Platelet-Activating Factor Enhances Production of Insulin-like Growth Factor Binding Proteins in a Human Adenocarcinoma Cell Line (HEC-1A)

GYNECOLOGIC ONCOLOGY ARTICLE NO.

61, 333–340 (1996)

0152

Platelet-Activating Factor Enhances Production of Insulin-like Growth Factor Binding Proteins in a Human Adenocarcinoma Cell Line (HEC-1A) S. GIANNINI, M. MAGGI, B. CRESCI, G. FINETTI, L. BONACCORSI, M. LUCONI, C. M. ROTELLA, G. FORTI, M. SERIO, AND E. BALDI1 Department of Clinical Pathophysiology, Endocrinology, Andrology, and Metabolism Units, University of Florence, viale Pieraccini 6, 50139 Florence, Italy Received June 5, 1995

INTRODUCTION We have recently demonstrated the existence of an autocrine growth loop driven by platelet-activating factor (PAF) in the human endometrial adenocarcinoma cell line HEC-1A. To investigate a possible cooperation between PAF and the insulin-like growth factor (IGF) system in this cell line, the effect of PAF on insulin-like growth factor binding protein (IGFBP) production as well as binding and biological activities of IGF-I, IGF-II, and the analog Des(1-3)IGF-I have been evaluated. Analysis of self- and cross-displacement curves of [125I]IGF-I binding to HEC-1A cells indicates the presence of a single class of binding sites, with affinity constants of 1–4 nM for IGF-I and IGF-II and 70 nM for Des(13)IGF-I, which binds to IGFBPs with lower affinity. Insulin does not apparently bind to this binding site. Moreover, the addition of increasing concentrations of IGF-I leads to a paradoxical increase of binding. These results indicate a similarity of this binding site to IGFBPs. The presence of IGFBPs has been demonstrated by Western ligand blot analysis of HEC-1A conditioned medium which shows the presence of two bands of 32–34 and 40–45 kDa. By Western immunoblotting analysis, the two bands were respectively identified as IGFBP-2 and IGFBP-3. Incubation with PAF (1 mM) highly increases the release of the two IGFBPs from the cells. Such an effect is inhibited by the PAF receptor antagonist L659,989, by the PKC inhibitor sangivamycin, and by the tyrosine kinase inhibitor genistein. PAF also induces a time-dependent increase of mRNA expression for IGFBP-3, suggesting an effect on synthesis of this protein. IGF-I and IGF-II (0.1–100 nM) are almost ineffective in inducing [3H]thymidine incorporation, whereas a slight proliferative effect is observed with Des(1-3)IGFI which also increases PAF synthesis. These data demonstrate a modulatory action of PAF on IGFBP secretion in HEC-1A cells and indicate that the IGF system plays a minor, if any, modulatory role on proliferation of this cell line. q 1996 Academic Press, Inc.

1 To whom correspondence should be addressed at Department of Clinical Pathophysiology, Andrology Unit, University of Florence, viale Pieraccini 6, I-50139 Firenze, Italy. Fax: //39.55.4221.848.

The regulation of uterine growth involves complex interactions between ovarian steroids and peptide growth factors [1]. In particular, increasing evidence demonstrates a role for the insulin-like growth factor (IGF) system in the modulation of growth and function of the mammalian uterus [1]. Indeed, IGFs and their receptors are present in the uterine tissue of several mammalian species [1–3] and are positively regulated by ovarian steroids [3, 4]. Moreover, the growthpromoting effect of IGF-I in the uterus is particularly evident in estrogen-primed animals [5], indicating an intimate cooperation between this growth factor and steroids in the regulation of uterine functions. Functionally active IGF-I receptors have been found in human endometrial stromal cells [6, 7] as well as in neoplastic endometrium [8], suggesting a role for IGFs in the growth of this tissue. The biological action of IGFs is modulated by the presence of several IGF-binding proteins (IGFBPs) which have been demonstrated in a variety of biological fluids, cell cultures, and tissues [9] including the uterus [10]. So far, six different binding proteins, ranging in molecular weight from approximately 22–31 to 53 kDa, have been characterized, cloned, and termed IGFBP1 through IGFBP-6 [11]. Although the exact mechanism of the IGFBP action has not been determined, they can both increase and decrease the IGF actions depending upon the nature of the IGFBP and the experimental conditions [12]. The presence of IGFBPs in uterine tissue as well as in the cultured media of human endometrial adenocarcinoma cell lines [13, 14], including the cell line HEC-1A [13, 15], has been demonstrated. However, their role in the regulation of growth and/or modulation of biological activity of IGF action in these cells has not been investigated in depth. We have recently demonstrated, in the endometrial adenocarcinoma cell line HEC-1A, the existence of an autocrine growth loop driven by the phospholipid platelet-activating factor

333

/ 6d0d$$4321

05-10-96 14:35:21

goa

0090-8258/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

AP: Gyn Onc

334

GIANNINI ET AL.

(PAF) [16]. In particular, we demonstrated production and biological activity of PAF as well as growth inhibition by a PAF receptor antagonist in this cell line [16, 17]. To establish possible cooperation between PAF and the IGF system in the regulation of HEC-1A cell proliferation, we studied the effect of PAF on IGFBP production by these cells. In addition, we evaluated the binding and biological activity of IGF-I, IGF-II, and the analog Des(1-3)IGF-I which binds to IGFBPs with lower affinity [18]. Our results demonstrate a positive modulatory effect of PAF on IGFBP production which may be important in limiting the growth-promoting effect of IGFs in this cell line. MATERIALS AND METHODS

Chemicals PAF, the stable analog cPAF (1-o-hexadecyl-2N-methylcarbamyl-sn-glycero-3-phosphocoline), and genistein were obtained from Calbiochem (La Jolla, CA). L659,989 was a generous gift of Merck Sharp and Dohme (Raway, NJ). Sangivamycin was obtained from the National Institute of Health Drug Synthesis and Chemistry Branch (Bethesda, MD). Human recombinant IGF-I and IGF-II were obtained from Boehringer-Mannheim (Mannheim, Germany). Des(13)IGF-I was obtained from Peninsula (Belmont, CA). The sodium salt of [3H]acetic acid (1.9 Ci/mmol) and [3H]PAF (60 Ci/mmol) were purchased from New England Nuclear (Boston, MA). [125I]IGF-I and -II (2000 Ci/mmol) were purchased from Amersham (Amity, Milan, Italy). High-performance TLC silica gel 60 plates were obtained from E. Merck (Darmstadt, Germany). Organic solvents were purchased from Carlo Erba (Milan, Italy). Bovine insulin, TLC phospholipid standards, BSA, NP-40, Tween 20, McCoy’s 5A medium, fetal calf serum, penicillin, and streptomycin were purchased from Sigma Chemical Co. (St. Louis, MO); acrylamide, bisacrylamide, and ammonium persulfate from BioRad Lab (Hercules, CA). Cells HEC-1A (obtained from ATCC, Bethesda, MD) is a cell line established in 1968 from explants of endometrial adenocarcinoma. The cells were grown in modified McCoy’s 5A (Sigma) supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY), penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 2.2 g/liter sodium bicarbonate as suggested by ATCC. Cell cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 377C. Medium was replaced every 2–3 days and the cells were split every 4–5 days using trypsin (0.05%) in Mg2/- and Ca2/-free PBS containing 0.02% EDTA. Collection of Conditioned Medium Under all of our experimental conditions, cells were seeded in 25-cm2 flasks (Corning Co., Corning, NY). To

/ 6d0d$$4321

05-10-96 14:35:21

goa

examine the production of IGFBPs, subconfluent cells were washed three times with PBS and maintained for 1 day in FCS-free culture medium. The cultures were then changed to fresh 0.1% BSA FCS-free culture medium with or without experimental stimuli (PAF and L659,989 were added simultaneously, whereas sangivamycin and genistein were added 30 min before addition of PAF) and incubated for a further 24 hr. Conditioned medium (CM) was then harvested and stored at 0757C in polypropylene tubes (Falcon, Becton– Dickinson, NJ) which were previously treated as reported [19] to decrease nonspecific binding of proteins to the tubes. Briefly, tubes were incubated at 377C for 3 hr in FCS-free medium containing 0.1% BSA and 20 mM Hepes, pH 7.2. In order to ensure an equal loading of CM in the gel, after the CM collection, the number of cells in each flask was determined with a hemocytometer and the volume of CM used for analysis with the ligand blot procedure was adjusted accordingly to the cell number. Northern Analysis For Northern analysis, HEC-1A cells were incubated for 4 and 24 hr with PAF (1 mM) and methyl carbamil-PAF (cPAF, 1 mM) in McCoy’s serum-free medium. Total cellular RNA was prepared by the hot-phenol method and quantitated by spectrofluorometric analysis at 260 nm. Twenty micrograms of RNA was fractionated in a 1% agarose–3% formaldehyde gel, transferred onto nylon membrane (Nytran, Schleicher & Schuell Inc.), and baked at 807C for 2 hr. The membranes were prehybridized for 1 hr and then hybridized overnight at 657C with a solution containing 10 mg/ml BSA, 7% SDS, 0.25 M Church & Gilbert buffer, 1 mM EDTA (pH 8), and 0.2 mg/ml hot-denatured sonicated herring sperm DNA. The probe used for RNA hybridization was a 475basepair (bp) gel-purified HindIII–EcoRI complementary DNA (cDNA) fragment for human IGFBP-3 (obtained from ATCC) labeled with deoxycytidine 5*-[a-32P]triphosphate by a random priming kit (Boheringer-Mannheim Italy, Milan, Italy), chromatographed (Nu-Clean D25 disposable spun columns, IBI, New Haven, CT), and hot-denatured before use. The membrane was washed four times with a solution containing 1% SDS, 0.2 M Church & Gilbert buffer, and 0.1 M EDTA (pH 8) at 657C for 10 min. The nylon membrane was then submitted to autoradiography using Kodak X-Omat AR film and Kodak X-Omatic regular intensifying screens at 0807C for 4 hr. Mitogenic Assay ([3H]Thymidine Incorporation) HEC-1A cells were plated into 96-well dishes (2 1 104 cells) in 200 ml of 10% FCS/McCoy’s and allowed to adhere for 12–18 hr. Cells were then maintained for 24 hr in serumfree medium, and stimuli or vehicles were added at the indicated concentrations and times. All treatments were performed in hexaplicate. Cells were pulsed with 1 mCi/well

AP: Gyn Onc

335

PAF-INDUCED IGFBP SECRETION IN HEC-1A CELLS

[3H]thymidine (New England Nuclear) for 4 hr and harvested by hypotonic lysis and filtration onto glass fiber filters using a Titertek cell harvester (Flow Lab., Costa Mesa, CA). Individual filters were counted by liquid scintillation in a bcounter. PAF Synthesis ([3H]Acetate Incorporation into PAF) [3H]PAF synthesis was measured by quantitative incorporation of [3H]acetate into PAF as reported previously [16]. Briefly, confluent cells were grown on six multi-well plates, washed twice in 0.1% BSA serum-free McCoy’s 5A medium, and preincubated in the same buffer containing 50 mCi/well [3H]acetate. Stimuli were then added at the indicated concentrations. The reaction was stopped by adding 3 ml of ice-cold chloroform:methanol:acetic acid (1:2:0.04, by volume), and lipids were extracted twice in chloroform:methanol according to the Bligh and Dyer extraction procedure [20]. The lower phases, containing the extracted lipids, were collected, washed once with methanol:water (10:9, v/v), evaporated under nitrogen, and resuspended in 200 ml of chloroform:methanol (9:1, v/v). Samples were then loaded on heat-activated high-performance TLC silica gel 60 plates and developed in chloroform:methanol:water (65:35:6, by volume). The labeled phospholipid products were identified by cochromatography with known standards. The Rf value of PAF was 0.2. The lipid fractions were visualized under UV lamp after 2-p-toluidinylnaphthylene sulfonate exposure; areas corresponding to PAF were scraped and the radioactivity was counted by liquid scintillation. Binding Experiments Confluent (100–150 1 103 cells/well in 24-well plates) HEC-1A cells were washed twice with PBS and incubated in 200 ml of binding medium (100 mM Hepes, 120 mM NaCl, 5 mM KCl, 1.3 mM MgSO4 , 1 mM EDTA, 10 mM glucose, 15 mM NaHCO3 , 1% BSA) with fixed concentrations of [125I]IGF-I and increasing concentrations of unlabeled IGF-I, IGF-II, Des(1-3)IGF-I, or insulin (10011 –1004 M). After incubation, cells were extensively washed with ice-cold PBS with 0.1% BSA and solubilized in 0.5 N NaOH, and the cell-bound radioactivity was determined. Measurements were obtained in triplicate. Cell counts routinely varied less than 10% between wells. Western Ligand Blot The method to detect the IGFBPs was carried out essentially according to the procedure described by Hossenlopp et al. [21]. Briefly, CM (100 ml) along with prestained molecular weight marker proteins (Bio-Rad Lab.) were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under nonreducing conditions. For blotting studies the proteins were transferred to nitrocellulose membranes (45 mm; Schleicher and Schuell, Dassel, Germany) at 0.08

/ 6d0d$$4321

05-10-96 14:35:21

goa

A for 16 hr using a transblotting cell apparatus (Bio-Rad Lab.). After transfer, the nitrocellulose membranes were prewashed with a Tris buffer (150 mM NaCl and 10 mM Tris– HCl, pH 7.4) containing 0.5 mg/ml sodium azide and 3% NP-40 for 30 min and then washed in Tris buffer with 1% BSA for 4 hr and in Tris buffer plus 0.1% Tween 20 for 10 min. The membranes were then incubated in a plastic bag for ligand blotting with [125I]IGF-II (Ç2 1 106 cpm) in Tris buffer containing 1% BSA and 0.1% Tween 20 for 2 hr at room temperature on a rocking platform. The membranes were washed three times with Tris buffer, dried, placed at 0807C in a bag, and autoradiographed by exposure to Kodak X-Omat RP film. For immunoblotting, the nitrocellulose, after protein transfer, was incubated at 47C for 2 hr with anti-hIGFBP-3, -2, -4, and -5 polyclonal antibodies (UBI, Lake Placid, NY) respectively at final dilutions of 1:350, 1:350, 1:500, and 1:200 in PBS containing 3% nonfat dry milk, followed by incubation (3 hr, 257C) with anti-rabbit IgG–alkaline phosphatase conjugate (Bio-Rad Lab.) at a final dilution of 1:20,000 in PBS containing 3% nonfat dry milk. The membrane was washed once with 0.1 M Tris–HCl buffer containing 0.1 M NaCl and 5 mM MgCl2 , detected by using the enhanced chemiluminescence system (ECL, Amersham, UK), and subjected to autoradiography by exposure to Kodak X-Omat RP film. Statistical Analysis Binding experiments were analyzed by the computer program LIGAND [22]. The program provides objective measures of goodness-of-fit and objective criteria for distinguishing between models of different complexity. The selection of the best model was based on a comparison of the weighted sum of squares and/or the root-mean square error. An F test based on the ‘‘extra sum of squares’’ principle was used. Results are expressed as means { SEM. Statistical comparisons were made by one-way ANOVA followed by Student’s t test. RESULTS

1. Binding Studies The binding reaction of [125I]IGF-I to HEC-1A cells was dependent on time and temperature of incubation (data not shown). Maximal specific binding was obtained after 18 hr at 47C. Accordingly, incubations for all the experiments were performed at 47C for 18 hr. In order to characterize the binding of [125I]IGF-I to HEC1A cells families of self- and cross-displacement curves between IGF-I, IGF-II, Des(1-3)IGF-I, and insulin, using different passages of HEC-1A cells, have been designed and performed. Simultaneous mathematical analysis of competition curves is highly suggestive for the presence of a homo-

AP: Gyn Onc

336

GIANNINI ET AL.

TABLE 1 Concentrations of Receptors (Sites/Cell) and Affinity (Kd , nM) for IGF-I, IGF-II, Des(1-3)IGF-I, and Insulin in HEC-1A Cells Binding capacity (sites/cell) 427,000 { 32,000

Affinity (Kd , nM) IGF-I IGF-II Des(1-3)IGF-I Insulin

1.1 { 0.1 4.2 { 0.5 75.6 { 15.1 ú10,000

Note. Values are expressed as means { SE and were obtained from computer modeling using the LIGAND program of three different families of competition curves (16 curves) obtained in HEC-1A cells using a onesite model.

geneous class of IGF binding sites. Indeed, in three separate experiments the fit for the one-site model appears consistently better than for the two-site model (P õ 0.0001). Table 1 summarizes the dissociation constants (Kd ) and binding capacity (Bmax ) for the best fitting model. Figure 1 graphically represents a typical family of competition curves and the predicted relationship for the one-site model. The IGF binding site binds with high-affinity IGF-I and IGF-II (Kd Å 1–4 nM), whereas the IGF-I agonist Des(1-3)IGF-I shows lower affinity (Kd Å 70 nM). Insulin does not apparently bind to this site even in the micromolar range. This site therefore does not correspond to the type I or II IGF receptors and shows similar characteristics to IGFBPs. The presence of IGFBPs was then confirmed when we performed binding studies in HEC-1A cells in later passages, as previously reported in different cell systems [23, 24]. In these cells we find a lower amount of [125I]IGF-I bound in ‘‘tracer’’ concentrations in comparison with cells at the earliest passages (see B/T ratio in Figs. 1 and 2). However, the addition of increasing concentrations of unlabeled IGF-I and -II leads to a paradoxical increase in bound counts. This increase

FIG. 1. Families of competition curves among [125I]IGF-I, the corresponding unlabeled peptide (open circles), IGF-II (solid circles), Des(1-3)IGF-I (open triangles), and insulin (solid triangles) in HEC-1A cells. Curves were generated by the LIGAND program from two typical experiments based on the predicted relationship for a one-site model as in Table 1.

/ 6d0d$$4321

05-10-96 14:35:21

goa

FIG. 2. Displacement of [125I]IGF-I binding to HEC-1A cells by the corresponding unlabeled peptide (open circles) and IGF-II (solid circles). The curves are the result of the cumulative analysis of two independent experiments and are derived from the predicted relationship for a positive cooperative model as in Munson and Rodbard [22]. Insulin up to millimolar concentrations did not affect [125I]IGF-I binding (not shown).

occurred in parallel with a competitive displacement by the unlabeled ligands IGF-I and IGF-II of [125I]IGF-I binding (Fig. 2). Conversely, neither insulin (up to 1 mM) nor the monoclonal antibody to the type I IGF receptor aIR-3 (up to 1 mM) affected [125I]IGF-I binding (data not shown). The paradoxical IGF-I-induced increase in bound [125I]IGF-I is maximal at nanomolar concentrations and is time and temperature dependent. At 47C, 1 nM IGF-I induces a 1200% increase in specific binding after 12 hr, whereas at 227C a similar increase is obtained after 4 hr. 2. Effect of PAF on IGFBP Production in the HEC-1A Cell Line Western ligand blot analysis of conditioned medium of HEC-1A cells shows the presence of two IGFBP bands: a small one in the molecular weight range of 32–34 kDa and a larger one in the range of 40–45 kDa (Figs. 3A and 3B). Twenty-four-hour stimulation with a 1 mM concentration of PAF induces an increase of these two protein bands (Figs. 3A and 3B), whereas 0.1 mM PAF was ineffective in inducing BP production by HEC-1A cells (Fig. 3A). The involvement of a specific PAF receptor in the action of PAF is suggested by the inhibitory action of equimolar concentrations of the PAF receptor antagonist L659,989 (Fig. 3A). Moreover, the effect of PAF on IGFBPs is inhibited by the tyrosine kinase inhibitor genistein (1 mM, Fig. 3B) and the protein kinase C inhibitor sangivamycin (1 mM, Fig. 3B), although the two substances were ineffective, by themselves, in modulating IGFBP release (Fig. 3B). An increase of the release of the two protein bands was also present after stimulation of HEC-1A cells with 0.1 mM IGF-I (Fig. 3B). In order to identify the two IGFBP bands, 100 ml of HEC1A conditioned medium was electrophoresed under nonreducing conditions and blotted with polyclonal anti-IGFBP antibodies. Except for incubation with anti-IGFBP-3 anti-

AP: Gyn Onc

PAF-INDUCED IGFBP SECRETION IN HEC-1A CELLS

337

FIG. 3. Autoradiogram of [125I]IGF-II Western ligand blot of conditioned media from HEC-1A cells. (A) Effect of 1 and 0.1 mM PAF on IGFBP production by HEC-1A cells. The effect of 0.1 mM PAF in the presence of equimolar concentration of the PAF antagonist L659,989 is also shown. (B) Effect of PAF (1 mM) in the presence or absence of the tyrosine kinase inhibitor genistein (Gen, 1 mM) or the protein kinase C inhibitor sangivamycin (Sang, 1 mM) on IGFBP production. The effect of IGF-I (0.1 mM) is also shown. Molecular weight markers are indicated on the left. Representative autoradiogram after 3 days exposure is shown. The arrows indicate the 30- to 32- and 40- to 45-kDa protein bands.

body, HEC-1A medium was concentrated six times, in order to enhance the protein concentration. As shown in Fig. 4, positive immunoreactive bands were detected for both antiIGFBP-3 and anti IGFBP-2 antibodies, respectively, in the molecular weight ranges of 40–45 and 30–32 kDa, indicating immunological identity of the higher molecular weight band with IGFBP-3 and the lower molecular weight band with IGFBP-2. No immunoreactive bands were detected after incubation with anti-IGFBP-4 and -5 (data not shown). The effect of PAF (1 mM) and its stable analog cPAF (1 mM) on IGFBP-3 mRNA steady-state levels in HEC-1A cells has also been investigated. As shown in Fig. 5, 4 hr

FIG. 4. Western immunoblot of conditioned media (100 ml) from HEC1A cells. Cells were electrophoresed on a 12% SDS–acrylamide gel and immunoblotted with anti-IGFBP-3 and anti-IGFBP-2 polyclonal antibodies. Molecular weight markers are indicated on the right. For immunoblotting with anti-IGFBP-2, conditioned medium was concentrated six times.

/ 6d0d$$4321

05-10-96 14:35:21

goa

stimulation with PAF and cPAF induces an increase of IGFBP-3 gene expression. However, such an increase was no longer present after 24 hr stimulation with both agonists. 3. Biological Effects of IGFs on HEC-1A Cells Figure 6A shows the effects of IGF-I and Des(1-3)IGFI on thymidine incorporation. IGF-I is almost ineffective in inducing DNA synthesis, demonstrating a weak, but significant effect only at 100 nM concentration (Fig. 6A). A rather similar effect is obtained with IGF-II (data not shown). On the contrary, the analog Des(1-3)IGF-I, which has a much lower affinity for IGFBPs (18), shows a sig-

FIG. 5. Effect of PAF (1 mM) and cPAF (1 mM) on IGFBP-3 mRNA steady-state transcript levels in HEC-1A cells at 4 and 24 hr stimulation. Ethidium bromide staining of total RNA loaded in each lane is shown below the Northern blot.

AP: Gyn Onc

338

GIANNINI ET AL.

FIG. 6. (A) Effect of 24 hr treatment with increasing concentrations of IGF-I (h) and Des(1-3)IGF-I (Ω) on [3H]thymidine incorporation in HEC1A cells. Error bars represent standard error of the indicated number of observations. (B) Effect of the monoclonal antibody aIR-3 (10 mg/ml) on basal and IGF-I (100 nM)-induced [3H]thymidine incorporation in HEC-1A cells. Error bars represent standard error of three different experiments performed in hexaplicate.

nificant effect on thymidine incorporation also at 1 and 10 nM concentrations (Fig. 6A). In order to prove that these effects are actually mediated through the type I IGF receptors, we tested the effect of the monoclonal antibody anti-IGF type I receptor aIR-3 on IGF-I-induced thymidine incorporation. As shown in Fig. 6B, in the presence of 10 mg/ml of the antibody, the stimulatory effect of IGFI (100 nM ) on thymidine incorporation was reduced to the control level. aIR-3 alone did not significantly modify thymidine incorporation (Fig. 6B). The effect of IGF-I and Des(1-3)IGF-I on PAF synthesis by HEC-1A cells (measured as [3H]acetate incorporation

FIG. 7. Synthesis of PAF (% increase over basal of [3H]acetate incorporation into PAF) in HEC-1A cells stimulated for 4 hr with IGF-I (100 nM) and Des(1-3)IGF-I (0.1–100 nM). Means { SEM of two different experiments are shown.

/ 6d0d$$4321

05-10-96 14:35:21

goa

into PAF) has also been studied. As shown in Fig. 7, PAF synthesis is stimulated by 2 hr incubation with 10 and 100 nM concentrations of Des(1-3)IGF-I, whereas IGF-I shows little stimulatory effect at 100 nM concentration. DISCUSSION

We have recently demonstrated an autocrine modulatory action of PAF in the endometrial adenocarcinoma cell line HEC-1A [16]. In the present study, we demonstrate for the first time a positive modulatory action of this lipid on IGFBP production by HEC-1A cells. Mathematical analysis of families of competition curves among [125I]IGF-I and analogs strongly indicates the presence of a homogenous class of binding sites apparently related to IGFBPs. A similar pattern of self- and cross-displacement curves has been reported in a different cell system, where the presence of IGFBP has been clearly shown [25]. The production and release of IGFBPs were demonstrated by Western blot analysis. Taken together, our results suggest the presence of IGFBPs both on the cell surface and in the conditioned medium. Indeed, ligand blot analysis identified two IGFBPs in HEC-1A cells, respectively, in the molecular weight ranges of 32–34 and 40–45 kDa, confirming previous results obtained by other authors [13, 15]. The larger 40- to 45-kDa protein band was identified as IGFBP-3 by immunoblot analysis using a polyclonal IGFBP-3 antiserum. Production of IGFBP-3 by these cells was also confirmed by detection of a specific mRNA for this protein by Northern blot analysis. The lower molecular weight IGFBP detected in our cells has been identified as IGFBP-2 by Western immunoblotting with anti-IGFBP-2 antibody, confirming

AP: Gyn Onc

PAF-INDUCED IGFBP SECRETION IN HEC-1A CELLS

previous results by Seppala et al. [26]. Incubation with micromolar concentrations of PAF induced an increase of the release of the two IGFBPs in the conditioned medium. Moreover, PAF induced a stimulation of IGFBP-3 gene expression, suggesting that the increased release of the 40- to 45kDa IGFBP might be accompanied by increased expression of the protein. This effect seems to be mediated by an interaction with a specific receptor, as indicated by the inhibitory effect of the PAF receptor antagonist L659,989. Two distinct classes of binding sites for PAF with affinity in the nanomolar and micromolar ranges were recently identified in HEC1A cells [16]. The effect of PAF on IGFBP production appears to be mediated by the low-affinity site, since nanomolar concentrations of PAF did not show any effect (Fig. 3A). With respect to the postreceptor mechanisms involved in the effect of PAF, we found that both the tyrosine kinase inhibitor genistein and the protein kinase C inhibitor sangivamycin were able to antagonize the effect of PAF, suggesting that multiple signaling pathways are involved in this action. Indeed, PAF has been shown to stimulate protein kinase C and tyrosine kinase activities in several cell types [27], and we have evidence for PAF-mediated increase of tyrosine phosphorylation of proteins in HEC-1A cells [28]. Based on these results, it is tempting to speculate that protein phosphorylation is an essential step in the regulation of IGFBP production and release by PAF in HEC-1A cells. In addition to PAF, release of IGFBPs by HEC-1A cells was also stimulated by IGF-I, as demonstrated by ligand blot and radioligand binding experiments. In particular, such an effect was more evident for the 40- to 45-kDa protein band. IGF-Imediated release of IGFBP-3 has been demonstrated in numerous other systems (29–32). As shown in several systems, the presence of IGFBPs strongly affects the biological activity of IGFs. In particular, IGFBP-3 has been shown to inhibit IGF-I action more commonly than it potentiates it [32, 33]. Furthermore, IGF-I binds with higher affinity to IGFBP-3 than to the IGF-I receptor [34]. In our cell line, the presence of IGFBPs is so relevant that the specific membrane receptor protein for IGF results is buried and not well characterized. In a previous report, the presence of [125I]IGF-I binding in HEC-1A cells has been shown; however, these binding sites were not characterized in such a study [13]. Moreover, in agreement with our finding, Kleinman et al. [36] recently reported, in a different endometrial cancer cell line, that about 95% of IGF-I binding to the cells is due to membrane-associated IGFBPs and that IGF-I is a weak stimulator of cell proliferation. In agreement with this observation, we report here that IGF-I and -II are very weak stimulators of thymidine incorporation and PAF synthesis in HEC-1A cells, whereas Des(1-3)IGF-I was more potent in inducing DNA and PAF synthesis than IGF-I. These findings indicate that the presence of IGFBPs in HEC-1A cells strongly inhibited the biological properties of IGFs. However, the biological effects

/ 6d0d$$4321

05-10-96 14:35:21

goa

339

of IGFs in HEC-1A cells are apparently mediated by specific IGF-I receptors, as suggested by the inhibitory effect of the anti-IGF-I receptor antibody aIR-3 on the slight mitogenic effect of IGF-I. In addition, IGF-I-induced higher release of IGFBPs may further limit its own action in these cells. Thus, the biological activity of the IGF system as growth-promoting factors in HEC-1A cells is questionable, especially when compared with that of PAF, which stimulates an approximately fourfold increase of thymidine incorporation [16]. In conclusion, present and previous results demonstrating a PAF-mediated autocrine loop in HEC-1A cells [16] support a major role for this phospholipid and a minor role for ‘‘classical’’ growth factors, such as IGFs, in the modulation of growth of this adenocarcinoma cell line. The physiological significance of this ‘‘unusual’’ PAF-mediated growth pathway remains unclear. ACKNOWLEDGMENTS This work was supported by grants from Consiglio Nazionale delle Ricerche (CNR, Rome, Targeted Project ACRO) and Associazione Italiana Ricerca sul Cancro (AIRC, Milan). Dr. Lorella Bonaccorsi is a recipient of a grant from AIRC.

REFERENCES 1. Murphy, L. J., and Ghahary, A. Uterine insulin-like growth factorI: Regulation of expression and its role in estrogen-induced uterine proliferation, Endocr. Rev. 11, 443–453 (1990). 2. Murphy, L. J., Bell, G. I., and Friesen, H. G. Tissue distribution of insulin-like growth factor I and II messenger ribonucleic acid in the adult rat, Endocrinology 120, 1279–1282 (1987). 3. Ghahary, A., and Murphy, L. J. Regulation of uterine insulin-like growth factor receptors by estrogen and variation throughout the estrous cycle, Endocrinology 125, 197–202 (1990). 4. Murphy, L. J., Murphy, L. C., and Friesen, H. G. Estrogen induces insulin-like growth factor I expression in the rat uterus, Mol. Endocrinol. 1, 445–450 (1987). 5. Nordstedt, G., Levinovitz, A., and Eriksson, H. Regulation of uterine insulin-like growth factor-I mRNA and insulin-like growth factor-II mRNA by estrogen in the rat, Acta Endocrinol. (Copenhagen) 120, 466–472 (1989). 6. Reynolds, R. K., Talavera, F., Roberts, J. A., Hopkins, M. P., and Menon, K. M. Regulation of epidermal growth factor and insulin-like growth factor I receptors by estradiol and progesterone in normal and neoplastic endometrial cell cultures, Gynecol. Oncol. 38, 396–406 (1990). 7. Von Eye Corleta, H., Strowitzki, T., Hellerer, M., and Haring, H. U. Insulin-like growth factor I-dependent tyrosine kinase activity in stromal cells of human endometrium in vitro, Am. J. Physiol. E 262, 863– 868 (1992). 8. Talavera, F., Reynolds, R. K., Roberts, J. A., and Menon, K. M. J. Insulin-like growth factor I receptors in normal and neoplastic human endometrium, Cancer Res. 50, 3019–3024 (1990). 9. Jones, J. J., and Clemmons, D. R. Insulin-like growth factors and their binding proteins: Biological actions, Endocr. Rev. 16, 3–34 (1995). 10. Seppala, M., Koistinen, R., and Rutanen, E. M. Uterine endocrinology and paracrinology: Insulin-like growth factor binding protein-I and placental protein 14 revisited, Hum. Reprod. 9, 917–925 (1994).

AP: Gyn Onc

340

GIANNINI ET AL.

11. Shimasaki, S., and Ling, N. Identification and molecular characterization of insulin-like growth factor binding proteins (IGFBP-1, -2, -3, 4, -5 and -6), Prog. Growth Factor Res. 3, 243–266 (1991). 12. Clemmons, D. R. Insulin-like growth factor binding proteins control secretion and mechanism of action, Adv. Exp. Med. Biol. 293, 113– 123 (1991). 13. Pekonen, F., Nyman, T., and Rutanen, E.-M. Human endometrial adenocarcinoma cell line HEC-1B and KLE secrete insulin-like growth factor binding protein-1 and contain IGF-I receptors, Mol. Cell. Endocrinol. 75, 81–87 (1991). 14. Gong, Y., Ballejo, G., Alkhalaf, B., Molnar, P., Murphy, L. C., and Murphy, L. J. Phorbol esters differentially regulate the expression of insulin-like growth factor binding proteins in endometrial carcinoma cells, Endocrinology 131, 2747–2754 (1992). 15. Lamson, G., Oh, Y., Pham, H., Giudice, L. C., and Rosenfeld, R. G. Expression of two insulin-like growth factor binding proteins in a human endometrial cancer cell line: Structural, immunological, and genetic characterization, J. Clin. Endocrinol. Metab. 69, 852–859 (1989).

25.

26.

27. 28.

16. Maggi, M., Bonaccorsi, L., Finetti, G., Carloni, V., Muratori, M., Laffi, G., Forti, G., Serio, M., and Baldi, E. Platelet-activating factor mediates an autocrine proliferative loop in the endometrial adenocarcinoma cell line HEC-1A, Cancer Res. 54, 4777–4784 (1994).

29.

17. Baldi, E., Bonaccorsi, L., Finetti, G., Luconi, M., Muratori, M., Susini, T., Forti, G., Serio, M., and Maggi, M. Platelet-activating factor in human endometrium, J. Steroid Biochem. Mol. Biol. 49, 359–363 (1994).

30.

18. Ross, M., Francis, G. L., Szabo, L., Wallace, J. C., and Ballard, F. J. Insulin-like growth factor (IGF) binding proteins inhibit the biological activities of IGF-I and IGF-II but not des-(1-3)-IGF-I, Biochem. J. 258, 267–272 (1989).

31.

19. Mohan, S., and Baylink, D. J. Evidence that the inhibition of TE85 human bone cell proliferation by agents which stimulate cAMP production may in part be mediated by changes in the IGF-II regulatory system, Growth Regul. 1, 110–118 (1991).

32.

20. Bligh, E. G., and Dyer, W. J. A rapid method of total lipid extraction and purification, Can. J. Biochem. Pharmacol. 37, 911–917 (1959). 21. Hossenlopp, P., Seurin, D., Segovia-Quinson, B., Ardouin, S., and Binoux, M. Analysis of serum insulin-like growth factor binding proteins using Western ligand blotting: Use of the method for titration of the binding proteins and competitive binding studies, Anal. Biochem. 154, 138–143 (1986). 22. Munson, P., and Rodbard, D. LIGAND: A versatile computerized approach for characterization of ligand-binding system, Anal. Biochem. 107, 220–239 (1980). 23. Goldstein, S., Moerman, E. J., Jones, R. A., and Baxter, R. C. Insulinlike growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblast, Proc. Natl. Acad. Sci. USA 88, 9680–9684 (1991). 24. Giannini, S., Mohan, S., Kasuya, J., Galli, G., Rotella, C. M., LeBon, T. R., and Fujita-Wamaguichi, Y. Characterization of insulin-like

/ 6d0d$$4321

05-10-96 14:35:21

goa

33.

34.

35.

36.

growth factor-binding proteins produced by cultured fibroblasts from patients with noninsulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus or obesity, J. Clin. Endocrinol. Metab. 79, 1824– 1830 (1994). Velez-Yanguas, M. C., Kalebic, T., Maggi, M., Chavez Kappel, C., Letterio, J., Uskokovic, M., and Helman, L. J. 1a-dihydroxy-16-ene23-yne-26,27-hexafluorocholecalciferol (Ro24-5531) modulation of IGFBP3 and induction of differentiation and growth arrest in a human osteosarcoma cell line, J. Clin. Endocrinol. Metab. 81, 93–99 (1996). Seppala, M., Angervo, M., Riittinen, L., Yajima, M., Julkunen, M., and Koistinen, R. Peptides and proteins in the human endometrium, Reprod. Med. Rev. 1, 37–55 (1992). Chao, W., and Olson, M. S. Platelet-activating factor: Receptors and signal transduction, Biochem. J. 292, 617–629 (1993). Bonaccorsi, L., Luconi, M., Finetti, G., Muratori, M., Maggi, M., Forti, G., and Baldi, E. Tyrosine kinase activation is involved in PAF-mediated proliferation of the endometrial adenocarcinoma cell line HEC1A, 4th International Symposium on Endocrinology under 35, Rome, May 25–27, Abstract Book, p. 50. Smith, E. P., Cheung, P. T., Ferguson, A., and Chernausek, S. D. Mechanism of Sertoli cell insulin-like growth factor (IGF)-binding protein-3 regulation by IGF-I and adenosine 3*,5*-monophosphate, Endocrinology 131, 2733–2741 (1992). Martin, J. L., Ballesteros, M., and Baxter, R. C. Insulin-like growth factor-I (IGF-I) and transforming growth factor-b1 release IGF-binding protein-3 from human fibroblasts by different mechanism, Endocrinology 131, 1703–1710 (1992). Oh, Y., Muller, H. L., Pham, H., Lamson, G., and Rosenfeld, R. G. Non-receptor mediated, post-transcriptional regulation of insulin-like growth factor binding protein (IGFBP)-3 in Hs578T human breast cancer cells, Endocrinology 131, 3123–3125 (1992). Camacho-Hubner, C., Busby, W. H., Jr., McCusker, R. H., Wright, G., and Clemmons, D. R. Identification of the forms of insulin-like growth factor binding proteins produced by human fibroblasts and the mechanisms that regulate their secretion, J. Biol. Chem. 267, 11949–11956 (1992). Conover, C. A., Ronk, M., Lombana, F., and Powell, D. R. Structural and biological characterization of bovine insulin-like growth factor binding protein-3, Endocrinology 127, 2795–2803 (1990). De Mellow, J. S. M., and Baxter, R. C. Growth hormone-dependent insulin-like growth factor (IGF) binding protein both inhibits and potentiates IGF-I stimulated DNA synthesis in human skin fibroblasts, Biochem. Biophys. Res. Commun. 156, 199–204 (1988). Conover, C. A. Glycosilation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: Evidence for processing of cell-bound IGFBP-3, Endocrinology 129, 3259–3268 (1991). Kleinman, D., Karas, M., Roberts, C. T., LeRoith, D., Phillip, M., Segev, Y., Levy, J., and Sharoni, Y. Modulation of insulin-like growth factor I (IGF-I) receptors and membrane-associated IGF-binding proteins in endometrial cancer cells by estradiol, Endocrinology 136, 2531–2537 (1995).

AP: Gyn Onc