0013-7227/92/1303-1716$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine Society
Vol. 130,No. 3 Printed
in U.S.A.
Presence of Epidermal Growth Factor, Platelet-Derived Growth Factor, and Their Receptors in Human Myometrial Tissue and Smooth Muscle Cells: Their Action in Smooth Muscle Cells in Vitro* MICHAEL
J. ROSSI,
NASSER
CHEGINI,
Department of Obstetricsand Gynecology,University Gainesville,Florida 32610
AND BYRON of
J. MASTERSON
Florida Collegeof Medicine,
alone (P c 0.05). PDGF-BB at 10 rig/ml also stimulated 3Hthymidine incorporation and ita effect was similar to that induced by PDGF-AB at the same concentration. 17@-Estradiol (Es) at 1 PM inhibited3H-thymidine incorporation by the smooth muscle cells (P < 0.05). Ez also reduced the stimulatory effect of EGF (15 rig/ml) and PDGF (3 rig/ml). Progesterone at 1 pi either alone or in combination with EP did not have any effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF. The effect of EGF and PDGF on cell mowth and 3H-thymidine incorporation by myometrial smooth muscle cells was independent of phases of the menstrual cycle. In summary, the results of present studies indicate that human mvometrial tissue and mvometrial smooth muscle cells in primary culture locally produce EGF and PDGF-AB and contain EGF and PDGF-B. but not Lu-recenters. Moreover. the myometrial smooth muscle cells in culture respond to the mitogenie action of EGF and PDGF. (Endocrinology130: 17161727,1992)
ABSTRACT. Immunohistochemical observations indicate that human myometrial smooth muscle cells express epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)AB and contain EGF and PDGF-8 receptors with no variation in intensity with phases of the menstrual cycle. Furthermore, immunofluorescent microscopic studies revealed that primary myometrial smooth muscle cell cultures also express EGF, PDGF-AB, and contain EGF and PDGF-8, but not a-receptor. Incubation of subconfluent smooth muscle cells in serum-free medium leads to quiescence within 48 h as demonstrated by ‘Hthymidine incorporation and labeling index. Exposure of quiescent cells to 10% fetal bovine serum stimulates resumption of DNA synthesis and proliferation in a time-dependent manner with a doubling time of 41.6 h. EGF (1.5-50 rig/ml) and PDGFAB (l-10 rig/ml) in a dose- and time-dependent manner significantly stimulated 3H-thymidine incorporation by quiescent myometrial smooth muscle cells (P < 0.05). Combinations of EGF (15 rig/ml) and PDGF-AB (10 rig/ml) significantly increased 3H-thymidine incorporation induced by either growth factor
I
C
ELLULAR proliferation and differentiation of the uterine tissue is considered to be regulated by ovarian steroids and mediated through the nuclear receptors present in both endometrial and myometrial cells (l-3). The response of the uterine tissue to steroids is complex involving many biochemical as well as morphological events resulting in uterine growth and differentiation (l3). Although the most profound mitogenic effect of estrogen appears to be in the endometrial compartment, myometrium is also sensitive to hypertrophic and hyperplastic actions of steroids (1, 2, 4-6). Myometrium is a quiescent tissue primarily composed of terminally differentiated smooth muscle cells (4); however, identification of mitosis (6), DNA synthesis (7), and the growth of both
benign (leiomyomata) and malignant (leiomyosarcoma) tumors suggests a steroid as well as growth factor-dependent growth potential (8-11). The mechanism(s) of sex steroid action(s) in proliferation and differentiation of various uterine cell types in uiuo are complex and not well defined (1,3). Additionally, in vitro observations have pointed out the lack of mitogenie action of steroids on a variety of uterine cell types (12-15). Recently however, it has been postulated that the mitogenic action of steroids in their target tissue is mediated through local production of growth factors, acting with paracrine and/or autocrine mechanisms (1618). Several in uiuo and in vitro studies in different systems, including uterus, have supported this hypothesis (16-21). Immunohistochemical and in situ hybridization studies have indicated the presence of epidermal growth factor (EGF) in mouse uterine epithelial cells, but not in ovariectomized animals (19-21). Estrogen treatment of ovariectomized animals stimulates the oc-
Received August 30, 1991. Address correspondence and requests for reprints to: Dr. Nasser Chegini, Department of OB/GYN, University of Florida, P.O. Box 100294, Gainesville, Florida 32610. * Funded in part by the University of Florida, Division of Sponsored Research. 1716
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EGF AND
PDGF ACTION
currence of EGF messenger RNA (mRNA) and its immunoreactive appearance (19-21). In contrast, all uterine cell types in the rat appear to contain immunoreactive EGF (22). Similar results have also been reported in the rat for insulin-like growth factor-I (IGF-I) and IGF-I receptor (10). The action of growth factors in their target tissue is mediated through specific cell surface receptors (10, 17). The presence of EGF receptors in all the major uterine cell types in human as well as rat and mouse have been reported (23-29). In the rat uterus the EGF receptor content has been well correlated with the phases of the cycle, with highest levels at proestrus as compared to the other days of the cycle (29). In the ovariectomized rat 17@-estradiol (Ez) administration up-regulates the uterine EGF receptor, presumably due to an increase in the levels of EGF receptor mRNA (30). However, in the human uterus no correlation exists between the EGF receptor content and the phases of menstrual cycle (23, 24, 26). Uterine tissue also contains platelet-derived growth factor (PDGF) receptors and responds to the mitogenic action of PDGF (31, 32). Porcine uterus has been demonstrated to be a major source of PDGF receptor, preferentially associated with endometrial stromal cells (32). PDGF receptor appears in two forms, with different affinities designated as (Y- and P-types (33). Porcine endometrial stromal cells, both in situ and in culture, have been shown to contain PDGF-@ receptor (34) and in situ hybridization, using complementary DNA to the PDGF-fl receptor, indicates the presence of mRNA preferentially associated with these cells (34). In contrast, porcine myometrial smooth muscle cells appear to contain a low level of PDGF+ receptors, but after 24-48 h in culture a majority of the cells, and after 1 week all the cells, express the PDGF-fi receptor mRNA and contain PDGF-P receptors (34). Human uterus has also been shown to contain PDGF receptors, however, the type and cellular distribution of PDGF receptor in this tissue has not been determined (31). Furthermore, there is no data available regarding the regulation of either PDGF or its receptors by steroids, although recent observation indicated that all human uterine cell types express mRNA for PDGF-B chain and a very low abundance of PDGF-A chain, without correlation with the phases of the cycle (35). The local production of growth factors and the presence of their specific receptors in myometrial smooth muscle cells suggest the possibility that these cells are the target for the mitogenic action of growth factors. Therefore, the present study was undertaken to elucidate the presence and cellular distribution of EGF and PDGFAB as well as EGF and PDGF-@ receptor in myometrial tissues and myometrial smooth muscle cells in culture.
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Additionally, to investigate the role of EGF and PDGF, either alone or in combination, and in the presence of steroids on 3H-thymicline incorporation by primary myometrial smooth muscle cell cultures.
Materials
and Methods
Material Dulbecco’s modified Eagle’s medium (DMEM), EDTA, trypsin-EDTA solution, and antibiotic-antimycotic solution were purchased from GIBCO Laboratories (Grand Island, NY). Fetal bovine serum (FBS), Ez, progesterone (P,), and phenol-red free medium were obtained from Sigma Chemical Company (St. Louis, MO). Collagenase was purchased from Worthington Biochemical (Freehold, NJ). Culture grade EGF, natural human PDGF-AB, recombinant PDGF-BB, and goat anti-human PDGF-AB antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal rabbit anti-human recombinant EGF was purchased from Biomedical Technologies, Inc. (Stoughton, MA), monoclonal antibodies to porcine PDGF-/3 receptor from Oncogene Science Inc. (Manhasset, NY), and monoclonal antibodies to human PDGF-(U and PDGF-/3 receptors from Genzyme Corporation (Cambridge, MA). Monoclonal antibodies to human cytokeratin 19 and human desmin were purchased from Dako Corporation (Carpinteria, CA) and monoclonal antibody to human vimentin was purchased from Sigma. Rhodamine (TRITC) conjugated affinipure donkey anti-rabbit and goat anti-mouse immunoglobulin G (IgG) were purchased from Jackson Laboratories, Inc. (West Grove, PA). Vectastain ABC Elite Kits were from Vector Laboratories (Burlingam, CA). Methyl-3H-thymidine (83 Ci/ mmol) was purchased from Amersham Co. (Arlington Heights, IL). The 75cm* flasks and the 24-well plates were purchased from Corning Glass Works (Corning, NY) and 8-well culture slides, either glass or plastic, from Nunc Inc. (Naperville, IL). Monoclonal antibodies to extracellular binding domain of the EGF receptor were a gift from Dr. W. A. Dunn, Jr. (Department of Anatomy and Cell Biology, University of Florida). Tissue collection and myometrial smooth muscle cells isolation Portions of uterine specimens from nonpregnant premenopausal women undergoing hysterectomy for medically indicated reasons, excluding endometrial cancer, were collected, placed in sterilized ice-cold PBS (pH 7.4) and immediately brought to the laboratory on ice. The tissues were collected at the University of Florida affiliated Shands Hospital, and collection and use of tissues for the purpose of these experiments was approved by the University of Florida Institutional Review Board. A total of 102 uterine tissues were collected of which 35 were from proliferative and 67 from secretory phase of menstrual cycle. Each uterine tissue was examined by the pathologist for histological evaluation and the dating of endometrium. The endometrial histological dating was performed according to Noyes et al. (36) and the patient’s last menstrual period. Myometrial tissues were dissected from endometrial cell layers, washed in PBS, cut into small pieces, and digested in 2% collagenase (wt/vol) at 37 C for 3-6 h. The isolated smooth muscle cells were collected by centrifugation at 460 X g for 5
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EGF AND PDGF ACTION ON HUMAN
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Endo Voll30.
l
1992 No 3
min, washedseveral times with DMEM containing 1% antibiotic-antimycotic solution and cultured. Myometrial smoothmusclecell culture The isolatedmyometrial smooth musclecells were grown as monolayersin 75-cm*flasks at approximate density of 2 x lo5 cells per flask. The cells were maintained in DMEM supplemented with 10%FBS (vol/vol) and 1% antibiotic-antimycotic solution (vol/vol) for 6 days at 37 C in a humidified atmosphere of 5% CO*-95% air and the mediawas changedevery 2-3 days. The subconfluent monolayer cells were subcultured at the above density after trypsinization with 0.1% trypsin-0.5% EDTA. The smooth musclecells after the third passagewere seededat approximately 5 x lo4 cells per well in 24 well dishes or 2 x lo4 cells per well in 8 well Lab-Tek slides in supplementedDMEM. The selectionof third passagewaschosenonly to obtain enough cells with which to perform experiments at the samepassage.It has been shown that human myometrial smoothmusclecells do not losetheir characteristics even after 16 passagesand being cultured for 1 yr (4). Cells that were
Time
f?
(hours)
2500
8% 2 =: 2000 8 42 IY1500 .- \ ge $ 2 X
m
1000 500
0
12
24
36
4E Time
~4
46
72
96
120
(hours)
FIG. 2. A shows the growth curve of smooth muscle cells incubated in the presence of 10% FBS (AA) and serum-free condition (O-0). The FBS stimulated cultures are significantly different from nonstimulated controls (P < 0.0001). B demonstrates the time dependency of 3H-thymidine incorporation by smooth muscle cells. The cells were cultured for 48 h in the presence of 10% FBS, then incubated in serum-free conditions (left-hand side, O0) indicating a gradual reduction in 3H-thymidine incorporation. Addition of 10% FBS to these quiescent cells stimulates 3H-thymidine incorporation in a timedependent manner (right-hnnd side, AA), however, the nonstimulated cells do not significantly incorporate 3H-thymidine (O0). Cell numbers in A and each point in B represent mean f SEM of three separate triplicate experiments from three individual patients.
grown in 8 well Lab-Tek slideswere only usedfor immunofluorescenceor autoradiographic studies. Trypan blue exclusion test determined the cell viability after trypsinization before each experiment. Growth curve Myometrial smooth muscle cells were plated into 30-mm culture dishesat approximately 2.4 X 10’ cells per plate and grown in DMEM supplementedwith 10% FBS. At various intervals cells were washedwith Ca*+-Me-free PBS, trypsinized, and counted usingCoulter Counter ZM (Coulter Electronics, Hialeah, FL). 3H-Thymidine incorporation
FIG. 1. A shows a Nomarski interference contrast micrograph of myometrial smooth muscle cells in primary culture and B shows immunofluorescence micrograph of smooth muscle cells stained with antibodies to human desmin. Magnification, X110.
Myometrial smooth muscle cells were initially plated in DMEM containing 10% FBS for 48 h, then washed,and incubated in serum-freeDMEM for an additional 48 h. The quiescent cells obtained under serum-freeconditions were returned to serum-supplementedDMEM containing 2 &i/ml 3H-thymidine for various periods ranging from 6-72 h. The cultures
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EGF AND
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FIG. 3. Light microscope autoradiographic Nomarski interference contrast photographs of myometrial smooth muscle cells. Quiescent cells were labeled with ‘H-thymidine for 48 h either in serum-free conditions (A) or in the presence of 10% FBS (B). Note the presence of few lightly labeled nuclei in A, whereas extensive nuclear labeling indicating significantly higher ‘H-thymidine incorporation in B. Magnification, ~161.
0.0
1.5
15.0
5.0
01
50.0
EGF hz./~)
0
1 PDGF
3
10
bdml)
800 i$ 5b 600 Bg 4oo
nX
0
24
4s Time
-
200
72
(hr)
FIG. 4. The dose dependency of EGF (A) and PDGF-AB (B) on 3H-thymidine incorporation by myometrial smooth muscle cells after 48 h of incubation. The effect of EGF at all concentrations and PDGF at 10 rig/ml on 3H-thymidine incorporation were significantly different from control (P < 0.01). C shows the time dependency of EGF at 15 rig/ml (6 -+), PDGF-AB at 10 rig/ml (A -A) and nonstimulated serumon 3H-thymidine incorporation. D indicates the 3H-thymidine incorporation by smooth muscle cells incubated in serumfree medium (0 -0) free medium (control) or in the presence of EGF (15 rig/ml), PDGF-AB (10 rig/ml), or combination of EGF and PDGF-AB (E + P) for 48 h. The action of EGF and PDGF on ‘H-thymidine incorporation were significantly different from the control (P < 0.01) and their combination different from either control (P < 0.02) or EGF or PDGF alone (P < 0.05). Points represent mean & SEM of three (A and C) and five (B and C) separate triplicate experiments from three to five individual patients. Disintegrations per min/103 cells for controls are; A: 674 f 19, B: 721 f 196, D: 604 f 171.
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EGF AND
1720
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ON HUMAN
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Endo. 1992 Voll30. No 3
using a LKB historange. The sectionswere deparaffnized and processed for immunohistochemical localization of EGF, PDGF-AB, EGF receptor, and PDGF-8 receptor aspreviously described(38). Frozen sectionsfrom 8 specimenswerealsoused for immunolocalization of PDGF-fl receptorsusinganti-porcine PDGF-,3 receptor antibody (34). The primary antibody dilutions usedin this study were: EGF (1:50), EGF receptor (l:lOO), PDGF-AB (l:lOO), anti-porcine PDGF-8 receptor (50 pg IgG/ ml), and anti-human PDGF-a and &receptor antibodies(5 pg IgG/ml). Fifty-seven of the 78 uteri fixed in Bouin’s solution and 8 from frozen uterine tissueswere used for the immunohistochemicalstudies. Immunofluorescencemicroscopicstudies
100 50
-
0 CONTROL
E2
P4
E2iP4
5. The effect of El and P, at 1 PM concentration and their combination (E2 + P,) on SH-thymidine incorporation by myometrial smooth muscle cells incubated for 48 h in the absence (A) or presence of 15 rig/ml EGF (B) and 3 rig/ml PDGF-AB (C). The controls in these figures are, serum-free condition (A), 15 rig/ml EGF (B), and 3 rig/ml PDGF-AB (C). Ez alone or in the presence of EGF and PDGF significantly inhibit ‘H-thymidine incorporation (P < 0.05). Points represent meanf SEM of sevenseparatetriplicate experimentsfrom either four (A andB) or three (C) individualpatients.Disintegrations permin/103 cellsfor controlsare,A: 645f 154,B: 1082f 234,C: 917f 171. FIG.
were then washedtwice with Ca*+-Me-free PBS and trypsinized. The cell suspensions(200 ~1total volume) from each well were usedto determine the cell number and the rate of incorporated 3H-thymidine. An aliquot of 60 ~1 was used for cell number determination. The remaining volume was retained on individual filters (Millipore 96-well filtration plates, Millipore Inc., Bedford, MA), washedonce with 10% trichloroacetic acid (TCA), twice with 5% TCA, and twice with 95% alcohol. Their radioisotopecontent was determined by a Beckman LS 5000 scintillation counter. SH-Thymidine autoradiography Myometrial smooth muscle cells grown in eight well LabTek slides under conditions described above were used to determine their labeling index. The level of 3H-thymidine incorporation by smoothmusclecells after incubation for various periods of time ranging from 6-72 h were determined by autoradiography using Kodak NTB-2 emulsion as previously described(37). Immurwhistochemicalstudies For immunohistochemical studies, portions of 78 of the uterine tissuescollected were washedwith PBS, cut into small pieces, and fixed in Bouin’s solution overnight. The tissue pieceswere washedwith PBS, dehydrated in a seriesof graded ethanol, paraffin embedded,and sectionedat 5-6 pm thickness
Myometrial smooth muscle cells grown on Lab-Tek slides for 48 h using DMEM supplemented with 10% FBS were washedwith Ca*+-M$+ free PBS and immediately fixed with ice-cold methanol for 5 min. The slideswere either usedimmediately for immunofluorescenceobservations or stored at -20 C until needed.The purity and homogeneityof myometrial smooth muscle cell cultures were analyzed using monoclonal antibodiesto human desmin (smoothmusclecells),cytokeratin 19 (epithelial cells), and vimentin (stromal cells). The slides were treated with 0.1% Tween 20 in PBS for 5 min, washedin PBS twice, and subsequently exposed to normal serum 1:50 dilution for 20 min, 1:lOOdilution of primary antibodiesfor 1 h, and then TRITC conjugatedsecondantibodiesfor 30 min in the dark. The cells were washedtwice with PBS and viewed in Olympus BH2 microscopeequipped for fluorescencemicroscopy. Similar cultures were alsousedfor cellular localization of EGF and PDGF-AB, and EGF, PDGF-a! and p-receptors at dilutions indicated in immunohistochemicalstudies. Statistical analysis Data are reported as the mean + SEM for the indicated experiments and analyzed by analysis of variance or Student’s t test. All the experiments were repeatedat least three times in triplicate.
Results Characterization of human myometrial smooth muscle cells
Collagenase treatment of myometrial tissue provided a pure population of isolated cells with smooth muscle cell characteristics without either stromal or glandular epithelial cell contamination (Fig. 1A). Immunofluorescence microscopic examination of myometrial smooth muscle cells cultured for 48 h after collection, or after their third passage using monoclonal antibody directed toward the muscle specific protein desmin, indicated immunostaining of more than 95% of the cells (Fig. 1B). These cells did not immunostain with antibodies to cytokeratin 19, a cytoskeletal protein specific for epithelial cells, or vimentin, a class of intermediate filament proteins present in fibroblasts, indicating their purity as smooth muscle cells (figures not shown). Cultures were monitored with these antibodies regularly and the pat-
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EGF AND
PDGF ACTION
ON HUMAN
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microFIG. 6. Immunofluorescence graphs of myometrial smooth muscle cells stained for EGF (A), EGF receptor (B), PDGF-AB (D), PDGF-fl receptor (E), and PDGF-CT receptor (anti-human PDGF-u and @-receptor antibodies were used in this presentations). Preabsorption of EGF receptor antibody with human term placental microvilli before application resulted in a considerable reduction in immunostaining (C). The controls for the other antibodies resulted in similar reduction in immunostaining (figures are not shown). Magnification, x110.
terns of immunostaining were consistent, suggesting the cells retained their smooth muscle nature under the culture conditions used in this study. DNA synthesis and growth of myometrial
smooth muscle
Myometrial smooth muscle cells grow exponentially in the presence of DMEM supplemented with 10% FBS, and reached confluency in 6 to 7 days with a doubling time of 41.6 h (Fig. 2A). The growth response of the cells did not change at various passage levels (up to 10 passages in this study) and were similar in smooth muscle cells isolated from different uteri at either proliferative or secretory phases of menstrual cycle. The smooth muscle cells grown in the presence of 10% FBS for 48 h were made quiescent by further incubation in serum-free DMEM for different lengths of time. The results indicated a gradual reduction in 3H-thymidine incorporation in a time-dependent manner reaching to approximately 95% inhibition after 36-48 h of incubation in serum-free medium (Fig. 2B). The quiescent smooth muscle cells obtained under this condition were able to
enter the cell cycle after serum stimulation, and after 48 h of incubation the incorporated 3H-thymidine was significantly (P c 0.001) higher than the nonstimulated cells (Fig. 2B). The labeling index of the smooth muscle cells grown in serum-free medium also indicated a gradual decrease in the number of labeled cells in a timedependent manner by approximately 90-95% after 48 h of incubation (Fig. 3A). Addition of serum to the media, similar to the TCA precipitation experiments, resulted in a gradual increase in the labeling index, and by 48 h more than 95% of the cells were labeled (Fig. 3B). All further studies examining the effect of EGF and PDGF on 3H-thymidine incorporation and cell proliferation were performed on quiescent smooth muscle cells grown in serum-free conditions for 48 h. Effect of EGF and PDGF on DNA synthesis EGF and PDGF-AB in a concentration dependent manner significantly stimulated 3H-thymidine incorporation in the quiescent smooth muscle cells, with maximal stimulation at 5 rig/ml and 10 rig/ml EGF and
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EGF
AND
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.. x
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EGF AND
PDGF ACTION
PDGF-AB, respectively (P < 0.01 and 0.02), compared to unstimulated serum-free conditions (Fig. 4, A and B). No significant stimulation occurred at higher concentrations of PDGF or EGF, although EGF at 15 rig/ml reduced the 3H-thymidine incorporation compared to 5 rig/ml. It was found not to be significant (P < 0.08, Fig. 4A). The action of EGF and PDGF on 3H-thymidine incorporation was time dependent (Fig. 4C). The effect of PDGF at 10 rig/ml on 3H-thymidine incorporation reached significant levels after 24 h of incubation (P C O.OOOl), whereas EGF at 15 rig/ml required 36-48 h (P < 0.005). The effect of PDGF-AB at 10 rig/ml was similar to that induced by EGF at 15 rig/ml concentration, and their combinations further enhanced the 3H-thymidine incorporation (P < 0.05, Fig. 4D). PDGF-BB at the same concentration as PDGF-AB (natural) induced a similar effect on 3H-thymidine incorporation (data not shown). Ez at 1 pM added to the serum-free media (Fig. 5A) inhibited 3H-thymidine incorporation by the smooth muscle cells (P < 0.05). Ez also inhibited the mitogenic effects of 15 rig/ml EGF (Fig. 5B) or 3 rig/ml PDGF (Fig. 5C). P4 at 1 pM either alone or in combination with E2 did not have any significant effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF (Fig. 5, A-C). Since phenol red has been shown to have a estrogen-like bioactivity (38), we have examined whether the pH indicator present in the medium mimics the biological effects of estradiol on 3H-thymidine incorporation. Serum-free, phenol red-free media with or without Ez (1 pm) had no significant effect on 3Hthymidine incorporation by these cells (not shown). Immunofluorescence
localization
Immunofluorescent microscopic observations indicated that myometrial smooth muscle cells in culture produce EGF and PDGF-AB and contain EGF and PDGF-P, but not PDGF-a receptors (Fig. 6). The immunostaining of EGF was very intense and noticeable around the nuclear periphery possibly associated with endoplasmic reticulum (Fig. 6A). The smooth muscle cells were also immunostained intensely for EGF receptors, uniformly distributed in the cytoplasmic region (Fig. 6B). The immunostaining of smooth muscle cells for PDGF-AB as well as PDGF-/3 receptor were uniform over these cells and with lower intensity than that observed for EGF and EGF receptor (Fig. 6, D and E). However, immunostaining for PDGF-(Y receptors was very low and sometimes not detectable in these cells (Fig. 6F). Preabsorption of antibodies with corresponding pro-
ON HUMAN
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1723
teins or replacing the primary antibodies with nonimmune sera show a considerable reduction in immunostaining intensity, indicating the specificity of the reactions (Fig. 6C). Immunohistochemical
localization
Uterine tissues from both proliferative and secretory phases of the menstrual cycle were used for immunohistochemical localization of EGF, PDGF-AB, as well as EGF and PDGF-8 receptors. The immunostaining for EGF was associated with elongated and circular smooth muscle cells of both phases without any differences in staining intensity (Fig. 7A). The endothelial and smooth muscle cells of the arterioles also immunostained for EGF (Fig. 7B). The pattern of immunostaining for EGF receptor was similar to that observed for EGF, but with more intensity (Fig. 7, D and E). The myometrial tissue also immunostained for PDGF-AB and PDGF-b receptor (Fig. 8). The immunostaining for PDGF-AB as well as PDGF-/3 receptors were associated with smooth muscle cells (Fig. 8, A and D) and endothelial and smooth muscle cells of the arterioles (Fig. 8, B and E). Preabsorption of EGF and PDGF-AB antibodies with the purified EGF and natural PDGF (Figs. 7C and 8C), and EGF receptor antibodies with purified human term placenta microvilli (Fig. 7F) resulted in considerable reduction in immunostaining intensity of myometrial tissues. Replacement of PDGF-/3 receptor antibody with normal serum also reduced the intensity of immunostaining (Fig. 8F). Discussion The immunocytochemical and immunofluorescence microscopic observations indicated the presence of EGF and PDGF-AB as well as EGF and PDGF-/3 receptors in human myometrial tissue and myometrial smooth muscle cells in primary culture. Although the presence of EGF receptor in human uterine tissue have previously been reported using biochemical, autoradiographic, and immunohistochemical approach (23-26), this is the first demonstration suggesting the local production of EGF and PDGF-AB as well as PDGF-fl receptors in both myometrium and myometrial smooth muscle cells in culture. The presence of immunoactive EGF protein and mRNA for EGF has previously been reported in mouse uterus, associated exclusively with uterine luminal epithelial cells (19-21). In contrast, present observations indicate that in human uterus, EGF immunoreactivity is
FIG. 7. Immunohistochemical localization of EGF (A-C) and EGF receptor (D-F) in human myometrial tissue. The immunostaining is associated with smooth muscle cells (A and D) and arterioles endothelial and smooth muscle cells (B and E). Preabsorption of EGF antibody with recombinant human EGF (C) and EGF receptor antibody with purified human placental microvilli (F) resulted in considerable reduction in immunostaining of the tissues. C and F are shown in Nomarski interference contrast. Magnification, ~151.
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EGF
AND
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ACTION
associated with all major myometrial as well as endometrial cell types (Chegini, N., M. J. Rossi, and B. J. Masterson, submitted for publication). This difference in the distribution of EGF to that reported in mouse is not clear, although the antibodies used in both studies were species specific. We have reported a similar pattern of distribution for EGF and transforming growth factor(Y (TGF+) in the rat (22) and TGF-a in human (39) uterus. Myometrial smooth muscle cells in primary cell culture derived from these tissues also express TGF-a (unpublished observation). We did not observe any variation in the intensity of immunostaining in relation to the phases of the cycle, although it has been reported that in rodents Ea regulates the occurrence of EGF protein and its mRNA (19-21). These observations indicate that human myometrial smooth muscle cells are an endogenous source of EGF and TGF-(u, suggesting that these growth factors may play a regulatory role in part by an autocrine pathway in this tissue. Myometrial tissues and isolated smooth muscle cells in primary culture also contain immunoreactive EGF receptors. Using specific monoclonal antibodies to the extracellular binding domain of EGF receptor, it was observed that both myometrial smooth muscle cells and the vasculature immunostain for EGF receptors. The pattern of immunostaining was similar to our previous autoradiographic observations with no variation due to the phases of the menstrual cycle, confirming our previously reported data (23,24). Immunohistochemical studies carried out in normal and malignant uteri also indicated a lack of correlation with histological grade, depth of myometrial invasion, estrogen-P4 receptor status, the presence of extrauterine metastases, or the development of recurrent disease (26). In addition to human uterus, the presence of EGF receptors in all the major uterine cell types in rodent has been reported (27,28). However, in the cycling rat EGF receptor content significantly increased in proestrus, and in the ovariectomized rat Ez treatment induced a 3-fold increase in EGF receptor and EGF receptor mRNA content, suggesting a role in EGF receptor regulation by estrogen (29, 30). Similar results have been reported for IGF-I and IGF-I receptor (10). In human uterus, although the presence of PDGF receptor has been demonstrated, the cellular distribution and distinction between the receptor types has not been determined (31). PDGF exists either as disulfide-linked homodimers (PDGF-AA and PDGF-BB) or heterodimer (PDGF-AB), with two separate receptors, the cy- and the @-type, with different affinities for the three dimeric
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forms of PDGF (33,34). The PDGF-(U receptor binds to all three dimeric forms of PDGF, whereas the P-type binds to the PDGF-BB with high affinity and to the PDGF-AB with lower affinity (33). Our observations provide the first evidence that human myometrial tissue contains specific immunoreactive PDGF-P receptors using antibody raised against human PDGF-/3 receptor. The myometrial smooth muscle cells in primary culture also immunostain for PDGF-P receptors but very low to nondetectable levels for PDGF+ receptors. Using antibody raised against porcine uterine PDGF-P receptors, our observations indicated a very low level of immunostaining in frozen sections of human myometrial tissue and a relatively strong staining of the smooth muscle in culture. These observations were similar to that previously reported in porcine using the same antibody to localize PDGF-P receptors in uterine tissue and myometrial smooth muscle cell cultures (34). It was suggested in this study that cell culturing may up-regulate the PDGF-P receptors in porcine myometrial smooth muscle cells (34). However, the antibody to human PDGF-/3 receptors not only immunostained frozen and fixed myometrial tissue (see Fig. 8), it gave similar intensity of immunostaining of cultured smooth muscle cells observed with the antibody to porcine PDGF-/3 receptor. This difference in immunostaining may simply lie in the nature of the antibodies, rather than the absence or down-regulation of PDGF-B receptors in human myometrial tissue. The presence of immunoreactive PDGF-/3 and a low level of PDGF-a receptors in human myometrial smooth muscle cells suggest that the major part of PDGF receptor content reported in human uterus is the PDGF-P. Furthermore, the results indicate that unlike porcine, cell culturing may not be required for up-regulation of PDGF-fl receptor in human myometrial smooth muscle cells as the PDGF-P receptor is already present in the myometrial tissue. The presence of low level of PDGF-a in myometrial smooth muscle cells is not surprising. It has been suggested that the PDGF-a receptor constitutes less than 10% of the total PDGF receptor in diploid human fibroblasts (40) and the major mitogenic action of PDGF in these cells is mediated through the PDGF-/3 receptor (33). In support of our observations is the recent report that human myometrial tissue expresses the mRNA for PDGF-B chain and low abundance of the PDGF-A chain mRNA, without a correlation to the phases of the cycle (35). No correlation was also observed in immunoreactive intensity of PDGF-P receptors in myometrial tissue in the present study. Our
FIG. 8. Immunohistochemical localization of PDGF-AB (A-C) and PDGF-8 receptor (D-F) in human myometrial tissue indicating the presence of immunostaining in association with smooth muscle cells (A and D), arterioles endothelial and smooth muscle cells (B and E). Replacement of antibodies with nonimmune normal serum resulted in considerable reduction in immunostaining of PDGF-AB (C) and PDGF-@ receptor (F). Magnification, ~151.
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1726
EGF AND PDGF ACTION ON HUMAN
results indicated that PDGF-BB and PDGF-AB, at the same concentration, were equally mitogenic in myometrial smooth muscle cells, suggesting that the action of PDGF in these cells is most likely mediated through the PDGF-P receptors. The results of the present study indicate that myometrial smooth muscle cells in culture are responsive to the mitogenic action of EGF and PDGF. EGF and PDGF, in a concentration and time-dependent manner, significantly stimulated 3H-thymidine incorporation in smooth muscle cells. The PDGF action on 3H-thymidine incorporation was similar to 10% FBS. Furthermore, EGF in the presence of PDGF significantly enhanced the 3Hthymidine incorporation compared to that observed with either EGF or PDGF alone. The action of EGF and PDGF were independent of phases of the menstrual cycle and equally stimulated 3H-thymidine incorporation by myometrial smooth muscle cells. In myometrial smooth muscle cultures established from different individual patients, the extend of 3H-thymidine incorporation varied in both nonstimulated serum-free as well as serum, or growth factor-stimulated cultures. Similar variation has been reported regarding the effect of EP and P4 on human endometrial stoma1 cells, although longer incubation times and earlier passages were used (15). Such differences concerning the effect of E2 on cell proliferation and efficacy of colony formation by human myometrial smooth muscle cells has been observed (41). Ez at the concentration used in this study inhibited 3H-thymidine incorporation by myometrial smooth muscle cells. EP also inhibited the mitogenic effect of EGF and PDGF. Pq, however, either alone or in combination with Ez, was ineffective in stimulation of 3H-thymidine incorporation by these cells and did not enhance the mitogenic action of EGF or PDGF. Although the Ez concentration used in our study may be considerably higher than physiological, it has been reported that Ez at 10-“-10-s M had no significant effect on epithelial cell proliferation (42). Similar in vitro results describing the ineffectiveness of estrogen and P4 in rodent and human endometrial epithelial, stromal, and myometrial smooth muscle cell proliferation have previously been reported (15-18, 42). However E2 at lo-’ M has been reported to stimulate both endometrial and myometrial smooth muscle cells proliferation in the presence of 20% serum (41). This was achieved only in cultures from 5th8th passages, without any effect on cells from 2nd-4th or 9th-12th subcultures (41). Although cell-cell interaction has been demonstrated to be important for mitogenie stimulation of endometrial cells (13), E2 even in uterine organ culture was ineffective in stimulating DNA synthesis (43). The additive or synergistic mechanism where cells are exposed to a variety of growth factors, i.e. EGF and
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Endo * 1992 Vol130*No3
PDGF, in cell proliferation is complex and not well understood. Quiescent fibroblasts require more than a single mitogen to stimulate DNA synthesis. PDGF stimulates these cells to become competent or to make the Go to G1 transition for subsequent cell division (44). However, EGF acts as a progression factor, stimulating the cells to continue through the cycle (44). PDGF has also been shown to influence the expression of TGF-/3 mRNA in human fibroblasts (45) which has multifunctional activities in a variety of cell types, including smooth muscle cells (46). Moreover, it has recently been shown that the mitogenic effect of all three PDGF isoforms on human fibroblasts is enhanced by TGF+?l through the induction of PDGF-a receptors (40). We have previously shown the presence of immunoreactive TGF-P in human myometrial tissue (39), which may suggest a possible interaction between TGF-P and PDGF in this tissue. Therefore the ultimate growth response to a given mitogen in the normal in viva condition of quiescent uterine myometrial tissue may involve both negative and positive regulatory mechanisms. However, under certain conditions, such as leiomyomata, the smooth muscle cells are able to resume their proliferative activity leading to the formation of these tumors. Acknowledgments The authors would like to thank the Department of OB/GYN Gynecologists for their help in the collection of the tissues. In addition, we thank Landis Young for her assistance in the preparation of this manuscript.
References 1. Clarke CL, Sutherland RL 1990 Progestin regulation of cellular proliferation. Endocr Rev 11:266-301 2. Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL, McCarty Jr KS 1988 Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cvcle. J Clin Endocrinol Metab 67~334-340 3. Soto AM, Sonnenschein C 1987 Cell proliferation of estrogensensitive cells: the case for negative control. Endocr Rev 844-52 4. Casey ML, MacDonald PC, Mitchell MD, Snyder JM 1984 Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture. In Vitro 20:396-403 5. Honore LH 1979 Menorrhagia, diffuse myometrial hypertrophy, and the intrauterine contraceptive device: a report of fourteen cases. Acta Obstet Gynecol Stand 58283-285 6. Hartz PH 1945 Proliferation of muscle cells in the mvometrium of the nonpregnant uterus. Arch Path01 39323-324 I. Cramer SF, Meyer JS, Kraner JF, Camel M, Mazur MT, Tenenbaum MS 1980 Metastasizing leiomyomata of the uterus- S phase fraction, estrogen receptor, and ultrastructure. Cancer 45:932-937 8. Rein MS, Friedman AJ, Pandian MR, Heffner LJ 1990 The secretion of insulin-like growth factors I and II by explant cultures of fibroids and myometrium from women treated with a gonadotropin-releasing hormone agonist. Obstet Gynecol76:388-394 9. HODDner JWM. Mosselman S. Roholl PJM. Lamberchats C. Slebos RJE, de Pagter-Holthuizen P, Lips CJM,‘Jansz HS, Sussenbach JS 1988 Expression of insulin-like growth factor-I and -11 genes in human smooth muscle tumors. EMBO J 21379-1385 10. Murphy LJ, Ghahary A 1990 Uterine insulin-like growth factor-I: Regulation of expression and its role in estrogen-induced uterine
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AND
PDGF
ACTION
proliferation. Endocr Rev 11:443-453 11. Quarmby VE, Korach KS 1984 The influence of 17-estradiol on pattern of cell division in the uterus. Endocrinology 114694-702 12. Pavlik EJ, Katzenellenbogen BS 1978 Human endometrial cells in primary tissue culture: estrogen interactions and modulation of cell proliferation. J Clin Endocrinol Metab 47~333-344 13. Iguchi T, Uchima F-D, Ostrander PL, Bern HA 1983 Growth of normal mouse vaginal epithelial cells in and on collagen gels. Proc Nat1 Acad Sci USA 80:3743-3747 14. Cooke PS, Uchima F-DA, Fujii DK, Bern HA, Cunha GR 1986 Restoration of normal morphology and estrogen responsiveness in cultured vaginal and uterine epithelial transplanted with stroma. Proc Nat1 Acad Sci USA 832109-2113 15. Irwin J, Kirk D, King RJB, Quigley MM, Gwatkin RBL 1989 Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertil Steril52:761-768 16. Lippman ME, Dickson RB, Knabbe C, Huff K, Swain S, McManaway M, Bronzert D, Kasid A, Gelmann EP 1986 Autocrine and paracrine growth regulation of human breast cancer. Breast Cancer Res Treat 7:59-70 17. Fisher DA, Lakshmanan J 1990 Metabolism and effects of epiderma1 growth factor and related growth factors in mammals. Endocr Rev 11:418-442 18. Traish AM, Wotiz HH 1987 Prostatic epidermal growth factor receptors and their regulation by androgens. Endocrinology 121:1461-1467 19. Huet-Hudson YM, Chakraborty C, De SK, Suzuki Y, Andrews GK, Dey SK 1990 Estrogen regulates the synthesis of epidermal growth factor in mouse uterine enithelial cells. Mol Endocrinol4:510-523 20. Gonzalez F, Lakshmanan J, Hoath S, Fisher DA 1984 Effect of estradiol-176 on uterine epidermal growth factor concentration in immature mice. Acta Endocrinol (Conenhl 105:425-428 21. DiAugustine RP, Petrusz P, Beil 61, Brown CF, Korach KS, McLachlan JA, Tseng CT 1988 Influence of estrogen on mouse epidermal growth factor precursor protein and messenger ribonucleic acid. Endocrinology 122:2355-2363 22. Simms JS, Chegini N, Williams RS, Rossi AM, Dunn Jr WA 1991 Identification of epidermal growth factor, transforming growth factor and epidermal growth factor receptor in surgically induced endometriosis in rat. Obstet Gynecol 78850-857 23. Chegini N, Rao ChV, Wakim N, Sanfilippo J 1986 Binding of lz51epidermal growth factor in human uterus. Cell Tissue Res 246:543548 24. Hoffmann GE, Rao ChV, Barrows GH, Schultz GS, Sanfilippo JS 1984 Binding sites for epidermal growth factor in human uterine tissues and leiomyomas.-J Clin Endocrinol Metab 58880-884 25. Tommola P, Pekonen F, Rutanen E-M 1989 Binding of epidermal growth factor and insulin-like growth factor I in human myometrium and leiomyomata. Obstet Gynecol74:658-662 26. Berchuck A. Soisson AP. Olt GJ. Saner JT. Clarke-Pearson DL. Bast Jr RB,‘McCarty Jr KS 1989 Epidermal growth factor receptor expression in normal and malignant endometrium. Am J Obstet Gynecol161:1247-1252 27. Bossert NL, Nelson KG, Ross KA, Takahashi T, McLachlan JA 1990 Epidermal growth factor binding and receptor distribution in the mouse reproductive tract during development. Dev Biol142:7585 28. Lin T-H, Mukku VR, Verner G, Kirkland JL, Stance1 GM 1988 Autoradiographic localization of epidermal growth factor receptors to all major uterine cell types. Biol Reprod 38403-411
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29. Gardner RM, Verner G, Kirkland JL, Stance1 GM 1989 Regulation of uterine epidermal growth factor (EGF) receptors by estrogen in the mature rat and during the estrous cycle. J Steroid Biocbem 32:339-343 30. Lingham R, Stance1 GM, Loose-Mitchell DS 1988 Estrogen induction of the epidermal growth factor receptor mRNA. Mol Endocrino1 2:230-235 31. Fayed YM, Tsibris JCM, Langenberg PW, Robertson AL 1989 Human uterine leiomyomata cells: binding and growth factor responses to epidermal growth factor, platelet-derived growth factor, and insulin. Lab Invest 60:30-37 32. Ronnstrand L, Beckmann MP, Faulders B, Ostman A, Ek B, Heldin CH 1987 Purification of the receptor for platelet-derived growth factor from norcine uterus. J Biol Chem 262:2929 33. Heldin CH, Backstrom G, Ostman A, Hammacher A, Ronnstrand L, Rubin K, Nister M, Westermark B 1988 Binding of different dimeric forms of PDGF to human fibroblasts: evidence for two separate receptor types. EMBO J 21387-1393 34. Terracio L, Ronnstrand L, Tingstrom A, Rubin K, Claesson-Welsh L, Funa K, Heldin CH 1988 Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing. J Cell Biol 107:1947-1957 35. Boehm KD, Daimon M, Gorodeski IG, Sheean LA, Utian WH, Ilan J 1990 Expression of the insulin-like and platelet-derived growth factor genes in human uterine tissues. Mol Reprod Dev 2793-101 36. Noyes RW, Hertig AT, Rock J 1951 Dating of endometrial biopsy. Fertil Steril 1:3-25 37. Chegini N, Safa AR 1987 Influence of mitoxantrone on nucleolar function in MDA-MB-231 human breast tumor cell line. Cancer Lett 37:327-336 38. Ernse M, Schmid C, Froesch ER 1989 Phenol red mimics biological action of estradiol: enhancement of osteoblast proliferation in uitro and of type I collagen gene expression in bone and uteri of rats in uiuo. J Steroid Biochem 33907-914 39. Williams RS, Chegini N, Localization of TGF-(U and TGF-8 in human uterine and ovarian tissue. Program of 46th Annual Meeting of the American Fertility Society, Washington DC 1990, p S5 (Abstract) 40. Ishikawa 0, LeRoy EC, Trojanowska M 1990 Mitogenic effect of transforming growth factor 61 on human fibroblasts involves the induction of platelet-derived growth factor a! receptors. J Cell Physiol 145:181-186 41. Chen L, Lindner HR, Lancet M 1973 Mitogenic action of oestradiol-178 on human myometrial and endometrial cells in long-term tissue cultures. J Endocrinol59:87-97 42. Tomooka Y, DiAugustine RP, McLaachian JA 1986 Proliferation of mouse uterine epithelial cells in vitro. Endocrinoloav-- 118:10111018 43. Ghahary A, Chakrabarti S, Murphy LJ 1990 Localization of the sites of synthesis and action of insulin-like growth factor-I in the rat uterus. Mol Endocrinol4:191-195 44. Pardee AB, Coppock DL, Yang HC 1986 Regulation of cell proliferation and the onset of DNA synthesis. J Cell Sci SUDD~ -- 4:171180 45. Paulsson Y, Hammacher A, Heldin C-H, Westermark B 1987 Possible positive autocrine feedback in the nrerenlicative nhase of human fibroblasts. Nature 328715-717 46. Barnard JA, Lyons RM, Moses HL 1990 The cell biology of transforming growth factor (3. Biochim Biophys Acta 1032:79-87
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Vol. 130,No. 3 Printed
in U.S.A.
Presence of Epidermal Growth Factor, Platelet-Derived Growth Factor, and Their Receptors in Human Myometrial Tissue and Smooth Muscle Cells: Their Action in Smooth Muscle Cells in Vitro* MICHAEL
J. ROSSI,
NASSER
CHEGINI,
Department of Obstetricsand Gynecology,University Gainesville,Florida 32610
AND BYRON of
J. MASTERSON
Florida Collegeof Medicine,
alone (P c 0.05). PDGF-BB at 10 rig/ml also stimulated 3Hthymidine incorporation and ita effect was similar to that induced by PDGF-AB at the same concentration. 17@-Estradiol (Es) at 1 PM inhibited3H-thymidine incorporation by the smooth muscle cells (P < 0.05). Ez also reduced the stimulatory effect of EGF (15 rig/ml) and PDGF (3 rig/ml). Progesterone at 1 pi either alone or in combination with EP did not have any effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF. The effect of EGF and PDGF on cell mowth and 3H-thymidine incorporation by myometrial smooth muscle cells was independent of phases of the menstrual cycle. In summary, the results of present studies indicate that human mvometrial tissue and mvometrial smooth muscle cells in primary culture locally produce EGF and PDGF-AB and contain EGF and PDGF-B. but not Lu-recenters. Moreover. the myometrial smooth muscle cells in culture respond to the mitogenie action of EGF and PDGF. (Endocrinology130: 17161727,1992)
ABSTRACT. Immunohistochemical observations indicate that human myometrial smooth muscle cells express epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)AB and contain EGF and PDGF-8 receptors with no variation in intensity with phases of the menstrual cycle. Furthermore, immunofluorescent microscopic studies revealed that primary myometrial smooth muscle cell cultures also express EGF, PDGF-AB, and contain EGF and PDGF-8, but not a-receptor. Incubation of subconfluent smooth muscle cells in serum-free medium leads to quiescence within 48 h as demonstrated by ‘Hthymidine incorporation and labeling index. Exposure of quiescent cells to 10% fetal bovine serum stimulates resumption of DNA synthesis and proliferation in a time-dependent manner with a doubling time of 41.6 h. EGF (1.5-50 rig/ml) and PDGFAB (l-10 rig/ml) in a dose- and time-dependent manner significantly stimulated 3H-thymidine incorporation by quiescent myometrial smooth muscle cells (P < 0.05). Combinations of EGF (15 rig/ml) and PDGF-AB (10 rig/ml) significantly increased 3H-thymidine incorporation induced by either growth factor
I
C
ELLULAR proliferation and differentiation of the uterine tissue is considered to be regulated by ovarian steroids and mediated through the nuclear receptors present in both endometrial and myometrial cells (l-3). The response of the uterine tissue to steroids is complex involving many biochemical as well as morphological events resulting in uterine growth and differentiation (l3). Although the most profound mitogenic effect of estrogen appears to be in the endometrial compartment, myometrium is also sensitive to hypertrophic and hyperplastic actions of steroids (1, 2, 4-6). Myometrium is a quiescent tissue primarily composed of terminally differentiated smooth muscle cells (4); however, identification of mitosis (6), DNA synthesis (7), and the growth of both
benign (leiomyomata) and malignant (leiomyosarcoma) tumors suggests a steroid as well as growth factor-dependent growth potential (8-11). The mechanism(s) of sex steroid action(s) in proliferation and differentiation of various uterine cell types in uiuo are complex and not well defined (1,3). Additionally, in vitro observations have pointed out the lack of mitogenie action of steroids on a variety of uterine cell types (12-15). Recently however, it has been postulated that the mitogenic action of steroids in their target tissue is mediated through local production of growth factors, acting with paracrine and/or autocrine mechanisms (1618). Several in uiuo and in vitro studies in different systems, including uterus, have supported this hypothesis (16-21). Immunohistochemical and in situ hybridization studies have indicated the presence of epidermal growth factor (EGF) in mouse uterine epithelial cells, but not in ovariectomized animals (19-21). Estrogen treatment of ovariectomized animals stimulates the oc-
Received August 30, 1991. Address correspondence and requests for reprints to: Dr. Nasser Chegini, Department of OB/GYN, University of Florida, P.O. Box 100294, Gainesville, Florida 32610. * Funded in part by the University of Florida, Division of Sponsored Research. 1716
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EGF AND
PDGF ACTION
currence of EGF messenger RNA (mRNA) and its immunoreactive appearance (19-21). In contrast, all uterine cell types in the rat appear to contain immunoreactive EGF (22). Similar results have also been reported in the rat for insulin-like growth factor-I (IGF-I) and IGF-I receptor (10). The action of growth factors in their target tissue is mediated through specific cell surface receptors (10, 17). The presence of EGF receptors in all the major uterine cell types in human as well as rat and mouse have been reported (23-29). In the rat uterus the EGF receptor content has been well correlated with the phases of the cycle, with highest levels at proestrus as compared to the other days of the cycle (29). In the ovariectomized rat 17@-estradiol (Ez) administration up-regulates the uterine EGF receptor, presumably due to an increase in the levels of EGF receptor mRNA (30). However, in the human uterus no correlation exists between the EGF receptor content and the phases of menstrual cycle (23, 24, 26). Uterine tissue also contains platelet-derived growth factor (PDGF) receptors and responds to the mitogenic action of PDGF (31, 32). Porcine uterus has been demonstrated to be a major source of PDGF receptor, preferentially associated with endometrial stromal cells (32). PDGF receptor appears in two forms, with different affinities designated as (Y- and P-types (33). Porcine endometrial stromal cells, both in situ and in culture, have been shown to contain PDGF-@ receptor (34) and in situ hybridization, using complementary DNA to the PDGF-fl receptor, indicates the presence of mRNA preferentially associated with these cells (34). In contrast, porcine myometrial smooth muscle cells appear to contain a low level of PDGF+ receptors, but after 24-48 h in culture a majority of the cells, and after 1 week all the cells, express the PDGF-fi receptor mRNA and contain PDGF-P receptors (34). Human uterus has also been shown to contain PDGF receptors, however, the type and cellular distribution of PDGF receptor in this tissue has not been determined (31). Furthermore, there is no data available regarding the regulation of either PDGF or its receptors by steroids, although recent observation indicated that all human uterine cell types express mRNA for PDGF-B chain and a very low abundance of PDGF-A chain, without correlation with the phases of the cycle (35). The local production of growth factors and the presence of their specific receptors in myometrial smooth muscle cells suggest the possibility that these cells are the target for the mitogenic action of growth factors. Therefore, the present study was undertaken to elucidate the presence and cellular distribution of EGF and PDGFAB as well as EGF and PDGF-@ receptor in myometrial tissues and myometrial smooth muscle cells in culture.
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Additionally, to investigate the role of EGF and PDGF, either alone or in combination, and in the presence of steroids on 3H-thymicline incorporation by primary myometrial smooth muscle cell cultures.
Materials
and Methods
Material Dulbecco’s modified Eagle’s medium (DMEM), EDTA, trypsin-EDTA solution, and antibiotic-antimycotic solution were purchased from GIBCO Laboratories (Grand Island, NY). Fetal bovine serum (FBS), Ez, progesterone (P,), and phenol-red free medium were obtained from Sigma Chemical Company (St. Louis, MO). Collagenase was purchased from Worthington Biochemical (Freehold, NJ). Culture grade EGF, natural human PDGF-AB, recombinant PDGF-BB, and goat anti-human PDGF-AB antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal rabbit anti-human recombinant EGF was purchased from Biomedical Technologies, Inc. (Stoughton, MA), monoclonal antibodies to porcine PDGF-/3 receptor from Oncogene Science Inc. (Manhasset, NY), and monoclonal antibodies to human PDGF-(U and PDGF-/3 receptors from Genzyme Corporation (Cambridge, MA). Monoclonal antibodies to human cytokeratin 19 and human desmin were purchased from Dako Corporation (Carpinteria, CA) and monoclonal antibody to human vimentin was purchased from Sigma. Rhodamine (TRITC) conjugated affinipure donkey anti-rabbit and goat anti-mouse immunoglobulin G (IgG) were purchased from Jackson Laboratories, Inc. (West Grove, PA). Vectastain ABC Elite Kits were from Vector Laboratories (Burlingam, CA). Methyl-3H-thymidine (83 Ci/ mmol) was purchased from Amersham Co. (Arlington Heights, IL). The 75cm* flasks and the 24-well plates were purchased from Corning Glass Works (Corning, NY) and 8-well culture slides, either glass or plastic, from Nunc Inc. (Naperville, IL). Monoclonal antibodies to extracellular binding domain of the EGF receptor were a gift from Dr. W. A. Dunn, Jr. (Department of Anatomy and Cell Biology, University of Florida). Tissue collection and myometrial smooth muscle cells isolation Portions of uterine specimens from nonpregnant premenopausal women undergoing hysterectomy for medically indicated reasons, excluding endometrial cancer, were collected, placed in sterilized ice-cold PBS (pH 7.4) and immediately brought to the laboratory on ice. The tissues were collected at the University of Florida affiliated Shands Hospital, and collection and use of tissues for the purpose of these experiments was approved by the University of Florida Institutional Review Board. A total of 102 uterine tissues were collected of which 35 were from proliferative and 67 from secretory phase of menstrual cycle. Each uterine tissue was examined by the pathologist for histological evaluation and the dating of endometrium. The endometrial histological dating was performed according to Noyes et al. (36) and the patient’s last menstrual period. Myometrial tissues were dissected from endometrial cell layers, washed in PBS, cut into small pieces, and digested in 2% collagenase (wt/vol) at 37 C for 3-6 h. The isolated smooth muscle cells were collected by centrifugation at 460 X g for 5
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Endo Voll30.
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1992 No 3
min, washedseveral times with DMEM containing 1% antibiotic-antimycotic solution and cultured. Myometrial smoothmusclecell culture The isolatedmyometrial smooth musclecells were grown as monolayersin 75-cm*flasks at approximate density of 2 x lo5 cells per flask. The cells were maintained in DMEM supplemented with 10%FBS (vol/vol) and 1% antibiotic-antimycotic solution (vol/vol) for 6 days at 37 C in a humidified atmosphere of 5% CO*-95% air and the mediawas changedevery 2-3 days. The subconfluent monolayer cells were subcultured at the above density after trypsinization with 0.1% trypsin-0.5% EDTA. The smooth musclecells after the third passagewere seededat approximately 5 x lo4 cells per well in 24 well dishes or 2 x lo4 cells per well in 8 well Lab-Tek slides in supplementedDMEM. The selectionof third passagewaschosenonly to obtain enough cells with which to perform experiments at the samepassage.It has been shown that human myometrial smoothmusclecells do not losetheir characteristics even after 16 passagesand being cultured for 1 yr (4). Cells that were
Time
f?
(hours)
2500
8% 2 =: 2000 8 42 IY1500 .- \ ge $ 2 X
m
1000 500
0
12
24
36
4E Time
~4
46
72
96
120
(hours)
FIG. 2. A shows the growth curve of smooth muscle cells incubated in the presence of 10% FBS (AA) and serum-free condition (O-0). The FBS stimulated cultures are significantly different from nonstimulated controls (P < 0.0001). B demonstrates the time dependency of 3H-thymidine incorporation by smooth muscle cells. The cells were cultured for 48 h in the presence of 10% FBS, then incubated in serum-free conditions (left-hand side, O0) indicating a gradual reduction in 3H-thymidine incorporation. Addition of 10% FBS to these quiescent cells stimulates 3H-thymidine incorporation in a timedependent manner (right-hnnd side, AA), however, the nonstimulated cells do not significantly incorporate 3H-thymidine (O0). Cell numbers in A and each point in B represent mean f SEM of three separate triplicate experiments from three individual patients.
grown in 8 well Lab-Tek slideswere only usedfor immunofluorescenceor autoradiographic studies. Trypan blue exclusion test determined the cell viability after trypsinization before each experiment. Growth curve Myometrial smooth muscle cells were plated into 30-mm culture dishesat approximately 2.4 X 10’ cells per plate and grown in DMEM supplementedwith 10% FBS. At various intervals cells were washedwith Ca*+-Me-free PBS, trypsinized, and counted usingCoulter Counter ZM (Coulter Electronics, Hialeah, FL). 3H-Thymidine incorporation
FIG. 1. A shows a Nomarski interference contrast micrograph of myometrial smooth muscle cells in primary culture and B shows immunofluorescence micrograph of smooth muscle cells stained with antibodies to human desmin. Magnification, X110.
Myometrial smooth muscle cells were initially plated in DMEM containing 10% FBS for 48 h, then washed,and incubated in serum-freeDMEM for an additional 48 h. The quiescent cells obtained under serum-freeconditions were returned to serum-supplementedDMEM containing 2 &i/ml 3H-thymidine for various periods ranging from 6-72 h. The cultures
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EGF AND
PDGF ACTION
ON HUMAN
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FIG. 3. Light microscope autoradiographic Nomarski interference contrast photographs of myometrial smooth muscle cells. Quiescent cells were labeled with ‘H-thymidine for 48 h either in serum-free conditions (A) or in the presence of 10% FBS (B). Note the presence of few lightly labeled nuclei in A, whereas extensive nuclear labeling indicating significantly higher ‘H-thymidine incorporation in B. Magnification, ~161.
0.0
1.5
15.0
5.0
01
50.0
EGF hz./~)
0
1 PDGF
3
10
bdml)
800 i$ 5b 600 Bg 4oo
nX
0
24
4s Time
-
200
72
(hr)
FIG. 4. The dose dependency of EGF (A) and PDGF-AB (B) on 3H-thymidine incorporation by myometrial smooth muscle cells after 48 h of incubation. The effect of EGF at all concentrations and PDGF at 10 rig/ml on 3H-thymidine incorporation were significantly different from control (P < 0.01). C shows the time dependency of EGF at 15 rig/ml (6 -+), PDGF-AB at 10 rig/ml (A -A) and nonstimulated serumon 3H-thymidine incorporation. D indicates the 3H-thymidine incorporation by smooth muscle cells incubated in serumfree medium (0 -0) free medium (control) or in the presence of EGF (15 rig/ml), PDGF-AB (10 rig/ml), or combination of EGF and PDGF-AB (E + P) for 48 h. The action of EGF and PDGF on ‘H-thymidine incorporation were significantly different from the control (P < 0.01) and their combination different from either control (P < 0.02) or EGF or PDGF alone (P < 0.05). Points represent mean & SEM of three (A and C) and five (B and C) separate triplicate experiments from three to five individual patients. Disintegrations per min/103 cells for controls are; A: 674 f 19, B: 721 f 196, D: 604 f 171.
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EGF AND
1720
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ON HUMAN
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using a LKB historange. The sectionswere deparaffnized and processed for immunohistochemical localization of EGF, PDGF-AB, EGF receptor, and PDGF-8 receptor aspreviously described(38). Frozen sectionsfrom 8 specimenswerealsoused for immunolocalization of PDGF-fl receptorsusinganti-porcine PDGF-,3 receptor antibody (34). The primary antibody dilutions usedin this study were: EGF (1:50), EGF receptor (l:lOO), PDGF-AB (l:lOO), anti-porcine PDGF-8 receptor (50 pg IgG/ ml), and anti-human PDGF-a and &receptor antibodies(5 pg IgG/ml). Fifty-seven of the 78 uteri fixed in Bouin’s solution and 8 from frozen uterine tissueswere used for the immunohistochemicalstudies. Immunofluorescencemicroscopicstudies
100 50
-
0 CONTROL
E2
P4
E2iP4
5. The effect of El and P, at 1 PM concentration and their combination (E2 + P,) on SH-thymidine incorporation by myometrial smooth muscle cells incubated for 48 h in the absence (A) or presence of 15 rig/ml EGF (B) and 3 rig/ml PDGF-AB (C). The controls in these figures are, serum-free condition (A), 15 rig/ml EGF (B), and 3 rig/ml PDGF-AB (C). Ez alone or in the presence of EGF and PDGF significantly inhibit ‘H-thymidine incorporation (P < 0.05). Points represent meanf SEM of sevenseparatetriplicate experimentsfrom either four (A andB) or three (C) individualpatients.Disintegrations permin/103 cellsfor controlsare,A: 645f 154,B: 1082f 234,C: 917f 171. FIG.
were then washedtwice with Ca*+-Me-free PBS and trypsinized. The cell suspensions(200 ~1total volume) from each well were usedto determine the cell number and the rate of incorporated 3H-thymidine. An aliquot of 60 ~1 was used for cell number determination. The remaining volume was retained on individual filters (Millipore 96-well filtration plates, Millipore Inc., Bedford, MA), washedonce with 10% trichloroacetic acid (TCA), twice with 5% TCA, and twice with 95% alcohol. Their radioisotopecontent was determined by a Beckman LS 5000 scintillation counter. SH-Thymidine autoradiography Myometrial smooth muscle cells grown in eight well LabTek slides under conditions described above were used to determine their labeling index. The level of 3H-thymidine incorporation by smoothmusclecells after incubation for various periods of time ranging from 6-72 h were determined by autoradiography using Kodak NTB-2 emulsion as previously described(37). Immurwhistochemicalstudies For immunohistochemical studies, portions of 78 of the uterine tissuescollected were washedwith PBS, cut into small pieces, and fixed in Bouin’s solution overnight. The tissue pieceswere washedwith PBS, dehydrated in a seriesof graded ethanol, paraffin embedded,and sectionedat 5-6 pm thickness
Myometrial smooth muscle cells grown on Lab-Tek slides for 48 h using DMEM supplemented with 10% FBS were washedwith Ca*+-M$+ free PBS and immediately fixed with ice-cold methanol for 5 min. The slideswere either usedimmediately for immunofluorescenceobservations or stored at -20 C until needed.The purity and homogeneityof myometrial smooth muscle cell cultures were analyzed using monoclonal antibodiesto human desmin (smoothmusclecells),cytokeratin 19 (epithelial cells), and vimentin (stromal cells). The slides were treated with 0.1% Tween 20 in PBS for 5 min, washedin PBS twice, and subsequently exposed to normal serum 1:50 dilution for 20 min, 1:lOOdilution of primary antibodiesfor 1 h, and then TRITC conjugatedsecondantibodiesfor 30 min in the dark. The cells were washedtwice with PBS and viewed in Olympus BH2 microscopeequipped for fluorescencemicroscopy. Similar cultures were alsousedfor cellular localization of EGF and PDGF-AB, and EGF, PDGF-a! and p-receptors at dilutions indicated in immunohistochemicalstudies. Statistical analysis Data are reported as the mean + SEM for the indicated experiments and analyzed by analysis of variance or Student’s t test. All the experiments were repeatedat least three times in triplicate.
Results Characterization of human myometrial smooth muscle cells
Collagenase treatment of myometrial tissue provided a pure population of isolated cells with smooth muscle cell characteristics without either stromal or glandular epithelial cell contamination (Fig. 1A). Immunofluorescence microscopic examination of myometrial smooth muscle cells cultured for 48 h after collection, or after their third passage using monoclonal antibody directed toward the muscle specific protein desmin, indicated immunostaining of more than 95% of the cells (Fig. 1B). These cells did not immunostain with antibodies to cytokeratin 19, a cytoskeletal protein specific for epithelial cells, or vimentin, a class of intermediate filament proteins present in fibroblasts, indicating their purity as smooth muscle cells (figures not shown). Cultures were monitored with these antibodies regularly and the pat-
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microFIG. 6. Immunofluorescence graphs of myometrial smooth muscle cells stained for EGF (A), EGF receptor (B), PDGF-AB (D), PDGF-fl receptor (E), and PDGF-CT receptor (anti-human PDGF-u and @-receptor antibodies were used in this presentations). Preabsorption of EGF receptor antibody with human term placental microvilli before application resulted in a considerable reduction in immunostaining (C). The controls for the other antibodies resulted in similar reduction in immunostaining (figures are not shown). Magnification, x110.
terns of immunostaining were consistent, suggesting the cells retained their smooth muscle nature under the culture conditions used in this study. DNA synthesis and growth of myometrial
smooth muscle
Myometrial smooth muscle cells grow exponentially in the presence of DMEM supplemented with 10% FBS, and reached confluency in 6 to 7 days with a doubling time of 41.6 h (Fig. 2A). The growth response of the cells did not change at various passage levels (up to 10 passages in this study) and were similar in smooth muscle cells isolated from different uteri at either proliferative or secretory phases of menstrual cycle. The smooth muscle cells grown in the presence of 10% FBS for 48 h were made quiescent by further incubation in serum-free DMEM for different lengths of time. The results indicated a gradual reduction in 3H-thymidine incorporation in a time-dependent manner reaching to approximately 95% inhibition after 36-48 h of incubation in serum-free medium (Fig. 2B). The quiescent smooth muscle cells obtained under this condition were able to
enter the cell cycle after serum stimulation, and after 48 h of incubation the incorporated 3H-thymidine was significantly (P c 0.001) higher than the nonstimulated cells (Fig. 2B). The labeling index of the smooth muscle cells grown in serum-free medium also indicated a gradual decrease in the number of labeled cells in a timedependent manner by approximately 90-95% after 48 h of incubation (Fig. 3A). Addition of serum to the media, similar to the TCA precipitation experiments, resulted in a gradual increase in the labeling index, and by 48 h more than 95% of the cells were labeled (Fig. 3B). All further studies examining the effect of EGF and PDGF on 3H-thymidine incorporation and cell proliferation were performed on quiescent smooth muscle cells grown in serum-free conditions for 48 h. Effect of EGF and PDGF on DNA synthesis EGF and PDGF-AB in a concentration dependent manner significantly stimulated 3H-thymidine incorporation in the quiescent smooth muscle cells, with maximal stimulation at 5 rig/ml and 10 rig/ml EGF and
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PDGF-AB, respectively (P < 0.01 and 0.02), compared to unstimulated serum-free conditions (Fig. 4, A and B). No significant stimulation occurred at higher concentrations of PDGF or EGF, although EGF at 15 rig/ml reduced the 3H-thymidine incorporation compared to 5 rig/ml. It was found not to be significant (P < 0.08, Fig. 4A). The action of EGF and PDGF on 3H-thymidine incorporation was time dependent (Fig. 4C). The effect of PDGF at 10 rig/ml on 3H-thymidine incorporation reached significant levels after 24 h of incubation (P C O.OOOl), whereas EGF at 15 rig/ml required 36-48 h (P < 0.005). The effect of PDGF-AB at 10 rig/ml was similar to that induced by EGF at 15 rig/ml concentration, and their combinations further enhanced the 3H-thymidine incorporation (P < 0.05, Fig. 4D). PDGF-BB at the same concentration as PDGF-AB (natural) induced a similar effect on 3H-thymidine incorporation (data not shown). Ez at 1 pM added to the serum-free media (Fig. 5A) inhibited 3H-thymidine incorporation by the smooth muscle cells (P < 0.05). Ez also inhibited the mitogenic effects of 15 rig/ml EGF (Fig. 5B) or 3 rig/ml PDGF (Fig. 5C). P4 at 1 pM either alone or in combination with E2 did not have any significant effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF (Fig. 5, A-C). Since phenol red has been shown to have a estrogen-like bioactivity (38), we have examined whether the pH indicator present in the medium mimics the biological effects of estradiol on 3H-thymidine incorporation. Serum-free, phenol red-free media with or without Ez (1 pm) had no significant effect on 3Hthymidine incorporation by these cells (not shown). Immunofluorescence
localization
Immunofluorescent microscopic observations indicated that myometrial smooth muscle cells in culture produce EGF and PDGF-AB and contain EGF and PDGF-P, but not PDGF-a receptors (Fig. 6). The immunostaining of EGF was very intense and noticeable around the nuclear periphery possibly associated with endoplasmic reticulum (Fig. 6A). The smooth muscle cells were also immunostained intensely for EGF receptors, uniformly distributed in the cytoplasmic region (Fig. 6B). The immunostaining of smooth muscle cells for PDGF-AB as well as PDGF-/3 receptor were uniform over these cells and with lower intensity than that observed for EGF and EGF receptor (Fig. 6, D and E). However, immunostaining for PDGF-(Y receptors was very low and sometimes not detectable in these cells (Fig. 6F). Preabsorption of antibodies with corresponding pro-
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teins or replacing the primary antibodies with nonimmune sera show a considerable reduction in immunostaining intensity, indicating the specificity of the reactions (Fig. 6C). Immunohistochemical
localization
Uterine tissues from both proliferative and secretory phases of the menstrual cycle were used for immunohistochemical localization of EGF, PDGF-AB, as well as EGF and PDGF-8 receptors. The immunostaining for EGF was associated with elongated and circular smooth muscle cells of both phases without any differences in staining intensity (Fig. 7A). The endothelial and smooth muscle cells of the arterioles also immunostained for EGF (Fig. 7B). The pattern of immunostaining for EGF receptor was similar to that observed for EGF, but with more intensity (Fig. 7, D and E). The myometrial tissue also immunostained for PDGF-AB and PDGF-b receptor (Fig. 8). The immunostaining for PDGF-AB as well as PDGF-/3 receptors were associated with smooth muscle cells (Fig. 8, A and D) and endothelial and smooth muscle cells of the arterioles (Fig. 8, B and E). Preabsorption of EGF and PDGF-AB antibodies with the purified EGF and natural PDGF (Figs. 7C and 8C), and EGF receptor antibodies with purified human term placenta microvilli (Fig. 7F) resulted in considerable reduction in immunostaining intensity of myometrial tissues. Replacement of PDGF-/3 receptor antibody with normal serum also reduced the intensity of immunostaining (Fig. 8F). Discussion The immunocytochemical and immunofluorescence microscopic observations indicated the presence of EGF and PDGF-AB as well as EGF and PDGF-/3 receptors in human myometrial tissue and myometrial smooth muscle cells in primary culture. Although the presence of EGF receptor in human uterine tissue have previously been reported using biochemical, autoradiographic, and immunohistochemical approach (23-26), this is the first demonstration suggesting the local production of EGF and PDGF-AB as well as PDGF-fl receptors in both myometrium and myometrial smooth muscle cells in culture. The presence of immunoactive EGF protein and mRNA for EGF has previously been reported in mouse uterus, associated exclusively with uterine luminal epithelial cells (19-21). In contrast, present observations indicate that in human uterus, EGF immunoreactivity is
FIG. 7. Immunohistochemical localization of EGF (A-C) and EGF receptor (D-F) in human myometrial tissue. The immunostaining is associated with smooth muscle cells (A and D) and arterioles endothelial and smooth muscle cells (B and E). Preabsorption of EGF antibody with recombinant human EGF (C) and EGF receptor antibody with purified human placental microvilli (F) resulted in considerable reduction in immunostaining of the tissues. C and F are shown in Nomarski interference contrast. Magnification, ~151.
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associated with all major myometrial as well as endometrial cell types (Chegini, N., M. J. Rossi, and B. J. Masterson, submitted for publication). This difference in the distribution of EGF to that reported in mouse is not clear, although the antibodies used in both studies were species specific. We have reported a similar pattern of distribution for EGF and transforming growth factor(Y (TGF+) in the rat (22) and TGF-a in human (39) uterus. Myometrial smooth muscle cells in primary cell culture derived from these tissues also express TGF-a (unpublished observation). We did not observe any variation in the intensity of immunostaining in relation to the phases of the cycle, although it has been reported that in rodents Ea regulates the occurrence of EGF protein and its mRNA (19-21). These observations indicate that human myometrial smooth muscle cells are an endogenous source of EGF and TGF-(u, suggesting that these growth factors may play a regulatory role in part by an autocrine pathway in this tissue. Myometrial tissues and isolated smooth muscle cells in primary culture also contain immunoreactive EGF receptors. Using specific monoclonal antibodies to the extracellular binding domain of EGF receptor, it was observed that both myometrial smooth muscle cells and the vasculature immunostain for EGF receptors. The pattern of immunostaining was similar to our previous autoradiographic observations with no variation due to the phases of the menstrual cycle, confirming our previously reported data (23,24). Immunohistochemical studies carried out in normal and malignant uteri also indicated a lack of correlation with histological grade, depth of myometrial invasion, estrogen-P4 receptor status, the presence of extrauterine metastases, or the development of recurrent disease (26). In addition to human uterus, the presence of EGF receptors in all the major uterine cell types in rodent has been reported (27,28). However, in the cycling rat EGF receptor content significantly increased in proestrus, and in the ovariectomized rat Ez treatment induced a 3-fold increase in EGF receptor and EGF receptor mRNA content, suggesting a role in EGF receptor regulation by estrogen (29, 30). Similar results have been reported for IGF-I and IGF-I receptor (10). In human uterus, although the presence of PDGF receptor has been demonstrated, the cellular distribution and distinction between the receptor types has not been determined (31). PDGF exists either as disulfide-linked homodimers (PDGF-AA and PDGF-BB) or heterodimer (PDGF-AB), with two separate receptors, the cy- and the @-type, with different affinities for the three dimeric
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forms of PDGF (33,34). The PDGF-(U receptor binds to all three dimeric forms of PDGF, whereas the P-type binds to the PDGF-BB with high affinity and to the PDGF-AB with lower affinity (33). Our observations provide the first evidence that human myometrial tissue contains specific immunoreactive PDGF-P receptors using antibody raised against human PDGF-/3 receptor. The myometrial smooth muscle cells in primary culture also immunostain for PDGF-P receptors but very low to nondetectable levels for PDGF+ receptors. Using antibody raised against porcine uterine PDGF-P receptors, our observations indicated a very low level of immunostaining in frozen sections of human myometrial tissue and a relatively strong staining of the smooth muscle in culture. These observations were similar to that previously reported in porcine using the same antibody to localize PDGF-P receptors in uterine tissue and myometrial smooth muscle cell cultures (34). It was suggested in this study that cell culturing may up-regulate the PDGF-P receptors in porcine myometrial smooth muscle cells (34). However, the antibody to human PDGF-/3 receptors not only immunostained frozen and fixed myometrial tissue (see Fig. 8), it gave similar intensity of immunostaining of cultured smooth muscle cells observed with the antibody to porcine PDGF-/3 receptor. This difference in immunostaining may simply lie in the nature of the antibodies, rather than the absence or down-regulation of PDGF-B receptors in human myometrial tissue. The presence of immunoreactive PDGF-/3 and a low level of PDGF-a receptors in human myometrial smooth muscle cells suggest that the major part of PDGF receptor content reported in human uterus is the PDGF-P. Furthermore, the results indicate that unlike porcine, cell culturing may not be required for up-regulation of PDGF-fl receptor in human myometrial smooth muscle cells as the PDGF-P receptor is already present in the myometrial tissue. The presence of low level of PDGF-a in myometrial smooth muscle cells is not surprising. It has been suggested that the PDGF-a receptor constitutes less than 10% of the total PDGF receptor in diploid human fibroblasts (40) and the major mitogenic action of PDGF in these cells is mediated through the PDGF-/3 receptor (33). In support of our observations is the recent report that human myometrial tissue expresses the mRNA for PDGF-B chain and low abundance of the PDGF-A chain mRNA, without a correlation to the phases of the cycle (35). No correlation was also observed in immunoreactive intensity of PDGF-P receptors in myometrial tissue in the present study. Our
FIG. 8. Immunohistochemical localization of PDGF-AB (A-C) and PDGF-8 receptor (D-F) in human myometrial tissue indicating the presence of immunostaining in association with smooth muscle cells (A and D), arterioles endothelial and smooth muscle cells (B and E). Replacement of antibodies with nonimmune normal serum resulted in considerable reduction in immunostaining of PDGF-AB (C) and PDGF-@ receptor (F). Magnification, ~151.
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EGF AND PDGF ACTION ON HUMAN
results indicated that PDGF-BB and PDGF-AB, at the same concentration, were equally mitogenic in myometrial smooth muscle cells, suggesting that the action of PDGF in these cells is most likely mediated through the PDGF-P receptors. The results of the present study indicate that myometrial smooth muscle cells in culture are responsive to the mitogenic action of EGF and PDGF. EGF and PDGF, in a concentration and time-dependent manner, significantly stimulated 3H-thymidine incorporation in smooth muscle cells. The PDGF action on 3H-thymidine incorporation was similar to 10% FBS. Furthermore, EGF in the presence of PDGF significantly enhanced the 3Hthymidine incorporation compared to that observed with either EGF or PDGF alone. The action of EGF and PDGF were independent of phases of the menstrual cycle and equally stimulated 3H-thymidine incorporation by myometrial smooth muscle cells. In myometrial smooth muscle cultures established from different individual patients, the extend of 3H-thymidine incorporation varied in both nonstimulated serum-free as well as serum, or growth factor-stimulated cultures. Similar variation has been reported regarding the effect of EP and P4 on human endometrial stoma1 cells, although longer incubation times and earlier passages were used (15). Such differences concerning the effect of E2 on cell proliferation and efficacy of colony formation by human myometrial smooth muscle cells has been observed (41). Ez at the concentration used in this study inhibited 3H-thymidine incorporation by myometrial smooth muscle cells. EP also inhibited the mitogenic effect of EGF and PDGF. Pq, however, either alone or in combination with Ez, was ineffective in stimulation of 3H-thymidine incorporation by these cells and did not enhance the mitogenic action of EGF or PDGF. Although the Ez concentration used in our study may be considerably higher than physiological, it has been reported that Ez at 10-“-10-s M had no significant effect on epithelial cell proliferation (42). Similar in vitro results describing the ineffectiveness of estrogen and P4 in rodent and human endometrial epithelial, stromal, and myometrial smooth muscle cell proliferation have previously been reported (15-18, 42). However E2 at lo-’ M has been reported to stimulate both endometrial and myometrial smooth muscle cells proliferation in the presence of 20% serum (41). This was achieved only in cultures from 5th8th passages, without any effect on cells from 2nd-4th or 9th-12th subcultures (41). Although cell-cell interaction has been demonstrated to be important for mitogenie stimulation of endometrial cells (13), E2 even in uterine organ culture was ineffective in stimulating DNA synthesis (43). The additive or synergistic mechanism where cells are exposed to a variety of growth factors, i.e. EGF and
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PDGF, in cell proliferation is complex and not well understood. Quiescent fibroblasts require more than a single mitogen to stimulate DNA synthesis. PDGF stimulates these cells to become competent or to make the Go to G1 transition for subsequent cell division (44). However, EGF acts as a progression factor, stimulating the cells to continue through the cycle (44). PDGF has also been shown to influence the expression of TGF-/3 mRNA in human fibroblasts (45) which has multifunctional activities in a variety of cell types, including smooth muscle cells (46). Moreover, it has recently been shown that the mitogenic effect of all three PDGF isoforms on human fibroblasts is enhanced by TGF+?l through the induction of PDGF-a receptors (40). We have previously shown the presence of immunoreactive TGF-P in human myometrial tissue (39), which may suggest a possible interaction between TGF-P and PDGF in this tissue. Therefore the ultimate growth response to a given mitogen in the normal in viva condition of quiescent uterine myometrial tissue may involve both negative and positive regulatory mechanisms. However, under certain conditions, such as leiomyomata, the smooth muscle cells are able to resume their proliferative activity leading to the formation of these tumors. Acknowledgments The authors would like to thank the Department of OB/GYN Gynecologists for their help in the collection of the tissues. In addition, we thank Landis Young for her assistance in the preparation of this manuscript.
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proliferation. Endocr Rev 11:443-453 11. Quarmby VE, Korach KS 1984 The influence of 17-estradiol on pattern of cell division in the uterus. Endocrinology 114694-702 12. Pavlik EJ, Katzenellenbogen BS 1978 Human endometrial cells in primary tissue culture: estrogen interactions and modulation of cell proliferation. J Clin Endocrinol Metab 47~333-344 13. Iguchi T, Uchima F-D, Ostrander PL, Bern HA 1983 Growth of normal mouse vaginal epithelial cells in and on collagen gels. Proc Nat1 Acad Sci USA 80:3743-3747 14. Cooke PS, Uchima F-DA, Fujii DK, Bern HA, Cunha GR 1986 Restoration of normal morphology and estrogen responsiveness in cultured vaginal and uterine epithelial transplanted with stroma. Proc Nat1 Acad Sci USA 832109-2113 15. Irwin J, Kirk D, King RJB, Quigley MM, Gwatkin RBL 1989 Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertil Steril52:761-768 16. Lippman ME, Dickson RB, Knabbe C, Huff K, Swain S, McManaway M, Bronzert D, Kasid A, Gelmann EP 1986 Autocrine and paracrine growth regulation of human breast cancer. Breast Cancer Res Treat 7:59-70 17. Fisher DA, Lakshmanan J 1990 Metabolism and effects of epiderma1 growth factor and related growth factors in mammals. Endocr Rev 11:418-442 18. Traish AM, Wotiz HH 1987 Prostatic epidermal growth factor receptors and their regulation by androgens. Endocrinology 121:1461-1467 19. Huet-Hudson YM, Chakraborty C, De SK, Suzuki Y, Andrews GK, Dey SK 1990 Estrogen regulates the synthesis of epidermal growth factor in mouse uterine enithelial cells. Mol Endocrinol4:510-523 20. Gonzalez F, Lakshmanan J, Hoath S, Fisher DA 1984 Effect of estradiol-176 on uterine epidermal growth factor concentration in immature mice. Acta Endocrinol (Conenhl 105:425-428 21. DiAugustine RP, Petrusz P, Beil 61, Brown CF, Korach KS, McLachlan JA, Tseng CT 1988 Influence of estrogen on mouse epidermal growth factor precursor protein and messenger ribonucleic acid. Endocrinology 122:2355-2363 22. Simms JS, Chegini N, Williams RS, Rossi AM, Dunn Jr WA 1991 Identification of epidermal growth factor, transforming growth factor and epidermal growth factor receptor in surgically induced endometriosis in rat. Obstet Gynecol 78850-857 23. Chegini N, Rao ChV, Wakim N, Sanfilippo J 1986 Binding of lz51epidermal growth factor in human uterus. Cell Tissue Res 246:543548 24. Hoffmann GE, Rao ChV, Barrows GH, Schultz GS, Sanfilippo JS 1984 Binding sites for epidermal growth factor in human uterine tissues and leiomyomas.-J Clin Endocrinol Metab 58880-884 25. Tommola P, Pekonen F, Rutanen E-M 1989 Binding of epidermal growth factor and insulin-like growth factor I in human myometrium and leiomyomata. Obstet Gynecol74:658-662 26. Berchuck A. Soisson AP. Olt GJ. Saner JT. Clarke-Pearson DL. Bast Jr RB,‘McCarty Jr KS 1989 Epidermal growth factor receptor expression in normal and malignant endometrium. Am J Obstet Gynecol161:1247-1252 27. Bossert NL, Nelson KG, Ross KA, Takahashi T, McLachlan JA 1990 Epidermal growth factor binding and receptor distribution in the mouse reproductive tract during development. Dev Biol142:7585 28. Lin T-H, Mukku VR, Verner G, Kirkland JL, Stance1 GM 1988 Autoradiographic localization of epidermal growth factor receptors to all major uterine cell types. Biol Reprod 38403-411
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29. Gardner RM, Verner G, Kirkland JL, Stance1 GM 1989 Regulation of uterine epidermal growth factor (EGF) receptors by estrogen in the mature rat and during the estrous cycle. J Steroid Biocbem 32:339-343 30. Lingham R, Stance1 GM, Loose-Mitchell DS 1988 Estrogen induction of the epidermal growth factor receptor mRNA. Mol Endocrino1 2:230-235 31. Fayed YM, Tsibris JCM, Langenberg PW, Robertson AL 1989 Human uterine leiomyomata cells: binding and growth factor responses to epidermal growth factor, platelet-derived growth factor, and insulin. Lab Invest 60:30-37 32. Ronnstrand L, Beckmann MP, Faulders B, Ostman A, Ek B, Heldin CH 1987 Purification of the receptor for platelet-derived growth factor from norcine uterus. J Biol Chem 262:2929 33. Heldin CH, Backstrom G, Ostman A, Hammacher A, Ronnstrand L, Rubin K, Nister M, Westermark B 1988 Binding of different dimeric forms of PDGF to human fibroblasts: evidence for two separate receptor types. EMBO J 21387-1393 34. Terracio L, Ronnstrand L, Tingstrom A, Rubin K, Claesson-Welsh L, Funa K, Heldin CH 1988 Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing. J Cell Biol 107:1947-1957 35. Boehm KD, Daimon M, Gorodeski IG, Sheean LA, Utian WH, Ilan J 1990 Expression of the insulin-like and platelet-derived growth factor genes in human uterine tissues. Mol Reprod Dev 2793-101 36. Noyes RW, Hertig AT, Rock J 1951 Dating of endometrial biopsy. Fertil Steril 1:3-25 37. Chegini N, Safa AR 1987 Influence of mitoxantrone on nucleolar function in MDA-MB-231 human breast tumor cell line. Cancer Lett 37:327-336 38. Ernse M, Schmid C, Froesch ER 1989 Phenol red mimics biological action of estradiol: enhancement of osteoblast proliferation in uitro and of type I collagen gene expression in bone and uteri of rats in uiuo. J Steroid Biochem 33907-914 39. Williams RS, Chegini N, Localization of TGF-(U and TGF-8 in human uterine and ovarian tissue. Program of 46th Annual Meeting of the American Fertility Society, Washington DC 1990, p S5 (Abstract) 40. Ishikawa 0, LeRoy EC, Trojanowska M 1990 Mitogenic effect of transforming growth factor 61 on human fibroblasts involves the induction of platelet-derived growth factor a! receptors. J Cell Physiol 145:181-186 41. Chen L, Lindner HR, Lancet M 1973 Mitogenic action of oestradiol-178 on human myometrial and endometrial cells in long-term tissue cultures. J Endocrinol59:87-97 42. Tomooka Y, DiAugustine RP, McLaachian JA 1986 Proliferation of mouse uterine epithelial cells in vitro. Endocrinoloav-- 118:10111018 43. Ghahary A, Chakrabarti S, Murphy LJ 1990 Localization of the sites of synthesis and action of insulin-like growth factor-I in the rat uterus. Mol Endocrinol4:191-195 44. Pardee AB, Coppock DL, Yang HC 1986 Regulation of cell proliferation and the onset of DNA synthesis. J Cell Sci SUDD~ -- 4:171180 45. Paulsson Y, Hammacher A, Heldin C-H, Westermark B 1987 Possible positive autocrine feedback in the nrerenlicative nhase of human fibroblasts. Nature 328715-717 46. Barnard JA, Lyons RM, Moses HL 1990 The cell biology of transforming growth factor (3. Biochim Biophys Acta 1032:79-87
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