- Email: [email protected]
Glucocorticoid receptors in carrageenin-induced granuloma in rats: Activation to dexamethasone-binding form by sulfhydryl compounds
Journal of Sreroid Biochemistry Vol. 14. pp. 1337 to 1345. 1981 Printed in Great Britain. All rights reserved
W22-4731’81 121337-09802.00,O Copynghl 0 1981 Pergdmon Press Ltd
GLUCOCORTICOID RECEPTORS IN CARRAGEENININDUCED GRANULOMA IN RATS: ACTIVATION TO DEXAMETHASONE-BINDING FORM BY SULFHYDRYL COMPOUNDS* YOSHIHIRO&Dot/l, EIICHI FUJIHIRA~, TSUTOMUARAKI~ and SUSUMU TSURUFUJI~ tDepartment of Toxicology, Hokkaido Institute of Pharmaceutical Sciences, 7-l Katsuraoka-cho.Otaru 047-02, Japan; fResearch Laboratory, Tokyo-Tanabe Seiyaku Co.. Ltd, 2-33-3 Akabane-kita. Kita-ku, Tokyo 115,Japan; PDepartment
of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku Aoba. Aramaki. Sendai 980, Japan (Received
30 Jmuar~
University.
1981)
SUMMARY The specific binding of glucocorticoids to the cytoplasmic receptor and nuclei in granuloma tissue was studied in in vitro experiments. Although 105,000 9 supernatants (cytosols) from liver and thymus homogenates indicate high affinity binding to both of cortisol and dexamethasone, granuloma cytosol was apparently lacking of the dexamethasone-binding capacity. However, a definite capacity to bind dexamethasone was demonstrated by the cytosol and nuclei of the granuloma tissue when the tissue was minced and subjected to the incubation with the steroid. Intracellular transport of dexamethasone to the nucleus oia cytosol in the incubation of the minced tissue was indicated to be a function of temperature and time. Demonstration of dexamethasone binding by the cytosol from the granuloma homogenate was achieved by adding sulfhydryl-protecting agents, such as dithiothreitol, glutathione and 2-mercaptoethano1 to the medium for homogenization and incubation. Treatment of cytosols from the liver and the granuloma with 5,5’-dithio-bis(2-nitro-benzoic acid) (DTNB) severely damaged their glucocorticoidbinding ability. The deteriorating effect of DTNB was antagonized by the above sulfbydryl agents. It is therefore concluded that glucocorticoid receptor, as demonstrated by dexamethasone-binding capacity. of the carrageenin-induced granuloma tissue involves active sulfhydryl group(s) which is sensitive to oxidizing agents
venous injection of radioactive cortisol [9]. However, biochemical properties of the glucocorticoid receptors from the granuloma tissues remain to be elucidated. This paper deals with specific binding as designated by Baxter and Tomkins[l l] of glucocorticoids (cortisol and dexamethasone) in the cytosol from rat carrageenin granuloma tissues and indicates that protection of the sulfhydryl groups is essential for measuring the binding activity of the granuloma cytosol.
INTRODUCTION It is generally accepted that the binding of glucocorticoids to the specific protein receptor in the cytoplasm of target cells is essential for the manifestation of their hormonal actions. The steroid-receptor complex undergoes a temperatureand energy-dependent transformation to an activated form capable of binding to acceptor sites in the cell nucleus. The association of the activated form of steroid-receptor complex with chromatin causes the synthesis of specific mRNAs to induce new synthesis of effector proteins. Such glucocorticoid receptors are found in the cytoplasmic fraction of various tissues responsive to glucocorticoids [l-7]. Recently, Tsurufuji et al. have proposed a concept that anti-inflammatory action of glucocorticoids is mediated by such “receptor mechanism”[8]. Koshihara et al. presented evidence for the existence of such a receptor-like protein for cortisol in the cytosol from an inflammatory tissues rat carrageenin granuloma, excised after intra-
MATERIALSAND METHODS Chemicals
* This study was supported in part by a research grant from the ministry of Education, Science and Culture of Japan, to Y.E. (Grant No. 477383). I(To whom correspondence should be sent. 1337
[1,2-3H]-Dexamethasone (40 Ci/mmol)
Radiochemical
Centre,
and
dithiothreitol
Chemical
(DTT)
Co., St Louis,
and
(26 Ci/mmol)
[1,23H]-cortisol
were
obtained
Amersham. were MO
from
the
Dexamethasone
purchased and
from
cortisol,
Sigma
glutathione
2-mercaptoethanol (ME), adenosine S-triphosphate disodium salt (ATP), and 5,5’-dithio-bis(2nitrobenzoic acid) (DTNB) were from Wako Junyaku Kogyo Co. Ltd, Tokyo.
(GSH),
Induction Male
of currageenin Sprague-Dawley
granuloma strain
pouch
rats
6-7
weeks
old.
1338
YOSHIHIR~ ENDO et ul.
weighing 17&2OOg, were used. They were fed on laboratory chow and tap water ad libitum. Granuloma pouch was induced by injecting a 2.0% (w/v) solution of carrageenin (Seakem No. 202, Marine colloid Inc., Springfield, NJ) in saline into the air sac prepared one day before on the dorsum of the rats, as described previously [lo]. The day of carrageenin-injection was designated as day 0. Preparation
of q,tosol fraction
On day 8, rats were killed by decapitation. Blood was collected in a beaker containing heparin solution. Plasma was separated by centrifugation at 1,000 g for 15 min at 2C. Granuloma and thymus were excised immediately and liver was removed after perfusion with ice-cold 0.9% NaCl through the portal vein, After removal of adhering fat and muscle, the tissues were washed in an ice-cold 20mM Tris-HCI buffer (pH 7.6) containing 0.4 mM EDTA, blotted, weighed and homogenized in five volumes of the Tris buffer in a Vir-Tris 45 homogenizer operated at 40 V for 1 min with 30-s intermission unless otherwise described. The homogenates were centrifuged at 2C at 105,OOOg for 60 min. Measurement of the spec$c coid to the receptor
binding of [3H]-glucocorti-
The specific binding of glucocorticoids to the receptor as designated by Baxter and Tomkins[ll] was determined according to their method with slight modification. Ten ~1 of an ethanolic [3H]-glucocorticoid (8 x lo-’ M) were pipetted into two sets of 15-ml centrifuging tubes and evaporated to dryness under nitrogen at room temperature. To one set of the tubes, the ethanolic non-labeled compound was added by the amount 1000 fold as much as the radioactive one and again evaporated to dryness. After chilling the tubes, 0.4 ml of ice-cold diluted cytosol or plasma were added, mixed throroughly and incubated in ice-water bath for 90 min by shaking frequently. Then five volumes of the suspension of 0.57” activated charcoal (Norit A, American Norit Co.) and 0.050/ dextran T-70 (Pharmacia Fine Chemicals, Sweden) in the Tris buffer were added and immediately agitated for 5 s on a Vortex mixer. After being allowed to stand for 10min in ice-water bath, the tubes were centrifuged at 800~ for 1Omin at 2-C. An aliquot of the clear supernatant (1 ml) was pipetted into a vial containing 10 ml of xylene based scintillation cocktail (PCS, Radiochemical Center, Amersham) and the radioactivity was counted after standing for more than 12 h in a Packard Tri-Carb model 2650 hquidscintillation spectrometer. The amount of labeled steroid specifically bound in cytosol or plasma was calculated by subtracting the radioactivity of the nonlabeled compound-containing sample from that of the radioactive compound alone-containing one. Experiments
with minced granuloma and lioer tissues
Specific binding to the receptor
and nuclear uptake
of glucocorticoids were investigated by the incubation of minced granuloma with the steroid according to the procedure demonstrated by Gianopoulos[lZ]. Granuloma and liver tissues are minced with scissors into 2 mm pieces and incubated at 0°C (ice-water bath), 25 or 37°C in the Krebs-Ringer bicarbonate buffer solution containing 1 x 10-s M [3H]-glucocorticoids with or without lOOO-fold corresponding non-labeled steroids in the atmosphere of 95”/] O,--Srl, C02. After the incubation for 120min, the flask was chilled in ice-water bath and the content was transferred into a 15-ml centrifuging tube, and centrifuged at 800 g for 5 min. The sedimented pellet of tissue pieces was washed three times with 0.25 M sucroseTKM buffer (TKM: 50 mM Tris-HCI, pH 7.5, containing 25 mM KC1 and 5 mM MgCl,) and homogenised mildly in a Vir-Tris 45 homogenizer at 20 V for 20 s in 9 ml of the buffer. The homogenate was filtered through four layers of gauze and a 0.5 ml portion of the filtrate was taken and kept in a deep freezer for the determination of DNA content. The remainder was centrifuged at 8OOg for 10 min and then 105,OOOg for 60 min. The pellet (cell nuclei) and supernatant (cytosol) were obtained respectively. The cytosol was treated with the dextran-coated charcoal and then measured for charcoal-resistant (unabsorbed) radioactivity. The specific binding of the steroid in the cytosol was calculated by subtracting the radioactivity of the sample with non-labeled compound from that of the sample without non-labeled compound. The nuclear fraction was purified from the pellet according to the modified method of Blobel and Potter[13] as follows. After washing twice by centrifuging in 5 ml of 0.25 M sucrose-TKM at 800 g for 5 min, the pellet was resuspended in 3.5 ml of 0.25 M sucrose-TKM. The three ml portion of the suspension was pipetted into a centrifuging tube and admixed thoroughly with 6ml of 2.3M sucroseTKM. The mixture was underlayered by 3 ml of 2.1 M sucroseeTKM and centrifuged at 124,000 g for 30min. The tube wall was then wiped dry with a spatula wrapped with tissue paper. The white nuclear pellet was resuspended in 0.25 M sucrose-TKM for the measurement of the radioactivity. Specific nuclear uptake was also calculated by subtracting the radioactivity of the sample with non-labeled compound from that of the sample without non-labeled compound. Thin layer chromatography nucleur fraction
qf the extract
fk~m the
To the “purified nuclear fraction” obtained from 4 g of granuloma incubated without addition of nonlabeled corticoids, equal volume of chilled chloroform containing 2 x lo-’ M non-labeled dexamethasone was added, and the mixture was shaken vigorously for 2 min, and centrifuged at 800 g for IO min to obtain the chloroform extract. The extraction was carried out further twice with chloroform and once with ethylacetate and the organic layers were combined and
Glucocorticoid
receptors in carrageenin granuloma
evaporated in vucuo. The residue was dissolved in 0.1 ml of chloroform and 20 ~1 was applied to a plate coated with silica gel (Kiesel gur G, type 60, E. Merck) and developed in methylene chloride-methanol-distilled water-glycerol (150: 10: 1:0.4, v/v). After air-dryness, the successive portions of silica gel in 0.5 cm width were scraped off from the plate and the radioactivity was measured. Non-labeled dexamethasone was run together as a standard and the location was visualized under ultraviolet light. DNA-drterminution DNA was extracted by the Schmidt-ThannhauserSchneider (STS) method [14] and determined by the method of Burton[ 151. Protein determination Protein Miller[16].
was
determined
by
the
method
binding of [3H]-glucocorticoids minced tissues
binding in granuloma
The cytosols from the granulation tissue, liver and thymus as well as the plasma were incubated with C3H]-cortisol and [3H]-dexamethasone, respectively, and assayed for the glucocorticoid-binding capacity as described in the section of Materials and Methods. C3H]-Cortisol was bound specifically in all of the plasma and cytosols tested (Table 1). However, the specific binding of [3H]-dexamethasone was not demonstrated in the granuloma cytosol and plasma, whereas dexamethasone binding was evident in the cytosols from the liver and thymus. In the literature the lack of dexamethasone-binding capacity has been reported for rat plasma [17]. Dexamethasone-binding was not demonstrated even in the granuloma cytosol obtained from bilaterally adrenalectomized rats in which endogenous steroids capable of competing with the labeled corticoids would have been depleted as indicated by the result with C3H]-cortisol
Specific binding (fmol/lOO peg DNA) Tissue
[3H]-Cortisol
Granuloma Liver
56.0 + 10.1 291.3 +_ 18.2
- (9)
+ (5)
Liver Thymus Plasma Granuloma Granuloma
Experiments
with minced grunulomu und liter
Granuloma and liver were minced and incubated with C3H]-cortisol and C3HJ-dexamethasone, respectively. Results summarized in Table 2 show that dexamethasone could be taken up rather more activity by the nuclei of these tissues than cortisol did. Based on those results, further experiments were done in order to examine effects of temperature and time on dexamethasone binding in the minced granuloma. The
C3H]-Cortisol
C3H]-Dexamethasone
0.157 * 0.011 0.059 +_0.005 0.997 * 0.121 0.172 + 0.038 1.308 + O.lllt
0.329 i 0.045 0.237 f 0.019 Not detectable Not detectable Not detectable
Each value is expressed as mean k SEM. adrenalectomized (+ ) or sham-operated (-) 2 days before sacrifice. Figures in parenthesis are number of rats used. t P i 0.001 compared to control granuloma. * Rats were bilaterally
111.5 + 15.9 888.3 f 148.1
The possibility of a receptor binding of dexamethasone was also tested by passing the granuloma cytosol incubated with labeled dexamethasone through a Sephadex G-25 column (1 x 15 cm, flow rate 0.4ml/min). No radioactivity was recorded in the fractions at the void volume, while in cases of the cytosols of liver and thymus significant radioactivity was recovered in the fractions at the void volume (data are now shown). In an attempt to make time interval from cell rupture to the start of the incubation as short as possible, the earliest incubation was performed 35 min after the homogenization. It still failed to show dexamethasone binding in granuloma cytosol.
Specific binding (pmol per mg protein) Sample
[3H]-Dexamethasone
One-gram aliquots of minced liver and granuloma tissues were incubated in IOml of Krebs-Ringer bicarbonate buffer solution with 1 x 10-s M radioactive steroids in the presence and absence of 1000-fold corresponding non-labeled steroids for 12Omin at 37’C under 95 O,-Spd COZ. After incubation, cell nuclei were separated and assayed for radioactivity and DNA as described in the text. “Specific binding” was calculated by subtracting the radioactivity for the sample incubated with non-labeled steroid from that without non-labeled steroid. Figures represent mean +_SEM from three determinations.
Table 1. Specific [3H]-glucocorticoid binding in tissue cytosols and blood plasma
Adrenalectomy*
in
of
RESULTS
Apparent Itrck of dexamethasone cytosol
Table 2. Nuclear
1339
1340
YOSHIHIRO
ENDO
et al.
Nuclei .-•
\ \
\
\
\
‘0
-.
-_
--0
cytoso1
00
0
IO
30
c ‘0
“.
30
20
lncubatlon
40
Temperature,“C
Fig. l(A). Relationship between incubation temperature and dexamethasone-binding in minced granuloma. An aliquot of minced granulation tissue (1 g) was incubated with [3H]-dexamethasone (1 x lo-’ M) for 60 min at 0, 25 and 37-C. respectively. After incubation the tissue was homogenized and the nuclear and cytosol fractions were prepared. Specifically bound radioactivity in these two fractions were determined separately. Specifically bound [‘HI-dexamethasone is expressed as fmol per 1OOpg of DNA of granuloma homogenate and 1OOc~g of nuclear DNA. respectively.
results are summarized in Figs 1A and IB. At 0°C nuclear uptake of dexamethasone hardly occurred (Fig. 1A). As the incubation temperature was raised, quantity of the steroid in the nuclear fraction increased, accompanying the decrease in the amounts
o.
Authentic
time,mln
kept in the cytosol. Inverse correlation in the amount of dexamethasone between the nuclear and the cytosol fractions was also observed in respect to the time change in the distribution of dexamethasone. Thin-layer chromatography of radioactive compounds extracted from the nuclei which were isolated from the minced granuloma after incubation with [3H]-dexamethasone indicated that the radioactivity localized in the nuclear fraction corresponds exclusively to unchanged dexamethasone (Fig. 2). Any radioactivity was not extracted from the nuclear residue remained after chloroform and ethylacetate extraction as described in Materials and Methods.
1 Segnents
120
Fig. l(B). Relationship between incubation time and dexamethasone-binding in minced granuloma. The experimental condition was same in Fig. l(A) except that the incubation temperature was constant at 37°C. DEX: dexamethasone.
n t Origin
60
rn
on ~~~eZ~~orLZ’”
cm ZZf
front
Fig. 2. Thin layer chromatogram of the extract of the nuclear fraction prepared from minced granuloma incubated with [3H]-dexamethasone. The experimental condition was same in Fig. 1. Details are under “Materials and Methods”.
Glucocorticoid
receptors
in carrageenin
granuloma
Table 3. Effects of four chemicals the granuloma
cytosol
added to the incubation on the binding of dexamethasone Bound
Test chemicals None Nat MOO, ATP EDTA DTT
Concentration (mM)
10 10 10 10
(-)
C3H]-DEX
Cold DEX
1341 mixture of (DEX)
(d.p.m.)*
(+) Cold DEX
2063 2114 2514 2396 3210
2152 2211 2731 2465 2236
Each additive was dissolved in redistilled water, neutralized if required, and diluted with redistilled water to required volume after adding a 0.1 volume of 0.2 M TrisHCl buffer containing 4mM EDTA, pH 7.6. A 0.3 ml portion of 20 mM solution of an additive was added into equal volume of the cytosol, thoroughly mixed, allowed to stand in ice-water bath for one hour with occasional shaking. After the above preincubation, a 0.4 ml portion of the mixture was pipetted into another tube containing 2 x lo-* M of C3H]-dexamethasone with or without l,OOO-fold cold dexamethasone and incubated for 90 min in ice-water bath. * All values represent mean of duplicate determinations and DEX means dexamethasone.
Table 4. Effects of four chemicals added to the medium for homogenizing granuloma tissues on the binding of dexamethasone (DEX) Bound Homogenizing 20 mM Tris-HCl-0.4 + 10 M Na,MoO, + 10 mM ATP + 10mM EDTA +lOmMDTT
medium mM EDTA
C3H]-DEX
(d.p.m.)*
(--) Cold DEX
(+) Cold DEX
3385 2683 3279 3476 19,468
3558 2876 3262 3735 3114
Granuloma were minced, divided into five equal parts and homogenized in two volumes of each homogenizing medium and the cytosols were prepared as described in the text. * All values represent mean of duplicate determinations and DEX means dexamethasone.
Protection by SH reagents of dexamethasone-binding capacity in cell-free systems Several chemicals such as sodium molybdate, adenosine 5’kphosphate (ATP), Ethylenediamine tetraacetic acid (EDTA) and dithiothreitol (DTT) are reported to influence binding of glucocorticoids with their receptors [l&25,29-31, 411. Therefore, these agents were tested by adding to the granuloma cytosol 1 h before the start of incubation with C3H]-dexamethasone. As indicated in Table 3, binding activity of granuloma cytosol for [3H]-dexamethasone was recovered by the addition of DTT to the final concentration of 1OmM. Table 4 shows that the protecting effect of DTT is enhanced by its addition to the homogenizing buffer. In addition, two suhhydryl agents, glutathione (GSH) and 2-mercaptoethanol (ME) also indicated a protecting effect similar to that of DTT (Table 5). The order of protecting potency was DTT 3 GSH > ME. S” I? 12
H
Table 5. Effect of dithiothreitol
(DTT), glutathione (GSH) and 2-mercaptoethanol (ME) on the dexamethasone-binding capacity of granuloma cytosol
Homogenizing medium Tris* + DTT, 1OmM 5mM 1mM + GSH, 1OmM 5mM 1mM + ME, IOmM 5mM 1mM
Amount of specifically bound [3H]-dexamethasone (pmol per mg protein)t Not detectable 0.083 0.072 0.022 0.074 0.069 0.049 0.041 0.013 0.003
* Tris: 20 mM Tris-HC1 containing 0.4 7.6. Granuloma were minced, divided into and homogenized in each medium and binding activities were measured in cytosols the text. t All values represent mean of duplicate
mM EDTA, pH ten equal parts dexamethasone as described in determinations.
YOSHIHIRO ENDO
1342 Table 6. Deleterious
et al.
effects of DTNB on glucocorticoid-binding the liver and granuloma cytosols
activity
of
Specifically bound steroid* (pmol/mg protein) Added Cytosols Liver
DTNB
(mM)
[3H]-Cortisol
None 10.0 7.5 5.0 2.5 None 5.0
Granuloma
[‘HI-Dexamethasone
0.150 0.034 0.048 0.068 0.084 0.271 0.220
0.318 ND ND ND ND 0.042 ND
Granuloma was homogenized in 20mM Tris-HCl containing 0.4mM EDTA and 1OmM dithiothreitol. Cytosols were preincubated with equal volume of the DTNB solution in 0.2 M Tris-HCI, pH 7.6, or vehicle for 1 h in ice-water bath before the incubation with labeled steroids. ND: not detectable. * All values represent mean of duplicate determinations.
Sodium molybdate, ATP and EDTA were all ineffective (Table 4). 5,5’-Dithio-bis (Znitrobenzoic acid) (DTNB), a chemical agent reacting with the SH group, was added to the liver and granuloma cytosols 1 h before the incubation with [3H]-dexamethasone. As indicated in Table 6, DTNB caused a complete loss of the dexamethasone-binding capacities of the cytosols and a marked reduction of the cortisol-binding capacity of the liver cytosol. The cortisol-binding capacity of the granuloma cytosol was not reduced as extensively as observed in the liver cytosol, probably because the former sample was fortified with DTT. Table 7 shows that the deleterious effect of DTNB
Table
can be antagonized pounds.
by addition
of the sulfhydryl
Determination of dissociation constant for dexamethasane receptor complex in yranuloma cytosol prepared in presence of 1OmM D7T In Figure 3, the amount of dexamethasone specifically bound to granuloma cytosol was plotted as a function of the [3H]-dexamethasone concentration in incubation medium. The values of specific binding were plotted by the method of Scatchard[42]. As shown in Fig. 3B, it gave a straight line, and it was indicated that there was only a single class of specific binding site. From this experiment, the dissociation
7. Antagonism with sulfhydryl agents glucocorticoid-binding capacity
against the DTNB-effect of liver cytosol
on
Specifically bound steroid* (pmol/mg protein) Pretreatment Vehicle DTNB,
5 mM
SH agents None None DTT GSH ME
com-
[‘HI-Cortisol 0.059 0.033 0.061 0.054 0.057
[3H]-Dexamethasone 0.109 ND 0.096 0.078 0.070
Liver cytosol was preincubated with equal volume of 1OmM DTNB in 0.2 M Tris-HCI, pH 7.6, or vehicle or 1 hr in ice-water bath, applied to a Sephadex G-25 column (1.6 x 33 cm) equilibrated with 20 mM Tris-HCI containing 0.4 mM EDTA, pH 7.6, and eluted with the same buffer at a rate of one ml per min. An aliquot of the macromolecule fraction separated was incubated for one hour in ice-water bath with equal volume of a solution containing 10 mM sulfhydryl compound and glucocorticoid-binding activity was measured. DTT: dithiothreitol; GSH: glutathione; ME: 2-mercaptoethanol; ND: not detectable. * All values represent mean of duplicate determinations.
Glucocorticoid
1343
receptors in carrageenin granuloma
0.05 -
. . . . . 0. -0
05
IO
50(x
I O“M
Dexomethasone concenlmtion
)
-0
100
50
Bound dexamethosone,f
mob?s/mg protein
Fig. 3. (A) Specific binding of [3H]-dexamethasone as a function of steroid concentration. (B). Scatchard plot of specific binding data shown in (A). Specific binding of C3H]-dexamethasone in granuloma cytosol, which was prepared in 10mM DTT-containing Tris buffer, was determined as described in “Materials and Methods”. Cytosol protein concentration was 2.6 mg/ml.
constant for the binding reaction was calculated at 1.953 x 10e9 M, the number of binding sites was at 9.397 X lo- l4 mol per mg protein, DISCUSSION
“Specific” binding, as designated by Baxter and Tomkin@ 11,ofdexamethasone and cortisol to macromolecular receptors in the cytoplasmic fraction (cytosol) of rat carrageenin granuloma tissues and liver was examined according to the procedure described [ll]. Although “specific” receptor(s) for both cortisol and dexamethasone were demonstrated in the liver cytosol, such a dexamethasone receptor was not detectable in the granuloma cytosol in which “specific” receptor for cortisol was clearly demonstrated (Table 1). On the other hand, the specific binding of dexamethasone in both the cytoplasmic and nuclear fractions of the granuloma was detectable when minced granuloma tissues were incubated with a labeled dexamethasone according to the method described by Gianopoulos[12] (Table 2 and Fig. 1A and 1B). The results of the incubation experiments for the minced granuloma with dexamethasone (Figs 1A and 1B) are quite consistent with the generally accepted concept that transfer of a steroid hormone specifically bound with the cytoplasmic receptor is gradually translocated through a temperature-dependent process into the nucleus [40]. Therefore, the above mentioned failure in the indication of the specific binding of dexamethasone in the cell-free experiments of the granuloma cytosols was considered to be due to some defects in the experimental conditions. Several factors inactivating the activity of glucocorticoid receptor in
the lung [18], thymus [21-23,303, muscle [29,31], brain [27], fibroblasts [3,22], lymphocytes [22,24] and mammary gland [41]. These include dephosphorylation by alkaline phosphatase [19-211, breakdown of phospholipid structure by phospholipases [22], and disappearance of free SH groups of the receptor molecule [18-231. Among various chemicals examined in this experiment, some SH compounds (DTT, GSH and ME) were effective to protect dexamethasone binding activity in the cytoplasmic fraction of the granuloma tissues (Tables 3 and 4). These facts suggest that cytoplasmic dexamethasone receptor of the granuloma tissues is easily inactivated by oxidation of SH groups in the molecule. Even in the cytosols of the liver, treatment of the cytosols with DTNB, a chemical agent reacting with SH group to produce disulfide bond, completely damages dexamethasone-binding activity (Table 7). SH-protective agents such as DTT. GSH and ME were effective also to protect the liver cytosols from deteriorating effects of DTNB. The granuloma cytosols apparently contain more cortisol-binding components than those for dexamethasone binding while vice oersa in the liver cytosols (Table 6). The cortisol-binding activity in the granuloma cytosol is rather resistant to 5 mM DTNB compared with the dexamethasone-binding activity. Many reports have indicated that there are plural glucocorticoid-binding components in tissue cytosols, that is, specific glucocorticoid receptor, corticosteroid-binding globulin (CBG), CBG-like macromolecule, cortisol metabolite binder and ligandin [2639]. It has been already known that cortisol binding activities of plasma CBG and tissue CBG-like macromolecule are relatively stable pro-
1344
YOSHIHIRO ENDO rt al.
teins which are ers [23,26,27,29-321. that cortisol binding blocking by DTNB in loma and the liver are components. Acknowledgements-The Miss Sachiko Tsuriva
insensitive to It may be therefore activities observed both the cytosols of due to CBG and/or
SH-blocksuggested after SHthe granuCBG-like
authors wish to acknowledge for her excellent technical assistance.
REFERENCES
1 Baxter J. D. and Forsham P. H.: Tissue effects of glucocorticoids. Am. J. Med. 53 (1972) 573-589. 2 Buller R. E. and O’Malley B. W.: The biology and mechanism of steroid hormone receutor interaction with the eukaryotic nucleus. Biochem: P~A~~Aco~. 25 (1976) l-12. 3. Pratt W. B.: The mechanism of glucocorticoid effects in fibroblasts. J. Invest. Dermatol. 71 (1978) 2435. 4. Kaiser N., Milholland R. J. and Rosen F.: Glucocorticoid receptors and mechanism of resistance in the cortisol-sensitive and -resistant lines of lymphosarcoma P1798. Cancer Res. 34 (1974) 621-626. 5. Beato M., Kalimi M. and Feigelson P.: Correlation between glucocorticoid binding to specific liver cytosol receptor and enzyme induction in uiuo. Biochem. Biophys. Rex Commun. 47 (1972) 14661472. H.: Comparison of corticoid 6. Tiara M. and Terayama receptor and other cytoplasmic factors among liver and hepatoma cell lines with different sensitivity to corticoid inhibition of cell growth. Biochim. biophys. AC~A 541 (1978) 4558. 7. Young D. A., Barnard T., Mendelsohn S. and Giddings S.: Early cordycepin-sensitive event in the action of glucocorticoid hormones on rat mRNA initiates the earliest metabolic effects of steroid hormones. Endocrine res. Commun. 1, (1974) 63/72. 8. Tsurufuji S., Sugio K. and Takemasa F.: The role of glucocorticoid receptor and gene expression in the anti-inflammatory action of dexamethasone. Nature 280 (1979) 408%410. 9. Koshihara Y., Yamagishi M., Murota S. and Tsurufuji S.: Cortisol-binding protein in the cytosol of rat carrageenin granuloma. Jpn. J. Pharmacol. 25 (1975) 271-280. 10. Tsurufuji S., Sugio K. and Endo Y.: Inhibition of vascular permeability by cycloheximide in granulomatous inflammation. Biochem. f’hArI?IAcOf. 26 (1977) 1131-1136. II. Baxter J. D. and Tomkins G. M.: Specific cytoplasmic glucocorticoid hormone receptors in hepatoma tissue culture cells. Proc. natn. Acad. Sci., U.S.A. 68 (1971) 932-937. G.: Ontogeny of elucocorticoid receptors 12. Gianopoulos in rat iiver. J. biol. Chem. iSO (1975) 5847-5851. . 13. Blobel G. and Potter V. R.: Nuclei from rat liver: Isolation method that combines purity with high yield. Science 154 (1966) 1662-1665. of nuclei acid. 14. Volkin E. and Cohn W. E.: Estimation In Methods 01 Biochemical At~~lpi~ (Edited by D. Glick). Interscience., New York, Vol. I (1954) p. 287. 15. Burton K.: Determination of DNA concentration with diphenylamine. In Methods in Enzymology (Edited by L. Grossman and K. Moldave). Academic Press, New York, Vol. XII, Part B (1968) p. 163. 16 Miller G. L.: Protein determination for large numbers of samples. Analyt. Chem. 31 (1959) 964. G. G., Baxter J. D. and Tomkins G. M.: 17. Rousseau
Glucocorticoid receptors: Relations between steroid binding and biological effects. J. mol. Biol. 67 (1972) 99-115. 18. Granberg J. P. and Ballard P. L.: The role of sulfhydryl groups in the binding of glucocorticoids by cytoplasmic receptors of lung and other mammalian tissues. Endocrinoloy~ 100 (1977) 1160-l 168. 19. Nielsen C. J., Vogel W. M. and Pratt W. B.: Inactivation of glucocorticoid receptors in cell-free preparations of rat liver. Comer Rex 37, (1977) 3420-3426. 20. Nielsen C. J.. Sando J. J. and Pratt W. B.: Evidence that dephosphorylation inactivates glucocorticoid receptors Proc. not. Acod. Aci. U.S.A. 74 (1977) 1398-1402. 21. Sando J. J., Hammond N. D., Stratford C. A. and Pratt W. B.: Activation of thymocyte glucocorticoid receptars to the steroid binding form. J. hiol. Chem. 254 (1979) 4779-4789. 22. Schulte H. F.. Nielsen C. J., Sand0 J. J. and Pratt W. B.: Evidence for a ohosoholiuid reauirement in the specific binding of &ucoiortidoids io receptors of fibroblasts and thymic lymphocytes. J. biol. Chem. 251 (1976) 2279-2289. 23. Rees A. M. and Bell P. A.: The involvement of receptor sulfhydryl groups in the binding of steroids to the cytoplasmic glucorticoid receptor from rat thymus. Biochim. biophys. AC~A 411 (1975) 121-132. 24. Sando J. J., La Forest A. C. and Pratt W. B.: ATPdependent activation of L cell glucocorticoid receptors to the steroid binding form. J. hiol. Chem. 254 (1979) 4772-4778. properties 25. Bell P. A. and Munck A.: Steroid-binding and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells. Biochem. J. 136 (1973) 97-107. 26. De Kloet E. R. and McEwen B. S.: A putative glucocorticoid receptor and a transcortin-like macromolecule in pituitary cytosol. Biochem. biophys. ACTA 421 (1976) 115123. 27. De Kloet E. R. and McEwen B. S.: Differences between cytosol receptor complexes with corticosterone and dexamethasone in hippocampal tissue from rat brain. Biochim. biophys. AC~A 421 (1976) 124132. 28. Werthamer S., Samuels A. J. and Amaral L.: Identification and partial purification of “transcortin”-like protein within human lymphocytes. J. hiol. Chem. 248 (1973) 6398-6407. 29. Mayer M., Kaiser N.. Milholland R. J. and Rosen F.: Cortisol binding in rat skeletal muscle. J. biol. Chem. 250 (1975) 1207-1211. 30. Schaumburg B. P.: Investigations on the glucocorticoid-binding protein from rat thvmocvtes. II. Stabilitv. kinetics. and specificity of binding of steroids. Biochim. hioph~.s. Acra 261 (19721 219-235. R. J. and Rosen F.: 31. Mayer M., Kaiser N., Milholland Binding of dexamethasone and triamcinolone acetonide to rlucocorticoid receptors in rat skeletal muscle. J. biol. Chrm. 249 (1974) 52365240. S. N., 32. Seleznev Yu M.. Dnilov S. M., Preobradienskv Volkova N. G.. Kuzentsova L. A., Smirn& V.-N. and Volchek A. G.: Glucocorticoid-binding proteins of rat heart cytosol and their possible role in the transfer of glucocorticoids into nuclei. J. mol. Cell. Cardio/. 10 (1978) 877-891. N.. 33. Litwack G., Filler R., Rosenfield S. A., Lichtash Wishman C. A. and Singer S.: Liver cytosol corticosteroid binder II, a hormone receptors. J. biol. Chem. 248 (1973) 7481-7486. of 34. Singer S., Morey K. S. and Litwack 0.: Properties the cortisol metabolite binding system in liver cvtosol. Phytiol. Chem. Physics 2 (1970) i17-126. _ 35. Litwack G. and Morey K. S.: Cortisol metabolite binder I: Identity with the dimethylaminoazobenzene
Glucocorticoid
36.
37.
38.
39.
receptors
binding protein of liver cytosol. Biochem. bioph_rs. Res. Commun. 38, (1970) 1141-1148. Singer S. and Litwack G.: Identity of corticosteroid binder I with the macromolecule binding 3-methylcholanthrene in liver cytosol in oiuo. Cancer Res. 31 (1971) 13641368. Litwack G. and Rosenfield S. A.: Liver cytosol corticosteroid binder IB, a new binding protein. J. biol. Chem. 250 (1975) 6799-6805. Beato M., Biesewig D., Braendle W. and Sekeris C. E.: On the mechanism of hormone action XV. Subcellular distribution and binding of [1,2-3H]-cortisol in rat liver. Biochim. biophJs. Actu 192 (1969) 494-507. Amaral L., Lin K., Samuels A. J. and Werthamer S.:
in carrageenin
granuloma
1345
Human liver nuclear transcortin. Its postulated role in glucocorticoid regulation of genetic activity. Biochim. biophys. Acta 362, (1974) 332-345. 40. Wira C. R. and Munck A.: Glucocorticoid-receptor complexes in rat thymus cells. “Cytoplasmic”-nuclear transformations. J. biol. Chem. 249 (1974) 53285336. 41. McBlain W. A. and Shyamala G.: Inactivation of mammary cytoplasmic glucocorticoid receptors under cell-free conditions. J. biol. Chem. 255 (1980) 38843891. 42. Scatchard G.: The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. St (1949) 660-672.
W22-4731’81 121337-09802.00,O Copynghl 0 1981 Pergdmon Press Ltd
GLUCOCORTICOID RECEPTORS IN CARRAGEENININDUCED GRANULOMA IN RATS: ACTIVATION TO DEXAMETHASONE-BINDING FORM BY SULFHYDRYL COMPOUNDS* YOSHIHIRO&Dot/l, EIICHI FUJIHIRA~, TSUTOMUARAKI~ and SUSUMU TSURUFUJI~ tDepartment of Toxicology, Hokkaido Institute of Pharmaceutical Sciences, 7-l Katsuraoka-cho.Otaru 047-02, Japan; fResearch Laboratory, Tokyo-Tanabe Seiyaku Co.. Ltd, 2-33-3 Akabane-kita. Kita-ku, Tokyo 115,Japan; PDepartment
of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku Aoba. Aramaki. Sendai 980, Japan (Received
30 Jmuar~
University.
1981)
SUMMARY The specific binding of glucocorticoids to the cytoplasmic receptor and nuclei in granuloma tissue was studied in in vitro experiments. Although 105,000 9 supernatants (cytosols) from liver and thymus homogenates indicate high affinity binding to both of cortisol and dexamethasone, granuloma cytosol was apparently lacking of the dexamethasone-binding capacity. However, a definite capacity to bind dexamethasone was demonstrated by the cytosol and nuclei of the granuloma tissue when the tissue was minced and subjected to the incubation with the steroid. Intracellular transport of dexamethasone to the nucleus oia cytosol in the incubation of the minced tissue was indicated to be a function of temperature and time. Demonstration of dexamethasone binding by the cytosol from the granuloma homogenate was achieved by adding sulfhydryl-protecting agents, such as dithiothreitol, glutathione and 2-mercaptoethano1 to the medium for homogenization and incubation. Treatment of cytosols from the liver and the granuloma with 5,5’-dithio-bis(2-nitro-benzoic acid) (DTNB) severely damaged their glucocorticoidbinding ability. The deteriorating effect of DTNB was antagonized by the above sulfbydryl agents. It is therefore concluded that glucocorticoid receptor, as demonstrated by dexamethasone-binding capacity. of the carrageenin-induced granuloma tissue involves active sulfhydryl group(s) which is sensitive to oxidizing agents
venous injection of radioactive cortisol [9]. However, biochemical properties of the glucocorticoid receptors from the granuloma tissues remain to be elucidated. This paper deals with specific binding as designated by Baxter and Tomkins[l l] of glucocorticoids (cortisol and dexamethasone) in the cytosol from rat carrageenin granuloma tissues and indicates that protection of the sulfhydryl groups is essential for measuring the binding activity of the granuloma cytosol.
INTRODUCTION It is generally accepted that the binding of glucocorticoids to the specific protein receptor in the cytoplasm of target cells is essential for the manifestation of their hormonal actions. The steroid-receptor complex undergoes a temperatureand energy-dependent transformation to an activated form capable of binding to acceptor sites in the cell nucleus. The association of the activated form of steroid-receptor complex with chromatin causes the synthesis of specific mRNAs to induce new synthesis of effector proteins. Such glucocorticoid receptors are found in the cytoplasmic fraction of various tissues responsive to glucocorticoids [l-7]. Recently, Tsurufuji et al. have proposed a concept that anti-inflammatory action of glucocorticoids is mediated by such “receptor mechanism”[8]. Koshihara et al. presented evidence for the existence of such a receptor-like protein for cortisol in the cytosol from an inflammatory tissues rat carrageenin granuloma, excised after intra-
MATERIALSAND METHODS Chemicals
* This study was supported in part by a research grant from the ministry of Education, Science and Culture of Japan, to Y.E. (Grant No. 477383). I(To whom correspondence should be sent. 1337
[1,2-3H]-Dexamethasone (40 Ci/mmol)
Radiochemical
Centre,
and
dithiothreitol
Chemical
(DTT)
Co., St Louis,
and
(26 Ci/mmol)
[1,23H]-cortisol
were
obtained
Amersham. were MO
from
the
Dexamethasone
purchased and
from
cortisol,
Sigma
glutathione
2-mercaptoethanol (ME), adenosine S-triphosphate disodium salt (ATP), and 5,5’-dithio-bis(2nitrobenzoic acid) (DTNB) were from Wako Junyaku Kogyo Co. Ltd, Tokyo.
(GSH),
Induction Male
of currageenin Sprague-Dawley
granuloma strain
pouch
rats
6-7
weeks
old.
1338
YOSHIHIR~ ENDO et ul.
weighing 17&2OOg, were used. They were fed on laboratory chow and tap water ad libitum. Granuloma pouch was induced by injecting a 2.0% (w/v) solution of carrageenin (Seakem No. 202, Marine colloid Inc., Springfield, NJ) in saline into the air sac prepared one day before on the dorsum of the rats, as described previously [lo]. The day of carrageenin-injection was designated as day 0. Preparation
of q,tosol fraction
On day 8, rats were killed by decapitation. Blood was collected in a beaker containing heparin solution. Plasma was separated by centrifugation at 1,000 g for 15 min at 2C. Granuloma and thymus were excised immediately and liver was removed after perfusion with ice-cold 0.9% NaCl through the portal vein, After removal of adhering fat and muscle, the tissues were washed in an ice-cold 20mM Tris-HCI buffer (pH 7.6) containing 0.4 mM EDTA, blotted, weighed and homogenized in five volumes of the Tris buffer in a Vir-Tris 45 homogenizer operated at 40 V for 1 min with 30-s intermission unless otherwise described. The homogenates were centrifuged at 2C at 105,OOOg for 60 min. Measurement of the spec$c coid to the receptor
binding of [3H]-glucocorti-
The specific binding of glucocorticoids to the receptor as designated by Baxter and Tomkins[ll] was determined according to their method with slight modification. Ten ~1 of an ethanolic [3H]-glucocorticoid (8 x lo-’ M) were pipetted into two sets of 15-ml centrifuging tubes and evaporated to dryness under nitrogen at room temperature. To one set of the tubes, the ethanolic non-labeled compound was added by the amount 1000 fold as much as the radioactive one and again evaporated to dryness. After chilling the tubes, 0.4 ml of ice-cold diluted cytosol or plasma were added, mixed throroughly and incubated in ice-water bath for 90 min by shaking frequently. Then five volumes of the suspension of 0.57” activated charcoal (Norit A, American Norit Co.) and 0.050/ dextran T-70 (Pharmacia Fine Chemicals, Sweden) in the Tris buffer were added and immediately agitated for 5 s on a Vortex mixer. After being allowed to stand for 10min in ice-water bath, the tubes were centrifuged at 800~ for 1Omin at 2-C. An aliquot of the clear supernatant (1 ml) was pipetted into a vial containing 10 ml of xylene based scintillation cocktail (PCS, Radiochemical Center, Amersham) and the radioactivity was counted after standing for more than 12 h in a Packard Tri-Carb model 2650 hquidscintillation spectrometer. The amount of labeled steroid specifically bound in cytosol or plasma was calculated by subtracting the radioactivity of the nonlabeled compound-containing sample from that of the radioactive compound alone-containing one. Experiments
with minced granuloma and lioer tissues
Specific binding to the receptor
and nuclear uptake
of glucocorticoids were investigated by the incubation of minced granuloma with the steroid according to the procedure demonstrated by Gianopoulos[lZ]. Granuloma and liver tissues are minced with scissors into 2 mm pieces and incubated at 0°C (ice-water bath), 25 or 37°C in the Krebs-Ringer bicarbonate buffer solution containing 1 x 10-s M [3H]-glucocorticoids with or without lOOO-fold corresponding non-labeled steroids in the atmosphere of 95”/] O,--Srl, C02. After the incubation for 120min, the flask was chilled in ice-water bath and the content was transferred into a 15-ml centrifuging tube, and centrifuged at 800 g for 5 min. The sedimented pellet of tissue pieces was washed three times with 0.25 M sucroseTKM buffer (TKM: 50 mM Tris-HCI, pH 7.5, containing 25 mM KC1 and 5 mM MgCl,) and homogenised mildly in a Vir-Tris 45 homogenizer at 20 V for 20 s in 9 ml of the buffer. The homogenate was filtered through four layers of gauze and a 0.5 ml portion of the filtrate was taken and kept in a deep freezer for the determination of DNA content. The remainder was centrifuged at 8OOg for 10 min and then 105,OOOg for 60 min. The pellet (cell nuclei) and supernatant (cytosol) were obtained respectively. The cytosol was treated with the dextran-coated charcoal and then measured for charcoal-resistant (unabsorbed) radioactivity. The specific binding of the steroid in the cytosol was calculated by subtracting the radioactivity of the sample with non-labeled compound from that of the sample without non-labeled compound. The nuclear fraction was purified from the pellet according to the modified method of Blobel and Potter[13] as follows. After washing twice by centrifuging in 5 ml of 0.25 M sucrose-TKM at 800 g for 5 min, the pellet was resuspended in 3.5 ml of 0.25 M sucrose-TKM. The three ml portion of the suspension was pipetted into a centrifuging tube and admixed thoroughly with 6ml of 2.3M sucroseTKM. The mixture was underlayered by 3 ml of 2.1 M sucroseeTKM and centrifuged at 124,000 g for 30min. The tube wall was then wiped dry with a spatula wrapped with tissue paper. The white nuclear pellet was resuspended in 0.25 M sucrose-TKM for the measurement of the radioactivity. Specific nuclear uptake was also calculated by subtracting the radioactivity of the sample with non-labeled compound from that of the sample without non-labeled compound. Thin layer chromatography nucleur fraction
qf the extract
fk~m the
To the “purified nuclear fraction” obtained from 4 g of granuloma incubated without addition of nonlabeled corticoids, equal volume of chilled chloroform containing 2 x lo-’ M non-labeled dexamethasone was added, and the mixture was shaken vigorously for 2 min, and centrifuged at 800 g for IO min to obtain the chloroform extract. The extraction was carried out further twice with chloroform and once with ethylacetate and the organic layers were combined and
Glucocorticoid
receptors in carrageenin granuloma
evaporated in vucuo. The residue was dissolved in 0.1 ml of chloroform and 20 ~1 was applied to a plate coated with silica gel (Kiesel gur G, type 60, E. Merck) and developed in methylene chloride-methanol-distilled water-glycerol (150: 10: 1:0.4, v/v). After air-dryness, the successive portions of silica gel in 0.5 cm width were scraped off from the plate and the radioactivity was measured. Non-labeled dexamethasone was run together as a standard and the location was visualized under ultraviolet light. DNA-drterminution DNA was extracted by the Schmidt-ThannhauserSchneider (STS) method [14] and determined by the method of Burton[ 151. Protein determination Protein Miller[16].
was
determined
by
the
method
binding of [3H]-glucocorticoids minced tissues
binding in granuloma
The cytosols from the granulation tissue, liver and thymus as well as the plasma were incubated with C3H]-cortisol and [3H]-dexamethasone, respectively, and assayed for the glucocorticoid-binding capacity as described in the section of Materials and Methods. C3H]-Cortisol was bound specifically in all of the plasma and cytosols tested (Table 1). However, the specific binding of [3H]-dexamethasone was not demonstrated in the granuloma cytosol and plasma, whereas dexamethasone binding was evident in the cytosols from the liver and thymus. In the literature the lack of dexamethasone-binding capacity has been reported for rat plasma [17]. Dexamethasone-binding was not demonstrated even in the granuloma cytosol obtained from bilaterally adrenalectomized rats in which endogenous steroids capable of competing with the labeled corticoids would have been depleted as indicated by the result with C3H]-cortisol
Specific binding (fmol/lOO peg DNA) Tissue
[3H]-Cortisol
Granuloma Liver
56.0 + 10.1 291.3 +_ 18.2
- (9)
+ (5)
Liver Thymus Plasma Granuloma Granuloma
Experiments
with minced grunulomu und liter
Granuloma and liver were minced and incubated with C3H]-cortisol and C3HJ-dexamethasone, respectively. Results summarized in Table 2 show that dexamethasone could be taken up rather more activity by the nuclei of these tissues than cortisol did. Based on those results, further experiments were done in order to examine effects of temperature and time on dexamethasone binding in the minced granuloma. The
C3H]-Cortisol
C3H]-Dexamethasone
0.157 * 0.011 0.059 +_0.005 0.997 * 0.121 0.172 + 0.038 1.308 + O.lllt
0.329 i 0.045 0.237 f 0.019 Not detectable Not detectable Not detectable
Each value is expressed as mean k SEM. adrenalectomized (+ ) or sham-operated (-) 2 days before sacrifice. Figures in parenthesis are number of rats used. t P i 0.001 compared to control granuloma. * Rats were bilaterally
111.5 + 15.9 888.3 f 148.1
The possibility of a receptor binding of dexamethasone was also tested by passing the granuloma cytosol incubated with labeled dexamethasone through a Sephadex G-25 column (1 x 15 cm, flow rate 0.4ml/min). No radioactivity was recorded in the fractions at the void volume, while in cases of the cytosols of liver and thymus significant radioactivity was recovered in the fractions at the void volume (data are now shown). In an attempt to make time interval from cell rupture to the start of the incubation as short as possible, the earliest incubation was performed 35 min after the homogenization. It still failed to show dexamethasone binding in granuloma cytosol.
Specific binding (pmol per mg protein) Sample
[3H]-Dexamethasone
One-gram aliquots of minced liver and granuloma tissues were incubated in IOml of Krebs-Ringer bicarbonate buffer solution with 1 x 10-s M radioactive steroids in the presence and absence of 1000-fold corresponding non-labeled steroids for 12Omin at 37’C under 95 O,-Spd COZ. After incubation, cell nuclei were separated and assayed for radioactivity and DNA as described in the text. “Specific binding” was calculated by subtracting the radioactivity for the sample incubated with non-labeled steroid from that without non-labeled steroid. Figures represent mean +_SEM from three determinations.
Table 1. Specific [3H]-glucocorticoid binding in tissue cytosols and blood plasma
Adrenalectomy*
in
of
RESULTS
Apparent Itrck of dexamethasone cytosol
Table 2. Nuclear
1339
1340
YOSHIHIRO
ENDO
et al.
Nuclei .-•
\ \
\
\
\
‘0
-.
-_
--0
cytoso1
00
0
IO
30
c ‘0
“.
30
20
lncubatlon
40
Temperature,“C
Fig. l(A). Relationship between incubation temperature and dexamethasone-binding in minced granuloma. An aliquot of minced granulation tissue (1 g) was incubated with [3H]-dexamethasone (1 x lo-’ M) for 60 min at 0, 25 and 37-C. respectively. After incubation the tissue was homogenized and the nuclear and cytosol fractions were prepared. Specifically bound radioactivity in these two fractions were determined separately. Specifically bound [‘HI-dexamethasone is expressed as fmol per 1OOpg of DNA of granuloma homogenate and 1OOc~g of nuclear DNA. respectively.
results are summarized in Figs 1A and IB. At 0°C nuclear uptake of dexamethasone hardly occurred (Fig. 1A). As the incubation temperature was raised, quantity of the steroid in the nuclear fraction increased, accompanying the decrease in the amounts
o.
Authentic
time,mln
kept in the cytosol. Inverse correlation in the amount of dexamethasone between the nuclear and the cytosol fractions was also observed in respect to the time change in the distribution of dexamethasone. Thin-layer chromatography of radioactive compounds extracted from the nuclei which were isolated from the minced granuloma after incubation with [3H]-dexamethasone indicated that the radioactivity localized in the nuclear fraction corresponds exclusively to unchanged dexamethasone (Fig. 2). Any radioactivity was not extracted from the nuclear residue remained after chloroform and ethylacetate extraction as described in Materials and Methods.
1 Segnents
120
Fig. l(B). Relationship between incubation time and dexamethasone-binding in minced granuloma. The experimental condition was same in Fig. l(A) except that the incubation temperature was constant at 37°C. DEX: dexamethasone.
n t Origin
60
rn
on ~~~eZ~~orLZ’”
cm ZZf
front
Fig. 2. Thin layer chromatogram of the extract of the nuclear fraction prepared from minced granuloma incubated with [3H]-dexamethasone. The experimental condition was same in Fig. 1. Details are under “Materials and Methods”.
Glucocorticoid
receptors
in carrageenin
granuloma
Table 3. Effects of four chemicals the granuloma
cytosol
added to the incubation on the binding of dexamethasone Bound
Test chemicals None Nat MOO, ATP EDTA DTT
Concentration (mM)
10 10 10 10
(-)
C3H]-DEX
Cold DEX
1341 mixture of (DEX)
(d.p.m.)*
(+) Cold DEX
2063 2114 2514 2396 3210
2152 2211 2731 2465 2236
Each additive was dissolved in redistilled water, neutralized if required, and diluted with redistilled water to required volume after adding a 0.1 volume of 0.2 M TrisHCl buffer containing 4mM EDTA, pH 7.6. A 0.3 ml portion of 20 mM solution of an additive was added into equal volume of the cytosol, thoroughly mixed, allowed to stand in ice-water bath for one hour with occasional shaking. After the above preincubation, a 0.4 ml portion of the mixture was pipetted into another tube containing 2 x lo-* M of C3H]-dexamethasone with or without l,OOO-fold cold dexamethasone and incubated for 90 min in ice-water bath. * All values represent mean of duplicate determinations and DEX means dexamethasone.
Table 4. Effects of four chemicals added to the medium for homogenizing granuloma tissues on the binding of dexamethasone (DEX) Bound Homogenizing 20 mM Tris-HCl-0.4 + 10 M Na,MoO, + 10 mM ATP + 10mM EDTA +lOmMDTT
medium mM EDTA
C3H]-DEX
(d.p.m.)*
(--) Cold DEX
(+) Cold DEX
3385 2683 3279 3476 19,468
3558 2876 3262 3735 3114
Granuloma were minced, divided into five equal parts and homogenized in two volumes of each homogenizing medium and the cytosols were prepared as described in the text. * All values represent mean of duplicate determinations and DEX means dexamethasone.
Protection by SH reagents of dexamethasone-binding capacity in cell-free systems Several chemicals such as sodium molybdate, adenosine 5’kphosphate (ATP), Ethylenediamine tetraacetic acid (EDTA) and dithiothreitol (DTT) are reported to influence binding of glucocorticoids with their receptors [l&25,29-31, 411. Therefore, these agents were tested by adding to the granuloma cytosol 1 h before the start of incubation with C3H]-dexamethasone. As indicated in Table 3, binding activity of granuloma cytosol for [3H]-dexamethasone was recovered by the addition of DTT to the final concentration of 1OmM. Table 4 shows that the protecting effect of DTT is enhanced by its addition to the homogenizing buffer. In addition, two suhhydryl agents, glutathione (GSH) and 2-mercaptoethanol (ME) also indicated a protecting effect similar to that of DTT (Table 5). The order of protecting potency was DTT 3 GSH > ME. S” I? 12
H
Table 5. Effect of dithiothreitol
(DTT), glutathione (GSH) and 2-mercaptoethanol (ME) on the dexamethasone-binding capacity of granuloma cytosol
Homogenizing medium Tris* + DTT, 1OmM 5mM 1mM + GSH, 1OmM 5mM 1mM + ME, IOmM 5mM 1mM
Amount of specifically bound [3H]-dexamethasone (pmol per mg protein)t Not detectable 0.083 0.072 0.022 0.074 0.069 0.049 0.041 0.013 0.003
* Tris: 20 mM Tris-HC1 containing 0.4 7.6. Granuloma were minced, divided into and homogenized in each medium and binding activities were measured in cytosols the text. t All values represent mean of duplicate
mM EDTA, pH ten equal parts dexamethasone as described in determinations.
YOSHIHIRO ENDO
1342 Table 6. Deleterious
et al.
effects of DTNB on glucocorticoid-binding the liver and granuloma cytosols
activity
of
Specifically bound steroid* (pmol/mg protein) Added Cytosols Liver
DTNB
(mM)
[3H]-Cortisol
None 10.0 7.5 5.0 2.5 None 5.0
Granuloma
[‘HI-Dexamethasone
0.150 0.034 0.048 0.068 0.084 0.271 0.220
0.318 ND ND ND ND 0.042 ND
Granuloma was homogenized in 20mM Tris-HCl containing 0.4mM EDTA and 1OmM dithiothreitol. Cytosols were preincubated with equal volume of the DTNB solution in 0.2 M Tris-HCI, pH 7.6, or vehicle for 1 h in ice-water bath before the incubation with labeled steroids. ND: not detectable. * All values represent mean of duplicate determinations.
Sodium molybdate, ATP and EDTA were all ineffective (Table 4). 5,5’-Dithio-bis (Znitrobenzoic acid) (DTNB), a chemical agent reacting with the SH group, was added to the liver and granuloma cytosols 1 h before the incubation with [3H]-dexamethasone. As indicated in Table 6, DTNB caused a complete loss of the dexamethasone-binding capacities of the cytosols and a marked reduction of the cortisol-binding capacity of the liver cytosol. The cortisol-binding capacity of the granuloma cytosol was not reduced as extensively as observed in the liver cytosol, probably because the former sample was fortified with DTT. Table 7 shows that the deleterious effect of DTNB
Table
can be antagonized pounds.
by addition
of the sulfhydryl
Determination of dissociation constant for dexamethasane receptor complex in yranuloma cytosol prepared in presence of 1OmM D7T In Figure 3, the amount of dexamethasone specifically bound to granuloma cytosol was plotted as a function of the [3H]-dexamethasone concentration in incubation medium. The values of specific binding were plotted by the method of Scatchard[42]. As shown in Fig. 3B, it gave a straight line, and it was indicated that there was only a single class of specific binding site. From this experiment, the dissociation
7. Antagonism with sulfhydryl agents glucocorticoid-binding capacity
against the DTNB-effect of liver cytosol
on
Specifically bound steroid* (pmol/mg protein) Pretreatment Vehicle DTNB,
5 mM
SH agents None None DTT GSH ME
com-
[‘HI-Cortisol 0.059 0.033 0.061 0.054 0.057
[3H]-Dexamethasone 0.109 ND 0.096 0.078 0.070
Liver cytosol was preincubated with equal volume of 1OmM DTNB in 0.2 M Tris-HCI, pH 7.6, or vehicle or 1 hr in ice-water bath, applied to a Sephadex G-25 column (1.6 x 33 cm) equilibrated with 20 mM Tris-HCI containing 0.4 mM EDTA, pH 7.6, and eluted with the same buffer at a rate of one ml per min. An aliquot of the macromolecule fraction separated was incubated for one hour in ice-water bath with equal volume of a solution containing 10 mM sulfhydryl compound and glucocorticoid-binding activity was measured. DTT: dithiothreitol; GSH: glutathione; ME: 2-mercaptoethanol; ND: not detectable. * All values represent mean of duplicate determinations.
Glucocorticoid
1343
receptors in carrageenin granuloma
0.05 -
. . . . . 0. -0
05
IO
50(x
I O“M
Dexomethasone concenlmtion
)
-0
100
50
Bound dexamethosone,f
mob?s/mg protein
Fig. 3. (A) Specific binding of [3H]-dexamethasone as a function of steroid concentration. (B). Scatchard plot of specific binding data shown in (A). Specific binding of C3H]-dexamethasone in granuloma cytosol, which was prepared in 10mM DTT-containing Tris buffer, was determined as described in “Materials and Methods”. Cytosol protein concentration was 2.6 mg/ml.
constant for the binding reaction was calculated at 1.953 x 10e9 M, the number of binding sites was at 9.397 X lo- l4 mol per mg protein, DISCUSSION
“Specific” binding, as designated by Baxter and Tomkin@ 11,ofdexamethasone and cortisol to macromolecular receptors in the cytoplasmic fraction (cytosol) of rat carrageenin granuloma tissues and liver was examined according to the procedure described [ll]. Although “specific” receptor(s) for both cortisol and dexamethasone were demonstrated in the liver cytosol, such a dexamethasone receptor was not detectable in the granuloma cytosol in which “specific” receptor for cortisol was clearly demonstrated (Table 1). On the other hand, the specific binding of dexamethasone in both the cytoplasmic and nuclear fractions of the granuloma was detectable when minced granuloma tissues were incubated with a labeled dexamethasone according to the method described by Gianopoulos[12] (Table 2 and Fig. 1A and 1B). The results of the incubation experiments for the minced granuloma with dexamethasone (Figs 1A and 1B) are quite consistent with the generally accepted concept that transfer of a steroid hormone specifically bound with the cytoplasmic receptor is gradually translocated through a temperature-dependent process into the nucleus [40]. Therefore, the above mentioned failure in the indication of the specific binding of dexamethasone in the cell-free experiments of the granuloma cytosols was considered to be due to some defects in the experimental conditions. Several factors inactivating the activity of glucocorticoid receptor in
the lung [18], thymus [21-23,303, muscle [29,31], brain [27], fibroblasts [3,22], lymphocytes [22,24] and mammary gland [41]. These include dephosphorylation by alkaline phosphatase [19-211, breakdown of phospholipid structure by phospholipases [22], and disappearance of free SH groups of the receptor molecule [18-231. Among various chemicals examined in this experiment, some SH compounds (DTT, GSH and ME) were effective to protect dexamethasone binding activity in the cytoplasmic fraction of the granuloma tissues (Tables 3 and 4). These facts suggest that cytoplasmic dexamethasone receptor of the granuloma tissues is easily inactivated by oxidation of SH groups in the molecule. Even in the cytosols of the liver, treatment of the cytosols with DTNB, a chemical agent reacting with SH group to produce disulfide bond, completely damages dexamethasone-binding activity (Table 7). SH-protective agents such as DTT. GSH and ME were effective also to protect the liver cytosols from deteriorating effects of DTNB. The granuloma cytosols apparently contain more cortisol-binding components than those for dexamethasone binding while vice oersa in the liver cytosols (Table 6). The cortisol-binding activity in the granuloma cytosol is rather resistant to 5 mM DTNB compared with the dexamethasone-binding activity. Many reports have indicated that there are plural glucocorticoid-binding components in tissue cytosols, that is, specific glucocorticoid receptor, corticosteroid-binding globulin (CBG), CBG-like macromolecule, cortisol metabolite binder and ligandin [2639]. It has been already known that cortisol binding activities of plasma CBG and tissue CBG-like macromolecule are relatively stable pro-
1344
YOSHIHIRO ENDO rt al.
teins which are ers [23,26,27,29-321. that cortisol binding blocking by DTNB in loma and the liver are components. Acknowledgements-The Miss Sachiko Tsuriva
insensitive to It may be therefore activities observed both the cytosols of due to CBG and/or
SH-blocksuggested after SHthe granuCBG-like
authors wish to acknowledge for her excellent technical assistance.
REFERENCES
1 Baxter J. D. and Forsham P. H.: Tissue effects of glucocorticoids. Am. J. Med. 53 (1972) 573-589. 2 Buller R. E. and O’Malley B. W.: The biology and mechanism of steroid hormone receutor interaction with the eukaryotic nucleus. Biochem: P~A~~Aco~. 25 (1976) l-12. 3. Pratt W. B.: The mechanism of glucocorticoid effects in fibroblasts. J. Invest. Dermatol. 71 (1978) 2435. 4. Kaiser N., Milholland R. J. and Rosen F.: Glucocorticoid receptors and mechanism of resistance in the cortisol-sensitive and -resistant lines of lymphosarcoma P1798. Cancer Res. 34 (1974) 621-626. 5. Beato M., Kalimi M. and Feigelson P.: Correlation between glucocorticoid binding to specific liver cytosol receptor and enzyme induction in uiuo. Biochem. Biophys. Rex Commun. 47 (1972) 14661472. H.: Comparison of corticoid 6. Tiara M. and Terayama receptor and other cytoplasmic factors among liver and hepatoma cell lines with different sensitivity to corticoid inhibition of cell growth. Biochim. biophys. AC~A 541 (1978) 4558. 7. Young D. A., Barnard T., Mendelsohn S. and Giddings S.: Early cordycepin-sensitive event in the action of glucocorticoid hormones on rat mRNA initiates the earliest metabolic effects of steroid hormones. Endocrine res. Commun. 1, (1974) 63/72. 8. Tsurufuji S., Sugio K. and Takemasa F.: The role of glucocorticoid receptor and gene expression in the anti-inflammatory action of dexamethasone. Nature 280 (1979) 408%410. 9. Koshihara Y., Yamagishi M., Murota S. and Tsurufuji S.: Cortisol-binding protein in the cytosol of rat carrageenin granuloma. Jpn. J. Pharmacol. 25 (1975) 271-280. 10. Tsurufuji S., Sugio K. and Endo Y.: Inhibition of vascular permeability by cycloheximide in granulomatous inflammation. Biochem. f’hArI?IAcOf. 26 (1977) 1131-1136. II. Baxter J. D. and Tomkins G. M.: Specific cytoplasmic glucocorticoid hormone receptors in hepatoma tissue culture cells. Proc. natn. Acad. Sci., U.S.A. 68 (1971) 932-937. G.: Ontogeny of elucocorticoid receptors 12. Gianopoulos in rat iiver. J. biol. Chem. iSO (1975) 5847-5851. . 13. Blobel G. and Potter V. R.: Nuclei from rat liver: Isolation method that combines purity with high yield. Science 154 (1966) 1662-1665. of nuclei acid. 14. Volkin E. and Cohn W. E.: Estimation In Methods 01 Biochemical At~~lpi~ (Edited by D. Glick). Interscience., New York, Vol. I (1954) p. 287. 15. Burton K.: Determination of DNA concentration with diphenylamine. In Methods in Enzymology (Edited by L. Grossman and K. Moldave). Academic Press, New York, Vol. XII, Part B (1968) p. 163. 16 Miller G. L.: Protein determination for large numbers of samples. Analyt. Chem. 31 (1959) 964. G. G., Baxter J. D. and Tomkins G. M.: 17. Rousseau
Glucocorticoid receptors: Relations between steroid binding and biological effects. J. mol. Biol. 67 (1972) 99-115. 18. Granberg J. P. and Ballard P. L.: The role of sulfhydryl groups in the binding of glucocorticoids by cytoplasmic receptors of lung and other mammalian tissues. Endocrinoloy~ 100 (1977) 1160-l 168. 19. Nielsen C. J., Vogel W. M. and Pratt W. B.: Inactivation of glucocorticoid receptors in cell-free preparations of rat liver. Comer Rex 37, (1977) 3420-3426. 20. Nielsen C. J.. Sando J. J. and Pratt W. B.: Evidence that dephosphorylation inactivates glucocorticoid receptors Proc. not. Acod. Aci. U.S.A. 74 (1977) 1398-1402. 21. Sando J. J., Hammond N. D., Stratford C. A. and Pratt W. B.: Activation of thymocyte glucocorticoid receptars to the steroid binding form. J. hiol. Chem. 254 (1979) 4779-4789. 22. Schulte H. F.. Nielsen C. J., Sand0 J. J. and Pratt W. B.: Evidence for a ohosoholiuid reauirement in the specific binding of &ucoiortidoids io receptors of fibroblasts and thymic lymphocytes. J. biol. Chem. 251 (1976) 2279-2289. 23. Rees A. M. and Bell P. A.: The involvement of receptor sulfhydryl groups in the binding of steroids to the cytoplasmic glucorticoid receptor from rat thymus. Biochim. biophys. AC~A 411 (1975) 121-132. 24. Sando J. J., La Forest A. C. and Pratt W. B.: ATPdependent activation of L cell glucocorticoid receptors to the steroid binding form. J. hiol. Chem. 254 (1979) 4772-4778. properties 25. Bell P. A. and Munck A.: Steroid-binding and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells. Biochem. J. 136 (1973) 97-107. 26. De Kloet E. R. and McEwen B. S.: A putative glucocorticoid receptor and a transcortin-like macromolecule in pituitary cytosol. Biochem. biophys. ACTA 421 (1976) 115123. 27. De Kloet E. R. and McEwen B. S.: Differences between cytosol receptor complexes with corticosterone and dexamethasone in hippocampal tissue from rat brain. Biochim. biophys. AC~A 421 (1976) 124132. 28. Werthamer S., Samuels A. J. and Amaral L.: Identification and partial purification of “transcortin”-like protein within human lymphocytes. J. hiol. Chem. 248 (1973) 6398-6407. 29. Mayer M., Kaiser N.. Milholland R. J. and Rosen F.: Cortisol binding in rat skeletal muscle. J. biol. Chem. 250 (1975) 1207-1211. 30. Schaumburg B. P.: Investigations on the glucocorticoid-binding protein from rat thvmocvtes. II. Stabilitv. kinetics. and specificity of binding of steroids. Biochim. hioph~.s. Acra 261 (19721 219-235. R. J. and Rosen F.: 31. Mayer M., Kaiser N., Milholland Binding of dexamethasone and triamcinolone acetonide to rlucocorticoid receptors in rat skeletal muscle. J. biol. Chrm. 249 (1974) 52365240. S. N., 32. Seleznev Yu M.. Dnilov S. M., Preobradienskv Volkova N. G.. Kuzentsova L. A., Smirn& V.-N. and Volchek A. G.: Glucocorticoid-binding proteins of rat heart cytosol and their possible role in the transfer of glucocorticoids into nuclei. J. mol. Cell. Cardio/. 10 (1978) 877-891. N.. 33. Litwack G., Filler R., Rosenfield S. A., Lichtash Wishman C. A. and Singer S.: Liver cytosol corticosteroid binder II, a hormone receptors. J. biol. Chem. 248 (1973) 7481-7486. of 34. Singer S., Morey K. S. and Litwack 0.: Properties the cortisol metabolite binding system in liver cvtosol. Phytiol. Chem. Physics 2 (1970) i17-126. _ 35. Litwack G. and Morey K. S.: Cortisol metabolite binder I: Identity with the dimethylaminoazobenzene
Glucocorticoid
36.
37.
38.
39.
receptors
binding protein of liver cytosol. Biochem. bioph_rs. Res. Commun. 38, (1970) 1141-1148. Singer S. and Litwack G.: Identity of corticosteroid binder I with the macromolecule binding 3-methylcholanthrene in liver cytosol in oiuo. Cancer Res. 31 (1971) 13641368. Litwack G. and Rosenfield S. A.: Liver cytosol corticosteroid binder IB, a new binding protein. J. biol. Chem. 250 (1975) 6799-6805. Beato M., Biesewig D., Braendle W. and Sekeris C. E.: On the mechanism of hormone action XV. Subcellular distribution and binding of [1,2-3H]-cortisol in rat liver. Biochim. biophJs. Actu 192 (1969) 494-507. Amaral L., Lin K., Samuels A. J. and Werthamer S.:
in carrageenin
granuloma
1345
Human liver nuclear transcortin. Its postulated role in glucocorticoid regulation of genetic activity. Biochim. biophys. Acta 362, (1974) 332-345. 40. Wira C. R. and Munck A.: Glucocorticoid-receptor complexes in rat thymus cells. “Cytoplasmic”-nuclear transformations. J. biol. Chem. 249 (1974) 53285336. 41. McBlain W. A. and Shyamala G.: Inactivation of mammary cytoplasmic glucocorticoid receptors under cell-free conditions. J. biol. Chem. 255 (1980) 38843891. 42. Scatchard G.: The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. St (1949) 660-672.