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Inhibitory action of steroid hormones on RNA synthesis of isolated thymus nuclei
562
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 97038
I N H I B I T O R Y ACTION OF S T E R O I D HORMONES ON RNA S Y N T H E S I S OF I S O L A T E D THYMUS N U C L E I ALEXANDRU
D. A B R A H A M " AND C O N S T A N T I N E .
SEKERIS
Physiologisch-Chemisches Institut, Philipps Universitdt Marbu~g/Lahn tGermany) (Received J u n e I6th, 1971)
SUMMARY
The effects of 12 steroids on RNA polymerase activity of aggregate enzyme preparations of isolated thymus nuclei were studied. Cortisol, cortisone, prednisolone, testosterone and dihydrotestosterone decreased RNA synthesis; the other structurally related steroids showed no effect. The inhibition of RNA synthesis was observed only if the assay for RNA polymerase activity was conducted with the sediment of lysed nuclei or with the aggregate enzyme preparations, but not with whole nuclei. The effects of the hormones in the presence of ~-amanitin suggest, that cortisol inhibits preferentially ribosomal RNA synthesis, whereas testosterone affects DNA like RNA.
INTRODUCTION
The ability of corticosteroids and androgens to increase RNA synthesis in their target organs such as liver or male accessory glands, has been widely documented 5,e,14,15,21,22,~,2e,3e-4°,~. Evidence has been presented that the steroids exert their primary action within the confines of the cell nucleus b y increasing the activity of chromatin to support RNA synthesis 17-19,37-~°. One other organ which responds to steroid hormones is the thymus. Both corticosteroids and androgens induce rapid involution processes in the thymus, characterized b y pronounced catabolism of nucleic acids and proteins 1-a,n,23,32,41,43. However, the mechanism of action of these steroids is still unknown. In order to explore the mechanism b y which steroids affect nucleic acid synthesis in the thymus we have studied their action on RNA synthesis of isolated thymus nuclei. It was demonstrated that corticosteroids and testosterone stimulate RNA synthesis of isolated rat liver nuclei in a similar way as they do in vivo 3e,3s,4°. We were therefore interested to see whether steroids which inhibit in vivo RNA synthesis of thymus nuclei could duplicate this effect in an in vitro system. MATERIAL AND METHODS
Male Wistar B R I I rats, weighing 9o-11o g were used. The animals were kept under standard conditions. The animals were killed b y cervical dislocation, thymi * Fellow of t h e " E u r o p e a n Molecular Biology Org&nisation" (197 o) a n d of t h e " A l e x a n d e r y o n H u m b o l d t F o u n d a t i o n " (1971).
Biochim. Biophys. Acta, 247 ti97 I) 562-569
HORMONAL INHIBITION OF THYMUSRNA SYNTHESIS
563
were quickly removed and placed into ice cold Tris-sucrose-salt buffer consisting of 0.25 M sucrose, 0.025 M KC1, o.oi M MgC12 in Tris-HC1 (pH 7.55), minced with scissors and homogenized in 5 vol. of Tris-sucrose-salt buffer by means of a PotterElvehjem homogenizator with a teflon pestle (clearence 300/~m) hand driven. All the following steps were performed at 0-2 ° unless otherwise mentioned. The homogenates were filtered successively through four and eight layers of cheese cloth and centrifuged at 75o×g for 7 rain. The supernatants were discarded and the crude nuclear pellets were further purified as follows. The nuclear pellets were resuspended in IO vol. of Tris-sucrose-salt buffer by gentle homogenisation and mixed with an equal volume of a 1 % solution of Triton X-Ioo in Tris-sucrose-salt buffer. The mixture was kept for 30 sec and the nuclei recovered by centrifugation at IOOO×g for 5 rain. The nuclear pellet was washed in 20 vol. of Tris-sucrose-salt buffer and the nuclei recovered b y centrifugation at IOOOx g for 7 rain. The purity of the nuclear preparations was checked b y phase-contrast or electron microscopy.
Administration o/steroids Analitically pure steroids: cortisol, cortisone, 5/5-preguanolone (5fl-pregnane3/5-ol-2o-one), II-deoxycorticosterone, testosterone, 4,5~-dihydrotestosterone and cyproterone (I~, 2c¢-methylene-pregna-4,6-diene-3,2o-dione-6-chloro-i7,¢-ol) were a kind gift of A.G. Schering (Berlin); prednisolone (11/5, I7~, 2I-trihydroxy-pregna-I, 4diene-3,2o-dione), tetrahydrocortisol (3~, II/5,I7~,2I-tetrahydroxy-5/5-pregnane-2oone), 5~-androsterone were purchased from E. Merck (Darmstadt) and dehydroepiandrosterone and ethiocholan-3~,6~-diol-i7-one were a kind gift of the Institute of Chemistry, Cluj (Romania). The steroids were dissolved in ethyleneglycol (p.a. E. Merck), and added in a final volume of o.oi ml to 5 ml of nuclear suspension. After IO min shaking in a water bath at 37 ° an equal volume of ice cold Tris-sucrosesalt buffer was added and the nuclei were recovered by centrifugation at IOOO×g for 5 rain. Control nuclei were treated with equal volume of ethyleneglycol (o.oi ml). Isolation o/the aggregate enzym# ~ and determination o/the R N A polymerase activity The control and steroid treated triton nuclear pellet recovered by centrifugation as described above were homogenized in IO vol. of 0.050 M Tris-HCl buffer (pH 7.4) and left at o ° for IO min. The sediment of lysed nuclei obtained after a centrifugation at 8000 x g for IO min was suspended in IO vol. of 0.067 M Tris-HC1 buffer (pH 7.9) and one quarter volume of 2 M KC1 was added (final concentration 0. 4 M KC1). This mixture was homogenized and left at o ° for 30 min. The precipitated fibrous material was recovered by centrifugation at 8000 Xg for IO rain. The preparation was resuspended and homogenized in 0. 5 ml Tris-HC1 (pH 7.9)- Quantiration of this preparation was performed on the basis of the amount of DNA, RNA and protein content determined according to BURTON 9, OGUR AND ROSENzl and LowRY et al. zs. The incorporation of E14C]UTP into acid precipitable material was taken as a measure of the activity of the RNA polymerase enzyme. The in vitro system consisted of 0.5/,mole each of 5'-ATP-Na2, GTP-NasH2, 5'-CTP-Na3, 0.2 #C 5'-E4-14C]UTP-NH 4 (specific activity, 51/,C//,mole), 5/*moles creatine phosphate-Na~, IO/,g creatine phosphokinase, 6/~moles/5-mercaptoethanol and 2/,moles M_.n2+ (MnSO4) in a final volume of 0.3 ml in Tris-HC1 buffer (pH 7.9). The aggregate enzyme preparaBiochim. Biophys. Acta, 247 (197I) 562-569
564
A.D.
ABRAHAM, C. E. SEKERIS
tion was added in amounts of 15o-2oo/~g DNA. Incorporation of labelled precursor was measured as described previously 5,26, using a Nuclear Chicago Mark I liquid scintillation counter. The experimental data were statistically evaluated applying the Chauvenet and Student's tests.
RESULTS
It is evident from Table I that cortisol, cortisone and prednisolone depress RNA synthesis b y the aggregate enzyme. Cortisol is the most active of the corticosteroids tested, depressing RNA polymerase activity up to 60 °/o at the concentration of lO -5 M and showing inhibitory effects up to concentrations of IO-v M. The effect is dose dependent in the range of 1o -~ to lO -5 M (see Fig. Ia). n-Deoxycorticosterone, tetrahydrocortisol and 5fl-pregnanolone had no significant effect on RNA synthesis.
I
TABLE EFFECT GATE
OF
CORTISOL
ENZYME
AND
RELATED
PREPARATION
STEROIDS
OBTAINED
ON
FROM
THE
ISOLATED
OF R N A
ACTIVITY RAT
THYMUS
POLYMERASE
Steroid
Final concentration (M)
pmoles [**C]UTP incorporated into R N A per rain per mg protein
n
%
Cortisol
Control i o -~ Control i o -e Control lO -5
9.4 4-0.3 7.4±0.8 I7.4:h2.i I i . O : h I. 3 9.4-4- o.9 3.8-4-1.o
6 6 9 ii 6 9
---21. 3 ---36.8 ---59.6
Control I°-S Control lO -5
9.04-1.5 5.44-0.5 13.44-1.4 8.9io. 9
5 5 15 16
---4 °.0 ---33.6
Control IO -e Control lO -5
I 2 . 7 4 - I.O 9.3:ho. 5 i6.4~i.6 io.o-ho. 3
6 6 9 IO
---26.8 ---38.2
Control IO -e Control lO -5
8.1 4- I .o 8.64-0. 3 19.6 i 1.4 19.84-2.8
9 6 6 9
-+ -+
Tetrahydrocortisol
Control lO -6 Control 10 -5
13.3 ± 3.2 I 4 . I - h 2.o 18.2 ~ 1,7 20.84-1.1
6 6 6 4
-+ 6.0 -+14"2
5fl-Pregnanolone
Control lO -6 Control 10--5
6.8 4- o. 7 7.1±1.o 6.8-t-o. 7 7 . 0 ± 1.2
7 9 7 5
-+ -+
Cortisone
Prednisolone
I i-Deoxycorticosterone
Biochim. Biophys. Acta,
247 ( I 9 7 I ) 5 6 2 - 5 6 9
OF
AGGRE-
NUCLEI.
P
<
0.05
<
0.05
<
O .OI
<
0.02
<
0.02
<
o.i o.ooi
6.1
> 0.25
o.i
> 0.25 > 0.25 > 0.05
4.7
> 0.25
3 .8
> 0.25
HORMONAL INHIBITION OF THYMUS R N A
565
SYNTHESIS
In a second series of experiments we studied the effects of androgenic and structurally related steroids on R N A polymerase activity of aggregate enzyme isolated from previously exposed rat thymus nuclei. As can be seen from the Table II testosterone and dihydrotestosterone at concentrations of IO-e and lO -5 M significantly inhibited R N A synthesis (See also Fig. Ib), whereas the other related steroids tested (5~-androsterone, ethiocholandiolone and the testosterone antagonist cyproterone 16) showed no effect. TABLE
II
E F F E C T OF T E S T O S T E R O N E AND R E L A T E D S T E R O I D S ON T H E A C T I V I T Y OF R N A
POLYMERASE OF
AGGREGATE ENZYME PREPARATION OBTAINED FROM ISOLATED RAT THYMUS NUCLEI
Steroid
Testosterone
Final
pmoles
concentration (M)
incorporated into RNA per rain per mg protein
n
%
P
Control
14.94-1.6 12.7+1.2 15.74-1. 5 lO.54-1. 4 17.o4-o. 9 lO.74-o.6
II 9 16 14 17 14
---14.8 ---33.1 ---37.1
-> --<
I2.3~I.O 9.7~ o.7 I2.3~I.O 9.1±o.6
16 17 16 18
---21.2 --26.1
-< -<
12.64-o. 9 iI.9~:o. 7 9.44-0.7 I°.4i°.5
6 6 8 9
--- 4.8 -+1o.6
-> 0.25 -> 0.05
Control
9.44-0.7 lO.9±o.5 i6.oi3.2
8 3 6
-+15.9 --
-~- o . I --
I°-8
15.°4-3.°
5
--
6. 3
>
Control
I5.9~ 1.3 13.94-o. 5 15.9± 1.3 14.54-1.1
6 5 6 6
---12.6 --- 8.8
-> ->
6.8 ± o.2
3
--
--
7.64- °.6 1 8 . 2 4- I . O 15.64-o. 7
3 9 8
+11.7 ---14. 3
lO-7
Control lO - s
Control lO -5 4,5c~-Dihydro -
testosterone
Control i o -~
Control IO - s
Dehydroepiandrosterone
Control lO -8
Control 1°-5
5~-Androsterone
Control lO-8
Ethiocholandiolone
IO - 8
Control IO -5
Cyproterone
Control I°-~
Control lO -5
[14C] U T P
o.i o.o2 o.ooi
o.05 o.oi
o.i
o.I 0.25
> o.i -> o.I
Addition of the steroids directly to the aggregate enzyme assay system had no effect of statistical significance on the precursor incorporation into RNA. Furthermore, we have studied the action of cortisol and testosterone on the rate of R N A synthesis of whole nuclei, lysed nuclei and aggregate enzyme derived from the same preparation of thymic nuclei preincubated in the presence of hormone in the same conditions as described above. As seen from the Table III no effect of these hormones on R N A synthesis by whole nuclei could be demonstrated, whereas in the lysed nuclei and aggregate enzyme preparation a decrease of R N A polymerase activity was observed. Biochim. Bio~hvs. Acta,
247 (1971) 562-569
566
A . D . ABRAHAM~ C. E. SEKERIS
TABLE
III
EFFECT OF 0¢-AMANITIN ON R N A PoLYMERA~E ACTIVITY OF STEROID PRETREATED RAT THYMUS NUCLEI, MEASURED IN WHOLE NUCLEAR, LYSED NUCLEAR OR AGGREGATE ENZYME PREPARATIONS F i n a l c o n c e n t r a t i o n o f s t e r o i d s : IO -~ mole/1. ~ - A m a n i t i n w a s a d d e d t o t h e R N A p o l y m e r a s e a s s a y s y s t e m a t lO -6 mole/1. T h e r e s u l t s a r e p r e s e n t e d i n p m o l e of E14C]UTP i n c o r p o r a t e d i n t o R N A i n 1 m i n / m g p r o t e i n . P1 r e p r e s e n t s t h e s i g n i f i c a n c e o f t h e d i f f e r e n c e s b e t w e e n c o n t r o l a n d o t h e r s e r i e s of e x p e r i m e n t s , P 2 b e t w e e n t h e , ¢ - a m a n i t i n a n d h o r m o n e + c ¢ - a m a n i t i n t r e a t e d o n e s .
Control
Cortisol
c~-Amanitin
Cortisol+c~-amanitin
Whole Nuclei
.~ ~:S.E. n % P1 P2
40.8 4.5 12 ----
49.2 5 .8 12 + 20.0 > 0.05 --
16.8 3 .6 6 -- 58.8 < o.ooi --
21.6 3.9 6 - - 47.1 < o.oi ~ 0"25
Lysed Nuclei
• ±S.E. n ~o P1 P2
36.7 4.4 8 ----
22.2 3.9 9 -- 39.5 < 0.05 --
12.6 3.5 5 -- 65.7 < o.oi --
3.3 I.I 6 -- 91.o < o.ooi < 0.02
Aggregate Enzyme
-~" ±S.E. n % P1 p~
32.5 2. 3 8 ----
15.o 1. 5 12 -- 53.9 < o.ooi --
16.o 2.1 6 -- 50.8 < o.ooi --
3.2 1.2 6 -- 90.2 < o.ooi < o.ooi
Control
Testosterone
c¢-Amanitin
Testosterone + e-amanitin
Whole Nuclei
_~ :j: S . E . n O/o P1 P.2
38.4 5.2 6 ----
38.9 4.6 6 + 1.3 > 0.25 --
12.9 i.i 6 - - 66. 4 < o.ooi --
13.6 4.o 6 -- 64.6 ( o.oi ~> 0"25
Lysed Nuclei
-~ IS.E. n O/o P1 P~
33.5 1. 4 6 ----
23 .2 5.0 6 -- 30.8 .< 0 . 0 5 __
l°.7 1.5 5 - - 68.1 < o.ooi __
9.5 3.9 5 -- 71.7 -~ o . o o i > 0.25
Aggregate Enzyme
-~ ±S.E. n °/o P1 p,
32.3 2. 7 5 ----
24. 7 1. 3 6 -- 23.5 < 0.05 --
14.3 i.i 5 -- 55-7 < O.Ol --
12.9 2.2 4 - - 6o.1 < o.ooi > 0.25
In
order
hormones,
we
inhibits
to differentiate conducted
transcription
A s,~°,~5. ~ - A m a n i t i n concentration merase
RNA
polymerase
with
a-amanitin.
RNA added
of 10 -6 M, enough
B. 0¢-Amanitin
or aggregate
by was
which
experiments
enzyme
accentuated preparation
polymerase directly to obtain the value obtained
Biochim. Biophys. Acta, 2 4 7 (1971) 5 6 2 - 5 6 9
B, but
to the
RNA
maximal
activity As
not
the
inhibitory
RNA assay
effects
in the
cortisol
known
with
polymerase
of inhibition from
that
is inhibited
well
case
exposed
by
the
~-amanitin polymerase system
on RNA of lysed thymus
at
a
polynuclei nuclei.
HORMONAL INHIBITION OF THYMUS RNA SYNTHESIS
567
Finalconcentratiofonsteroids 10-7
-20
10-6
10-5
10-7
M
1~ 6
1~ 5
M
rednisolone -20
~Testosterone ~ stosterone
c
a
g
-4C
v
~ -6C 0-
-4£
~,
-6c
Fig. I. Per cent inhibition of RIqA polymerase activity of aggregate enzyme preparation obtained from the isolated rat thymus nuclei depending on the doses of steroids. No significant differences could be seen in the case of st-amanitin and testosterone+ c¢-amanitin treated whole nuclei, lysed nuclei or aggregate enzyme (Table III).
DISCUSSION From the work of NAKAGAWA AND WHITE 29'3°, WHITE et al. 4s, I~UNCK AND BRICK-JOHNSON28, DREWSTMand DREWS AND WAGNERTM, it is known that in vivo application of corticosteroids decreases RNA synthesis in the thymus. Although m a n y hypotheses have been proposed to explain this phenomenon, no clear-cut evidence has been presented as yet in their favour. The inhibition of RNA synthesis under physiological conditions is probably only lO-2O %, but is enough to initiate a general catabolic process, which is characteristic for thymus involution z-4,x°,~s,32,41. From our results we can conclude that the inhibition of RNA polymerase activity is a very rapid phenomenon and can be considered as one of the primary effects of the hormones, preceeding the inhibition of oxidative processes 1° or of the glucose uptake ~7,zs. In the case of cortisol the steroid dosage needed to induce the effects on the thymus nuclei is at least ten times less than that needed to stimulate RNA synthesis of isolated rat liver nuclei 7,3s,3s. This m a y very well be due to the presence in the thymus nuclei of receptors taking up steroids, which render the presence of cytosol transport systems unnecessary. Such receptors have been demonstrated b y SCHAUMBURG84 and WIRA AND I~UNCKaS. One argument in favour of this hypothesis is that the direct administration of steroids to the aggregate enzyme preparations showed no effects on its RNA polymerase activity. The effects of steroids is probably mediated b y a receptor system present in the nuclei, but lost during the preparation of the aggregate enzyme. Biochim. Biophys. Acta, 247 (1971) 562-569
568
A. D. ABRAHAM, C. E. SEKERIS
Recent findings of NAKAGAWAAND WHITE 3°, which came to our attention during the preparation of our manuscript, show a decreased activity of the RNA polymerase activity extracted from thymus nuclear preparations obtained from pretreated rats with corticosteroids. They ascribed this to a reduced activity of the RNA polymerase of the hormone-treated animals. One other possibility is that the amount of the enzyme bound to the nucleochromosomal apparatus is less, reflecting a decreased availability of enzyme binding sites on the genome as a consequence oJ hormone action. It is interesting to note that RNA polymerase activity of whole nuclei preincubated with steroids is not modified in comparison to controls. However, when the assays were performed on lysed nuclei or aggregate enzyme preparation, the hormones reduced RNA polymerase activity. One explanation for these findings could be, that due to the preparation procedures, the lysed nuclear sediment or the aggregate enzyme preparation contains no soluble RNA polymerase, but only bound and transcribing enzymes. In whole nuclei a great quantity of soluble RNA polymerase is present which under the in vitro conditions of the assay, could unspecifically bind to DNA and synthesize RNA. The experiments with ~-amanitin were performed in order to differentiate the nature of the enzyme activity affected b y the hormones. It is well known, that in vitro ~-amanitin inhibits the RNA polymerase involved in the synthesis of DNA like RNA, whereas the enzyme presumably transcribing the ribosomal genes is not affected s,2°,85. Our results showed that cortiso] inhibits the RNA polymerase synthesizing ribosomal RNA, as has been proposed previously b y DREWS 12, DREWS AND WAGNER13, on the basis of the nearest neighbor frequency spectrum of E3~PIRNA synthesized b y isolated thymus nuclei in the presence of Mg 2+ from rats which had received prednisolone. These authors showed that administration of prednisolone, however, did not modify the quantity or the quality of the product formed b y thymus nuclei in the Mn2+-activated system, which findings are in good corelation with our data concerning the action of cortisol on thymus nuclei. Testosterone, on the other hand, preferentially decreases the synthesis of DNA like RNA, whereas no additive effect of testosterone+~-amanitin was observed. It is probable that the effect of various groups of steroids on RNA synthesis of thymus nuclei is specific. Glucocorticoids are potent inhibitors of RNA polymerase activity and cause rapid changes in the thymus RNA metabolism. Hypersecretion of androgens causes atrophy of the thymus and castration prevents this atrophy. This points to specific relations between thymus and the genital organs. In this paper the specific inhibitory effect of testosterone and dihydrotestosterone on RNA synthesis was demonstrated. Not all the substances which posses a steran skeleton had inhibitory effect on RNA polymerase activity of thymus nuclei, but only those with a certain well defined chemical structure and steric conformation, a fact accentuated b y us in other papers 1,3, too. ACKNOWLEDGEMENTS
We t h a n k Professor P. Karlson for his encouraging interest in our work and Dr. K. H. Seifart for m a n y helpful discussions. We are grateful to Mrs. Ch. Pfeiffer, Miss G. Frbhlich, and Mrs. U. Weiser for their skillfull assistance. One of us {A.D.A.) Biochim. Biophys, Acta, 247 (1971) 562-569
HORMONAL INHIBITION OF THYMUS
RNA
SYNTH,~ISIS
569
thanks the European Molecular Biology 0rganisation and "Alexander von Humboldt Foundation" for providing research scholarships. This work was supported by the Deutsche Forschungsgemeinschaft. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43 44 45
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Biochim. Biophys. Acta, 247 (1971) 562-569
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 97038
I N H I B I T O R Y ACTION OF S T E R O I D HORMONES ON RNA S Y N T H E S I S OF I S O L A T E D THYMUS N U C L E I ALEXANDRU
D. A B R A H A M " AND C O N S T A N T I N E .
SEKERIS
Physiologisch-Chemisches Institut, Philipps Universitdt Marbu~g/Lahn tGermany) (Received J u n e I6th, 1971)
SUMMARY
The effects of 12 steroids on RNA polymerase activity of aggregate enzyme preparations of isolated thymus nuclei were studied. Cortisol, cortisone, prednisolone, testosterone and dihydrotestosterone decreased RNA synthesis; the other structurally related steroids showed no effect. The inhibition of RNA synthesis was observed only if the assay for RNA polymerase activity was conducted with the sediment of lysed nuclei or with the aggregate enzyme preparations, but not with whole nuclei. The effects of the hormones in the presence of ~-amanitin suggest, that cortisol inhibits preferentially ribosomal RNA synthesis, whereas testosterone affects DNA like RNA.
INTRODUCTION
The ability of corticosteroids and androgens to increase RNA synthesis in their target organs such as liver or male accessory glands, has been widely documented 5,e,14,15,21,22,~,2e,3e-4°,~. Evidence has been presented that the steroids exert their primary action within the confines of the cell nucleus b y increasing the activity of chromatin to support RNA synthesis 17-19,37-~°. One other organ which responds to steroid hormones is the thymus. Both corticosteroids and androgens induce rapid involution processes in the thymus, characterized b y pronounced catabolism of nucleic acids and proteins 1-a,n,23,32,41,43. However, the mechanism of action of these steroids is still unknown. In order to explore the mechanism b y which steroids affect nucleic acid synthesis in the thymus we have studied their action on RNA synthesis of isolated thymus nuclei. It was demonstrated that corticosteroids and testosterone stimulate RNA synthesis of isolated rat liver nuclei in a similar way as they do in vivo 3e,3s,4°. We were therefore interested to see whether steroids which inhibit in vivo RNA synthesis of thymus nuclei could duplicate this effect in an in vitro system. MATERIAL AND METHODS
Male Wistar B R I I rats, weighing 9o-11o g were used. The animals were kept under standard conditions. The animals were killed b y cervical dislocation, thymi * Fellow of t h e " E u r o p e a n Molecular Biology Org&nisation" (197 o) a n d of t h e " A l e x a n d e r y o n H u m b o l d t F o u n d a t i o n " (1971).
Biochim. Biophys. Acta, 247 ti97 I) 562-569
HORMONAL INHIBITION OF THYMUSRNA SYNTHESIS
563
were quickly removed and placed into ice cold Tris-sucrose-salt buffer consisting of 0.25 M sucrose, 0.025 M KC1, o.oi M MgC12 in Tris-HC1 (pH 7.55), minced with scissors and homogenized in 5 vol. of Tris-sucrose-salt buffer by means of a PotterElvehjem homogenizator with a teflon pestle (clearence 300/~m) hand driven. All the following steps were performed at 0-2 ° unless otherwise mentioned. The homogenates were filtered successively through four and eight layers of cheese cloth and centrifuged at 75o×g for 7 rain. The supernatants were discarded and the crude nuclear pellets were further purified as follows. The nuclear pellets were resuspended in IO vol. of Tris-sucrose-salt buffer by gentle homogenisation and mixed with an equal volume of a 1 % solution of Triton X-Ioo in Tris-sucrose-salt buffer. The mixture was kept for 30 sec and the nuclei recovered by centrifugation at IOOO×g for 5 rain. The nuclear pellet was washed in 20 vol. of Tris-sucrose-salt buffer and the nuclei recovered b y centrifugation at IOOOx g for 7 rain. The purity of the nuclear preparations was checked b y phase-contrast or electron microscopy.
Administration o/steroids Analitically pure steroids: cortisol, cortisone, 5/5-preguanolone (5fl-pregnane3/5-ol-2o-one), II-deoxycorticosterone, testosterone, 4,5~-dihydrotestosterone and cyproterone (I~, 2c¢-methylene-pregna-4,6-diene-3,2o-dione-6-chloro-i7,¢-ol) were a kind gift of A.G. Schering (Berlin); prednisolone (11/5, I7~, 2I-trihydroxy-pregna-I, 4diene-3,2o-dione), tetrahydrocortisol (3~, II/5,I7~,2I-tetrahydroxy-5/5-pregnane-2oone), 5~-androsterone were purchased from E. Merck (Darmstadt) and dehydroepiandrosterone and ethiocholan-3~,6~-diol-i7-one were a kind gift of the Institute of Chemistry, Cluj (Romania). The steroids were dissolved in ethyleneglycol (p.a. E. Merck), and added in a final volume of o.oi ml to 5 ml of nuclear suspension. After IO min shaking in a water bath at 37 ° an equal volume of ice cold Tris-sucrosesalt buffer was added and the nuclei were recovered by centrifugation at IOOO×g for 5 rain. Control nuclei were treated with equal volume of ethyleneglycol (o.oi ml). Isolation o/the aggregate enzym# ~ and determination o/the R N A polymerase activity The control and steroid treated triton nuclear pellet recovered by centrifugation as described above were homogenized in IO vol. of 0.050 M Tris-HCl buffer (pH 7.4) and left at o ° for IO min. The sediment of lysed nuclei obtained after a centrifugation at 8000 x g for IO min was suspended in IO vol. of 0.067 M Tris-HC1 buffer (pH 7.9) and one quarter volume of 2 M KC1 was added (final concentration 0. 4 M KC1). This mixture was homogenized and left at o ° for 30 min. The precipitated fibrous material was recovered by centrifugation at 8000 Xg for IO rain. The preparation was resuspended and homogenized in 0. 5 ml Tris-HC1 (pH 7.9)- Quantiration of this preparation was performed on the basis of the amount of DNA, RNA and protein content determined according to BURTON 9, OGUR AND ROSENzl and LowRY et al. zs. The incorporation of E14C]UTP into acid precipitable material was taken as a measure of the activity of the RNA polymerase enzyme. The in vitro system consisted of 0.5/,mole each of 5'-ATP-Na2, GTP-NasH2, 5'-CTP-Na3, 0.2 #C 5'-E4-14C]UTP-NH 4 (specific activity, 51/,C//,mole), 5/*moles creatine phosphate-Na~, IO/,g creatine phosphokinase, 6/~moles/5-mercaptoethanol and 2/,moles M_.n2+ (MnSO4) in a final volume of 0.3 ml in Tris-HC1 buffer (pH 7.9). The aggregate enzyme preparaBiochim. Biophys. Acta, 247 (197I) 562-569
564
A.D.
ABRAHAM, C. E. SEKERIS
tion was added in amounts of 15o-2oo/~g DNA. Incorporation of labelled precursor was measured as described previously 5,26, using a Nuclear Chicago Mark I liquid scintillation counter. The experimental data were statistically evaluated applying the Chauvenet and Student's tests.
RESULTS
It is evident from Table I that cortisol, cortisone and prednisolone depress RNA synthesis b y the aggregate enzyme. Cortisol is the most active of the corticosteroids tested, depressing RNA polymerase activity up to 60 °/o at the concentration of lO -5 M and showing inhibitory effects up to concentrations of IO-v M. The effect is dose dependent in the range of 1o -~ to lO -5 M (see Fig. Ia). n-Deoxycorticosterone, tetrahydrocortisol and 5fl-pregnanolone had no significant effect on RNA synthesis.
I
TABLE EFFECT GATE
OF
CORTISOL
ENZYME
AND
RELATED
PREPARATION
STEROIDS
OBTAINED
ON
FROM
THE
ISOLATED
OF R N A
ACTIVITY RAT
THYMUS
POLYMERASE
Steroid
Final concentration (M)
pmoles [**C]UTP incorporated into R N A per rain per mg protein
n
%
Cortisol
Control i o -~ Control i o -e Control lO -5
9.4 4-0.3 7.4±0.8 I7.4:h2.i I i . O : h I. 3 9.4-4- o.9 3.8-4-1.o
6 6 9 ii 6 9
---21. 3 ---36.8 ---59.6
Control I°-S Control lO -5
9.04-1.5 5.44-0.5 13.44-1.4 8.9io. 9
5 5 15 16
---4 °.0 ---33.6
Control IO -e Control lO -5
I 2 . 7 4 - I.O 9.3:ho. 5 i6.4~i.6 io.o-ho. 3
6 6 9 IO
---26.8 ---38.2
Control IO -e Control lO -5
8.1 4- I .o 8.64-0. 3 19.6 i 1.4 19.84-2.8
9 6 6 9
-+ -+
Tetrahydrocortisol
Control lO -6 Control 10 -5
13.3 ± 3.2 I 4 . I - h 2.o 18.2 ~ 1,7 20.84-1.1
6 6 6 4
-+ 6.0 -+14"2
5fl-Pregnanolone
Control lO -6 Control 10--5
6.8 4- o. 7 7.1±1.o 6.8-t-o. 7 7 . 0 ± 1.2
7 9 7 5
-+ -+
Cortisone
Prednisolone
I i-Deoxycorticosterone
Biochim. Biophys. Acta,
247 ( I 9 7 I ) 5 6 2 - 5 6 9
OF
AGGRE-
NUCLEI.
P
<
0.05
<
0.05
<
O .OI
<
0.02
<
0.02
<
o.i o.ooi
6.1
> 0.25
o.i
> 0.25 > 0.25 > 0.05
4.7
> 0.25
3 .8
> 0.25
HORMONAL INHIBITION OF THYMUS R N A
565
SYNTHESIS
In a second series of experiments we studied the effects of androgenic and structurally related steroids on R N A polymerase activity of aggregate enzyme isolated from previously exposed rat thymus nuclei. As can be seen from the Table II testosterone and dihydrotestosterone at concentrations of IO-e and lO -5 M significantly inhibited R N A synthesis (See also Fig. Ib), whereas the other related steroids tested (5~-androsterone, ethiocholandiolone and the testosterone antagonist cyproterone 16) showed no effect. TABLE
II
E F F E C T OF T E S T O S T E R O N E AND R E L A T E D S T E R O I D S ON T H E A C T I V I T Y OF R N A
POLYMERASE OF
AGGREGATE ENZYME PREPARATION OBTAINED FROM ISOLATED RAT THYMUS NUCLEI
Steroid
Testosterone
Final
pmoles
concentration (M)
incorporated into RNA per rain per mg protein
n
%
P
Control
14.94-1.6 12.7+1.2 15.74-1. 5 lO.54-1. 4 17.o4-o. 9 lO.74-o.6
II 9 16 14 17 14
---14.8 ---33.1 ---37.1
-> --<
I2.3~I.O 9.7~ o.7 I2.3~I.O 9.1±o.6
16 17 16 18
---21.2 --26.1
-< -<
12.64-o. 9 iI.9~:o. 7 9.44-0.7 I°.4i°.5
6 6 8 9
--- 4.8 -+1o.6
-> 0.25 -> 0.05
Control
9.44-0.7 lO.9±o.5 i6.oi3.2
8 3 6
-+15.9 --
-~- o . I --
I°-8
15.°4-3.°
5
--
6. 3
>
Control
I5.9~ 1.3 13.94-o. 5 15.9± 1.3 14.54-1.1
6 5 6 6
---12.6 --- 8.8
-> ->
6.8 ± o.2
3
--
--
7.64- °.6 1 8 . 2 4- I . O 15.64-o. 7
3 9 8
+11.7 ---14. 3
lO-7
Control lO - s
Control lO -5 4,5c~-Dihydro -
testosterone
Control i o -~
Control IO - s
Dehydroepiandrosterone
Control lO -8
Control 1°-5
5~-Androsterone
Control lO-8
Ethiocholandiolone
IO - 8
Control IO -5
Cyproterone
Control I°-~
Control lO -5
[14C] U T P
o.i o.o2 o.ooi
o.05 o.oi
o.i
o.I 0.25
> o.i -> o.I
Addition of the steroids directly to the aggregate enzyme assay system had no effect of statistical significance on the precursor incorporation into RNA. Furthermore, we have studied the action of cortisol and testosterone on the rate of R N A synthesis of whole nuclei, lysed nuclei and aggregate enzyme derived from the same preparation of thymic nuclei preincubated in the presence of hormone in the same conditions as described above. As seen from the Table III no effect of these hormones on R N A synthesis by whole nuclei could be demonstrated, whereas in the lysed nuclei and aggregate enzyme preparation a decrease of R N A polymerase activity was observed. Biochim. Bio~hvs. Acta,
247 (1971) 562-569
566
A . D . ABRAHAM~ C. E. SEKERIS
TABLE
III
EFFECT OF 0¢-AMANITIN ON R N A PoLYMERA~E ACTIVITY OF STEROID PRETREATED RAT THYMUS NUCLEI, MEASURED IN WHOLE NUCLEAR, LYSED NUCLEAR OR AGGREGATE ENZYME PREPARATIONS F i n a l c o n c e n t r a t i o n o f s t e r o i d s : IO -~ mole/1. ~ - A m a n i t i n w a s a d d e d t o t h e R N A p o l y m e r a s e a s s a y s y s t e m a t lO -6 mole/1. T h e r e s u l t s a r e p r e s e n t e d i n p m o l e of E14C]UTP i n c o r p o r a t e d i n t o R N A i n 1 m i n / m g p r o t e i n . P1 r e p r e s e n t s t h e s i g n i f i c a n c e o f t h e d i f f e r e n c e s b e t w e e n c o n t r o l a n d o t h e r s e r i e s of e x p e r i m e n t s , P 2 b e t w e e n t h e , ¢ - a m a n i t i n a n d h o r m o n e + c ¢ - a m a n i t i n t r e a t e d o n e s .
Control
Cortisol
c~-Amanitin
Cortisol+c~-amanitin
Whole Nuclei
.~ ~:S.E. n % P1 P2
40.8 4.5 12 ----
49.2 5 .8 12 + 20.0 > 0.05 --
16.8 3 .6 6 -- 58.8 < o.ooi --
21.6 3.9 6 - - 47.1 < o.oi ~ 0"25
Lysed Nuclei
• ±S.E. n ~o P1 P2
36.7 4.4 8 ----
22.2 3.9 9 -- 39.5 < 0.05 --
12.6 3.5 5 -- 65.7 < o.oi --
3.3 I.I 6 -- 91.o < o.ooi < 0.02
Aggregate Enzyme
-~" ±S.E. n % P1 p~
32.5 2. 3 8 ----
15.o 1. 5 12 -- 53.9 < o.ooi --
16.o 2.1 6 -- 50.8 < o.ooi --
3.2 1.2 6 -- 90.2 < o.ooi < o.ooi
Control
Testosterone
c¢-Amanitin
Testosterone + e-amanitin
Whole Nuclei
_~ :j: S . E . n O/o P1 P.2
38.4 5.2 6 ----
38.9 4.6 6 + 1.3 > 0.25 --
12.9 i.i 6 - - 66. 4 < o.ooi --
13.6 4.o 6 -- 64.6 ( o.oi ~> 0"25
Lysed Nuclei
-~ IS.E. n O/o P1 P~
33.5 1. 4 6 ----
23 .2 5.0 6 -- 30.8 .< 0 . 0 5 __
l°.7 1.5 5 - - 68.1 < o.ooi __
9.5 3.9 5 -- 71.7 -~ o . o o i > 0.25
Aggregate Enzyme
-~ ±S.E. n °/o P1 p,
32.3 2. 7 5 ----
24. 7 1. 3 6 -- 23.5 < 0.05 --
14.3 i.i 5 -- 55-7 < O.Ol --
12.9 2.2 4 - - 6o.1 < o.ooi > 0.25
In
order
hormones,
we
inhibits
to differentiate conducted
transcription
A s,~°,~5. ~ - A m a n i t i n concentration merase
RNA
polymerase
with
a-amanitin.
RNA added
of 10 -6 M, enough
B. 0¢-Amanitin
or aggregate
by was
which
experiments
enzyme
accentuated preparation
polymerase directly to obtain the value obtained
Biochim. Biophys. Acta, 2 4 7 (1971) 5 6 2 - 5 6 9
B, but
to the
RNA
maximal
activity As
not
the
inhibitory
RNA assay
effects
in the
cortisol
known
with
polymerase
of inhibition from
that
is inhibited
well
case
exposed
by
the
~-amanitin polymerase system
on RNA of lysed thymus
at
a
polynuclei nuclei.
HORMONAL INHIBITION OF THYMUS RNA SYNTHESIS
567
Finalconcentratiofonsteroids 10-7
-20
10-6
10-5
10-7
M
1~ 6
1~ 5
M
rednisolone -20
~Testosterone ~ stosterone
c
a
g
-4C
v
~ -6C 0-
-4£
~,
-6c
Fig. I. Per cent inhibition of RIqA polymerase activity of aggregate enzyme preparation obtained from the isolated rat thymus nuclei depending on the doses of steroids. No significant differences could be seen in the case of st-amanitin and testosterone+ c¢-amanitin treated whole nuclei, lysed nuclei or aggregate enzyme (Table III).
DISCUSSION From the work of NAKAGAWA AND WHITE 29'3°, WHITE et al. 4s, I~UNCK AND BRICK-JOHNSON28, DREWSTMand DREWS AND WAGNERTM, it is known that in vivo application of corticosteroids decreases RNA synthesis in the thymus. Although m a n y hypotheses have been proposed to explain this phenomenon, no clear-cut evidence has been presented as yet in their favour. The inhibition of RNA synthesis under physiological conditions is probably only lO-2O %, but is enough to initiate a general catabolic process, which is characteristic for thymus involution z-4,x°,~s,32,41. From our results we can conclude that the inhibition of RNA polymerase activity is a very rapid phenomenon and can be considered as one of the primary effects of the hormones, preceeding the inhibition of oxidative processes 1° or of the glucose uptake ~7,zs. In the case of cortisol the steroid dosage needed to induce the effects on the thymus nuclei is at least ten times less than that needed to stimulate RNA synthesis of isolated rat liver nuclei 7,3s,3s. This m a y very well be due to the presence in the thymus nuclei of receptors taking up steroids, which render the presence of cytosol transport systems unnecessary. Such receptors have been demonstrated b y SCHAUMBURG84 and WIRA AND I~UNCKaS. One argument in favour of this hypothesis is that the direct administration of steroids to the aggregate enzyme preparations showed no effects on its RNA polymerase activity. The effects of steroids is probably mediated b y a receptor system present in the nuclei, but lost during the preparation of the aggregate enzyme. Biochim. Biophys. Acta, 247 (1971) 562-569
568
A. D. ABRAHAM, C. E. SEKERIS
Recent findings of NAKAGAWAAND WHITE 3°, which came to our attention during the preparation of our manuscript, show a decreased activity of the RNA polymerase activity extracted from thymus nuclear preparations obtained from pretreated rats with corticosteroids. They ascribed this to a reduced activity of the RNA polymerase of the hormone-treated animals. One other possibility is that the amount of the enzyme bound to the nucleochromosomal apparatus is less, reflecting a decreased availability of enzyme binding sites on the genome as a consequence oJ hormone action. It is interesting to note that RNA polymerase activity of whole nuclei preincubated with steroids is not modified in comparison to controls. However, when the assays were performed on lysed nuclei or aggregate enzyme preparation, the hormones reduced RNA polymerase activity. One explanation for these findings could be, that due to the preparation procedures, the lysed nuclear sediment or the aggregate enzyme preparation contains no soluble RNA polymerase, but only bound and transcribing enzymes. In whole nuclei a great quantity of soluble RNA polymerase is present which under the in vitro conditions of the assay, could unspecifically bind to DNA and synthesize RNA. The experiments with ~-amanitin were performed in order to differentiate the nature of the enzyme activity affected b y the hormones. It is well known, that in vitro ~-amanitin inhibits the RNA polymerase involved in the synthesis of DNA like RNA, whereas the enzyme presumably transcribing the ribosomal genes is not affected s,2°,85. Our results showed that cortiso] inhibits the RNA polymerase synthesizing ribosomal RNA, as has been proposed previously b y DREWS 12, DREWS AND WAGNER13, on the basis of the nearest neighbor frequency spectrum of E3~PIRNA synthesized b y isolated thymus nuclei in the presence of Mg 2+ from rats which had received prednisolone. These authors showed that administration of prednisolone, however, did not modify the quantity or the quality of the product formed b y thymus nuclei in the Mn2+-activated system, which findings are in good corelation with our data concerning the action of cortisol on thymus nuclei. Testosterone, on the other hand, preferentially decreases the synthesis of DNA like RNA, whereas no additive effect of testosterone+~-amanitin was observed. It is probable that the effect of various groups of steroids on RNA synthesis of thymus nuclei is specific. Glucocorticoids are potent inhibitors of RNA polymerase activity and cause rapid changes in the thymus RNA metabolism. Hypersecretion of androgens causes atrophy of the thymus and castration prevents this atrophy. This points to specific relations between thymus and the genital organs. In this paper the specific inhibitory effect of testosterone and dihydrotestosterone on RNA synthesis was demonstrated. Not all the substances which posses a steran skeleton had inhibitory effect on RNA polymerase activity of thymus nuclei, but only those with a certain well defined chemical structure and steric conformation, a fact accentuated b y us in other papers 1,3, too. ACKNOWLEDGEMENTS
We t h a n k Professor P. Karlson for his encouraging interest in our work and Dr. K. H. Seifart for m a n y helpful discussions. We are grateful to Mrs. Ch. Pfeiffer, Miss G. Frbhlich, and Mrs. U. Weiser for their skillfull assistance. One of us {A.D.A.) Biochim. Biophys, Acta, 247 (1971) 562-569
HORMONAL INHIBITION OF THYMUS
RNA
SYNTH,~ISIS
569
thanks the European Molecular Biology 0rganisation and "Alexander von Humboldt Foundation" for providing research scholarships. This work was supported by the Deutsche Forschungsgemeinschaft. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43 44 45
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Biochim. Biophys. Acta, 247 (1971) 562-569