Synthesis of specific estradiol-induced protein by surviving rat uteri and cell-free uterine extracts

Molecular and Cellular Endocrinology

1 (1974) 77-88. @ North-Holland

Publ. Comp.

SYNTHESIS OF SPECIFIC ESTRADIOL-INDUCED PROTEIN BY SURVIVING RAT UTERI AND CELL-FREE UTERINE EXTRACTS

Dalia StiMJEN*, Department

Alvin

of Biodynamics,

M. KAYE

The Weizmann

and Hans R. LINDNER Institute

Received 27 November

of Science,

Rehovot,

Israel

1973

A cell-free system was developed for the synthesis of the specific ‘induced protein’ (IP) which is stimulated in the rat uterus within the first hour after treatment with estradiol-17p. Surviving uteri or uterine extracts were incubated with [3H]-labeled (estrogen-treated) or with [I%]- or [%I-labeled (controls) leucine, glutamic acid or methionine, and the 150,000 g,, supernatant solution was analyzed by electrophoresis on cellulose acetate. IP was detected by determining the isotope incorporation ratio. The 12,000 g max post-mitochondrial supernatant suspension from uteri of lo- or 20-day old rats, prepared 1 h after injection of estradiol-17/3 (4 or 5 pg/rat), was found to synthesize IP. Treatment of surviving uteri with 30 nM estradiol17(3for 1 h at 37 “C also permitted the extraction of a post-mitochondrial supernatant suspension capable of synthesizing IP. The electrophoretic distribution of radiolabeled soluble proteins was different in the cellfree system and surviving uteri. However, both preparations showed a single protein band with increased incorporation of tritiated amino acid after estradiol treatment. This band had an electrophoretic mobility relative to bovine serum albumin of approximately 1.1, characteristic of IP. Appearance of this band was prevented by addition of antibodies to estradiol or of cordycepin during incubation of the uteri with estradiol. The induction of IP in surviving uteri followed by IP synthesis in a cell-free system provides a versatile model for the more detailed analysis of the regulation of specific protein synthesis by estradiol. Keywords:

protein synthesis;

induced protein; oestradiol-17!3; actions uterus.

A cell-free system for the synthesis of a specific protein induced by estradiol would be advantageous for the elucidation of the regulation of protein synthesis by estrogens. The first step towards such a system was taken by Notides and

* In partial fulfilment of the requirements for the Ph. D. Degree of the Feinberg Graduate School of the Weizmann Institute of Science. This work was supported by grants from the Ford Foundation and the Population Council, New York.

18

D. Siimjen et al.

Gorski (1966) who detected by starch gel electrophoresis a single protein band from an extract of rat uteri, the rate of synthesis of which was increased by estradiol. The protein was called ‘induced protein’ (IP); its synthesis increased within 30-40 min after estradiol administration (Barnea and Gorski, 1970; Mayo1 and Thayer, 1970) and was inhibited by cycloheximide (Notides and Gorski, 1966) and actinomycin D (DeAngelo and Gorski, 1970; Mayo1 and Thayer, 1970). We found that JP is a cytoplasmic component with an apparent molecular weight of 39 x lo3 dalton and isoelectric point of 4.5 (Sijmjen et al., 1973~; King et al., 1974). These parameters were independently estimated recently as 45 x lo3 dalton and 4.7 respectively, by Iacobelli et al. (1973). Synthesis of IP in vitro has also been demonstrated by incubation of surviving immature rat uteri in solutions containing estradiol-17P (Mayo1 and Thayer, 1970; Wira et al., 1971 ; Katzenellenbogen and Gorski, 1972). Evidence favouring translational control of ‘induced protein’ synthesis has recently been presented by DeAngelo and Fujimoto (1973). Baulieu et al. (1972) have proposed that IP is the ‘key intermediate protein’ in the stimulation of RNA synthesis by estradiol, following its interaction with estrogen receptor (see review by Jensen and DeSombre, 1973). In this paper, we describe a cell-free system capable of synthesizing IP and compare the characteristics of synthesis of IP in this system with its synthesis in surviving uteri. A preliminary report on part of this work has been presented at the 13th annual meeting of the American Society for Cell Biology (Somjen et al., 1973a).

MATERIALS

AND

METHODS

Animals Female rats of the Biodynamics Department colony, derived from Wistar stock, were used at ages of 10 or 20 days. The colony was housed in air-conditioned quarters lit from 05.00-19.00 h and had free access to pelleted food (Purina Laboratory Chow, Ralston Purina Co., St. Louis, MO., U.S.A.) and water. The days on which pups were found with their mother was designated day 1. Materials Estradiol-17P (58 Ci/mmole), 3 %-methionine glutamic acid

was a product of Organon, Oss, The Netherlands. 3H-leucine 14C-leucine (342 mCi/mmole), 3H-methionine (7.61 Ci/mmole), (185 Ci/mmole), 3G-glutamic acid (1.7 Ci/mmole), 14C(10 mCi/mmole), and 3H-adenosine (12 Ci/mmole), were

Synthesis of esfrogen-indticedprotein

79

purchased from the Radiochemical Centre, Amersham, Bucks., U. K. Dithiothreitol, creatine phosphate, amino acids, Aquacide, Tris and Hepes buffers were obtained from Calbiochem, Los Angeles, Calif., U.S.A. ‘Cellogel’ strips (cellulose acetate gel) were obtained from Chemetron, Milan, Italy. Soluene sample solubilizer and scintillation reagents were products of Packard Instrument Co., Downers Grove, Ill., U.S.A. Bovine serum albumin, cordycepin and the sodium salts of ATP, GTP and CTP were obtained from Sigma Chemical Co., St. Louis, MO., U.S.A., creatine kinase (41.8 Ujmg) from Worthington Biochemical Corp., Freehold, N. J., U.S.A., concanavalin A, twice crystallized, from Miles Yeda Ltd., Rehovot, Israel and sodium periodate from BDH Chemicals Ltd., England. Administration of’est~adiol-17P A stock solution of estradiol-l7B in ethanol at a concentration of 1 mg/ml was stored at 4 “C and diluted with double distilled water before use. Control animals were treated with dilute ethanol. For in vivo induction, the hormone or the vehicle was injected intraperitoneally in 0.5 ml or less. For in vitro stimulation, 3 uteri of 20-day old rats, or 6 uteri of IO-day old rats, were incubated for 1 h at 37 “C in 1 ml of phosphate-buffered saline (PBS) medium (Dulbecco and Vogt, 1954) containing 30 nM estradiol-17P (Katzenellenbogen and Gorski, 1972) or vehicle with agitation in a Dubnoff shaker under an atmosphere of 02, CO, (95:5). ~eterm~~atio~ of’ ind~&~dprotein Uteri of control (ethanol-treated) rats were incubated in 1 ml PBS containing 5 uCi of l 4C-leucine or 3 %-methionine or 14C-glutamic acid; estradiol-17B treated uteri were incubated in 1 ml PBS in the presence of 25 uCi of tritiated leucine, methionine or glutamic acid. Incubation, homogenization in 0.25 M sucrose in TKM medium (Blobel and Potter, 1966), separation and analysis by electrophoresis on cellogel strips and quantitation by the isotope-ratio method were performed as previously described (Sijmjen et al., 1973d). Electrophoreses were run for times up to 75 min. Antibodies Antisera to estradiol-17~ were prepared according to Lindner et al. (1972) by Dr. Sara Bauminger of our Department and had a binding capacity of approximately 0.2 nmol/ml. Sera against histone fraction F, (Bustin and Stollar, 1972) were prepared by Dr. M. Bustin of this Institute.

80

Synthesis

D.

Siimjen et al.

of induced protein in a cell-free extract

Fifteen uteri from IO-day old or 8 from 20-day old rats, exposed to estradiol for 1 h in vivo or in vitro, were homogenized in 10 mM Hepes buffer, pH 7.5, containing 10 mM KCl, 1.5 mM magnesium acetate and 7 mM P-mercaptoethanol. After homogenization, 0.05 vol of 20 mM Hepes buffer, pH 7.5, containing 125 mM KC1 and 5 mM magnesium acetate were added. Uteri after estradiol treatment were homogenized in the presence of 30 nM estradiol. The homogenates were centrifuged for 10 min at 12,000 g,,, to obtain post-mitochondrial supernatant suspensions. The incorporation mixture contained : 2 mM MgCl,, 50 mM KCI, 1 mM ATP, 0.1 mM GTP, 0.5 mM CTP, 10 mM creatine phosphate, 0.16 mg creatine kinase, 16 amino acids (excluding the labeled species) 50 mM each, and uterine post-mitochondrial supernatant suspension (SO-150 ug protein), in a total volume of 250 ul. Extracts from contol uteri were incubated for 45’ at 37 “C in the presence of 3 uCi ’ 4C-leucine or 35S-methionine and those from estradiol treated uteri in the presence of 30 nM estradioI-17~ and 10 uCi of the corresponding tritiated amino acid. Incubation was stopped by cooling and addition of disodium EDTA to a final concentration of 0.05%. The control and experimental incubation mixtures were combined and centrifuged for 50 min at 150,000 g,,. The supernatant solution was dialyzed for 2 h against 20 vol 0.05% disodium EDTA, concentrated with Aquacide and analyzed by electrophoresis on cellogel strips as described previously (SSmjen et al., 1973d; King et al., 1974). Protein determination This was done by the method albumin (BSA) as the standard.

of Lowry

et al. (1951) with bovine

serum

Liquid scintillation counting For counting 3H and “C-labeled samples on cellogel strips 0.5 ml of Soluene was added to each vial which contained 5 ml of 0.5% 2,5-diphenyloxazole (PPO) and 0.03 % of dimethyl 1,4-his-(S-phenyloxazolyl-2-)-benzene (dimethyl POPOP) in toluene. Sufficient counting time was allowed to reduce the statistical error to less than 3%. Stimulation of mouse spleen cell suspensions Mouse spleen cell suspensions were stimulated for 3 h with concanavalin A (2 pg/ml) or 1 mM sodium periodate according to Novogrodsky and Katchalski (1972). Incorporation of 3H- and ’ 4C-leucine and electrophoresis of labeled proteins was carried out as for the induced protein.

Svnthesis of estrogen-induced protein

RESULTS Optimization

AND

81

DISCUSSION

of conditions jbr IP synthesis in surviving uteri

In order to obtain a post-mitochondrial suspension containing the maximum amount of polyribosomes programmed with the mRNA for IP, we sought the dose of estradiol that would give maxima1 synthesis of IP in uteri excised 1 h after injection of the hormone. Synthesis of IP was detected at doses of estradiol178 ranging from 50 ng/rat (a 20% increase in synthetic rate) to 50 ug/rat (a 150 ‘A increase). Over this range the response was almost directly proportional to the logarithm of the dose (fig. I). A dose of 5 ug per rat produced a sufficiently large and reproducible increase (115 %) in the rate of IP synthesis, and hence was routinely used for in vivo induction. Katzenellenbogen and Gorski (1972) found a 50% increase in the rate of IP synthesis after incubation of surviving uteri in 1O-9 M estradiol, a maxima1 rate of synthesis at 2 to 3 x lo-* M and a decrease at lO-5 M estradiol.

150

100

50

LL

1

ti

z 8

o&A 5

I

I

50 DOSE

5x102

I

I

5x103

5x104

OF ESTRADIDL

(ng/rot)

Fig. 1. Synthesis of IP by uteri from 20-day old rats as a function of dose of estradiol-17S. Estradiol or vehicle was injected 1 h before the rats were killed. The uteri were incubated with 3H-leucine (estrogeh-treated) or 1%~leucine (controls). IP was separated by electrophoresis and estimated by the isotope ratio method as described in Materials and Methods. The vertical bar indicates the 95% confidence interval of the mean (9 observations).

It might be expected that the detection of IP would be improved by use of ovariectomized rats in which the synthesis of other proteins migrating in the IP region of the electropherogram would be reduced. However, uteri from rats ovariectomized at 20 days and tested at 28 days showed the same response as intact rats. We also sought to improve the detection of IP by substituting glutamic acid

D. S6mjen et al.

82

for leucine as the labeled precursor, since Iacobelli et al. (1973) found that this amino acid occurred at the highest concentration in IP. We also tested labeled with approximately methionine, because of the availability of 3 ?S-methionine 500 x higher specific activity than the 14C-leucine available. Both pairs of radioactive amino acids, 3H- and 14C-labeled glutamic acid and 3H- and 35S-labeled methionine, were capable of serving as precursors of IP, but no more efficiently than the pair 3H- and 14C-leucine previously used (fig. 2). The usefulness of glutamic acid was limited by its low specific activity, resulting in extracts with relatively low total counting rates, while the incorporation of methionine was apparently limited by the low concentration of this amino acid in IP. Leucine was therefore routinely used as the labeled precursor of IP.

u * -\ m=

6

6

u

4 2

i

0

20

40

60

00

MIGRATION

0

20

40

60

80

(mm1

Fig. 2. Comparison of IP synthesis by surviving uteri following induction by estradiol in vivo or in vitro, using different amino acids as precursors. a) and b), uteri were collected from untreated 20-day old rats and incubated for 1 h with or without 30 nM estradiol; c) and d), uteri were collected from 20-day old rats 1 h after injection of estradiol (5 ug/rat) or vehicle. The uteri were incubated with the labeled amino acid pairs indicated; the tritiated species served as precursors for the estrogen-treated preparations. Shown is the distribution of the isotope ratio on Cellogel electropherograms of the soluble uterine proteins, prepared as described in Materials and Methods. Arrow, position of BSA.

Induction of IP synthesis in vitro The ability to induce IP in vitro provided a sensitive assay for putative inhibitors and inducers. This system was used to demonstrate that antibodies to estradiol are able to neutralize a direct biological response to estradiol: the in vitro induction of IP synthesis by estradiol was prevented by the presence of anti-estradiol rabbit serum in the incubation medium (fig. 3). Unrelated antibodies (directed against histone fraction F,) had no effect on IP synthesis. Cordycepin, which blocks the formation of poly-A ends of mRNA molecules

Synthesis of estrogen-induced

protein

83

and their subsequent transfer to the cytoplasm (review by Darneil et al., 1973), inhibits the induction of IP (fig. 4). The rate of IP synthesis in surviving uteri as measured by the isotope ratio technique, was reduced by 70%, while the incorporation of 3H-adenosine into RNA nucleotides (analyzed as described previously by Kaye et al., 1972) was reduced by 50%. This experiment suggests that the mRNA for IP belongs to the class of mammalian mRNA molecules which contain poly-A ends.

Fig. 3. Inhibition of the induction of IP by antibodies against estradiol-17@. Uteri from 20-day old rats were incubated for 1 h with or witbout estradiol (30 nM) in medium containing a) 10% normal rabbit serum or b) 10% rabbit antiserum to estradiol-17[3. This was followed by incubation with 3H-leu (estrogen-treated) or 14C-leu (controls), Shown is the distribution of the isotope ratio on Cellogel efectropherograms of the soluble uterine proteins. Arrow, position of BSA.

Fig. 4. Inhibition of the induction of IP by cordycepin. Uteri from 20-day old rats were incubated for 0.5 h in PBS (a) or in PBS containing 100 pg/ml cordycepin (b). Incubation was continued for 1 h in fresh medium with or without estradiol (30 nM) and for a further I h with 3H-leu (estrogentreated or **C-Ieu (controls). In experiment b), cordycepin (100 UrJfml) was present throughout the three skges of the incubation. Shown is the distribution of isotope ratio in the soluble uterine proteins separated by Celiogel electrophoresis. Arrow, position of BSA.

I-

I 0

I

I

I

20

40

60

MIGRATION

I

I

3-

a.

“= 3-

b.

(mm)

I

I

I

If \ c

II

I

I

I

I

0

20

40

60

80

MIGRATION

tmmf

Isoproterenol (2 pg/ml), which leads to the formation of cyclic AMP uterus (Sanborn et al., 1973; Zor et al., 1973) was ineffective in inducing has previously been shown that exogenous cyclic AMP and dibutyryl AMP do not stimulate IP formation (Katzenellenbogen and Gorski,

in the IP. It cyclic 1972).

D. Siimjen et al.

84

Tissue specificity of IP synthesis

Apart from uterus, IP has thus far been detected only in estrogen stimulated vagina (Katzman et al., 1971). No newly synthesized proteins were detected in immature rat ovaries 1 h after treatment with luteinizing hormone, nor was IP synthesized in rat liver following estradiol treatment (King et al., 1974). In order to test the hypothesis that proteins such as IP are made in preparation for eventual cell division, we also investigated regenerating liver but found no evidence for IP induction. Another induction system involving rapid proliferation is the transformation of lymphocytes by lectins. In collaboration with Dr. A. Novogrodsky we have tested for the presence of IP 3 h after the stimulation of spleen lymphocyte suspensions with concanavalin A or sodium periodate (Novogrodsky and Katchalsky, 1973) treatments which lead to blastogenesis. In neither case was any newly synthesized protein detected by the standard double-label assay for IP. Cell-free

synthesis of IP

Post-mitochondrial suspensions of immature rat uteri prepared as described in Materials and Methods were found to incorporate amino acids into acidinsoluble material as a linear function of protein concentration in the suspension, up to 150 pg of protein per 250 ~1 incubation mixture. The incorporation

Table 1 Relative efficiency of synthesis of IP in surviving uteri and cell-free extracts. Hormone treatments, incubation with labeled leucine and estimation of IP by the isotope ratio method were performed as described in Materials and Methods. Induction

phase

Protein synthesis phase

IP formed

Estradiol treatment

Age of rat

In vivo In vitro

20 20

Surviving uteri Surviving uteri

9 9

1141 4 69 ,L 3

In vivo In vitro

10 10

Surviving uteri Surviving uteri

3 5

64 = 3 58 + 1

In vivo In vitro

20 20

Cell-free Cell-free

3 3

22 +3 24 i 5

In vivo In vitro

10 10

Cell-free Cell-free

4 6

50 zt 5 49 % 3

System

(days)

Number of experiments

(‘A increase + SEM)

was linear with time up to 45 min. The efficiency for protein synthesis of the post-mitochondrial supernatant suspension of immature rat uterus is comparable to that of the system described by Teng and Hamilton (1967), who used uterine ribosomes and soluble factors from rat liver. No synthesis of IP by post-mitochondrial supernatant suspensions, measured by the isotope ratio method after electrophoresis on Cellogel, could be detected at 0 “C. There was significant synthesis of IP at 25 “C and the rate rose over the range 25 to 37 “C, the highest temperature tested (fig. 5). Extracts prepared from uteri excised 1 or 2 h after estradiol injection synthesized IP at the same rate, but similar post-mitochondrial supernatant suspensions from uteri taken 0.5 h after hormone injection failed to synthesize IP.

Fig. 5. Rate of cell-free synthesis of IP measured by the isotope ratio method, as a function of temperature. Uteri were excised from IO-day old rats 1 h after injection of estradiol (4 ,ug/rat) or vehicle. Cell-free extracts, prepared as described in Materials and Methods, were incubated for 45 min with 3H-leu (hormone-treated~ or %Xeu (controls) at the temperatures indicated. The vertical bar indicates the 95% confidence interval of the mean.

TEMP. (“Cl

Table 2 Electrophoretic mobility of uterine estradiolinduced protein (IP) from immature rats. Hormone treatments, incubation with labeled teucine and estimation of IP by the isotope ratio method were performed as described in Materials and Methods. RA= mobility relative to BSA. Protein synthesis phase Induction by estradiol

System

--

RA

Number of experiments -.___

___ _..

_

Mean f 95% confidence interval ~ ~~~ ._

Range

In vivo

Surviving uteri

28

1.1 I *to.01

1.06.-l. 15

In vitro

Surviving uteri

19

1.10*0.02

1.05-1.19

In vivo

Cell-free

12

1.13*0*09

1.08-l .20

D. Smjen

86

et al.

Since direct measurement of the amount of IP synthesized is not yet possible, an approximation of the relative incorporation ratios into IP in different systems was made. In surviving uteri, the increase in IP following incubation of the uteri in estradiol (table 2) was approximately 60% for 20-day old rats, and 90% for IO-day old rats, of the increase found when the estradiol was administered in vivo. Katzenellenbogen and Gorski (1972) reported a maximal value of 85% for 20-day old rats. The increase in IP found in the post-mitochondrial suspension from 20-day old rats (24%) was one fifth of the maximal value for surviving uteri (114%) after in vivo induction. However, the use of lo-day old rats as the source of the post-mitochondrial suspensions raised this value to 49%. The higher efficiency of extracts of lo-day old rats may be due to the fact that the rate of incorporation of amino acids into proteins reaches a peak at this age (Kaye et al., 1974) while the uterus at this age shows no response to estradiol in terms of general protein synthesis (Siimjen et al., 1973b). The IP synthesizing capacity of uteri from IO-day old rats also differs from that in 20-day old rats in that the response was the same at 1 and 4 h after estradiol injection while in 20-day old rats the response at 4 h is less than at 1 h (Barnea and Gorski, 1970; DeAngelo and Fujimoto, 1973; King et al., 1974). r’, Ten-day old rats were given intraperitoneal injections of 4 ug of estradiol-17P, and the synthesis of IP by surviving uteri and by post-mitochondrial suspensions from homogenates of such uteri were compared. TheIpattern of newly synthe-

0

20

40

60 MIGRATION

(mm)

Fig. 6. Comparison of IP synthesis in surviving uteri and in a cell-free system after induction in vivo. Uteri were collected from IO-day old rats 1 h after an intraperitoneal injection of estradiol-17S (4 ug/rat). Intact uteri (a) or post-mitochondrial supernatant suspensions (b) were incubated for 1 h (a) or 45 min (b) with 3H-leu (estrogen-treated) or “C-leu (controls). The distribution of radioactivity on electropherograms of the soluble uterine proteins is shown. Arrow, position of BSA.

Synthesis of estrogen-induced protein

0

20

40

60

I 00

MIGRATION

I 0

20

40

60

00

=

(mm)

Fig. 7. IP synthesis in surviving uteri and in a cell-free system after induction in vitro. Uteri from IO-day old rats were incubated with or without estradiol (30 nM) for 1 h at 37 “C. Surviving uteri (a) or post-mitochondrial supernatant suspensions (b) were incubated with labeled leucine and the labeled proteins analyzed as described in the legend to fig. 6.

sized proteins revealed by Cellogel electrophoresis differed between the two systems : the proteins synthesized in the surviving uteri showed a wider spread of migration rates (fig. 6). Nevertheless, both systems showed a single peak with a mobility relative to BSA characteristic of IP. The mobility of IP was independent of the age of rat used as the source of the uterus. The electrophoretic pattern and amount of IP synthesized by the post-mitochondrial suspension was virtually the same whether induction was by the injection of estradiol in vivo or by incubation with estradiol in vitro (compare figs. 6 and 7, table 2). The combination of in vitro induction of IP synthesis in surviving uteri with subsequent cell-free synthesis by a post-mitochondrial suspension prepared from these, uteri is the nearest approach available at present to a completely cell-free system for the induction and synthesis of IP. Such a model should assist the analysis of the regulation of protein synthesis by estradiol.

ACKNOWLEDGEMENTS We thank Shalom Joseph for the management of our animal colony. A.M.K. is the Herbert Sidebotham Senior Research Fellow and H.R.L. is the Adlai E. Stevenson Professor of Endocrinology and Reproductive Biology at the Weizmann Institute of Science.

D. S6mjen et al.

88

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