Stimulation of DNA synthesis in rat uterine cells by growth factors and uterine extracts

Molecular and Cellular Endocrinology, 84 (1992) 109-118 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303.7207/92/$05.00

MOLCEL

109

02712

Stimulation of DNA synthesis in rat uterine cells by growth factors and uterine extracts Candace

A. Beck ’ and Charles W. Garner

Department of Biochemistry and Molecular Biology, Texas Tech Unicersity Health Sciences Center, Lubbock, TX 79430, USA (Received

11 November

1991; accepted

22 November

Key words; Estradiol; Cell culture; Uterus; Thymidine incorporation; Insulin; factor; Insulin-like growth factor-I; Fibroblast growth factor

Epidermal

1991)

growth

factor;

Platelet-derived

growth

Summary

Replicative DNA synthesis, as measured by thymidine incorporation, has been measured in rat uterine cells in primary culture in response to growth factors. Insulin, insulin-like growth factor-I (IGF-I), multiplication-stimulating activity (MSA) and platelet-derived growth factor (PDGF) stimulated DNA synthesis, while estradiol, epidermal growth factor (EGF), transforming growth factor-p (TGF-P), basic fibroblast growth factor (bFGF) and relaxin did not stimulate or did so weakly and only at very high concentrations. Uterine acid extracts also stimulated DNA synthesis. IGF-I stimulated at concentrations consistent with its acting through the IGF-I receptor; however, insulin stimulated at concentrations higher than expected for its acting through its receptor and thus its action may be mediated through the IGF-I receptor. IGF-I was found in uterine tissue by radioimmunoassay (RIA). There was a 5- to lo-fold increase in IGF-I in the uteri from ovariectomized rats that had been treated with estradiol 24 h earlier. This is analogous to the increase in growth factor activity found previously in rat uterus after 24-h estradiol treatment (Beck, C.A. and Garner, C.W. (1989) Mol. Cell. Endocrinol. 63, 93-101). These data are consistent with the hypothesis that estradiol effects in the uterus are in part mediated through IGF-I,

Introduction

One of the major actions of estrogens in the reproductive tract of mammals is the stimulation of uterine cell growth. In the adult ovariec-

Correspondence to: Charles W. Garner, Department of Biochemistry and Molecular Biology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA. Tel. 806743-2500, Fax 806-743-2990. This work was supported by NIH grant HD-17590. ’ Present address: Department of Pathology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

tomized rat, the growth response is apparently a consequence of interaction of estrogen with specific high affinity nuclear receptors (Toft and Gorski, 1966; Gorski and Gannon, 1976). The biochemical changes that occur in the uterus in response to estrogens suggest that receptor binding results in gene activation (Gorski and Gannon, 1976; Yamanota and Alberts, 1976). This model of hormone action is consistent with many observed responses and is supported by much evidence. However, the sequential biochemical events that lead to stimulation of cell proliferation by estrogens are not yet known. Although receptor binding and enhanced transcription are

1IO

certainly involved, it is possible that estrogen stimulation of metabolic responses and of proliferation occurs indirectly through the autocrine/ paracrine mitogenic action of polypeptide growth factors (Sirbasku and Benson, 1979; Lippman et al., 1987). Evidence for a role of autocrine/paracrine factors in hormone-mediated responses is provided from studies of estrogen-regulated tissues. Estrogen-induced growth factor activity has been found in crude homogenates of rat uterus, liver and kidney (Ikeda and Sirbasku, 1984). Reports from a variety of sources suggest that multiple factors are associated with the mitogenic property of crude uterine homogenates (Sirbasku et al., 1981; Gonzalez et al., 1984; Bronzert et al., 1987; Beck and Garner, 1989). Insulin-like growth factor-1 (IGF-I) expression (Murphy et al., 1987; Murphy and Friesen, 1988) and epidermal growth factor (EGF) precursor expression in rat uterus are elevated after exposure to estradiol. Conditioned media from breast cancer cells exposed to estradiol contain elevated levels of several growth factors including platelet-derived growth factor (PDGF) (Dickson et al., 1986b) and transforming growth factor-p (TGF-P) (Dickson et al., 1986a; Huff et al., 1986). TGF-P has an inhibitory action on epithelial cells and its secretion is depressed after estradiol treatment. IGF-I is secreted from breast cancer cells and initially showed no hormonal regulation (Huff et al., 1986) but recent evidence suggests that there is an estradiol induction of IGF-I in breast cancer cells (Lippman et al., 1987). The ovarian follicle, whose growth is regulated by gonadotropins and estradiol, has been shown to secrete growth factors. Porcine granulosa cells secrete IGF-I in response to estradiol stimulation in vitro (Hammond et al., 1985). In addition, granulosa cells have been shown to secrete basic fibroblast growth factor (bFGF) (Neufield et al., 1987) and are regulated by TGF-/3 produced in the surrounding theta cells (Skinner et al., 1987). The role of steroid hormone in the latter two obse~ations has not been investigated. Because of the growing evidence for growth factor action via a paracrine/autocrine mechanism in the estradiol stimulation of cell growth in uterus, we have established rat uterine cells in culture and describe a system that allows quanti-

tative assessment of the rate [‘Hlthymidine incorporation under serum-free conditions and in the absence of phenol red (Berthais et al., 1986). We have used this system to evaluate the effects of a variety of growth factors and stimulator peptides on DNA synthesis. In addition, we have compared the effect of acid extracts of rat uterus with purified growth factors on uterine cells in culture. Materials and methods Ma terids

Epidermal growth factor (EGF), lot 0001 and lot 96F8862, was obtained from Collaborative Research (Bedford, MA, USA) and from Sigma Chemical Co. (St. Louis, MO, USA), respectively. Insulin-like growth factor (IGF-I), lots 88-1035 and 87-1237, multiplication-stimulating activity (MSA), lot 87-1471 and transforming growth factor-@ (TGF-/3>, lot 87-1307, were purchased from Collaborative Research (Bedford, MA, USA). According to the manufacturer’s specifications, IGF-I was mitogenic for a variety of cell types in the range of 5-20 ng/ml; MSA was mitogenic for chick embryo fibroblasts in the range of S-250 ng/ml and TGF-P demonstrated a colony-forming response with normal rat kidney cells in the range of 0.01-10 ngfml. Basic fibroblast growth factor (bFGF), lot CO6721 1, was purchased from JR Scientific (Woodland, CA, USA) and, according to specifications, was mitogenic for human dermal fibroblasts in the range of 30-300 pg/ml. Platelet-derived growth factor (PDGF), lots 20270 and 23130, was purchased from ICN Biomedicals (Cleveland, OH, USA). According to the manufacturer’s specifications, PDGF gave 50% maximal stimulation in a DNA synthesis assay using Swiss 3T3 cells at 0.5 U/ml. Porcine relaxin (NIH-RXN-Pl; 3000 U/me> was obtained from the National Hormone and Pituitary Program, NIAMDD. Porcine pancreas insulin (24 U/mg) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Collagenase, type 1 (199 U/mg) (lot 44N6782) was purchased from Cooper Biomedical (Malvern, PA, USA). [Methyl3H]thymidine (20 Ci/mmol) was purchased from ICN Radiochemicals (Irvine, CA, USA). Other chemicals not listed were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

111

Animals

Sprague-Dawley rats were obtained from Small Animal Supply Co. (Omaha, NE, USA) at 160180 g. Ovariectomy was accomplished through a dorsal incision while the animals were under light ether anesthesia. Animals were not used until 3 weeks after surgery. Frozen rabbit uterus was obtained from Pel-Freeze (Rogers, AR, USA).

bursts with 15-s rests between bursts. The homogenized tissue was centrifuged at 20,000 x g for 30 min at 4°C. The supernatant was neutralized with 0.855 M Tris base at a ratio of 0.5 ml supernatant per 0.2 ml Tris base. A precipitate usually formed which was removed by centrifugation at 2000 x g for 15 min. Protein was determined by the method of Bradford (Bradford, 1976).

Cell isolation and culture

Optimization of thymidine incorporation

Cells were isolated from 3-week ovariectomized rats (Miiller and Wotiz, 1979). Briefly, the rat uteri were excised and trimmed of fat. The uteri were minced into 1 mm pieces and rinsed with Hanks’ balanced salt solution (HBSS), and transferred to a tube with 2.0 mg/ml collagenase and 0.2 mg/ml DNAase in HBSS. The tube was allowed to rotate at 37°C for 30 min. After incubation, the cells were dispersed by pulling the tissue in and out of a Pasteur pipette 20-50 times. The fluid was filtered through a 100 Frn mesh and the tissue remaining was treated with collagenase and DNAase a second time. After the final treatment, the fluid was filtered through the 100 ,um mesh, added to the first collection and centrifuged to remove the enzyme solution. The cells were resuspended in Dulbecco’s modified Eagle’s medium (DME) with 5% calf serum and plated into 2-cm2 wells at a concentration of 5-10 X lo4 cells per well. All primary uterine cell cultures were maintained in DME supplemented with 5% calf serum plus additions of 25 mM Hepes, 2.2 g/l sodium bicarbonate, 100 mg/l streptomycin sulfate, 1 X lo5 U/l penicillin, 2.5 pg/l amphotericin B. Cultures were maintained at 37°C in a humidified atmosphere of 90% air plus 5% CO,. The media were changed every 2 days.

Preliminary experiments were undertaken to establish the optimum conditions for uterine extract stimulation of thymidine incorporation into DNA of cultured uterine cells, after serum had been removed 24 h earlier. Incorporation was very low at early times after the cells were exposed to uterine extracts but began to be elevated at 18 h and peaked at 24 h. By 36 h, the rate of incorporation had returned to the initial level. Similar results were observed when 10% fetal bovine serum (FBS) was used as the stimulating agent. These time courses were consistent with a relatively synchronized population of cells that underwent a single round of replicative activity during the 48 h period.

Preparation of acid ethanol uterine extracts

Uterine tissue was extracted with a mixture of ethanol and HCl as described (Daughaday et al., 1980). Briefly, ovariectomized rats were injected subcutaneously with 10 pg of 17P-estradiol and 24 h later the uteri were collected. Uterine tissue was homogenized in a mixture of 87.5% ethanol and 12.5% 2 N hydrochloric acid at a ratio of 1 g tissue to 4 ml liquid. Homogenization was performed with a Tekmar Tissumizer using four 15-s

Optimized growth conditions

Cultures were most responsive to extract stimulation on day 7 of culture. Cultures were significantly (P < 0.05) stimulated on days 5 through 9 but by day 12, the cultures were unresponsive to extract stimulation. The same pattern of responsiveness was observed with stimulation by 10% FBS. The growth and incorporation conditions used in this study were a 5-day growth period to establish cultures, a 24-h period of serum deprivation (starvation) followed by a 24-h treatment with extract or other stimulating agent, the last 4 h of which was a radioactive labeling period. No attempt was made to determine the percentage of cells in a particular culture that were stimulated to divide. Radioimmunoassay (RIA) for IGF-I

IGF-I RIA was conducted as described (Furlanetto et al., 1977), except that the nonequilibrium period was 1 h at room temperature before adding the radioactive tracer (Daughaday et al., 19801, and the incubation continued overnight at

112

4°C before the addition of the second antibody. Briefly, the standard or unknown was added to the assay tube followed by addition of IGF-I antiserum. After 1 h at room temperature, [‘2”I]IGF-I was added and the assay tube was incubated for 16 h at 4°C. The reaction was terminated by the addition of second antibody. Equal volumes of acid-ethanol were added to the standard and blank tubes. Antiserum was provided by Drs. J.J. Van Wyk and L. Underwood and was distributed by the National Hormone and Pituitary Institute. Iodinated tracer was obtained from Amersham (Arlington Heights, IL, USA) and human recombinant IGF-I used as standards was obtained from Collaborative Research (Bedford, MA, USA). Goat anti-rabbit IgG was obtained from Sigma (St. Louis, MO, USA) and was used in a second antibody precipitation at l/40 dilution in buffer with 1% normal rabbit serum. Estrogen receptor assays

Primary uterine cells were analyzed for estradiol binding by the cytosol assay method (Kassis et al., 1984). Briefly, cells were harvested, broken and centrifuged for 30 s in a Beckman centrifuge. The supernatant was spun at 100,000 X g for 60 min to obtain a high speed supernatant (HSS). HSS was combined with various concentrations of [ “Hlestradiol with or without a ZOO-fold excess of unlabeled 17p-estradiol. Samples were incubated overnight at 4°C and then treated with hydroxyapatite slurry. Pellets were extracted with ethanol and specific binding was calculated. [ “HI ~hymjdine i~zcorporatio~ assay

Incorporation of [“Hlthymidine into DNA was measured upon the addition of thymidine to medium (1 PCi in 0.2 ml) for a period of 4 h. Incorporation was terminated by the addition of methanol plus acetic acid (3: 1). After 1 h, cells were washed 2 times with 80% methanol to remove unbound thymidine. Cells were solubilized with 0.5 ml of 1% sodium dodecyl sulfate (SDS). The contents of each well were transferred to scintillation vials and the radioactivity was determined in 4.5 ml of Betaphase fluid. Counting efficiency was approximately 20%.

Results Characterization of primary cultures

The purposes of this study were (a) to determine if uterine cells in primary culture respond to several growth factors which are possibly found in the uterus by using thymidine incorporation as a measure of DNA synthesis; (b) to determine if IGF-I is found in the uterus; and (c> if IGF-I is found in the uterus, to determine if it is expressed in response to estradiol. To do this we have prepared uterine cells by collagenase digestion of uterus from 21-day ovariectomized rats that have the same morphological and biochemical characteristics as previously reported (Miiller and Wotiz, 19791. No attempt was made to separate the uterine cells into individual cell types. The yield was approximately 25-30 X 10” cells/uterus and the viability of greater than 90% as judged by trypan blue exclusion. By inspection after one day of culturing, the cultures appeared to be of mixed morphology. The majority of cells were fibroblastic in appearance with clumps of epithelial-like cells interspersed throughout. After 3 days, the epitheliallike ceils began to disappear or to alter their characteristics and the cultures became nearIy homogeneous and fibroblastic in appearance. The cells were cultured in phenol red-free DME with 5% calf serum that had been treated with dextran-coated charcoal to remove culture medium-borne estrogenic substances. The primary cultures on day 5 were found to contain 22,000 I 6000 receptors per cell. This is similar to previously reported values (Kassis et al., 1984). Even though these cells had estrogen receptors they were not growth-responsive to estradiol. Cultures were treated with estradiol in the absence of serum over a wide concentration range (lo-(’ to lo-” M) for up to 7 days with no increase in DNA, protein or cell number. Estradiol also had no effect on DNA synthesis when added to culture medium containing serum in several concentrations. Thymidine incorporation into replicative DNA synthesis

In order to determine whether the [“Hlthymidine incorporation assay used in these experi-

113

6 .;ii

6000

is ‘0

13

-.gs

z* 111

e

5

6000

4000

2000

CL 0

Blank

100 pg/ml EX

10% FBS

Fig. 1. Effect of 1 mM hydroxyurea (OH-urea) on [“Hlthymidine incorporation into DNA of serum- and extract-stimulated uterine cells. Primary cultures of rat uterine cells were prepared as described in Materials and methods. Some of the cultures were stimulated with either 10% FBS or 100 @g/ml rabbit uterine extracted protein (100 fig/ml EX) or were kept under serum-free conditions (Blank). These are indicated by the stippled bars and are labeled DME. The others were kept under serum-free conditions (Blank) or were treated with either 10% FBS or 100 Kg/ml rabbit uterine extracted protein In DME plus 10 mM hydroxyurea (OH-urea). These are indicated by the solid bars and are labeled DME+OH-urea. Results represent the mean + SD; n = 3.

ments was measuring replicative DNA synthesis rather than repair incorporation, the effects of hydroxyurea were investigated. Hydroxyurea inhibits ribonucleotide reductase and hence inhibits replicative DNA synthesis but does not inhibit repair synthesis (Collins and Oates, 1987). The results (Fig. 1) show that hydroxyurea reduced incorporation of [3H]thymidine into serum- or uterine extract-stimulated cells down to that observed with basal cells. Incorporation into basal cells was not inhibited by hydroxyurea. This suggests that the stimulation in L3H]thymidine incorporation measured in the presence of uterine extract and serum represents an increase in replicative DNA synthesis and not repair. Primary cultures were established for 2 days in serum and then placed in serum-free medium for up to 2 weeks to determine if the basal level of incorporation of thymidine was hydroxyurea-inhibitable and therefore replicative. DNA content of each culture well was then measured daily and found not to change for the entire 16day period. However, there was a low level of [3H]thymidine incorporation which was not hydroxyurea-inhibitable, consistent with repair incorporation in the

absence of replicative synthesis (data not shown). When cells, which had been cultured in the absence of serum for 4 days, were given serum or uterine extract, [3H]thymidine incorporation increased which was hydroxyurea-inhibitable. [ “HlThymidine incorporation increased in parallel with an increase in DNA content (Beck and Garner, 1989). These data suggest that the stimulation of [ 3H]thymidine incorporation into uterine cells is a measure of replicative DNA synthesis. [ “H]Thymidine incorporation was also measured after an incubation of uterine cells with various amounts of unlabeled thymidine for 20 h before addition of [“Hlthymidine in order to determine if there were significant alterations in thymidine pool size by estradiol treatment and by 10% FBS treatment. Incubation of cells with significant amounts of unlabeled thymidine would have increased the size of the thymidine pools and would have overcome any potential difference in pool size induced by FBS or uterine extracts. Incorporation of [“Hlthymidine was inhibited in the presence of unlabeled thymidine as expected (not shown). However, as shown in Fig. 2, the ratios of incorporation in the presence of extracts (or serum) to the incorporation in the absence of extract (or serum) were constant at all concentrations of unlabeled thymidine tested indicating that the differences in incorporation caused by extract proteins or by FBS were not a reflection of differences in the pool size.

Effect of various growth factors on DNA synthesis

Table 1 shows the effects of various growth factors in the incorporation of [3H]thymidine into DNA of primary cultures of rat uterine cells. Basic FGF was tested over a range of 1 pg/ml to 3 ng/ml. EGF was analyzed over a range of 1 pg/ml to 1 pug/ml. TGF-P was tested over the range of 1 pg/ml to 3 ng/ml. No significant change in incorporation was detected with bFGF, EGF, or TGF-P. With certain cell types, TGF-P is reported to have a prolonged lag phase with a peak of DNA synthetic activity at 30-36 h (KeskiOja et al., 1987). Therefore, the TGF-/3 effect on uterine cells was analyzed over a 40-h period with radioactive labeling during the last 20 h. No stim-

n Extract Ratio q 10% FBS Ratio 8000

0

(3s P9fmt1

0.001

0.01

TdR

(Mgiml)

0.1

Fig. 2. Ratio of radioactivity incorporated by uterine cells in the presence of FBS or uterine extract in the presence of various amounts of unlabeled thymidine (TdR). Cells were prepared as described in Materials and methods. Cells were treated with FBS or extract, as indicated, in the presence of different amounts of unlabeled thymidine for 20 h. [“H]Thymidine (36 pg/ml of thymidine) was added as described in Materials and methods and the incubation was continued for 4 h. The amount of radiolabeled thymidine incorporated was determined and the ratio of cpm incorporated in the presence of FBS (or extract) to the amount incorporated in the control is shown in each bar.

ulation or inhibition of extract stimulation was observed (data not shown). Relaxin has been shown to have uterotrophic effects (Vasilenko et al., 1986; Vasilenko and Mead, 1987). [ ‘H]Thymidine incorporation was measured after incubation of uterine cells with relaxin over a range of 0.1 ng/ml to 100 pg/ml. Increased [ 3H]thymidine incorporation was observed at only the highest concentration (Table l), which is about 10” times higher than the K, reported for binding to the receptor (MercadoSimmen et al., 1980). There was no effect of relaxin on extract-stimulated [3H]thymid~ne incorporation (data not shown). Porcine PDGF also stimulated [ 3H]thymidine incorporation in uterine cells (Table 1 and Fig. 3) at 1-3 units/ml. One unit of pure PDGF is equivalent to approximateIy 28 ng/ml protein. Insulin and IGF-I also stimulated ~~HJthymidine incorporation into DNA of uterine cells (Table 1 and Fig. 4) in the range of 10-1000 ng/ml. Only at 100 ng/ml was MSA stimulation significantly (P < 0.05) above control. Fig. 5 shows the dose response of acid extracts prepared from rat and rabbit uterus. Acid extract

PDGF (Units/ml) Fig. 3. Effect of PDGF on [‘Hlthymidine incorporation. Primary cultures of rat uterine ceils were prepared as described in Materials and methods. PDGF at the indicated concentrations was added to the cells and radioactive thymidine incorporated into DNA was measured as described. The bars at the right indicate the stimulation observed under serum-free conditions (Blk), with 100 pg/ml extracted uterine protein (Extract) and with 10% FBS. Results represent the mean f SD; n=3.

from estradiol-treated rat uterus showed greater activity at lower concentrations than acid extracts from rabbit uterus. Both acid extracts were maximally active at 30 and 300 pg/ml (estradioltreated rat and nontreated rabbit, respectively).

TABLE 1 EFFECT OF GROWTH FACTORS ON THYMIDINE INCORPORATION BY UTERINE CELLS IN CULTURE Primary cultures of rat uterine cells were prepared as described in Materials and methods. [ 3HJThymidine incorporation into DNA was measured after stimulation of the cells with the growth factors listed below. The maximum concentration used is indicated in the table; stimulation was analyzed over at Least a 103-fold concentration range for each growth factor. Values are the mean f SD; n = 3. Effector None bFGF TGF-JI EGF Relaxin Relaxin Insulin IGF-I MSA PDGF Extract FBS

Concentration

cpm/pg

3 ng/ml 3 ng/ml l pg/ml IO Ctg/ml 100 pg/ml 100 ng/mi 10 ng/ml 100 ng/ml 3 U/ml 100 pg/ml 10% (v/v)

1,795* 60 1,762 t 369 2,110&578 1,768 rt 225 2,509+312 3,731 rt 168 6,585 + 334 6,719*431 3,569 & 761 9,147 * 5.59 6,396 j, 444 13,363k 486

DNA

Significance NS NS

NS NS P < 0.05 P < 0.05

P P P P P

< 0.05 < 0.05 < 0.05 < 0.05 < 0.05

-

10000

-o-

c

.z!

xi

log Acid -2

Ins

Ethanol -1

Extract 0

(~1) 1

IGF-1

6000

“0

9;

6000

00, -cd. 1 an

4000

Is'"

2000

P CL?.,

115

ll

0

10-2 10-l 100 10'

102 103 104 105

Fig. 4. Effect of insulin and IGF-I on thymidine incor~ration of primary cultures. Primary cultures of rat uterine cells were prepared as described in Materials and methods. Concentrations of insulin (0) and IGF-I (0) indicated above were added to the cells and thymidine incorporation was measured as described. Incorporation under serum-free conditions (Blank) and with 100 pg/ml extracted rat uterine protein (100 kg/ml Extract) is indicated by the bars on the right of the graph. Results represent the mean i SD; n = 3.

Detection of IGF-l in uterine extracts Previous work has shown that acid extracts of uterus from estradiol-treated ovariectomized rats

1

1000

Protein

Stds

E2 Control

A

Effector (ngiml)

Uterin?

o 0

‘~~,~,~

Fig. 5. Effect of acid-extracted uterine protein on primary cultures of rat uterine cells. Primary cultures were prepared as described in Materials and methods. Uterine extract was prepared from rats (0) that had been injected 24 h previously with a single injection of 10 pg 17&estradiol. The uteri were homogenized in 10 mM H$O, at a ratio of 1 g tissue to 4 ml acid. The particulate material was removed by centrifugation at 20,000~ g for 30 min. The supernatant was filtered through hvo layers of cheese-cloth. Rabbit uterine extracts (0) were prepared by the same procedure using frozen uteri. Protein determinations were done by the dye binding method using bovine serum albumin as standard as described in the Materials and methods section. 10% FBS was used as a positive control and gave a stimulation of 11,506+ 1033 cpm/ pg DNA.

-3

-2

-1

log

0

1

2

2

IGF-1fng)

Fig. 6. Analysis of iIGF-I in acid-ethanol extracts of estradiol-treated and control rat uteri. Rats were injected with 10 pg 17~~estradiol or an equal volume of PBS 24 h before they were killed. Acid-ethanol extraction and RIA was done as described in Materials and methods. The binding of tracer radioligand (B) in the presence of acid-ethanol extracts from estradiol-treated rat uteri (01 and control uteri (A 1 and in the presence of IGF-I standards (0) is presented as a fraction of initial binding (B,,) on a log-logit plot. The displacement curves for acid-ethanol extracts are plotted against the top axis. The lines were fit by linear regression.

stimulate glucose transport and that this property is associated with a low molecular weight protein (Conrad-Kessel et al., 1988). Since IGF-I stimulates glucose transport in uterine primary cultures (data not shown), studies were undertaken to determine, by immunological methods, the concentration of IGF-I in control and estradioltreated extracts and to determine if IGF-I could be responsible for the stimulation of glucose transport and thymidine incorporation. A single injection of estradiol resulted in an increase in immunoreactive IGF-I (iIGF-I) extracted from rat uterus 24 h later. Fig. 6 shows the results of a competitive binding assay using antiserum to iIGF-I. The log-logit plots of estradiol and control acid-ethanol extracts are parallel to the plot of IGF-I standards indicatjng the simiIarity between the rat and human forms of iIGF-I. IGF II, somatomedin A, and insulin are recognized by the antiserum but with only 5%, 3% and less than 0.0001% binding respectively (Furlanetto et al., 1977). Table 2 shows the relative concentrations of iIGF-I present in estradiol and control uterine

116 TABLE

2

EFFECT OF ESTRADIOL EXTRACT

ON IIGF-I

IN RAT UTERINE

Rats were injected with 10 Kg 17p-estradiol or PBS 24 h before they were killed. Acid-ethanol extracts of uteri were prepared and analyzed for iIGF-I by RIA as described in Materials and methods. The relative concentrations of iIGF-I were calculated from RIA data and represent the mean f SD; II = 4. Treatment

Amount

of iIGF-I

m/gwet

in uterus ng/uterus

ng/mg protein

23 k 2 236 +I3 10.2+ 1.1

48 * 4 229 k13 4x* 0.5

weight None (N) Estradiol (El Ratio of E/N

334 i 30 2.046 k262 6.1 i 1.0

extracts and depicts the relative estradiol administration. Estradiol sults in a 5 to lo-fold increase uterine extracts.

increase with injection reon iIGF-I in

Discussion We have used the incorporation of [“Hlthymidine into DNA to measure the effects of estradiol, uterine extracts and growth factors on uterine cell replicative DNA synthesis. Cultures of rat uterine cells show no increase in DNA or [ ‘Hlthymidine incorporation in response to a wide range of estradiol concentrations (lOph to lo-” M) over a 3- to lo-day period after establishment of cultures. These results constitute a paradox between in vivo and in vitro observations. We have shown that acid extracts of rat uterus contain one or more substances which are increased after estradiol treatment and that this substance(s) can affect glucose transport and DNA synthesis in cultured uterine tumor cells in much the same way that estradiol affects these processes in vivo (Conrad-Kessel et al., 1988). From these and other observations, we have assumed the model that estradiol induces production and secretion of positive growth effecters that act in an autocrine/paracrine manner to affect cellular processes such as glucose transport and DNA synthesis. Before testing this hypothesis, we sought to determine which growth factors could stimulate uterine cells to increase in replicative DNA syn-

thesis. We have targeted TGF-P, EGF, bFGF, relaxin, MSA (IGF-II), PDGF, IGF-I and insulin. TGF-P, EGF and bFGF did not stimulate DNA synthesis in primary cultures of uterine cells. Reports indicate that TGF-/3 has both stimulatory and inhibitory effects on cell proliferation depending on cell type and experimental conditions. It it generally inhibitory to epithelial cell types especially if they are responsive to EGF and is stimulatory to fibroblasts (Keski-Oja et al., 1987). An effect of TGF-P on uterine cells has not been previously reported; however, TGF-/3 does inhibit EGF stimulation of granulosa cells (Skinner et al., 1987) and can regulate granulosa cell steroidogenesis. TGF-/3 is reported to stimulate anchorage-independent growth of many cells in soft agar but the mitogenicity is comprised under anchorage-dependent assay conditions (Fernandez-Pol et al., 1986). No attempt was made to analyze the effect of TGF-P on anchorage-independent growth of uterine cells. Gospodarowicz et al. (1987) suggested that uterine-derived growth factor is synonymous with bFGF. Although stimulatory for many cell types, no effect on DNA synthesis could be shown with bFGF in primary cultures of rat uterine cells. The results of EGF on uterine cells in primary cultures were unexpected. EGF concentrations in immature mouse uterus have been reported to be elevated after seven sequential estradiol injections, but not 24 h after a single injection (Gonzalez et al., 1984). In another report, mature mice, after receiving a single dose of estradiol, showed a rapid (within 15 min) increase in uterine EGF but the concentrations fell to control levels by 30 min (DiAugustine et al., 19881. EGF receptors are found in immature rat uterus and are under estradiol regulation (Mukku and Stanccl, 1985). Localization of EGF receptors to all major cell types within the rat (Lin et al., 1988) and human (Chegini et al., 1986) uterus has been reported. Tomooka et al. (1986) have reported that primary cultures of epithelial cells from immature CD-l mice are responsive to EGF stimulation of cell proliferation. In addition, Nelson et al. (1991) found that administration of EGF to ovariectomized mice replaced estrogen in growth and differentiation of the uterus. They also showed that injection of an antibody against EGF

117

prevented estrogen effects. Our results consistently showed that primary cultures of ovariectomized rat uterus do not respond to EGF under several experimental conditions. No attempt was made to determine if these cultures contain EGF receptors. The discrepancy may be a result of the difference between immature and mature rodents. However, EGF did stimulate glucose transport in primary cultures of uterine cells albeit at high concentrations and to a small extent when compared to uterine acid extracts (Conrad-KesseI et al., 1988). Relaxin (l-30 ~g/animal) has been reported to have uterotrophic effects when injected into immature and mature rodents (Vasilenko et al., 1986; Vasilenko and Mead, 1987). Receptors for relaxin have been identified in both the myometrium and endometrium and are under estrogen regulation (Weiss and Bryant-Greenwood, 1982). Relaxin has a stimulatory effect (207% of control) at high concentrations when assayed for [ “Hlthymidine incorporation in primary cultures of rat uterus. Even though relaxin has little sequence homology to insulin, it can adopt a conformation identical to that of insulin (Blundell and Wood, 1982). Thus the insulin-like activity of relaxin may be due to its ability to cross-react with the insulin or the IGF-I receptors at high concentrations. MSA, the rat homologue of human IGF-II (Adams et al., 1983), is slightly mitogenic for rat uterine cells at 100 ng/ml. Since there has been reported (Rechler and Nissley, 1985) as much as lo-100% cross-reactivity of MSA with the type I IGF receptor, the stimmation seen here may be the result of cross-reactivi~. PDGF, IGF-I, and insulin were the major stimulatory factors for DNA synthesis in cultured rat uterine cells. Insulin (0.1-10 pg/ml) has been reported to stimulate growth of almost all cell types in culture and is often used as a supplement in serum-free media (Barnes and Sato, 1980). It has been found to be stimulatory to uterine cells in that range. PDGF was also mitogenic for uterine cells in the same range as reported for other cell types. Whether PDGF is synthesized in the uterus or PDGF receptor increase, thereby increasing PDGF sensitivity, is not yet known. IGF-I stimulated rat uterine cells in the range of lo-100

ng/ml similar to the mitogenic effect on other cell types (Decks et al., 1988). IGF-I mRNA expression is increased in response to estradiol in immature rat uteri (Murphy et al., 1987; Murphy and Friesen, 1988) and, corresponding to this, immunoreactive IGF-I protein is elevated in acid extracts of estradiol-treated mature rat uteri (Fig. 6). One might question whether estradiol treatment resulted in an increase in local IGF-I synthesis rather than an increased delivery of IGF-I to the uterus from the plasma. A well documented response to estradiot is increased blood flow to the uterus. Also, Murphy et al. (1987) measured serum levels of IGF-I in rats and found them to be higher than acid extractable uterine levels. Several lines of evidence argue against circulating factors being brought in by the increased blood supply. Estradiol induces IGF-I gene expression in uterine tissue 14-fold but has no effect on hepatic or renal IGF-I gene expression and has no effect on circulating serum levels of IGF-I (Murphy et al., 1987; Murphy and Friesen, 1988). Estradiol stimulates glucose transport in the uterus within 2 h (Meier and Garner, 19871, but only if the estradiol is administered in vivo. DNA synthesis is also increased but at about 24 h (Stack and Gorski, 1984). Growth factor activity in acid extracts of uterus, as measured by the stimulation of glucose transport and by thymidine incorporation into uterine cells, was also increased in the uterus in response to estradiol. However, the increase was observed only after 12-18 h. Therefore if the growth factor activity in the uterus is responsible for the stimulation of glucose transport and DNA synthesis by estradiol, one may postulate that there is an estradiol-stimulated release of the growth factor into the extracellular space during the first 2-h response period with replenishment of the stored form of the growth occurring after 12 h. Substantiation of this idea must await a demonstration that specific growth factor receptors are activated in response to estradiol in a manner consistent with Iigand binding. References Adams, S.O., Nissley, S.P., Greenstein, L.A., Yang, Y.W.-H. and Rechler, M.M. (1983) Endocrinology 112, 979-987.

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