/.[lO]. Aliquots (100 ~1) of labeled cytosol were incubated in duplicate for 45 min at 04’C with 50~1 packed DNA-cellulose prepared as previously described [21]. After the incubation, 2 ml of cold TE buffer (10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid [EDTA], pH 8.0 at 04’C) were added to each tube. The suspensions were then agitated and centrifuged for 5 min at 600 g in a clinical centrifuge at 4 ‘C. The DNA-cellulose pellets obtained after three additional washes with the same buffer were resuspended in 0.8 ml TE buffer, and an aliquot (0.5 ml) was assayed for radioactivity after being added to 5 ml of Liquiscint Scintillation Cocktail. DEAE-cellulose
chromatography
Diethylaminoethyl (DEAE)-cellulose (DE-52, Whatman, Inc., Clifton, NJ) columns were prepared in 5 cm3 plastic disposable syringes (3 ml bed volume) and equilibrated with KPD buffer (5 mM potassium phosphate, 0.5 mM dithiothreitol, pH 7.6 at 04°C). A 0.5 ml aliquot of labeled cytosol was applied to the column and was washed for 10 min with KPD buffer. Bound radioactivity was eluted with a linear 5 to 400 mM KPD gradient as previously described [22]. Twenty-five 1 ml fractions were collected, and 0.5 ml aliquots of each fraction were added to 5 ml of Liquiscint Scintillation Cocktail for the determination of radioactivity. The KPD concentration was determined by measuring the conductivity of appropriate fractions with a Markson model 10 conductivity meter and comparison made with a standard curve. All chromatographic procedures were performed at 4°C. 85 + 9% of the added radioactivity was routinely recovered from the columns. RESULTS pH Induced [‘HITA-receptor
activation complexes
und the binding to DNA -cellulose
of
The effects of increasing pH on activation as measured by the binding of [‘HITA-receptor complexes to DNA-cellulose is summarized in Table 1. The pH of the cytosol was adjusted as described in Materials and Methods. Because the volume change after the pH adjustment was small (less than 1.59/,), dilution activation was insignificant [8, 141; and despite the low salt concentration of the incubation buffer (50 mM Tris-HCI) [18], the pH of the cytosol remained relatively after adjustment constant throughout the length of the experiments (_t 0.1 pH units). Aliquots of the pH adjusted cytosol were then incubated for 30 min at 15°C after which they were returned to 0°C and 10mM Na,MoO, (20pl/ml) was added to prevent further activation of the
pH Induced
activation
of steroid-receptor
complexes
685
Table 1. The effects of pH induced activation on the binding of complexes to [‘HITA-receptor DNA-cellulose PH 6.5 7.5 8.5
% Bound to DNA-cellulose* 4.1 (kO.2) 18.4 ( f 2.0) 39.2 (k0.4)
-
Aliquots of [3H]TA-labeled cytosol (prepared as described in Materials and Methods) were incubated for 30 min at 15°C and at the various pH’s. The pH adjustments of the cytosol were accomplished as described in Materials and Methods. 10 mM Na,MoO, was added to each aliquot at the end of the incubations, and they were then returned to 0°C. DNA-cellulose
and hydroxylapatite binding was then determined in duplicate as described in Materials and Methods. The results presented represent the average experiments. of two such *(DNA-cellulose binding/HAP binding) x 100.
steroid-receptor complexes [23-251. At this point, DNA-cellulose and HAP binding assays were performed as described in Materials and Methods. When cytosol was incubated at pH 6.5 prior to assay, only 4.1% (kO.2) of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% ( f 0.4) bound to DNA-cellulose when the cytosol was incubated at pH 8.5; 18.4% (f2.0) bound when the pH of the cytosol was left at 7.5, i.e. not adjusted (control). Thus, we were able to show increased activation of the glucocorticoid-receptor complex with increasing pH using the DNA-cellulose binding assay [l l-131. When aliquots of previously heat-activated cytosol (25°C 30 min) [3,69] were adjusted to pH 6.5 or 8.5, or left at pH 7.5 prior to the DNA-cellulose and HAP binding assays, the percent of the [‘HITA-receptor complexes bound to DNA-cellulose was the same (40-45x; data not shown) indicating that the binding of fully activated complexes to DNA-cellulose was not pH dependent in this range. The amount of [3H]TA-receptor complexes that bound to HAP was also constant in this pH range, indicating that steroid was not preferentially dissociating from the receptor in the pH range of 6.5 to 8.5 (data not shown). pH Induced matography
activation and DEAE-cellulose
chro-
Analysis of pH induced activation using DEAEcellulose chromatography is shown in Fig. 1. In Panel A, an aliquot of [‘HITA-receptor complexes was adjusted to pH 6.5 and then incubated at 15°C for 30 min. A 1 ml sample was then removed, returned to O”C, and 10mM Na,MoO, was added. A 0.5 ml aliquot of this sample was then processed for DEAEcellulose chromatography as described in Materials
Fig. 1. Analysis of pH induced activation of [3H]TAreceptor complexes using DEAE-cellulose chromatography. Ahquots of [‘HITA-labeled cytosol (prepared as described in Materials and Methods) were incubated at pH 6.5 (15°C 30 min) (panel A), or incubated at pH 6.5 (15°C 30 min) followed by incubation at pH 8.5 (15°C 60 min) (panel B). At the end of each incubation, 1ml samples were removed, returned to 0°C and 10 mM Na,MoO, was added. One-half ml was then removed from each sample and processed for DEAE-cellulose chromatography as described in Materials and Methods. The bound radioactivity was eluted in 25 (1 ml) fractions with a linear 5 to 400 mM KPD gradient. The pH adjustments for panel B were performed as described in Materials and Methods, and required 3-5 min to be completed. 8.4 x 10’ dpm were applied to the column in panel A, and 5. I x 10’ dpm were applied to the column in panel B.
and Methods; 76.4% of the total radioactivity originally added to the column was recovered. The chromatograph in panel A shows that only 19.6% of the recovered [3H]TA-receptor complexes are in the activated peak (eluted at 50 mM KPD) [22], while only 3.9% of these labeled steroid-receptor complexes bound to DNA-cellulose (data not shown). The remaining cytosol, which had been returned to 0°C at the end of the first incubation in order to minimize the possibility of further activation of steroid-receptor complexes [3,6-91, was then adjusted from pH 6.5 to pH 8.5 as described in Materials and Methods, and incubated for an additional 60 min at 15°C. A 1 ml sample was then removed and treated as described above. A 0.5 ml aliquot of this sample was then processed for DEAEcellulose chromatography as described in Materials and Methods. The results of this experiment are presented in panel B of Fig. 1; 79.6% of the total radioactivity added to the column was recovered. The chromatograph shows that 90.1 y0 of the recovered [3H]TApreceptor complexes are in the activated position (eluted at 50mM KPD), while 38% of the labeled steroid-receptor complexes bound to DNAthe cellulose (data not shown). Thus, [3H]TA-receptor complexes not activated at pH 6.5 were activated when they were subsequently incubated at pH 8.5. It is important to note that 39% less radioactivity was applied to the column in panel B than the one in panel A. This is most likely due to dissociation of some steroid-receptor complexes
686
PETER V. BODINE rt al.
which has been previously shown to occur at equal unactivated activated and rates for both Conseglucocorticoid-receptor complexes [ 171. quently, it can be assumed that the loss of radioactivity was not preferentially from the unactivated peak (eluted at 200 mM KPD). Also, the total radioactivity in the activated peak of panel B does increase 2.5-fold over the activated peak in panel A.
i;
I51 PH change
I
s
/
Following the same format as in Fig. 2, DEAEcellulose chromatography was performed (as described in Materials and Methods) in order to visualunactivated ize activated and both the [“HITA-receptor complexes after each of the pH adjustments. The results of this experiment are shown in Fig. 3. Panel A of Fig, 3 is a DEAE-cellulose chromatograph performed at the end of the first 30min incubation (pH 8.5, 15 C); 67.5”,, of the bound [‘HITA-receptor complexes are in the activated pos-
I
change I I
I
30
0 Msn
Analysis qf‘ udjustments in pH on the elution qj [‘HITA-receptor complexes ,fiom DEAE-cellulosr
pH
;
/
20
c
To test for the reversal of pH induced activation [18], the following format was followed. An aliquot of [3H]TA-receptor complexes was initially adjusted to pH 8.5 and incubated for 30 min at 15°C at which time a 1 ml sample was removed, returned to D”C, 10 mM Na?MoO, was added, and the sample was then processed for DNA-cellulose and HAP binding as described in Materials and Methods. The remaining cytosol was returned to O‘C, and the pH was adjusted from 8.5 to 6.5 as described in Materials and Methods. The cytosol was then incubated for 30 min at 15 C, at which time another 1 ml sample was removed and treated as above. The remaining cytosol was again returned to 0°C. and its pH was adjusted back to 8.5. This was followed by another 30 min incubation at IS C, at which time a third 1 ml sample was removed and treated as above. The results of this experiment are presented in Fig. 2. After 30 min at 15 C and pH 8.5, 39.2’fd (iO.4) of the [iH]TA--receptor complexes bound to DNAcellulose. When an aliquot of the same cytosol was then adjusted to pH 6.5, 47.0’:;, (& 7.2) of the labeled steroid-receptor complexes bound to DNA-cellulose after another 30 min incubation at 15 C; 43.9?: (+ I .9) bound when the pH of the cytosol was left at pH 8.5 during the second incubation (control). Thus, as determined by DNA-cellulose binding, pH induced activation of the glucocorticoid--receptor complex was irreversible. When the cytosol at pH 6.5 was returned to pH 8.5 and a third incubation at 15°C was performed, 40.57, (i 1.3) of the pH]TA-receptor complexes bound to DNA-cellulose; 52.2”;, ( + 7. I) of the labeled steroid-receptor complexes were bound under control conditions.
I 2nd
I
I
I 10
/ 0
I 30
a+ 15°C
Fig. 2. The effects nf adjustments in pH on the binding of [‘HITA-receptor complexes to DNA~cellulose. An ahGot of [‘HlTA-labeled cvtosol (nrenared as described in Materials-and Methods) was initi‘ally-adjusted to pH 8.5 (- a--) and then incubated at 15°C for 30 min, at which time a 1ml sample was removed, returned to O’C, 1OmM Na2Mo0, was added, and the sample was then processed for DNAcellulose and HAP binding as described in Materiais and Methods. The remaining cytosol was divided into two aliquots and both were returned to 0°C. One aliquot was adjusted to pH 6.5 (-a-), the other was left at pH 8.5 (-•-_), after which both were incubated a second time for 30min at 15°C. At the end of this second incubation. another 1 ml sample was removed from each aliquot and treated as above. The remaining cytosoi of both aliyuots were again returned to WC. The one at pH 6.5 was adjusted back to pH 8.5 (--a-), and the aliquot at pH 8.5 was left at pH 8.5 (-+--), after which both were incubated a third time at 15°C for 30 min, at which time another I ml sample was removed from each aliquot and treated as above. The data presented represent the average of two such experiments. The pH adjustments were performed as described in Materials and Methods and required 3-5 min to be completed; this time is not included in the time scale (abcissa of Figure). ition (eluted at 65 mM KPD), and 76.20/, of the added radioactivity was recovered from the column. The remaining cytosol from this step was adjusted to pH 6.5 and another 30 min incubation was performed at 15C. The DEAE-cellulose chromatograph performed at the end of this second incubation is shown in pane1 B of Fig. 3; 74.4% of the bound [3H]TA-receptor complexes are in the activated peak (65 mM KPD), and 94.6% of the added radioactivity was recovered from the column. Thus, there was no shift of [‘HITA-receptor complexes from the activated (eluted at 65 mM KPD) to the unactivated peak (eluted at 250mM KPD) when the pH of the cytosol was shifted from 8.5 to 6.5 for the second incubation, i.e. there was no reversal of activation. The remaining cytosol from this step was then returned to pH 8.5 and a third 30 min. 15°C incubation was performed. The DEAE-cellulose chromatograph from the end of this incubation is shown in panel C of Fig. 3; 92.4% of the bound [‘HITA-receptor complexes are in the activated position (eluted at 55 mM KPD), and 89.2% of the added radioactivity was recovered from the column. Thus, the amount of [iH]TA-receptor complexes associated with the activated peak of DEAE-cellulose increased continuously with increasing incubation at 15°C from an initial value of 67.5”,/, after the first incubation
pH Induced
activation
of steroid-receptor
.J 60
Froctlon
number
( I ml/fraction)
Fig. 3. Analysis of adjustments in pH on [)H]TA-receptor complex activation using DEAE-cellulose chromatography. The experimental procedure followed was the same as in Fig. 2 except that DEAE-cellulose chromatography was performed at the end of each 30 min incubation at 15’C. One ml samples were removed at the end of each incubation, returned to 0°C 10 mM Na,MoO, was added, and 0.5 ml of each sample was removed and processed for DEAEcellulose chromatography as described in Materials and Methods. The bound radioactivity was eluted in 25- to 30-( 1 ml) fractions with a linear 5 to 400 mM KPD gradient. The pH adjustments were performed as described in Materials and Methods and required 3-5 min to be completed. Panel A: end of first 30mit-1, 15°C incubation at pH 8.5; Panel B: end of second 30 min, 15°C incubation at pH 6.5; Panel C: end of third 30 min, 15°C incubation at pH 8.5. 1.3 x IO6 dpm were applied to the column in panel A, I .O x lo6 dpm were applied to the column in panel B, and 8.3 x 10Sdpm were applied to the column in pane1 C.
(pH 8.5) to 74.4 and 92.4% after the second (pH 6.5) and third (pH 8.5) incubations, respectively. DISCUSSION
In the present study, the possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. When [3H]TA-labeled cytosol was activated at pH 8.5 (15°C for 30 min), there was a 9.6-fold increase in the binding of [‘HITA-receptor complexes to DNA-cellulose as compared with cytosol incubated under same conditions at pH 6.5 (Table 1). Likewise, 3.4-fold more [3H]TA-receptor com-
complexes
687
plexes were in the activated peak on DEAE-cellulose (eluted at 65 mM KPD) when cytosol was incubated at pH 8.5 (Fig. 3A) than cytosol that was incubated under the same conditions at pH 6.5 (Fig. IA). This confirms previous reports which demonstrated that the percentage of activation of the rat hepatic glucocorticoid-receptor complex is greater at higher pH (11-13 and 18) and that anion-exchange chromatography can be used to study pH induced activation [l 1, 121. Analysis on DEAE-cellulose also showed that incubation at pH 8.5 resulted in an increase in activation when compared to cytosol previously incubated at pH 6.5 (Fig. 1). This confirms an earlier report for pH induced activation of the glucocorticoid-receptor complex using the isolated nuclear binding assay [ 181. When previously pH activated cytosol (pH 8.5, 15°C 30 min) was incubated a second time at pH 6.5, no loss of binding to DNA-cellulose was detected (Fig. 2). Similarly. no conversion of activated [3H]TA-receptor complexes to unactivated complexes as measured by DEAE-cellulose chromatography was detected when previously pH activated cytosol was incubated a second time under the same conditions at pH 6.5 (Fig. 3A and 3B). Thus, by these in tiitro pH induced activation of the criteria, glucocorticoid-receptor complex was determined to be irreversible. This conclusion contradicts a previous study by Milgrom and his colleagues [ 181 which showed that pH induced activation, as measured by the binding of rat hepatic [‘HITA-receptor complexes to isolated nuclei, was reversible. Although it might be argued that the DNA-cellulose binding assay under these circumstances may not be fulfilling the requirements of the isolated nuclei technique [6, 181, the results obtained from DEAE-cellulose chromatography cannot be ignored: there clearly was no conversion of [3H]TA-receptor complexes from the activated to the unactivated form on DEAE-cellulose when the pH was adjusted from 8.5 to 6.5 (Fig. 3). In this study we define reversal of glucocorticoid-receptor complex activation as the following: (i) the failure of previously activated [3H]TA-receptor complexes to bind to DNAcellulose, and (ii) the conversion of previously activated [3H]TA-receptor complexes from the activated to the unactivated form as measured by DEAEcellulose chromatography. We feel that both of these criteria must be satisfied before activation can be concluded as being reversible. Hutchens et a/.[261 recently reported that RNA induces reversal of glucocorticoid-receptor complex activation. However, in their study, activation was defined in terms of the ability of steroid-receptor complexes to bind to ATP-agarose. Since RNA inhibits the binding of the activated estrogenreceptor ccmplex to DNA [27], it is not surprising that RNA, being a polyanion, combines with activated glucocorticoid-receptor complexes and thus inhibits their binding to other polyanions, such as ATP-agarose or DNA-cellulose.
688
PETER V. BODINE et al.
Consequently, this finding does not prove that the activation step itself is reversed. Likewise, pyridoxal 5’-phosphate (PLP) has been shown to inhibit the binding of activated glucocorticoid-receptor as several other steroid complexes, as well hormone-receptor complexes to both DNA-cellulose and isolated nuclei [28-321. By this criteria alone (i.e. DNA-cellulose and nuclei binding), activation would be concluded as being reversible in the presence of PLP. However, a later report from this laboratory showed that activation, as measured by DEAEcellulose chromatography, was actually potentiated by PLP [33]. Thus, by our criteria for reversibility, activation is not reversed by PLP. Recently, Raaka and Samuels[34] explored the reversibility of in uiuo glucocorticoid receptor activation in GH, cells, a rat pituitary tumor cell line. Using dense amino acid labeling and sucrose gradient centrifugation, they demonstrated that an equilibrium exists between a high molecular weight unactivated steroid-receptor complex (sedimentation coefficient of 10s) and a low molecular weight activated complex (sedimentation coefficient of 4s). The 10s complex is postulated as being an oligomer, and the 4S complex is though to be the active monomer which translocates to the nucleus and binds to DNA. The authors showed that as the concentration of steroid increased, the amounts of the 4S complex in the cytosol and nucleus increased, while the amount of the 10s complex in the cytosol decreased. But, when steroid was removed, the amounts of the cytosolic and nuclear 4S complex decreased, and the amount of the cytosolic 10s complex increased. The authors conclude from this that in viuo activation of the glucocorticoid-receptor complex is reversible. However, they admit that although their data suggests that each step of the activation process is reversible, it may be possible, for one or more of the steps, that the apparent reversibility is due to separate reaction pathways in each direction. Thus, their results do not necessarily contradict our conclusion that in vitro pH induced activation of the glucocorticoid-receptor complex is irreversible, because the step that we conclude is irreversible is the monomeric conversion of the unactivated steroid-receptor complex to the activated form, perhaps via a covalent modification such as a dephosphorylation [23,24, 351. However, although we have concluded that this individual step is irreversible, other steps of the activation pathway could result in activated nuclear steroid-receptor complexes appearing in the cytoplasm as unactivated complexes; this would be true, for example, if recycling of the receptor occurred after nuclear binding [ 11. Vedeckis[36] has also proposed a subunit dissociation model for the activation of the glucocorticoid receptor in mouse AtT-20 cells, a pituitary tumor cell line. In this study, activation was accomplished in vitro by warming cytosol for 1 h at 25 C and then passing it over a Sephadex G-25
column. Aliquots of the cytosol were then incubated with or without 20mM Na,MoO, at 0_4”C, and samples were then analyzed via DEAE-cellulose chromatography. No significant conversion of the activated form to the unactivated form was seen on DEAE-cellulose when activated cytosol was incubated with or without Na,MoO, prior to chromatography. Thus, this supports our results for the in vitro pH induced activation of the rat hepatic glucocorticoid-receptor complex. However, when similar studies were performed in vivo using sucrose gradient centrifugation to measure activation (S. B. Eastman and W. V. Vedeckis, unpublished observations), an apparent reversibility of activation was seen when cytosol was incubated with Na,MoO,. The author postulates that in vitro heat activation may destroy a necessary component of the in viva reversal process. However, although this may be possible, it seems unlikely that in our case such a factor is destroyed simply by pH induced activation of the cytosol at 15°C. Our conclusion that pH induced activation of the glucocorticoid-receptor complex is irreversible supports a previous report by Goidl et a/.[171 which implied that heat activation of the rat hepatic glucocorticoid-receptor complex was also irreversible, as measured by DNA-cellulose binding. The present study, however, is the first known reported time that DEAE-cellulose chromatography has been reversibility of pH induced used to study activation [l]. The use of this procedure to visualize both activated and unactivated [3H]TA-receptor complexes has allowed us to directly demonstrate that in vitro pH induced activation, at least, is irreversible. Acknowledgements-This work was supported by Research Grants: AM13531 from NIADDK, NIH and by PCM 8215844 from the NSF and by CA 12227 from the NCI, NIH. to the Fels Research Institute. P.V.B. is a reciuient of a Graduate School Fellowship, and is supported by an NIH Basic Research Support Grant to the Department of Biochemistry, Temple University School of Medicine.
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activation
of steroid-receptor
the thermal activation of glucocorticoid hormone receptor complex. J. biol. Chem. 251 (1976) 47584762. Baxter J. D., Rousseau G. G., Benson M. C., Garcea R. L., Ito J. and Tomkins G. M.: Role of DNA and specific cytoplasmic receptors in glucocorticoid action. Proc. natn Acad. Sci.. U.S.A. 69 (1972) 1892-1896. Higgins S. J., Rousseau G. G.; Baxter J. D. and Tomkins G. M.: Early events in glucocorticoid action. Activation of steroid receptor and its subsequent specific nuclear binding studied in a cell-free system. J. biol. Chem. 248 (1973) 5866-5872. Kalimi M., Beato M.‘and Feigelson P.: Interaction of glucocorticoids with rat liver nuclei. I. Role of cytosol proteins. Biochemistry 12 (1973) 3365-3371. Kalimi M., Colman P. and Feigelson P.: The “activated” hepatic glucocorticoid-receptor complex. Its generation and properties. J. biol. Chem. 250 (1975) 108~1086. Parchman L. G., Goidl J. and Litwack G.: Basic amino the stimulate the activation of acids glucocorticoid-receptor complex. FEBS Lerl. 79 (1977) 25-28. Parchman L. G. and Litwack G.: Resolution of activated and unactivated forms of the glucocorticoid receptor from rat liver. Archs biochem. Biophys. 183 (I 977) 374382. Parchman L. G., Markovic R. D. and Litwack G.: Multiple forms of the glucocorticoid receptor determined by its state of activation. In Multiple Molecular Forms qf Steroid Hormone Receptors (Edited by M. K. Agarwal). Elsevier/North Holland, New York (1977) pp. 1-15. Goidl J. A., Dolan K. P., Cake M. H., Parchman L. G. and Litwack G.: Reversible activation of the glucocorticoid receptor. Fedn Proc. 35 (1976) 1365a (abstract). Bailly A., Sallas N. and Milgrom E.: A low molecular weight inhibitor of steroid receptor activation. J. biol. Chem. 252 (1977) 858-863. Cake M. H., Goidl J. A., Parchman L. G. and Litwack G.: Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor. Biochem. biophys. Res. Commun. 71 (1976) 45-52. Goidl J. A., Cake M. H., Dolan K. P., Parchman L. G. and Litwack G.: Activation of the rat liver glucocorticoid-receptor complex. Biochemistry 16 (1977) 2125-2130. Bailly A., Sovouret J.-F., Sallas N. and Milgrom E.: Factors modifying the equilibrium between activated and non-activated forms of steroid-receptor complexes. Eur. J. Biochem. 88 (1978) 623-632. Erdos T., Best-Belpomme M. and Bessada R.: A rapid assay for binding of estradiol to uterine receptor(s). Analyt. Biochem. 37 (1970) 244252. LeFevre B., Bailly A., Sallas N. and Milgrom E.: Activated steroid-receptor complex. Comparison of assays using DNA-cellulose or homologous nuclei. Biochim. biophys. Acta 585 (1979) 266-272.
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B. and Herrick G.: DNA-cellulose chro21. Alberts matography. Meth. Enzym. 21 (1971) 198-217. E. B.: Characteristics of two 22. Sakaue Y. and Thomoson forms of glucocorticoid hormone-receptor complex separated bi DEAE-cellulose column chromatography. Biochem. biovhvs. Res. Commun. 77 (1977) 533-541. 23. Bamett C. A., Schmidt T. J. and Litwack G.: Elfects of calf intestinal alkaline phosphatase, phosphatase inhibitors and phosphorylated compounds on the rate of activation of glucocorticoid receptor complexes. Biochemistrv 19 (1980) 5446-5455. N. D., Sando 24. Leach K. L., Dahmer M. K., Hammond J. J. and Pratt W. B: Molvbdate inhibition of glucocorticoid receptor inactivation and transformation. J. biol. Chem. 254 (1979) 1188411890. J. M. and Thompson E. B.: 25. Schmidt T. J., Harmon “Activation-labile“ glucocorticoid-receptor complexes of a steroid-resistant variant of CEM-C7 human lymphoid cells. Nature, London 286 (1980) 507-510. F. S. and Hawkins E. F.: 26. Hutchens W. T., Markland RNA induced reversal of glucocorticoid receptor activation. Biochem. biophys. Res. Commun. 105 (1982) 2&27. 27. Feldman M., Kallos J. and Hollander P.: RNA inhibits estrogen receptor binding to DNA. J. biol. Chem. 256 (1981) 1145-l 148. 28. Cake M. H., DiSorbo D. M. and Litwack G.: Effect of pyridoxal phosphate on the binding site of the activated hepatic glucocorticoid receptor. J. biol. Chem. 253 (1978) 48864891. 29. Cake M. H. and Litwack G.: Effect of methylxanthines on binding of the glucocorticoid receptor- to DNAcellulose and nuclei. Eur. J. Biochem. 82 (1978) 97-103. T. G. and Cidlowski J. ~A.: Specific 30. Muldoon modification of rat uterine estrogen receptor by pyridoxal 5’-phosphate. J. biol. Chem. 255 (1980) 310&3107. 31. Miiller R. E., Traish A. and Wotiz H. H.: Effects of pyridoxal 5’-phosphate on uterine estrogen receptor. I. Inhibition of nuclear binding in cell-free system and intact uterus. J. biol. Chem. 255 (1980) 40624067. 32. DiSorbo D. M. and Litwack G.: ‘The use of pyridoxal 5’-phosphate as a tool in the study of steroid receptors. In Biochemical Actions of Hormones (Edited by G. Litwack). Academic Press, New York, Vol. 9 (1982) pp. 205-219. 33. Sekula B. C., Schmidt T. J., Oxenham E. A., DiSorbo D. M. and Litwack G.: Dual effects of pyridoxal 5’-phosphate on glucocorticoid-receptor complexes. Biochemistry 21 (1982) 2915-2922. 34. Raaka B. M. and Samuels H. H.: The glucocorticoid receptor in GH, cells. J. biol. Chem. 258 (1983) 417425. 35. Sando J. J., Hammond N. D., Stratford C. A. and Pratt W. B.: Activation of thymocyte glucocorticoid receptors to the steroid binding form. The roles of reducing agents, ATP, and heat stable factors. J. biof. Chem. 254 (1979) 47794789. 36. Vedeckis W. V.: Subunit dissociation as a possible mechanism of glucocorticoid receptor activation. Biochemistry 22 (1983) 1983-1989.
Vol.
20, No.
3, pp. 683-689,
1984
0022-4731184 $3.00 +O.OO
Printed in Great Britain. All rights reserved
Copyright 0 1984Pergamon Press Ltd
EVIDENCE THAT pH INDUCED ACTIVATION OF THE RAT HEPATIC GLUCOCORTICOID-RECEPTOR COMPLEX IS IRREVERSIBLE PETER V. BODINE*, THOMAS J. SCHMIDTQ and GERALD LtTwAck*t$ Department of Biochemistry* and The Fels Research Institute?, Temple University School of Medicine, Philadelphia, PA 19140, U.S.A. (Received 22 June 1983) Summary-The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15°C 30 min), and then returning it to pH 6.5 for a second incubation (15°C 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide @HITA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [rH]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [‘HITA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and been used to study pH induced activation of the DEAE-cellulose chromatography have glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.
INTRODUCTION
The formation of the cytoplasmic glucocorticoid-receptor complex is thought to initiate a 2-step process that ultimately leads to the translation of mRNA into specific proteins. The first step, activation [for review see 1 and 21, is believed to involve a conformational change of the steroid-receptor complex which results in the exposure of basic amino acid residues on the surface of the complex [3]. The second step, translocation, involves the movement of the activated complex from the cytoplasm to the nucleus where it then is believed to interact with acceptor sites within the chromatin. Activation has been shown to occur in viuo under physiological conditions [4, 51, and can be achieved in vitro by elevated temperature [3,6-91, increased ionic strength [3,8, lo], increased pH [l l-131, dilution [8, 141, or gel filtration [15-171. Bailly et a/.[ 181 previously presented evidence which suggested that in vitro pH induced activation of the rat hepatic glucocorticoid-receptor is reversible. They showed that incubating previously pH activated steroid-receptor complexes at a lower pH reduced the binding of those complexes to isolated nuclei; similar results were also obtained using activafPresent address: Department of Physiology and Biophysics, College of Medicine, University of Iowa, 5432 Bowen Science Building, Iowa City, Iowa 52242, U.S.A. §To whom s B 20,13-A
correspondence
should
tion induced by increased ionic strength. From this study the authors postulated that there exists an equilibrium between the unactivated and activated steroid-receptor complexes, and that one can shift this equilibrium by simply reversing the conditions. However, activation in this study was based solely on the binding of steroid-receptor complexes to isolated nuclei. We sought to further study the possible reversibility of pH induced activation of the glucocorticoid-receptor complex by using DNAcellulose and hydroxylapatite (HAP) binding to quantitate activation, and DEAE-cellulose chromatography to visualize both activated and unactivated steroid-receptor complexes. Although the combination of DNA-cellulose binding and chromatography on DEAE-cellulose has been used extensively to various aspects of glucostudy corticoid-receptor complex activation [ 11, this is the first known report of their combined usage to study possible reversibility of pH induced activation. By these criteria, we demonstrate that in vitro pH induced activation of the glucocorticoid-receptor complex is irreversible. MATERIALS
Animals
and preparations
AND METHODS
of cytosol
Adrenalectomized male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington,
be addressed. 683
684
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MA) were used 4-8 days after surgery. The 150 to 175 g rats were fed a normal chow diet and maintained on 0.9% NaCl. The animals were killed by decapitation and the livers were perfused in situ through the portal vein with cold 0.9% NaCl. The livers were removed and homogenized in an equal volume of TSM buffer (50 mM Tris-HCl, 0.25 M sucrose, 3 mM MgCl,, pH 8.0 at 0-4”C). The homogenate was centrifuged at 105,OOOg for 1 h at 04°C in a Beckman L5-50 ultracentrifuge (Beckman Instruments, Palo Alto, CA). The upper lipid layer was discarded and the cytosol was removed and stored under a liquid nitrogen gas phase until further use. Steroid binding to glucocorticoid receptors in the frozen cytosol was stable for at least 1 month. Cytosolic ([‘HITA)’
binding of [‘Hltriamcinolone and pH adjustments
acetonide
Aliquots of cytosol were diluted 2: 1 with cold TSM buffer (pH 7.2 at 0-4”C) and incubated for 2 h at 0-4”C with 60 nM [6,7-‘HITA (31.3 Ci/mmol; New England Nuclear Corp., Boston, MA) in the presence or absence of lOOO-fold excess of nonradioactive TA (Sigma Chemical Co., St. Louis, MO) to determine nonspecific binding to DNA-cellulose and hydroxylapatite. The initial pH of the cytosol was 7.5 & 0.2. pH adjustments of the cytosol were accomplished by adding 1 N HCl or 1 N NaOH (lo-15 pi/ml) at t&&C to bring the cytosol to pH 6.5 f 0.1 or 8.5 + 0.1, respectively. Hydroxylapatite
(HAP)
binding assay
The total amount of [‘HITA-receptor complexes present was measured by the hydroxylapatite technique [ 191. Aliquots (50 ~1) of the labeled cytosol were incubated in duplicate for 45 min at 04°C with 400~1 of 10% (w/v) HAP (Bio Rad Laboratories, Richmond, CA) in a KP buffer (50 mM potassium phosphate, pH 7.0 at 04C). After the incubation, 2 ml of the cold KP buffer were added to each tube. The suspensions were agitated and centrifuged for 5 min at 600g in a clinical centrifuge at 4°C. The HAP pellets obtained after three additional washes with the same buffer were resuspended in 5 ml of Liquiscint Scintillation Cocktail (National Diagnostics, Somerville, NJ). Radioactivity was determined with an average counting efficiency of 30% for tritium. DNA-cellulose
binding assay
LeFevre et aZ.[20] have previously shown that DNA-cellulose binding can be employed to differentiate between nonactivated and activated glucocorticoid-receptor complexes. This technique facilitates the measurement maximally of only 5&70x of the activated complexes. However, in a given experiment using the same batch of DNA‘Triamcinolone acetonide is the trivial name for 9-fluoro118,21 -dihydroxy- 16a,17- 1-[-methylethylidenebis(oxy)] pregna- 1,4-diene-3,20-dione.
cellulose, this maximal binding remains relatively constant, and therefore this batch assay may be employed for comparative measurements. In the present study, the binding of previously activated [‘HITA-receptor complexes to DNA-cellulose was determined by the procedure of Kalimi er a/.[lO]. Aliquots (100 ~1) of labeled cytosol were incubated in duplicate for 45 min at 04’C with 50~1 packed DNA-cellulose prepared as previously described [21]. After the incubation, 2 ml of cold TE buffer (10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid [EDTA], pH 8.0 at 04’C) were added to each tube. The suspensions were then agitated and centrifuged for 5 min at 600 g in a clinical centrifuge at 4 ‘C. The DNA-cellulose pellets obtained after three additional washes with the same buffer were resuspended in 0.8 ml TE buffer, and an aliquot (0.5 ml) was assayed for radioactivity after being added to 5 ml of Liquiscint Scintillation Cocktail. DEAE-cellulose
chromatography
Diethylaminoethyl (DEAE)-cellulose (DE-52, Whatman, Inc., Clifton, NJ) columns were prepared in 5 cm3 plastic disposable syringes (3 ml bed volume) and equilibrated with KPD buffer (5 mM potassium phosphate, 0.5 mM dithiothreitol, pH 7.6 at 04°C). A 0.5 ml aliquot of labeled cytosol was applied to the column and was washed for 10 min with KPD buffer. Bound radioactivity was eluted with a linear 5 to 400 mM KPD gradient as previously described [22]. Twenty-five 1 ml fractions were collected, and 0.5 ml aliquots of each fraction were added to 5 ml of Liquiscint Scintillation Cocktail for the determination of radioactivity. The KPD concentration was determined by measuring the conductivity of appropriate fractions with a Markson model 10 conductivity meter and comparison made with a standard curve. All chromatographic procedures were performed at 4°C. 85 + 9% of the added radioactivity was routinely recovered from the columns. RESULTS pH Induced [‘HITA-receptor
activation complexes
und the binding to DNA -cellulose
of
The effects of increasing pH on activation as measured by the binding of [‘HITA-receptor complexes to DNA-cellulose is summarized in Table 1. The pH of the cytosol was adjusted as described in Materials and Methods. Because the volume change after the pH adjustment was small (less than 1.59/,), dilution activation was insignificant [8, 141; and despite the low salt concentration of the incubation buffer (50 mM Tris-HCI) [18], the pH of the cytosol remained relatively after adjustment constant throughout the length of the experiments (_t 0.1 pH units). Aliquots of the pH adjusted cytosol were then incubated for 30 min at 15°C after which they were returned to 0°C and 10mM Na,MoO, (20pl/ml) was added to prevent further activation of the
pH Induced
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685
Table 1. The effects of pH induced activation on the binding of complexes to [‘HITA-receptor DNA-cellulose PH 6.5 7.5 8.5
% Bound to DNA-cellulose* 4.1 (kO.2) 18.4 ( f 2.0) 39.2 (k0.4)
-
Aliquots of [3H]TA-labeled cytosol (prepared as described in Materials and Methods) were incubated for 30 min at 15°C and at the various pH’s. The pH adjustments of the cytosol were accomplished as described in Materials and Methods. 10 mM Na,MoO, was added to each aliquot at the end of the incubations, and they were then returned to 0°C. DNA-cellulose
and hydroxylapatite binding was then determined in duplicate as described in Materials and Methods. The results presented represent the average experiments. of two such *(DNA-cellulose binding/HAP binding) x 100.
steroid-receptor complexes [23-251. At this point, DNA-cellulose and HAP binding assays were performed as described in Materials and Methods. When cytosol was incubated at pH 6.5 prior to assay, only 4.1% (kO.2) of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% ( f 0.4) bound to DNA-cellulose when the cytosol was incubated at pH 8.5; 18.4% (f2.0) bound when the pH of the cytosol was left at 7.5, i.e. not adjusted (control). Thus, we were able to show increased activation of the glucocorticoid-receptor complex with increasing pH using the DNA-cellulose binding assay [l l-131. When aliquots of previously heat-activated cytosol (25°C 30 min) [3,69] were adjusted to pH 6.5 or 8.5, or left at pH 7.5 prior to the DNA-cellulose and HAP binding assays, the percent of the [‘HITA-receptor complexes bound to DNA-cellulose was the same (40-45x; data not shown) indicating that the binding of fully activated complexes to DNA-cellulose was not pH dependent in this range. The amount of [3H]TA-receptor complexes that bound to HAP was also constant in this pH range, indicating that steroid was not preferentially dissociating from the receptor in the pH range of 6.5 to 8.5 (data not shown). pH Induced matography
activation and DEAE-cellulose
chro-
Analysis of pH induced activation using DEAEcellulose chromatography is shown in Fig. 1. In Panel A, an aliquot of [‘HITA-receptor complexes was adjusted to pH 6.5 and then incubated at 15°C for 30 min. A 1 ml sample was then removed, returned to O”C, and 10mM Na,MoO, was added. A 0.5 ml aliquot of this sample was then processed for DEAEcellulose chromatography as described in Materials
Fig. 1. Analysis of pH induced activation of [3H]TAreceptor complexes using DEAE-cellulose chromatography. Ahquots of [‘HITA-labeled cytosol (prepared as described in Materials and Methods) were incubated at pH 6.5 (15°C 30 min) (panel A), or incubated at pH 6.5 (15°C 30 min) followed by incubation at pH 8.5 (15°C 60 min) (panel B). At the end of each incubation, 1ml samples were removed, returned to 0°C and 10 mM Na,MoO, was added. One-half ml was then removed from each sample and processed for DEAE-cellulose chromatography as described in Materials and Methods. The bound radioactivity was eluted in 25 (1 ml) fractions with a linear 5 to 400 mM KPD gradient. The pH adjustments for panel B were performed as described in Materials and Methods, and required 3-5 min to be completed. 8.4 x 10’ dpm were applied to the column in panel A, and 5. I x 10’ dpm were applied to the column in panel B.
and Methods; 76.4% of the total radioactivity originally added to the column was recovered. The chromatograph in panel A shows that only 19.6% of the recovered [3H]TA-receptor complexes are in the activated peak (eluted at 50 mM KPD) [22], while only 3.9% of these labeled steroid-receptor complexes bound to DNA-cellulose (data not shown). The remaining cytosol, which had been returned to 0°C at the end of the first incubation in order to minimize the possibility of further activation of steroid-receptor complexes [3,6-91, was then adjusted from pH 6.5 to pH 8.5 as described in Materials and Methods, and incubated for an additional 60 min at 15°C. A 1 ml sample was then removed and treated as described above. A 0.5 ml aliquot of this sample was then processed for DEAEcellulose chromatography as described in Materials and Methods. The results of this experiment are presented in panel B of Fig. 1; 79.6% of the total radioactivity added to the column was recovered. The chromatograph shows that 90.1 y0 of the recovered [3H]TApreceptor complexes are in the activated position (eluted at 50mM KPD), while 38% of the labeled steroid-receptor complexes bound to DNAthe cellulose (data not shown). Thus, [3H]TA-receptor complexes not activated at pH 6.5 were activated when they were subsequently incubated at pH 8.5. It is important to note that 39% less radioactivity was applied to the column in panel B than the one in panel A. This is most likely due to dissociation of some steroid-receptor complexes
686
PETER V. BODINE rt al.
which has been previously shown to occur at equal unactivated activated and rates for both Conseglucocorticoid-receptor complexes [ 171. quently, it can be assumed that the loss of radioactivity was not preferentially from the unactivated peak (eluted at 200 mM KPD). Also, the total radioactivity in the activated peak of panel B does increase 2.5-fold over the activated peak in panel A.
i;
I51 PH change
I
s
/
Following the same format as in Fig. 2, DEAEcellulose chromatography was performed (as described in Materials and Methods) in order to visualunactivated ize activated and both the [“HITA-receptor complexes after each of the pH adjustments. The results of this experiment are shown in Fig. 3. Panel A of Fig, 3 is a DEAE-cellulose chromatograph performed at the end of the first 30min incubation (pH 8.5, 15 C); 67.5”,, of the bound [‘HITA-receptor complexes are in the activated pos-
I
change I I
I
30
0 Msn
Analysis qf‘ udjustments in pH on the elution qj [‘HITA-receptor complexes ,fiom DEAE-cellulosr
pH
;
/
20
c
To test for the reversal of pH induced activation [18], the following format was followed. An aliquot of [3H]TA-receptor complexes was initially adjusted to pH 8.5 and incubated for 30 min at 15°C at which time a 1 ml sample was removed, returned to D”C, 10 mM Na?MoO, was added, and the sample was then processed for DNA-cellulose and HAP binding as described in Materials and Methods. The remaining cytosol was returned to O‘C, and the pH was adjusted from 8.5 to 6.5 as described in Materials and Methods. The cytosol was then incubated for 30 min at 15 C, at which time another 1 ml sample was removed and treated as above. The remaining cytosol was again returned to 0°C. and its pH was adjusted back to 8.5. This was followed by another 30 min incubation at IS C, at which time a third 1 ml sample was removed and treated as above. The results of this experiment are presented in Fig. 2. After 30 min at 15 C and pH 8.5, 39.2’fd (iO.4) of the [iH]TA--receptor complexes bound to DNAcellulose. When an aliquot of the same cytosol was then adjusted to pH 6.5, 47.0’:;, (& 7.2) of the labeled steroid-receptor complexes bound to DNA-cellulose after another 30 min incubation at 15 C; 43.9?: (+ I .9) bound when the pH of the cytosol was left at pH 8.5 during the second incubation (control). Thus, as determined by DNA-cellulose binding, pH induced activation of the glucocorticoid--receptor complex was irreversible. When the cytosol at pH 6.5 was returned to pH 8.5 and a third incubation at 15°C was performed, 40.57, (i 1.3) of the pH]TA-receptor complexes bound to DNA-cellulose; 52.2”;, ( + 7. I) of the labeled steroid-receptor complexes were bound under control conditions.
I 2nd
I
I
I 10
/ 0
I 30
a+ 15°C
Fig. 2. The effects nf adjustments in pH on the binding of [‘HITA-receptor complexes to DNA~cellulose. An ahGot of [‘HlTA-labeled cvtosol (nrenared as described in Materials-and Methods) was initi‘ally-adjusted to pH 8.5 (- a--) and then incubated at 15°C for 30 min, at which time a 1ml sample was removed, returned to O’C, 1OmM Na2Mo0, was added, and the sample was then processed for DNAcellulose and HAP binding as described in Materiais and Methods. The remaining cytosol was divided into two aliquots and both were returned to 0°C. One aliquot was adjusted to pH 6.5 (-a-), the other was left at pH 8.5 (-•-_), after which both were incubated a second time for 30min at 15°C. At the end of this second incubation. another 1 ml sample was removed from each aliquot and treated as above. The remaining cytosoi of both aliyuots were again returned to WC. The one at pH 6.5 was adjusted back to pH 8.5 (--a-), and the aliquot at pH 8.5 was left at pH 8.5 (-+--), after which both were incubated a third time at 15°C for 30 min, at which time another I ml sample was removed from each aliquot and treated as above. The data presented represent the average of two such experiments. The pH adjustments were performed as described in Materials and Methods and required 3-5 min to be completed; this time is not included in the time scale (abcissa of Figure). ition (eluted at 65 mM KPD), and 76.20/, of the added radioactivity was recovered from the column. The remaining cytosol from this step was adjusted to pH 6.5 and another 30 min incubation was performed at 15C. The DEAE-cellulose chromatograph performed at the end of this second incubation is shown in pane1 B of Fig. 3; 74.4% of the bound [3H]TA-receptor complexes are in the activated peak (65 mM KPD), and 94.6% of the added radioactivity was recovered from the column. Thus, there was no shift of [‘HITA-receptor complexes from the activated (eluted at 65 mM KPD) to the unactivated peak (eluted at 250mM KPD) when the pH of the cytosol was shifted from 8.5 to 6.5 for the second incubation, i.e. there was no reversal of activation. The remaining cytosol from this step was then returned to pH 8.5 and a third 30 min. 15°C incubation was performed. The DEAE-cellulose chromatograph from the end of this incubation is shown in panel C of Fig. 3; 92.4% of the bound [‘HITA-receptor complexes are in the activated position (eluted at 55 mM KPD), and 89.2% of the added radioactivity was recovered from the column. Thus, the amount of [iH]TA-receptor complexes associated with the activated peak of DEAE-cellulose increased continuously with increasing incubation at 15°C from an initial value of 67.5”,/, after the first incubation
pH Induced
activation
of steroid-receptor
.J 60
Froctlon
number
( I ml/fraction)
Fig. 3. Analysis of adjustments in pH on [)H]TA-receptor complex activation using DEAE-cellulose chromatography. The experimental procedure followed was the same as in Fig. 2 except that DEAE-cellulose chromatography was performed at the end of each 30 min incubation at 15’C. One ml samples were removed at the end of each incubation, returned to 0°C 10 mM Na,MoO, was added, and 0.5 ml of each sample was removed and processed for DEAEcellulose chromatography as described in Materials and Methods. The bound radioactivity was eluted in 25- to 30-( 1 ml) fractions with a linear 5 to 400 mM KPD gradient. The pH adjustments were performed as described in Materials and Methods and required 3-5 min to be completed. Panel A: end of first 30mit-1, 15°C incubation at pH 8.5; Panel B: end of second 30 min, 15°C incubation at pH 6.5; Panel C: end of third 30 min, 15°C incubation at pH 8.5. 1.3 x IO6 dpm were applied to the column in panel A, I .O x lo6 dpm were applied to the column in panel B, and 8.3 x 10Sdpm were applied to the column in pane1 C.
(pH 8.5) to 74.4 and 92.4% after the second (pH 6.5) and third (pH 8.5) incubations, respectively. DISCUSSION
In the present study, the possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. When [3H]TA-labeled cytosol was activated at pH 8.5 (15°C for 30 min), there was a 9.6-fold increase in the binding of [‘HITA-receptor complexes to DNA-cellulose as compared with cytosol incubated under same conditions at pH 6.5 (Table 1). Likewise, 3.4-fold more [3H]TA-receptor com-
complexes
687
plexes were in the activated peak on DEAE-cellulose (eluted at 65 mM KPD) when cytosol was incubated at pH 8.5 (Fig. 3A) than cytosol that was incubated under the same conditions at pH 6.5 (Fig. IA). This confirms previous reports which demonstrated that the percentage of activation of the rat hepatic glucocorticoid-receptor complex is greater at higher pH (11-13 and 18) and that anion-exchange chromatography can be used to study pH induced activation [l 1, 121. Analysis on DEAE-cellulose also showed that incubation at pH 8.5 resulted in an increase in activation when compared to cytosol previously incubated at pH 6.5 (Fig. 1). This confirms an earlier report for pH induced activation of the glucocorticoid-receptor complex using the isolated nuclear binding assay [ 181. When previously pH activated cytosol (pH 8.5, 15°C 30 min) was incubated a second time at pH 6.5, no loss of binding to DNA-cellulose was detected (Fig. 2). Similarly. no conversion of activated [3H]TA-receptor complexes to unactivated complexes as measured by DEAE-cellulose chromatography was detected when previously pH activated cytosol was incubated a second time under the same conditions at pH 6.5 (Fig. 3A and 3B). Thus, by these in tiitro pH induced activation of the criteria, glucocorticoid-receptor complex was determined to be irreversible. This conclusion contradicts a previous study by Milgrom and his colleagues [ 181 which showed that pH induced activation, as measured by the binding of rat hepatic [‘HITA-receptor complexes to isolated nuclei, was reversible. Although it might be argued that the DNA-cellulose binding assay under these circumstances may not be fulfilling the requirements of the isolated nuclei technique [6, 181, the results obtained from DEAE-cellulose chromatography cannot be ignored: there clearly was no conversion of [3H]TA-receptor complexes from the activated to the unactivated form on DEAE-cellulose when the pH was adjusted from 8.5 to 6.5 (Fig. 3). In this study we define reversal of glucocorticoid-receptor complex activation as the following: (i) the failure of previously activated [3H]TA-receptor complexes to bind to DNAcellulose, and (ii) the conversion of previously activated [3H]TA-receptor complexes from the activated to the unactivated form as measured by DEAEcellulose chromatography. We feel that both of these criteria must be satisfied before activation can be concluded as being reversible. Hutchens et a/.[261 recently reported that RNA induces reversal of glucocorticoid-receptor complex activation. However, in their study, activation was defined in terms of the ability of steroid-receptor complexes to bind to ATP-agarose. Since RNA inhibits the binding of the activated estrogenreceptor ccmplex to DNA [27], it is not surprising that RNA, being a polyanion, combines with activated glucocorticoid-receptor complexes and thus inhibits their binding to other polyanions, such as ATP-agarose or DNA-cellulose.
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PETER V. BODINE et al.
Consequently, this finding does not prove that the activation step itself is reversed. Likewise, pyridoxal 5’-phosphate (PLP) has been shown to inhibit the binding of activated glucocorticoid-receptor as several other steroid complexes, as well hormone-receptor complexes to both DNA-cellulose and isolated nuclei [28-321. By this criteria alone (i.e. DNA-cellulose and nuclei binding), activation would be concluded as being reversible in the presence of PLP. However, a later report from this laboratory showed that activation, as measured by DEAEcellulose chromatography, was actually potentiated by PLP [33]. Thus, by our criteria for reversibility, activation is not reversed by PLP. Recently, Raaka and Samuels[34] explored the reversibility of in uiuo glucocorticoid receptor activation in GH, cells, a rat pituitary tumor cell line. Using dense amino acid labeling and sucrose gradient centrifugation, they demonstrated that an equilibrium exists between a high molecular weight unactivated steroid-receptor complex (sedimentation coefficient of 10s) and a low molecular weight activated complex (sedimentation coefficient of 4s). The 10s complex is postulated as being an oligomer, and the 4S complex is though to be the active monomer which translocates to the nucleus and binds to DNA. The authors showed that as the concentration of steroid increased, the amounts of the 4S complex in the cytosol and nucleus increased, while the amount of the 10s complex in the cytosol decreased. But, when steroid was removed, the amounts of the cytosolic and nuclear 4S complex decreased, and the amount of the cytosolic 10s complex increased. The authors conclude from this that in viuo activation of the glucocorticoid-receptor complex is reversible. However, they admit that although their data suggests that each step of the activation process is reversible, it may be possible, for one or more of the steps, that the apparent reversibility is due to separate reaction pathways in each direction. Thus, their results do not necessarily contradict our conclusion that in vitro pH induced activation of the glucocorticoid-receptor complex is irreversible, because the step that we conclude is irreversible is the monomeric conversion of the unactivated steroid-receptor complex to the activated form, perhaps via a covalent modification such as a dephosphorylation [23,24, 351. However, although we have concluded that this individual step is irreversible, other steps of the activation pathway could result in activated nuclear steroid-receptor complexes appearing in the cytoplasm as unactivated complexes; this would be true, for example, if recycling of the receptor occurred after nuclear binding [ 11. Vedeckis[36] has also proposed a subunit dissociation model for the activation of the glucocorticoid receptor in mouse AtT-20 cells, a pituitary tumor cell line. In this study, activation was accomplished in vitro by warming cytosol for 1 h at 25 C and then passing it over a Sephadex G-25
column. Aliquots of the cytosol were then incubated with or without 20mM Na,MoO, at 0_4”C, and samples were then analyzed via DEAE-cellulose chromatography. No significant conversion of the activated form to the unactivated form was seen on DEAE-cellulose when activated cytosol was incubated with or without Na,MoO, prior to chromatography. Thus, this supports our results for the in vitro pH induced activation of the rat hepatic glucocorticoid-receptor complex. However, when similar studies were performed in vivo using sucrose gradient centrifugation to measure activation (S. B. Eastman and W. V. Vedeckis, unpublished observations), an apparent reversibility of activation was seen when cytosol was incubated with Na,MoO,. The author postulates that in vitro heat activation may destroy a necessary component of the in viva reversal process. However, although this may be possible, it seems unlikely that in our case such a factor is destroyed simply by pH induced activation of the cytosol at 15°C. Our conclusion that pH induced activation of the glucocorticoid-receptor complex is irreversible supports a previous report by Goidl et a/.[171 which implied that heat activation of the rat hepatic glucocorticoid-receptor complex was also irreversible, as measured by DNA-cellulose binding. The present study, however, is the first known reported time that DEAE-cellulose chromatography has been reversibility of pH induced used to study activation [l]. The use of this procedure to visualize both activated and unactivated [3H]TA-receptor complexes has allowed us to directly demonstrate that in vitro pH induced activation, at least, is irreversible. Acknowledgements-This work was supported by Research Grants: AM13531 from NIADDK, NIH and by PCM 8215844 from the NSF and by CA 12227 from the NCI, NIH. to the Fels Research Institute. P.V.B. is a reciuient of a Graduate School Fellowship, and is supported by an NIH Basic Research Support Grant to the Department of Biochemistry, Temple University School of Medicine.
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