Ribonuclease-induced transformation of progesterone receptor from rabbit uterus

J. steroid Biochem.

Vol. 24,

No. 2, pp. 505-511, 1986

0022-473 l/86 $3.00 + 0.00 Copyright 0 1986 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

RIBONUCLEASE-INDUCED TRANSFORMATION OF PROGESTERONE RECEPTOR FROM RABBIT UTERUS THRESIA THOMAS and DAVID T. KIANG Section of Medical Oncology, Department of Medicine, University of Minnesota, Box 168, Mayo Memorial Building, 420 Delaware Street SE., Minneapolis, MN 55455, U.S.A. (Receioed 9 April 1985)

Summary-The effect of RNase on the transformation of progesterone receptor from rabbit uterus was studied by density-gradient centrifugation and DNA-cellulose binding assay. The 75 form of the receptor in crude cytosol was RNase sensitive, and converted to the 4s form after RNase treatment. This reaction was prevented by an RNase inhibitor and reversed by the addition of ribosomal RNA. RNase treatment also caused a two-fold increase in the DNA binding of cytosohc receptor, and reduced the time required for heat-induced transformation. However, sucrose-gradient-purified progesterone receptor (7s) did not undergo transformation by warming unless exogenous RNase was added, thereby suggesting that a cytosolic factor, which might be endogenous RNase, is necessary for the heat-induced transformation of progesterone receptor. Furthermore, degradation of the receptors which occurred after prolonged warming at 25°C in the presence of RNase could be prevented by the addition of DNA-cellulose to the reaction mixture. These results indicate that RNA is associated with the 7s form of progesterone receptor, and that its hydrolysis by RNase might be involved in the transformation of this receptor.

buffers at low temperature, and has a low affinity for DNA. However, it undergoes a reduction in size to The steroid hormones, progestins, estrogens, gluthe 4s form during transformation by a variety of cocorticoids and androgens act as signaling molprocesses including warming at 25”C, dilution and ecules, regulating a variety of functions in their exposure to high ionic strength [16, 17. An increase respective target cells [l-4]. Progesterone induces the in DNA binding is observed with the transformation synthesis of specific proteins in uterus and other of the receptor to 4s form. target organs, altering their morphological and bioRecent studies suggest the involvement of RNA in chemical characteristics [l, 561. These effects are the structural organization and transformation of mediated through specific, intracellular “receptor” estrogen and glucocorticoid receptors. Thus, the diproteins which are present only in target cells 171. gestion of the 7s form of glucocorticoid receptor with Progesterone receptors from chick oviduct [S-IO], RNase converted it to the 45 form, with a concomirabbit uterus [I l-131 and human breast cancer cell tant increase in DNA binding. Other investigators line T47D [14] have been extensively studied to 122-241 have also confirmed the association of RNA of action. The understand their mechanism with glucocorticoid receptors. We have recently progesterone-receptor complex is known to interact shown that estrogen receptors also exist in a form with specific DNA sequences in the regulatory reassociated with RNA [25]. RNase-sensitive and gions of the genes under its control [15]. Several RNase-resistant forms of estrogen receptors were high-affinity binding sites for progesterone receptor identified in rat and rabbit uterine cytosol. In conhave been identified in the 5’ parts of ovalbumin and tinuation of our studies on estrogen receptors, we conalbumin genes of the chick oviduct 19, 151, and have now investigated the effects of RNase on the that of the uteroglobin gene of the rabbit uterus [I I]. transfo~ation of progesterone receptors from rabbit Even though it is generally accepted that prouterus and the results are presented in this paper. gesterone and other steroid receptors interact with DNA after their transformation in the presence of the EXPERIMENTAL respective hormones (16, 171, precise details of the mechanism of transformation of these receptors are Chemicals not clear. The progesterone analog [3H]R5020, The progesterone receptor has been identified in bovine serum albumin and human y-globulin were different molecular forms characterized by the sedipurchased from New England Nuclear (Boston, mentation coefficient, Stokes radius, molecular weight and other physico-chemical properties Mass.). RNase A and RNase T, were purchased from [l&20]. The cytosolic receptor sediments in the 7-10s Wo~hington Bi~hemi~ls (Freehold, N.J.). Human region of sucrose gradients in low ionic strength placental ribonuclease inhibitor was obtained from 505

THRESIATHOMASand DAVID T. KIANG

506

Bethesda Research Labs (Bethesda, Md) and Promega Biotec (Madison Wis.). Unlabeled steroids and calf thymus DNA were obtained from Sigma Chemical Co. (St Louis, MO.). Ribosomal RNA (calf liver) was purchased from P. L. Biochemicals (Milwaukee, Wis.).

Preparation

of progesterone receptor

Uterine tissue was collected from rabbits (S-6lb, New Zealand White). The tissue was homogenized at 4~C in IO vol of TED [IOmM Tris-HCI (pH 7.5) 1 mM EDTA and 1 mM dithiothreitol] buffer using a Polytron IOST homogenizer (Brinkmann Instruments, Steinhofhalde, Switzerland) [25]. The tissue homogenate was centrifuged at 105,OOOg to obtain cytosol. Cytosol protein concentrations were in the range of 3-4mg/ml as determined by the Lowry procedure [26]. The cytosolic progesterone receptor was labeled by incubating with 10 nM [3H]R5020 (sp. act. 87 Ci/mmol) in the presence or absence of a 200-fold excess of nonradioactive R5020 for 2 h at 4°C. For most experiments on the effect of RNase, labeled cytosolic receptor was used directly. Sucrosegradient-purified receptor was used for a selected number of DNA binding experiments. Sucrose solutions were prepared in TED buffer, and linear l&30% (w/v) gradients were made in 5 ml tubes using Buchler automatic gradient former. Aliquots (0.5 ml) of labeled cytosol treated with dextrancoated charcoal (0.25% dextran, 0.25% charcoal, 15 min, 4°C) were layered on the top of the gradients. Centrifugation was at 234,000g for 18 h. Six-drop fractions were collected from the bottom of the tubes by gravity flow. Equivalent fractions from 4-5 gradients were pooled and 50-~1 samples from each fraction were used for the determination of bound [3H]R5020. Samples containing excess of nonradioactive R5020 were processed similarly to prepare nonspecifically-bound complexes. By this procedure, a four-fold purification of the receptor was achieved. RNase treatment

Analytical sucrose-gradient ultracentrifugation

Aliquots of labeled cytosol was layered on 3.8 ml, l&30% gradients. Centrifugation was for 17 h at 256,OOOg, and 5-drop fractions were collected. 14C-Labeled bovine serum albumin (4.5s) and y-glubulin (7.1 S) were used as internal markers. RESULTS

The effect of RNase on the sedimentation pattern of progesterone receptor from rabbit uterus is shown in Fig. 1. After incubating the receptor solution with 0.5 pg/ml of RNase for 5 min at 25°C the receptor peak shifted from the 7s to the 4s region. However, a small portion of the receptor remained in the 6-7s region. Similar results were obtained by incubating

x

A stock solution of RNase A (5 mglml) was prepared in TED buffer and heated at 100°C for 15 min to eliminate heat-labile proteases. RNase solution containing less than 1 mg/ml of the enzyme was stabilized by the addition of 1 mg/ml of bovine serum albumin. The concentration, time and temperature of incubation were varied for different experiments as described in the legends to the figures. The stock solution of RNase T, consisted of 1.4 mg/ml, which was diluted IO-fold just before use. RNase T, was used for the few selected experiments identified in the text. For all other experiments, RNase A was used. Thus, unless specifically noted, RNase refers to RNase A in this paper. DNA binding assay

DNA binding was determined

centrifugation assay (27,281. DNA-cellulose was obtained from Sigma Chemical Co. and contained about 6 mg DNA/g cellulose. DNA+ellulose was soaked in TED buffer for about 12 h, and washed to remove any free DNA. Equal volumes of receptor preparation and DNAc!ellulose slurry containing about 50 pg DNA were mixed and incubated at 4 C for 90 min with frequent vortexing. Neither KC1 nor NaCl was included in the reaction mixture to avoid the contribution of salt-induced transformation. The reaction mixtures were then centrifuged and the supernatant was removed. The DNA-cellulose was washed twice with TED buffer to remove unbound receptors, and incubated with 2 ml of ethanol at 25-C for 12 h. The ethanol extract was mixed with IO ml of Aqueosol (New England Nuclear), and radioactivity determined using a Beckman LS 2800 scintillation counter.

by DNA-cellulose

g3 % P2 Z 1

4

0

12

16

20

24

Fraction Nwnber

Fig. I. Effect of RNase on the sedimentation pattern of progesterone receptor. Cytosolic receptor (0.2 ml) labeled with [‘H]RS020 was warmed at 25°C for 5min without (O---O) and with (O---O) 0.1 pg of RNase and with 0.1 pg RNase + 50 U of RNase inhibitor (A---&. Sedimentation of an aliquot incubated in the presence of 200-fold excess of nonradioactive R5020 (m---W) is also shown.

RNase-induced transformation of progesterone receptor

I 7s

I

4 5s

Fraction Number

Fig. 2. Effect of RNA on the sedimentation profile of RNase-treated progesterone receptor. Cytosolic receptor (0.2 ml) labeled with [‘H]R5020 was warmed at 25°C without (O-PPO) and with (O---O) 0. I pg RNase for 10 min. An RNase-treated aliquot was incubated with 25 ~I(150 pg) or rRNA for IO min at 4C (A---& and another aliquot was incubated in the presence of excess nonradioactive R5020 (O--PO).

the receptor sample with 100 pg/ml of RNase at 4°C for 1 h. The possibility that the change in sedimentation constant could be caused by a protease contamination of RNase preparation was examined using the placental ribonuclease inhibitor. RNase treatment of the cytosol in the presence of 250 U/ml

of the inhibitor prevented the conversion of the 7s receptor to the 4S form (Fig. I), indicating that the reduction in receptor size was caused by RNase. Similar experiments were done with RNase T,, which also caused the transformation of the 7S receptor to the 4s form. We have further examined the reconstitution of 7s progesterone receptor from the 4s form by the addition of nicked ribosomal RNA, and the results are given in Fig. 2. The addition of 150 pg of rRNA to the RNase-treated receptor solution caused an increase in sedimentation constant which might be due to the association of the 4s receptor with RNA. Attempts to inhibit the RNase-induced transformation by including yeast tRNA, calf liver rRNA etc. in the cytosol were not successful, since addition of excess of RNA caused changes in the sedimentation of the 7s receptor. If the untransformed receptor is complexed with RNA, it might be necessary to remove this RNA to convert the receptor to the DNA-binding form. Thus, RNA hydrolysis can have a stimulatory effect on transformation. The effect of RNase treatment on DNA binding of the receptor is shown in Fig. 3. At 4°C the receptor binding to DNAcellulose for different cytosol preparations was in the range of 13 + 6%. After the addition of RNase (100 pg/ml), the binding increased to about 22 + 5%. The maximal increase in DNA binding of 51 k 5% was obtained by RNase treatment of the receptor at 25°C while in the absence of RNase, DNA binding due to warming alone was 27 + 8% (P < 0.01). The involvement of RNase in the transformation of progesterone receptor was also studied by experiments using sucrose-gradient-purified receptor. The 7s receptor was collected from cytosol samples not treated with RNase, and the 4s form was obtained from samples treated with RNase at 4’C for 1 h.

3 I

40

n P 30 0 5 20 $ CL 10 a T LL CL

Fig. 3. Effect of warming and RNase treatment on the DNA
507

.

1Ii J

L25'CJ

Fig. 4. Effect of warming and RNase treatment on the DNA~ellulose binding of sucrose-gradient-purified 7s and 4S progesterone receptors. Aliquots of receptor solution were warmed at 25°C for 15 min with or without RNase. Controls without warming are marked 4°C. The sedimentation cofficient and the presence of RNase are represented by different shades as follows: 7S form without RNase (N); 4S form without RNase (0); and 7S (N) and 4S (m) forms with 100 ,ug/ml of RNase. DNA binding of the receptor was determined as described in the Experimental section. Error bars represent +SD in three separate experiments each carried out in triplicate.

508

THRESIATHOMASand DAVIUT. KIANC Table I. DNA

binding of progesterone receptor after treatment with RNase and placental RNase inhibitor

Sample

Additions

DNA binding (%)

Heated cytosol

None RNase inhibitor (500 U/ml)

100 68 + 2.2

Unheated cytosol

N0ne RNase inhibitor (SO0 U/ml)

68 I? 5 59 _‘4

Heated cytosol

RNase RNnse RNase RNase

A A T, T!

(0.5 #g/ml) (0.5 pgiml) + RNase inhibitor (500 Uiml) (2.5ygiml) (2.5 pgjml) + RNase inhibitor (1100 U/ml)

222 + 8 152+4 142 t 3.2 lOOt6

Sucrose-gradient-purified receptor Heated

Unheated

None RNase RNase RNase RNase

A A A A

(0.5 (0.5 (2.5 (2.5

100 346 + 21 126 + 9.5 3661?:13

~pirnl) fig/ml) + RNase inhibitor (1100 U/ml) pg/ml) pg/ml) + RNase inhibitor (I 100 U/ml)

113+ I5 95 + 8

None

Progesterone receptor preparations were heated at 25’ C for 15 min in the presence or absence of the additives. DNA cellulose binding was carried out by incubation at 4°C for 60min. Nonspecific binding in the presence of excess nonradioactive R5020 was deducted in each case. Results represent i SD from two separate experiments, each carried out in triplicate.

Aliquots of receptor solution from gradients were mixed with DNA-cellulose and the DNA binding was determined. Figure 4 shows that only 12 9~3% of purified 7s receptor was bound to DNA even after warming at 25”C, while the DNA binding increased to 40 t_ 3% after the 7s receptors were treated with RNase. On the other hand, purified 4S receptor showed 45 + 5% binding at 4°C and no significant change was observed on warming at 25°C. Since purified 7s receptor did not undergo heatinduced transformation, it appeared that cytosolic factors, especially endogenous RNase, might have a roie in heat-induced transfo~ation. This hypothesis was tested by examining heat-induced transformation of receptors from crude cytosol in the presence of RNase inhibitor. Results are presented in Table 1. RNase inhibitor effectively suppressed DNA binding caused by warming at 25°C to the level of unheated cytosol. However, the inhibitor did not affect DNA binding of unheated cytosol. RNase inhibitor was also used to inhibit the increase in DNA binding caused by exogenous RNase. The increase in DNA binding caused by RNase A and RNase T, was suppressed by the RNase inhibitor, indicating that in both of these cases, the changes are caused by RNase and not by proteolysis. The inhibitor was effective in inactivating RNase in experiments using crude cytosol as well as those using sucrose-gradient-purified receptor. Figure 5 shows a correlation between the increase in 4s receptor and DNA binding. Aliquots of cytosol were treated with increasing amounts of RNase (100-1000 ng/ml) and analyzed for DNA binding, as well as sedimentation pattern. Reaction mixtures were incubated for 5 min at 25°C. The amount of 45 receptor and the extent of DNA binding increased concomitantly with RNase concentration. A comparison of sucrose gradient patterns showed that at iOOng/mI of RNase concentration, a form of recep-

tor sedimenting near the bottom of the gradient is converted to the 7s form and no increase in DNA binding was observed at this concentration. Figure 6 shows the results of experiments designed to test the effect of RNase on the kinetics of transformation as well as the stability of the DNA-binding site of the transformed receptor. In some experiments, RNase was added and the reaction mixtures were incubated at 25°C. Samples were transferred to

40

80

120

160

200

RNase. ng1200#l Fig. 5. Correlation of increase in 4.5 receptor, and increase in DNA binding. Aliquots of labeled cytosol were treated with the designated amounts of RNase for 5 min at 25’C. and DNA+ellulose binding was carried out as described in the Experimental section. Equivalent aliquots treated with RNase were used to determine a series of sucrose-gradient patterns. The sum of radioactivity from three fractions in the 4S region {determined by the position of internal marker BSA) was taken as representative of the 45 peak. Curves represent percentage increases in 4s receptor (O---O) and DNA binding (a---e) based on the values of these parameters for control samples without RNase. Similar results were obtained in three separate experiments. Concentration

of

RNase-induced transformation

of progesterone receptor

10

I

I

1

10

20

I

I

30 40 Time (minutes)

I

1

50

60

Fig. 6. Effect of RNase on the kinetics of receptor transformation. DNAcellulose binding was carried out using cytosolic receptor as described in the Experimental section. Time of incubation at 25 C is plotted against percentage of total receptors capable of binding to DNA. Curves represent samples containing: 100 pg/ml of RNase (O---O); no RNase (O---O), RNase and DNA-cellulose added at the beginning of the incubation (A---A); DNA+ellulose added at IO min after the addition of RNase and the beginning of the incubation (m---m); the DNA+ellulose alone added at the beginning of the incubation (OPPPO). This figure is representative of three experiments, each carried out in triplicate. Nonspecific binding was subtracted in each case.

4’.C at various time periods, DNA+ellulose was added and the incubation continued at 4°C for 60min. Control samples were treated similarly, but no RNase was added. Results showed that the time required for transformation was reduced from 60 min in the control samples to IO min in the RNase-treated samples. Thus, RNase catalyses the transformation reaction. These experiments also revealed that receptors transformed by RNase were losing their capacity to bind to DNA soon after achieving the maximal binding. Thus, the DNA binding of RNase-treated receptors dropped from 45% at 10 min of incubation to 20% at 60 min of incubation. It appeared that removal of RNA caused the DNA binding site to be extremely labile. Hence, it was thought that addition of DNA at the beginning of incubation would prevent the loss of DNA binding capacity of transformed receptor. It was found that if DNAcellulose and RNase were added at the same time, no loss in binding capacity was observed at 60min of incubation. A partial loss of DNA binding capacity was observed if DNAcellulose was added 10 min after starting the incubation at 25°C. Control experiments using plain cellulose had no stabilization effect on the receptors transformed by RNase. Similarly, controls in which DNA+ellulose was added in the beginning of incubation, but RNase was avoided, did not show significant transformation.

20

30 40 Time (minutes)

509

50

60

Fig. 7. Effect of RNase on the ligand binding of progesterone receptor. Aliquots of labeled cytosolic receptor were incubated at 25°C without RNase (O---O) with lOO~g/ml of RNase (@-P-O) and with lOO~g/ml of RNase and 250pg/ml sonicated calf thymus DNA (UP--O). Results represent *SD from three separate experiments, each carried out in triplicate.

We have also studied the effect of RNase on the stability of progesterone receptor in terms of ligand binding (R5020). Figure 7 shows that RNA hydrolysis caused a faster degradation of progesterone receptor at 25°C. About 70% of the RNase-treated receptors were recovered by dextran-coated charcoal assay at 60min of incubation compared to 85% in the controls. However, if sonicated DNA (250 pg/ml) was added to the RNase-treated samples, the kinetics of degradation was the same as that of controls leading to the recovery of 85% of the receptor at 60 min of incubation. DISCUSSION

Recent studies have supported the hypothesis that RNA is associated with glucocorticoid [21-241, estrogen [25,30-321, androgen [33-361 and vitamin D receptors [37]. Similar results were obtained on these receptors using sucrose-gradient analysis and DNA binding. The native 7-10s form was transformed to 4S form with a concomitant increase in DNA binding. A more direct analysis of the association of RNA with glucocorticoid receptor was reported recently [24]. Photochemical cross-linking of intact cells labeled with [3H]uridine, and analysis of the glucocorticoid receptor by polyacrylamide gel electrophoresis showed the presence of labeled ribonucleotides on the receptor. In estrogen receptor systems, a low molecular weight RNA associated with the receptors was isolated from the breast cancer cell lines MCF7 and T47D [32]. In androgen receptor systems, studies using polyribonucleotides showed sequence-specific RNA-receptor interactions [34-361. In vitamin D receptor, poly G and poly I were more active in displacing receptor from DNAcellulose than double-stranded DNA [37]. In conformity with

510

THRESIA

THOMASand DAVIDT. KIANG

these results, we have obtained data on the RNaseinduced transformation of progesterone receptor, suggesting that RNA is associated with the untransformed form of this receptor. The RNase-induced transformation of 7s progesterone receptor to the 4s form could occur due to a number of factors: (1) protease contamination of the RNase preparations (2) ionic interaction between RNase and the receptor; or (3) hydrolysis of an RNA component of the 7s receptor. Since an RNase inhibitor prevents the change in sedimentation constant and that in DNA binding, proteolysis of the receptor might not be responsible for transformation of the receptor. Since RNase A, a basic protein with an isoelectric point of 9.45 and RNase ‘I,, an acidic protein with an isoelectric point of 2.9, are capable of inducing this transfo~ation, it is unlikely that ionic interaction between RNase and the receptor could induce transfo~ation of the receptor. Hence. the RNA hydrolytic activity of these enzymes appears to be responsible for the transformation induced by RNase. The reversal of transformation by the addition of RNA further supports this mechanism. In the reconstitution experiment shown in Fig. 2, addition of rRNA has caused an unusually broad peak. However, sedimentation of “C-labeled internal markers showed sharp peaks, indicating that the gradients were not disturbed. Thus, the broadening of the receptor peak might be due to the heterogeneity of receptor forms. This heterogeneity could be caused by the presence of the 7s form in conjunction with a 5.5s form which is reported to be an intermediate in the transformation of progesterone receptor. Although purified 7s receptor binding to DNA was unaffected by warming, a two-fold increase in binding was observed by warming crude cytosolic receptor. This transfo~ation could be inhibited by RNase inhibitor, suggesting the involvement of endogenous RNase in the crude cytosol. The observation of endogenous RNase (equivalent to about 25-50ng/ml of RNase A) in rabbit uterine cytosol supports this view [25]. The correlation of RNaseinduced formation of 4s receptor and the increase in DNA binding to the RNase concentration (Fig. 5) is in agreement with the involvement of RNA hydrolysis during transformation. In the absence of exogenous RNase, transFo~ation is known to occur by a variety of treatments such as dilution, ammonium sulphate precipitation etc. It is conceivable that these methods of transformation might involve the dissociation of the RNA component. Possibility of the dissociation of RNA during ammonium sulphate precipitation is indicated by the observation that the precipitated receptor was stabilized by the addition of RNA, whereas the cytosolic receptor was not affected [38]. It was also found that binding to nucleic acids restored progesterone receptor’s ligand binding site to the functional state 1381.Our results, shown in Figs 6 and 7. are in agreement with this concept.

Using crude cytosol, maximal effect of RNase on the DNA binding of progesterone receptor was achieved only if the samples were warmed briefly at 25-C (Fig. 3). However, incubation of the cytosolic receptor with RNase at 4°C was sufficient to convert it from the 75 to the 45 form (results not shown). Thus, 4s receptor in crude cytosoi required a temperature-induced change for achieving maximum DNA-binding capacity. However. with sucrosegradient-purified 4s receptor, warming was not necessary to obtain maximal binding (Fig. 4). Lowaffinity interactions between cytosolic proteins and the receptor might account for the lower binding of cytosolic 4s receptor to DNA. The results presented in this paper indicate that RNA is associated with the 7s form of progesterone receptor from rabbit uterus. However. the functional significance of this association is not known at present. It can be argued that association of RNA with the receptors could be a consequence of their affinity for DNA. However, there is evidence to suggest that binding of the receptor to RNA and DNA might have functional specificity. Thus, some of the polyribonucleotides have a higher affinity for receptor than the corresponding deoxypolyribonucleotides 1391. In androgen receptor systems, a 3s receptor was identified which lacks the specific domain or confo~ation necessary for binding to DNA but retains a high affinity for certain forms of RNA [36]. Although the present experiments do not rule out artifactual association of RNA with the receptor, these studies indicate that receptor-RNA interaction could occur regardless of its capacity to bind to DNA. A possible physiological role could be a feedback mechanism in which the association of the receptor with nascent mRNA chains makes it unavailable for further initiation of transcription. Transfo~ation of the RNAassociated receptor then leads to another cycle of transcription. Receptor-RNA binding may also contribute to the stability of mRNAs 1401. Other protein-RNA interactions that might give an insight into the role of receptor-RNA interaction include posttranscriptional processing of secretory proteins [41] and the regulation of 5s RNA synthesis [42]. Acknowledgements-This work was supported by NIH Grant No. CA-30350 and by the Minnesota Medical Foundation. REFERENCES

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