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Endocrinology Vol. 140, No. 2 997-1004
Copyright © 1999 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factor Binding Protein-1 Expression in Baboon Endometrial Stromal Cells: Regulation by Filamentous Actin and Requirement for de Novo Protein Synthesis

J. Julie Kim1, R. C. Jaffe and A. T. Fazleabas

Departments of Obstetrics and Gynecology (J.J.K., A.T.F.) and Physiology and Biophysics (R.C.J., A.T.F.), University of Illinois at Chicago, Chicago, Illinois 60212-7313

Address all correspondence and requests for reprints to: Dr. A. T. Fazleabas, Department of Obstetrics and Gynecology, University of Illinois at Chicago, 820 South Wood Street (M/C 808), Chicago, Illinois 60612. E-mail: asgi{at}uic.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stromal fibroblasts in the primate endometrium undergo dramatic morphological and biochemical changes in response to pregnancy. This transformation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1). Stromal cells from the baboon endometrium of nonpregnant animals were cultured and subsequently treated with cytochalasin D to disrupt actin filaments. In response to cytochalasin D treatment, cells contracted and became rounded as early as 10 min after the initiation of treatment. When cytochalasin D was removed, cells reverted back to their original fibroblastic shape within 1 h. After cells were treated with cytochalasin D for 5 h, addition of (Bu)2cAMP and/or hormones (estradiol, medroxyprogesterone acetate, and relaxin) resulted in the expression of IGFBP-1 messenger RNA and protein within 24 h. Cells with an intact cytoskeleton did not express detectable levels of IGFBP-1 in response to hormones and/or (Bu)2cAMP. Furthermore, the addition of cycloheximide inhibited expression of IGFBP-1 in cytochalasin D-treated cells. Stromal cells were also isolated from early pregnant and simulated pregnant animals. Within 48 h, cells from both the pregnant and simulated pregnant animals produced IGFBP-1 in response to hormones and/or (Bu)2cAMP. In these studies, IGFBP-1 expression was also inhibited by cycloheximide. These studies suggest that induction of IGFBP-1 requires an intermediary protein and that alterations in the cytoskeleton may be involved.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE GROWTH FACTOR binding protein-1 (IGFBP-1) is the major secretory product of the human and baboon decidualized endometrium (1, 2) and, thus, has served as a biochemical marker for this process. The decidual response, which is characterized by the differentiation of fibroblast-like mesenchymal cells in the uterine stroma to epithelioid-like cells, is initiated in the human during the luteal phase under the influence of estradiol, progesterone, and relaxin (3). Stromal edema is observed on day 23 of the menstrual cycle and is followed 3–4 days later by a predecidual reaction that begins around the spiral arteries and spreads through the upper two-thirds of the endometrium. In contrast, the baboon does not undergo a predecidual reaction during the menstrual cycle (4, 5). However, after implantation, the stromal fibroblasts undergo extensive modification to form the decidua.

The sequence of biochemical and molecular events associated with the transformation of a stromal fibroblast to a secretory decidual cell remains unclear. We have previously demonstrated that the baboon endometrium undergoes a complex series of changes during the establishment of pregnancy and that the conceptus is essential to regulate these changes (6, 7). Specifically, stromal fibroblasts sequentially express {alpha}-smooth muscle actin ({alpha}SMA), a cytoskeletal protein, followed by IGFBP-1 (6, 7, 8). {alpha}SMA and IGFBP-1 are not expressed in stromal fibroblasts during the menstrual cycle in the baboon.

Cytoskeletal proteins are important in mitosis, cell growth, changes in cell shape, and for the regulation of protein secretion (9). Several studies have demonstrated the importance of the actin cytoskeleton or actin binding proteins in cellular differentiation of granulosa cells (10), chondrocytes (11), and HL-60 cells (12). In the baboon, specific staining for {alpha}SMA was observed in the stromal fibroblasts during early pregnancy or simulated pregnancy (8). As cells subsequently expressed IGFBP-1 as part of the decidualization process, {alpha}SMA staining disappeared. This phenomenon has also been demonstrated in vitro (13). Stromal cells from nonpregnant baboons expressed {alpha}SMA spontaneously after attachment and spreading. After 17 days of continuous hormone and (Bu)2cAMP treatment, significant levels of IGFBP-1 were produced with a corresponding decrease in {alpha}SMA expression. This transitory expression of {alpha}SMA demonstrated in vivo and in vitro and its association with an alteration in cell morphology and the appearance of IGFBP-1 in pregnant baboons suggested that {alpha}SMA is associated with the transformation of a stromal fibroblast to a decidual cell. We hypothesize that restructuring of the cytoskeleton is required for expression of the IGFBP-1 gene. In the present study, we have demonstrated that disruption of the cytoskeleton in vitro potentiates the expression of IGFBP-1. Furthermore, this expression requires de novo protein synthesis. As a comparison, the expression of IGFBP-1 in stromal cells from pregnant and simulated pregnant animals was also analyzed. In vivo priming of these cells by pregnancy-associated factors induces the expression of {alpha}SMA in the stromal fibroblasts (8). Induction of IGFBP-1 and the associated down-regulation of {alpha}SMA occurs at the site of implantation and requires the presence of the conceptus (13, 14). The in vitro studies described herein also demonstrate that (Bu)2cAMP potentiates IGFBP-1 expression in stromal cells that are expressing {alpha}SMA in vivo with a requirement for de novo protein synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All cell culture supplies were obtained from Gibco BRL (Gaithersburg MD). Other reagents of cell culture grade were purchased from Fisher Scientific International, Inc. (Itasca, IL), Sigma Chemical Co. (St. Louis, MO), or Boehringer Mannheim (Indianapolis, IN).

Cell culture
Endometrial tissues were obtained from cycling (day 9–10 postovulation), human CG (hCG)-treated (simulated pregnant), and day 22 pregnant baboons (Papio anubis) by endometriectomy or after hysterectomy. All animal studies were approved by the Animal Care Committee of the University of Illinois at Chicago. The day of ovulation was designated to be 2 days after the estradiol surge and was confirmed by prospective measurement of peripheral serum levels of estradiol and progesterone (14). To simulate pregnancy, normally cycling animals received increasing dosages of recombinant hCG (Serono Laboratories, Inc., Norwell, MA). This treatment regimen resulted in peripheral levels of hormones comparable to those measured on day 18 of pregnancy (14).

The tissue was minced thoroughly in calcium- and magnesium-free HBSS and placed in an enzyme solution containing 0.5% collagenase and 0.02% deoxyribonuclease (DNase). They were incubated for 20 min at 37 C in a shaking water bath. The supernatant was recovered and placed at 4 C. The remaining tissue was further digested in an enzyme solution consisting of 0.5% collagenase, 0.02% DNase, 0.1% hyaluronidase, and 0.1% pronase and processed as described above. The cell suspensions from the first and second digestions were centrifuged at 1,500 x g for 5 min, and the pellet was resuspended in HBSS. The cells were then passed through a 95-µm nylon mesh folllowed by a 20-µm nylon mesh (Small Parts, Inc., Miami Lakes, FL). The filtrates were pooled and centrifuged again at 1,500 x g for 5 min. The pellet was resuspended in 30% Percoll solution and overlayed on top of a 60% Percoll solution. After centrifugation at 800 x g for 30 min, the cells that sedimented at the 30%–60% interface were collected. The cells were then resuspended in phenol red-free RPMI-1640 supplemented with 0.1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS depleted of steroids by treatment with dextran-coated charcoal (stripped). Cells were seeded at 1 x 105 cells/cm2 and incubated overnight at 37 C and 5% C02. The medium was changed the next day and subsequently every 3 days. Cell purity was assessed by immunocytochemistry using antibodies against cytokeratin (Dako Corp., Carpenteria, CA), vimentin (Zymed Laboratories, Inc., San Francisco, CA) and Factor VIII (Dako Corp.). The purity of the stromal cell preparations used in these studies was more than 97%.

Treatment of cells
At approximately 80% confluency, the cell culture medium was changed to RPMI-1640 supplemented with sodium pyruvate, penicillin/streptomycin, 10% stripped FBS, with or without 5 µM cytochalasin D. Cytochalasin D is a specific inhibitor of the actin microfilament assembly (15). Photographs of cells were taken at 10 min, 30 min, 50 min, 100 min, 2 h, 3 h, 4 h, and 5 h of cytochalasin D treatment. Cells were also treated with 6.7 µM nocodazole, a microtubule-dissociating drug, for 5 h. Medium was then changed to RPMI-1640, 2% stripped FBS, 5 µM cytochalasin D, with or without hormones (36 nM 17ß-estradiol, 1 µM MPA, and 100 ng/ml highly purified porcine relaxin, kindly provided by Dr. David Sherwood, University of Illinois, Urbana), and with or without 0.1 mM (Bu)2cAMP. Treatment continued for an additional 24 or 48 h. For cycloheximide studies, 5 h after initiation of cytochalasin D treatment, cells were pretreated with cycloheximide for 1 h by changing medium to RPMI-1640 plus 2% stripped FBS, 5 µM cytochalasin D, and 10 µg/ml cycloheximide. The concentrations of cytochalasin D and nocodazole used in these studies were based on previous reports describing actin-mediated gene transcription in fibroblasts (15, 16). The concentrations were confirmed as being nontoxic to the cells by the measurement of lactate dehydrogenase (LDH) in the media at various time points. Cyclohexamide was used at a concentration that had been previously reported to inhibit IGFBP-1 synthesis in vitro (17).

After pretreatment, the medium was changed again to RPMI-1640, 2% stripped FBS, 10 µg/ml cycloheximide, 5 µM cytochalasin D, with or without hormones, with or without 0.1 mM (Bu)2cAMP for 24 and 48 h. The medium was collected and IGFBP-1 present in the culture medium was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Diagnostic Systems Laboratories, Inc., Webster, TX). Cells were lysed with TriReagent (Molecular Research Center, Inc., Cincinnati, OH) and RNA and protein were extracted using the protocol provided by the manufacturer. Cellular protein content was measured using the Micro BCA protein assay kit (Pierce, Rockford, IL).

RT-PCR
The RNA preparation isolated from cells was treated with 1 U/µl DNase (Promega Corp.). RT was then performed in a final volume of 20 µl with 1 µg RNA and 50 U/µl MuLV reverse transcriptase at 42 C for 30 min. PCR amplification was performed with the RT product, the appropriate primers (50 pmol/tube), 0.5 µl of 5 U/ml Taq polymerase (Gibco BRL), and 0.25 µl of 10 mCi/ml [{alpha}32P]deoxycytosine triphosphate. After an initial incubation at 94 C for 10 min, 24 amplification cycles consisting of 94 C (1 min), 60 C (2 min), and 72 C (3 min) were performed followed by 15 min of final extension at 72 C. Primers for H3.3, IGFBP-1, and {alpha}SMA, described in Kim et al. (13), were added together in a single tube so that amplification of the cDNAs for H3.3, IGFBP-1, and {alpha}SMA occurred in the same tube. The PCR products were electrophoresed in 1.5% agarose gels, and the gels were dried and exposed to film.

Immunofluorescent staining
Stromal cells isolated from baboons on day 10 post ovulation were grown on glass coverslips. Cells were treated with cytochalasin D, as described above, followed by incubation in the presence or absence of 0.1 mM (Bu)2cAMP for 72 h. Control cells were not treated with cytochalasin D. The additional 24-h incubation time for the immunocytochemical studies was to ensure that we could detect an effect of (Bu)2cAMP on IGFBP-1 synthesis in the control cells (13).

At the end of the incubation period the medium was removed and cells were washed three times with PBS. Cells were fixed in 4% paraformaldehyde for 10 min and washed three times with PBS. The cells were then permeabilized with 0.1% Triton X-100/0.1% deoxycholate in PBS for 10 min. After a PBS wash the permeabilized cells were incubated with a filtered solution of 1% BSA in PBS for 10 min. A monoclonal IGFBP-1 antibody [1:1000 in 1% BSA in PBS (17)] was added to the cells and incubated overnight at 4 C. Cells were washed three times with PBS and incubated with a fluorescein-conjugated antimouse IgG second antibody (10 µg/ml; Vector Laboratories, Inc., Burlingame, CA). In conjuction with the fluorescein-conjugated second antibody, rhodamine-labeled phalloidin (1:100; Sigma Chemical Co.) was also added. Incubations were done in the dark at room temperature for 1 h. The cells were washed with PBS and the cover slips were mounted on glass microscope slides with a drop of Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA). Fluorescence corresponding to the antigen-antibody complexes for actin and IGFBP-1 were photographed using an inverted IM35 fluorescent microscope (Carl Zeiss, Thornwood, NY). Double exposures for the colocalization of actin and IGFBP-1 were created by superimposing the rhodamine signal (exposure time 1 sec) on the IGFBP-1 fluorescence signal (exposure time 5 sec) using Kodak Gold 200 ASA color film (Eastman Kodak Co., Rochester, NY).

Statistical analysis
Data were analyzed by least-squares ANOVA using the SuperANOVA software package (Abacus Concepts, Inc., Berkeley, CA). Sources of variation included experiment (defined as tissues from different animals), treatment (presence or absence of hormones/(Bu)2cAMP), drug (presence or absence of cytochalasin D or nocodazole), and their interactions. Data were log transformed, and orthogonal contrasts were used to compare the means of the different treatments with their corresponding controls. The pattern of IGFBP-1 protein expression at 24 h and 48 h were the same and thus the data at these two time points were pooled (Fig. 4AGo).



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  		Figure 4. Effect of the disruption of the actin filaments and microtubules on IGFBP-1 expression. Near-confluent stromal cells from a day 10 postovulatory endometrium were treated with either 5 µM cytochalasin D or 6.7 µM nocodazole for 5 h. Medium was changed to fresh RPMI-1640 + 2% stripped FBS, 5 µM cytochalasin D, or 6.7 µM nocodazole, with or without 0.1 mM (Bu)2cAMP, with or without hormones (36 nM estradiol, 1 µM MPA, and 100 ng/ml relaxin) for 24 or 48 h. A, IGFBP-1 protein released into the culture medium was measured using ELISA. Data are expressed as least squares means ± SEM of three experiments. * Denotes values statistically different from the corresponding controls (P <= 0.05). B, Total RNA from cells treated as described in Materials and Methods was isolated and subjected to RT-PCR using primers for {alpha}SMA, IGFBP-1, and the internal standard H3.3. cDNA products were run on 1.5% agarose gels. Treatments: culture medium, lanes 1, 5, and 9; (Bu)2cAMP only, lanes 2, 6, and 10; hormones only, lanes 3, 7, and 11; (Bu)2cAMP + hormones, lanes 4, 8, and 12. Note the marked increase in mRNA expression and protein synthesis in response to (Bu)2cAMP after the disruption of the cytoskeleton (lanes 6 and 8). C, Addition of cycloheximide (10 µg/ml) after 5 h of incubation with cytochalasin D to the treatment paradigms described in lanes 1–8 in Fig. 4BGo. The isolated RNA was subjected to RT-PCR. Treatments: culture medium, lanes 1 and 5; (Bu)2cAMP only, lanes 2 and 6; hormones only, lanes 3 and 7; (Bu)2cAMP + hormones, lanes 4 and 8. Note the inhibition of IGFBP-1 gene transcription in the presence of cyclohexamide (compare lanes 6 and 8 in B and C).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Morphological changes of baboon stromal cells in response to cytochalasin D
Stromal cells from the baboon endometrium grown in culture exhibited fibroblast-like morphology (Fig. 1AGo) similar to endometrial stromal cells from other species. Within 10 min of the addition of 5 µM cytochalasin D, cytoplasmic contractions were apparent under the phase-contrast microscope (Fig. 1BGo). Cells continued to contract (Fig. 1Go, C–I) and by 5 h, cells had lost their fibroblast-like shape and had become rounded with numerous thin filopodia (Fig. 1IGo). Cell viability at this time was assessed by assaying for LDH in the culture media. No LDH activity was detected.



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  		Figure 1. Effect of cytochalasin D treatment on the morphology of baboon endometrial stromal cells. Near-confluent stromal cells from a day 10 postovulatory baboon endometrium were treated with 5 µM cytochalasin D for 0 min (A), 10 min (B), 30 min (C), 50 min (D), 100 min (E), 2 h (F), 3 h (G) 4 h (H), and 5 h (I). Note the complete disruption of the actin microfilaments at the end of the 5 h. Magnification, x200.

 
After 5 h of cytochalasin D treatment, the drug was removed and replaced with fresh medium containing no cytochalasin D (Fig. 2Go). Within 1 h of drug removal, cells reverted back to their fibroblastic shape (Fig. 2FGo), although the morphology was not identical to untreated stromal cells (Fig. 1AGo), i.e. stress fibers (Fig. 2FGo) were not as evident as those of the untreated cells (Fig. 1AGo). To determine whether these morphological changes were specifically associated with the disruption of the actin filaments, cells were also treated with the microtubule dissociating drug, nocodazole (6.7 µM). The morphological changes associated with this treatment (Fig. 3BGo) were not significantly different from those observed in untreated cells (Fig. 3AGo). Cells remained fibroblastic.



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  		Figure 2. Effects of the removal of cytochalasin D on stromal cell morphology. After 5 h of cytochalasin D treatment, medium was changed to RPMI-1640 + 2% stripped FBS, and photographs were taken at 0 min (A), 10 min (B), 20 min (C), 30 min (D), 40 min (E), and 60 min (F). The cells revert back to their original fibroblastic morphology after the removal of cytochalasin D. Magnification, x200.

 


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  		Figure 3. Effect of nocodazole treatment on the morphology of baboon endometrial stromal cells. Near-confluent stromal cells from a day 10 postovulatory baboon endometrium were treated with 6.7 µM nocodazole for 5 h. Untreated (A) and nocodazole-treated (B) cells are shown. Treatment with a microtubule inhibitor has no effect on the cytoskeletal morphology. Magnification, x200.

 
Disruption of actin filaments and microtubules induces IGFBP-1 expression
We previously demonstrated that baboon stromal cells isolated from nonpregnant baboons required at least 6 days of continuous treatment with the hormones estradiol, MPA, and relaxin, in addition to (Bu)2cAMP, to initiate decidualization as assessed by expression and synthesis of IGFBP-1 (13). IGFBP-1 mRNA was not detectable by RT-PCR or protein by ELISA in stromal cells cultured for 24 h with hormones and/or (Bu)2cAMP (Fig. 4Go, A and B). In contrast, when cells were treated with cytochalasin D, further treatment with (Bu)2cAMP only (P <= 0.05), hormones only (P <= 0.05), and hormones in conjunction with (Bu)2cAMP (P <= 0.05), increased IGFBP-1 protein (Fig. 4AGo) and mRNA (Fig. 4BGo) expression within 24 and 48 h. Cells with disrupted actin filaments that were treated with hormones alone produced lower levels of IGFBP-1 compared with treatments with (Bu)2cAMP alone and hormones plus (Bu)2cAMP. When cells resumed their fibroblastic shape after the withdrawal of cytochalasin D, IGFBP-1 was induced in response to hormones and (Bu)2cAMP, irrespective of repolymerization of actin microfilaments, albiet to a significantly lower concentration (6.7 ng/ml) than in the presence of cytochalasin D (26 ng/ml). The protein concentrations were reflective of the IGFBP-1 mRNA levels as determined by semiquantitative RT-PCR (data not shown).

The localization of IGFBP-1 in cells treated with (Bu)2cAMP with or without the disruption of the cytoskeleton is shown in Figure 5Go. As we had previously demonstrated, incubation of stromal fibroblasts with (Bu)2cAMP induces low levels of IGFBP-1 [Fig. 5BGo (13)]. In contrast, the disruption of the cytoskeleton with cytochalasin D results in a marked increase in IGFBP-1 immunolocalization (Fig. 5DGo). In the absence of (Bu)2cAMP, no staining for IGFBP-1 is evident (Fig. 5Go, A and C).



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  		Figure 5. Immunofluorescent colocalization of actin and IGFBP-1. Near-confluent stromal cells obtained at day 10 post ovulation were treated with (C and D) or without (A and B) cytochalasin D for 5 h. Fresh RPMI 1640 + 2% stripped FBS with (B and D) or without (A and C) (Bu)2cAMP was added to the cells. Cells shown in panels A and B are controls and cells shown in panels C and D were treated with cytochalasin D throughout the 72-h incubation period. In the presence of cytochalasin D, cells treated with (Bu)2cAMP showed intense staining for IGFBP-1 (arrowheads in panel D). In the absence of cytochalasin D, a few stromal cells showed the characteristic perinuclear localization of IGFBP-1 (arrowhead, panel B, and Ref. 6). Magnification, x2200.

 
To compare the effects of actin filament vs. microtubule disruption on IGFBP-1 expression, cells were treated with the microtubule-dissociating drug, nocodazole. IGFBP-1 expression was evident in nocodazole-treated cells after exposure to hormones in conjunction with (Bu)2cAMP (P <= 0.05). IGFBP-1 expression in response to (Bu)2cAMP alone and hormones alone did not significantly differ from control cells with the same treatments. In addition, levels of IGFBP-1 expression in nocodazole-treated cells in response to hormones plus (Bu)2cAMP were much lower than that observed for cytochalasin D- treated cells.

IGFBP-1 expression requires de novo protein synthesis
After treatment with cytochalasin D, cycloheximide was added to the cells together with the different hormone treatments. If cells in which the actin filaments were disrupted by cytochalasin D treatment were exposed to cycloheximide at the same time as hormones and/or (Bu)2cAMP addition, the IGFBP-1 mRNA levels remained below the limits of detection. This suggests that de novo protein synthesis is required for IGFBP-1 mRNA to be expressed in response to hormones and/or (Bu)2cAMP.

Physiological priming in vivo promotes IGFBP-1 induction in vitro
Our data show that IGFBP-1 is induced in response to hormones and (Bu)2cAMP in stromal cells isolated from nonpregnant baboons within 24–48 h after the disruption of the cytoskeleton. We hypothesized that in response to pregnancy or simulated pregnancy, stromal cells are modulated in vivo, which allows for the expression of IGFBP-1 in vitro. Stromal cells from early pregnant and simulated pregnant (long-term hCG treatment) baboons were isolated and grown to 80% confluency. They were then treated with (Bu)2cAMP alone, hormones alone, and hormones plus (Bu)2cAMP. Control cells were treated with media alone. Control cells from pregnant baboons expressed low levels of IGFBP-1 (Fig. 6Go). Within 48 h, cells that were treated with (Bu)2cAMP alone, hormones alone, and hormones plus (Bu)2cAMP expressed IGFBP-1. Cycloheximide treatment inhibited IGFBP-1 expression, implicating the involvement of an intermediate proteinaceous factor(s). Stromal cells isolated from long-term hCG-treated baboons also expressed IGFBP-1 mRNA and protein in response to (Bu)2cAMP, hormones, and hormones plus (Bu)2cAMP (Fig. 7Go). Cycloheximide inhibited IGFBP-1 expression in all treatment groups. In addition, the absence of measurable IGFBP-1 protein in the conditioned media (Figs. 6AGo and 7AGo) also indicates that cyclohexamide was effective in blocking any additional protein synthesis downstream of the transcriptional response to (Bu)2cAMP. It is interesting to note that in both pregnant and hCG-treated baboons, the stromal cells produced more IGFBP-1 in response to hormones alone than in response to (Bu)2cAMP alone. In cytochalasin D-treated cells from nonpregnant baboons, IGFBP-1 production was greater with (Bu)2cAMP treat-ment alone than with hormones alone.



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  		Figure 6. Effect of pregnancy on IGFBP-1 expression. Near-confluent stromal cells from a day 22 pregnant endometrium were treated with or without 0.1 mM (Bu)2cAMP and with or without hormones (36 nM estradiol, 1 µM MPA, and 100 ng/ml relaxin) for 48 h. A, IGFBP-1 protein released into the culture medium was measured using ELISA. B, Total RNA from cells treated as described above was isolated and subjected to RT-PCR using primers for {alpha}SMA, IGFBP-1, and H3.3 (lanes 1–4). Cells were also treated with cycloheximide together with the above treatments (lanes 5–8). Treatments: culture medium, lanes 1 and 5; (Bu)2cAMP only, lanes 2 and 6; hormones only, lanes 3 and 7; (Bu)2cAMP + hormones, lanes 4 and 8.

 


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  		Figure 7. Effect of long-term hCG treatment in vivo on IGFBP-1 expression in vitro. Near-confluent stromal cells from an hCG-treated baboon were treated with or without 0.1 mM (Bu)2cAMP, and with or without hormones (36 nM estradiol, 1 µM MPA, and 100 ng/ml relaxin) for 48 h. A, IGFBP-1 protein released into the culture medium was measured using ELISA. Data are expressed as least squares means ± SEM of two experiments. B, Total RNA from cells treated as described above was isolated and subjected to RT-PCR using primers for {alpha}SMA, IGFBP-1, and H3.3 (lanes 1–4). Cells were also treated with cycloheximide together with the above treatments (lanes 5–8). Treatments: culture medium, lanes 1 and 5; (Bu)2cAMP only, lanes 2 and 6; hormones only, lanes 3 and 7; (Bu)2cAMP + hormones, lanes 4 and 8.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The decidualization process both in vivo and in vitro is characterized by the expression of the decidual marker IGFBP-1. It has been shown that IGFBP-1 expression requires days or weeks to manifest itself under the appropriate hormonal stimulus (18, 19, 20). In the baboon, this process is mediated by the long-term treatment with hormones and (Bu)2cAMP and is associated with the down-regulation of {alpha}SMA (13). In this study we demonstrated that IGFBP-1 can be induced by (Bu)2cAMP within 24–48 h after the disruption of the cytoskeleton suggesting that the presence of actin inhibits IGFBP-1 gene expression. This was shown by both pharmacologically disrupting the cytoskeleton in vitro and by activating stromal cells isolated during pregnancy or hormonally simulated pregnancy that are expressing {alpha}SMA in vivo. Furthermore, we showed that the expression of IGFBP-1 under these experimental conditions requires the synthesis of an intermediary protein(s).

Decidualization in the baboon requires the presence of the conceptus (6). Furthermore, stromal cells isolated from a receptive baboon endometrium require at least 12–17 days of continuous hormone (estradiol, MPA, relaxin) and (Bu)2cAMP treatment to produce significant levels of IGFBP-1 (13). What happens within these cells in response to hormones over such an extended time span remains an enigma. Here, we showed that the disruption of the actin filaments or microtubules with cytochalasin D or nocodazole, respectively, increased the level of IGFBP-1 expression in response to (Bu)2cAMP. The role of the cytoskeleton in regulating gene expression has been reported by other investigators. Carbajal and Vitale (21) suggested that the cortical actin cytoskeleton of cultured lactotropes is involved in the control of PRL secretion. Local dismantling of the cortical actin network permits the excluded granules to reach the plasma membrane for exocytosis. Tomasek et al. (15) demonstrated that disruption of the cytoskeleton by cytochalasin D in fibroblasts dramatically increased gelatinase A activity, a matrix metalloproteinase involved in both connective tissue remodeling and tumor invasion. Nocodazole treatment, however, did not promote gelatinase A activation. Disruption of the cytoskeleton has also been shown to induce transcription of the c-fos gene within 15 min of cytochalasin D treatment (22). Whether the disruption of the cytoskeleton using pharmacological agents and the events that follow mirrors normal physiological processes associated with decidualization is not known at this time. The association of the cytoskeleton with IGFBP-1 gene regulation is possible, given that the cytoskeleton is important in cell growth, changes in cell shape, regulation of protein secretion, and cell signaling (9). Furthermore, {alpha}SMA, a cytoskeletal actin protein that is induced in predecidualizing baboon endometrial stromal cells disappears when the cells express IGFBP-1 (6, 8).

Baboon endometrial stromal cells in culture require continuous exposure to hormones and (Bu)2cAMP for 6 days before detectable levels of IGFBP-1 are observed (13). Disruption of the actin filaments and microtubules promoted IGFBP-1 expression within 24 h of hormone and (Bu)2cAMP treatment. Assuming that (Bu)2cAMP is acting on protein kinase A (PKA) in baboon stromal cells, as has been shown in human endometrial stromal cells (23), it is possible that the cytoskeleton inhibits PKA from acting on target molecules. Lester et al. (24) demonstrated that the hormone, glucagon-like peptide 1, induces insulin secretion from pancreatic cells via the cAMP-signaling pathway and that the correct subcellular location of PKA was necessary for this process. The selectivity of PKA action has been attributed to proteins that bind and anchor PKA to specific subcellular structures, termed A-kinase anchor proteins (AKAPs) (reviewed in Ref. 25). In neuronal and nonneuronal cells, AKAPs are associated with the cytoskeleton (26, 27). Perhaps the anchoring of PKA through AKAPs to the correct subcellular location or the release of PKA from the cytoskeletal complex is important in the cAMP-signaling pathway for the regulation of IGFBP-1 expression.

The mechanism of induction of the IGFBP-1 gene by (Bu)2cAMP is not known. Telgmann et al. (23) demonstrated that the PRL promoter, when transfected into human endometrial stromal cells, is activated by cAMP in two steps. There is an initial weak induction after 12 h of stimulation and a subsequent, more pronounced induction. While the first induction depended on the cAMP-response element (CRE), the second induction was not driven by the CRE. They proposed that an intermediate endometrial stromal cell-specific factor(s) was involved in inducing the second major transcriptional response of the PRL promoter. In our study, we demonstrated that inhibition of de novo protein synthesis by cycloheximide inhibits the expression of IGFBP-1 in response to hormone/(Bu)2cAMP treatments. This was shown in both the cytochalasin D- treated cells and in cells from pregnant and simulated pregnant baboons. It is possible that cAMP is required to induce an intermediary protein, acting as a transcription factor(s) either directly or indirectly on the IGFBP-1 promoter to regulate its expression. It has been demonstrated that hepatocyte nuclear factor-1 and the glucocorticoid receptor synergistically activate transcription of the IGFBP-1 gene in the rat (28). The Sp3 protein, found in human decidual cells, has been implicated in the transcriptional regulation of IGFBP-1 by acting on distal promoter regions [~-2.6 kb (29)]. Finally, given that cAMP induces IGFBP-1 expression in our system, it is possible that the transcription factor CREB (CRE-binding protein) may be the required protein that is synthesized. However, Gellersen et al. (30) demonstrated that CREB was equally expressed in both nondecidualized and in decidualized human endometrial stromal cells. Furthermore, Mukherjee et al. (31) demonstrated that in the rat ovary, CREB mRNA and protein levels change only minimally in response to gonadotropins but that the phosphorylation of preexisting CREB protein on serine 133 is regulated by gonadotropins. It is possible that although CREB appears to be fairly ubiquitously expressed in a number of cell systems, its regulation by other molecules is necessary for activating gene expression through the CRE. We are currently investigating which protein(s) is involved in activating the IGFBP-1 gene in baboon stromal cells during decidualization.

Finally, it was interesting to note a difference in the pattern of IGFBP-1 expression in response to hormones/(Bu)2cAMP treatment in cells with disrupted actin filaments vs. cells from pregnant or simulated pregnant animals. Cytochalasin D-treated cells exhibited significantly elevated expression of IGFBP-1 in response to (Bu)2cAMP only (P <= 0.05) compared with controls. IGFBP-1 expression was higher in these cells than when cells were treated with hormones alone. Alternatively, cells from pregnant or simulated pregnant animals expressed higher levels of IGFBP-1 in response to hormones alone than (Bu)2cAMP alone. This observation suggests that the events involved with IGFBP-1 gene regulation in cytochalasin D-treated cells are not identical to that of the in vivo primed cells. However, both groups of cells did require de novo protein synthesis for the expression of IGFBP-1. We have previously shown that IGFBP-1 is hormonally regulated in glandular epithelial cells (17). IGFBP-1 staining was evident in the deep basal glands of the baboon endometrium at day 10 post ovulation and increased thereafter (17). In stromal cells, IGFBP-1 staining is absent throughout the menstrual cycle. However, as shown in this report, cells taken from an early pregnant or simulated pregnant animal expressed IGFBP-1 in response to hormones only. It is possible that in response to an embryonic signal(s), stromal cell physiology changes, allowing steroids to increase the expression of the IGFBP-1 gene. Alternatively, cytochalasin D-treated cells have not been exposed to the embryonic signal and thus are not physiologically identical to cells that have seen this signal. IGFBP-1 expression in cytochalasin D-treated cells after (Bu)2cAMP exposure may be a response that provides possible clues toward the mechanisms associated with the structural differentiation of the endometrial stromal cell. Perhaps in vivo, the factors associated with pregnancy are conducive to the restructuring of the cytoskeleton, which in turn potentiate stromal cells to induce IGFBP-1 in response to the appropriate stimulus. Moreover, long-term hormone/cAMP treatment in vitro of stromal cells from a nonpregnant animal may alter the cytoskeletal arrangement, which then promotes IGFBP-1 expression in response to hormones and/or (Bu)2cAMP in vitro. It has been shown, in mouse endometrial stromal cells, that stress fibers or bundles of microfilaments that run parallel to the long axis of the cell become shorter and thicker, running in many different orientations and concentrating at the periphery with stromal cell differentiation (32). Furthermore, Babiarz et al. (32) have demonstrated parallel remodeling of the extracellular matrix with changes in cell morphology, both of which occur during the decidualization process in vivo.

In summary, it is apparent that IGFBP-1, a marker for decidualizing cells, can be regulated by altering the cytoskeleton and by exposing the cells to a pregnant or simulated pregnant environment. The investigation of the association of the cytoskeleton with IGFBP-1 gene regulation is a novel approach to identifying the biochemical and molecular events involved in the decidualization process.


   Acknowledgments
 
We are grateful to Dr. Jeffrey Fortman for surgical assistance and Dr. Terry Unterman for helpful discussions on the manuscript. We thank Dr. Primal de Lanerolle for assistance with the immunofluorescent studies.


   Footnotes
 
1 Supported by a postdoctoral fellowship from the Ernst Schering Foundation, Berlin, Germany. Back

Received June 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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