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Biology of Reproduction 60, 567-572 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Vitamin D Regulates Human Ectocervical Epithelial Cell Proliferation and Insulin-Like Growth Factor-Binding Protein-3 Level1

Chapla Agarwala, Adam Lamberta, Roshantha A.S. Chandraratnaf,g, Ellen A. Rorkee, and Richard L. Eckert2,a,b,c,d

a Departments of Physiology and Biophysics, b Dermatology, c Reproductive Biology, d Biochemistry, and e Environmental Health Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970 f Retinoid Research, Departments of Chemistry and g Biology, Allergan Pharmaceuticals, Irvine, California 92713


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The differentiation status of the cervical epithelial cell has an important influence on responsiveness to estrogens and progestins. Several agents, including glucocorticoids and retinoids, are known to influence cervical cell differentiation. However, the effects of vitamin D have not been examined. Vitamin D is known to regulate cell proliferation and gene expression in a variety of epithelial cells. In the present study we investigated the ability of 1{alpha}25-dihydroxyvitamin D3 (D3) to regulate cell proliferation and expression of insulin-like growth factor-binding protein-3 (IGFBP-3) in human ectocervical epithelial cells. ECE16-1, a non-tumorigenic cervical cell line, was growth inhibited by D3 with maximal inhibition at 1000 nM. IGFBP-3 levels increased in parallel with the growth inhibition. IGFBP-3 levels were half-maximally increased at approximately 10-100 nM and maximally increased (10- to 30-fold) at 1000 nM D3. These studies show that vitamin D regulates cervical epithelial cell gene regulation and cell proliferation and that IGFBP-3 may be an in vivo marker of vitamin D action in the cervix.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human cervix is a stratifying epithelial tissue that responds to estrogens, progestins, and other agents that regulate differentiation. The differentiation status of the epithelium is an important determinant of responsiveness to estrogen and other regulatory hormones. Glucocorticoids and vitamin A compounds have been shown to participate as determiners of the differentiation set point in cervical epithelial cells [1, 2]. For example, retinoids regulate epidermal growth factor receptor function [3], the synthesis of growth-inhibitory cytokines [4], and insulin-like growth factor-binding protein-3 (IGFBP-3) gene expression [5–7]. IGFBPs are a family of secreted proteins that bind IGF to inhibit or enhance insulin-like growth factor (IGF) activity. Inhibition is thought to involve formation of an IGF/IGFBP complex that makes the IGF unavailable to the cell [8, 9]. We have previously shown that IGFBP-3 is produced and secreted by cervical epithelial cells. In the ECE16-1 cell line, IGFBP-3 levels are increased by treatment with retinoic acid receptor-selective retinoids [10–13]. These changes in IGFBP-3 protein level are mediated by changes in the level of mRNA encoding IGFBP-3 [11, 12]. Thus, IGFBP-3 is a marker of retinoid action in cervical tissues.

Vitamin D-related ligands have a regulatory role in a variety of cell types [14, 15]. 1{alpha}25-Dihydroxyvitamin D3 (D3) has been shown to inhibit the proliferation of human breast cancer cells [16, 17]. Other cell types also respond to vitamin D and vitamin D derivatives [14, 18–22]. In the present study, we show that vitamin D3 inhibits the proliferation of ECE16-1 cervical epithelial cells. We also show that D3 increases production of IGFBP-3 via regulation of IGFBP-3 mRNA level.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Fetal calf serum, insulin, BSA, hydrocortisone, 3,3'5-triiodothyronine (T3), transferrin, and adenine were obtained from Sigma Chemical Co. (St. Louis, MO). Cholera toxin was purchased from ICN Biomedical (Costa Mesa, CA). Human recombinant epidermal growth factor (EGF) was from Upstate Biotechnology, Inc. (Lake Placid, NY); (3-[125I]iodotyrosyl)IGF-1 (2000 Ci/mmol) and [32P]dCTP were purchased from Amersham (Arlington Heights, IL). Dulbecco's Modified Eagle's medium, F-12 medium, nonessential amino acids, L-glutamine, trypsin, and antibiotics were purchased from Gibco (Grand Island, NY). 1{alpha}25-Dihydroxyvitamin D3 (D3) was purchased from Calbiochem (La Jolla, CA). TTNPB, a potent synthetic retinoid, was produced in the Department of Chemistry, Allergan Inc. (Irvine, CA). The structure is presented in our previous report [10]. In ECE16-1 cells, TTNPB is 10-fold more active than all-trans-retinoic acid [10].

Cell Proliferation Studies

ECE16-1 cells were cultured as previously described [12] in Dulbecco's Modified Eagle's medium:F-12 medium (3:1) containing 5% fetal calf serum, 5 µg/ml insulin, 0.1 nM cholera toxin, 5 g/ml transferrin, 1 nM T3, 10 ng/ml EGF, 0.18 nM adenine, nonessential amino acids, L-glutamine, and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, and 5 µg/ml of gentamicin). For growth studies, cells were plated at 10 000 cells per square centimeter in 35-mm dishes in growth medium. After overnight attachment, the cells were shifted to DM. DM is Dulbecco's Modified Eagle's medium:F-12 (3:1) supplemented with 1 mg/ml of BSA, nonessential amino acids, L-glutamine, 5 µg/ml transferrin, 1 nM T3, 0.18 nM adenine, 50 µg/ml ascorbic acid, and antibiotics. After a 24-h incubation in DM, the cells were shifted to fresh DM or DM-EGF (DM containing 20 ng/ml EGF) supplemented with the appropriate test compounds. Fresh test medium was added at 24-h intervals, and the cells were harvested and counted after the third day of treatment. The cells were fixed and counted using a Coulter (Hialeah, FL) counter as previously described [6]. Medium, conditioned by cells for 24 h, was collected, centrifuged to remove cell debris, and stored at -20°C. This medium was subsequently assayed for IGFBP content. Differences in cell number were compared using the Student's t-test.

Detection of IGFBPs

The type and quantity of IGFBPs released by cells into the culture medium were determined by ligand blot assay according to the method of Hossenlopp et al. [23] as outlined previously [12]. In brief, an equivalent amount of each sample of conditioned medium, normalized based on cell number, was loaded in each gel sample lane. The volume loaded ranged from 5000 to 10 000 cell equivalents per lane in different experiments. The samples were heated at 100°C and electrophoresed through a 10% polyacrylamide gel [24] under nonreducing conditions. Although not shown in the gel profiles, a standard amount of human cord serum, a rich source of IGFBPs, was loaded onto each gel as an IGFBP migration standard. The gels were transferred to nitrocellulose and, after the membrane was blocked, incubated with 125I-IGF-1 for 18 h at 4°C. After unbound 125I-IGF-1 was washed away, the gels were exposed on x-ray film for 3–8 days at -80°C.

Nucleic Acid Methods

Polyadenylated RNA was isolated using oligo dT-cellulose columns, and 5 µg/lane was electrophoresed on a formaldehyde-containing agarose gel and transferred to Biodyne A membrane (ICN, Irvine, CA) as previously described [12, 25]. The membrane was hybridized with cDNAs encoding human IGFBP-3 [26] or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [27], each labeled with [32P]dCTP (6000 Ci/mmol; New England Nuclear, Boston, MA) by random priming (Amersham). Hybridization was detected by exposure on x-ray film. The hybridization and wash conditions were as previously described [12, 25].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
D3 Inhibited Cervical Cell Proliferation

ECE16-1 cells depend upon EGF for proliferation [12]. To test for the effect of D3 on ECE16-1 cell proliferation, cells were grown in DM-EGF supplemented with increasing concentrations of D3 (0–1000 nM) or 1000 nM all-trans-retinoic acid. The DM group received DM not supplemented with EGF. As shown in Figure 1, D3 treatment produced a concentration-dependent inhibition of the EGF-dependent cell proliferation. Suppression was half-maximal at 100 nM and maximal at 1000 nM. The suppression observed at 1000 nM D3 was comparable to that observed with 1000 nM all-trans-retinoic acid (RA), and cell number was nearly reduced to the level observed when cells were grown in the absence of EGF (DM). No further growth suppression was observed for 10 µM D3 (not shown).



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  		FIG. 1. D3 inhibits cervical epithelial cell proliferation. ECE16-1 cells were seeded at 10 000 cells per square centimeter in 35-mm dishes and allowed to attach overnight. After a 24-h equilibration in DM, cells were treated for 3 days with DM (DM, open bar) or DM-EGF (solid and cross-hatched bars). As indicated, the cells were incubated with 0–1000 nM D3 (cross-hatched bars) or 1000 nM RA (solid bar). Fresh test medium was added at 24-h intervals, and the cells were harvested and counted after the third day of treatment. The cell numbers shown here are the averages of five separate experiments ± SEM. Values for the 100 nM D3 treatment are significantly different from control (zero D3) at p <= 0.1, and those for the 1000 nM D3 and 1000 nM RA treatments are significantly different from control at p < 0.05.

D3 Increased IGFBP-3 Levels

IGFBP-3 level is altered in response to several agents that regulate cervical cell proliferation [7, 11, 12]. To evaluate its effects, ECE16-1 cells were treated with D3, and IGFBP-3 levels in the culture medium was assayed by ligand blot. All groups were treated with DM-EGF except for the control group, which received DM. Cells grown in DM produced a low level of IGFBP-3, and this level decreased after treatment with EGF (Fig. 2). As previously reported [12], the predominant bands were 38–42 kDa. Treatment with 0–1000 nM D3 produced a concentration-dependent increase in IGFBP-3 level. As measured by laser densitometry in four separate experiments and as compared to no treatment, 1000 nM D3 increased IGFBP-3 levels by 10- to 30-fold.



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  		FIG. 2. D3 increases IGFBP-3 release into the cell culture medium. ECE16-1 cells were grown for 3 days with daily addition of fresh medium supplemented with the indicated ligands. Medium from the final 24 h of treatment was harvested and assayed for IGFBPs by ligand blot. All groups were treated with DM-EGF except for the DM group. The D3 concentrations are indicated as nanomoles per liter. A bracket indicating the migration of IGFBP-3 and molecular weight markers (Mr x 10-3) are shown at left.

D3 Regulated IGFBP-3 Level by Increasing the Level of mRNA Encoding IGFBP-3

To evaluate the mechanism by which D3 regulates IGFBP-3 level, we measured the level of IGFBP-3 mRNA. Figure 3 shows that the IGFBP-3 mRNA level was increased by 100 nM and 1000 nM D3. The results of four similar experiments indicated a range of increase from 10- to 30-fold. GAPDH, shown as a control, is not regulated by D3.



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  		FIG. 3. D3 increases IGFBP-3 mRNA production. ECE16–1 cells were treated for 4 days with DM supplemented with 20 ng/ml EGF (DM-EGF) in the presence of 100 or 1000 nM D3. The cells were harvested, and poly(A)+ RNA was prepared and electrophoresed (5 µg/lane) on a denaturing agarose gel. Preparation of the formaldehyde agarose gel and the electrophoresis conditions have been described [12, 25]. The fractionated RNA was then transferred to Biodyne A membrane and hybridized with 32P-labeled cDNAs encoding GAPDH or IGFBP-3. Migration of the 2.6-kilobase (kb) IGFBP-3 and the 1.0-kb GAPDH mRNAs is indicated, as is migration of the 18S (1.9 kb) and 28S (4.7 kb) rRNA species.

D3 and Retinoids Regulated IGFBP-3 Level and Cell Proliferation by Different Mechanisms

Figure 4 shows that EGF-dependent ECE16-1 cell proliferation (EGF, open bar) was suppressed in a concentration-dependent manner by D3. This suppression was not antagonized by AGN193109 (cross-hatched bars), a specific antagonist of vitamin A-related ligands [28]. However, the retinoid (TTNPB)-dependent suppression was reversed by the presence of AGN193109.



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  		FIG. 4. The D3-dependent suppression of proliferation is not mediated via a retinoid-dependent pathway. Cells were grown as described in the legend to Figure 1. After 3 days of treatment, the cells were harvested and counted. TTNPB and AGN193109 were added simultaneously; concentrations were 100 and 1000 nM, respectively. The cell numbers shown here are the mean ± SEM of four experiments. The mean was significantly reduced (compared to zero D3) at 100 nM (p <= 0.1) and 1000 nM (p <= 0.05) D3 and at 100 nM TTNPB (p <= 0.05).

Figure 5A shows that 1000 nM D3 and 100 nM TTNPB increased IGFBP-3 levels compared to those in the EGF-treated group. AGN193109 had no effect on IGFBP-3 level when given alone and inhibited the retinoid (TTNPB)-dependent increase in IGFBP-3 level. However, AGN193109 did not affect the D3-dependent increase. Figure 5B shows that these changes are reflected at the IGFBP-3 mRNA level. AGN193109 inhibited the TTNPB- but not the D3-dependent increase in IGFBP-3 levels.



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  		FIG. 5. The D3-dependent increase in IGFBP-3 expression does not depend upon a retinoid-signaling pathway. A) ECE16-1 cells were grown for 3 days with daily addition of fresh medium supplemented with the indicated ligands. Medium from the final 24 h of treatment was harvested and assayed for IGFBPs by ligand blot. All groups were treated with DM-EGF except for the DM group. The bracket indicates the migration of IGFBP-3. Molecular weight markers (Mr x 10-3) are shown at left. AGN193109 was present at a concentration of 1000 nM. B) ECE16-1 cells were treated for 4 days in DM-EGF supplemented with 1 µM AGN193109 in the presence of 1 µM D3 or 100 nM TTNPB. The cells were harvested, and poly(A)+ RNA was prepared and electrophoresed on a denaturing agarose gel. The fractionated RNA was then transferred to Biodyne A membrane and hybridized with 32P-labeled cDNAs encoding GAPDH or IGFBP-3. Migration of the 2.6-kb IGFBP-3 and the 1.0-kb GAPDH mRNAs is indicated, as is migration of the 18S (1.9 kb) and 28S (4.7 kb) rRNA species.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Vitamin D Regulation of ECE16-1 Cell Proliferation

The effects of vitamin D are mediated by the vitamin D receptor (VDR), a member of the ligand-activated receptor family of trans-acting factors [29]. Biologically active forms of vitamin D, and related analogues, have been shown to inhibit the proliferation of cells derived from the breast [17, 19, 30–34], prostate [15, 18, 35–37], bone marrow [20, 35], colon [21, 38–40], brain [22], and epidermis [14, 41]. ECE16-1 cells provide a model cell line for the study of relatively normal cervical cell differentiation [42]. These cells are immortal, but they do not form tumors in nude mice or grow in soft agar [42]. Retinoids and interferon suppress [10, 11] and EGF increases ECE16-1 cell proliferation [12].

In the present paper we examine the role of vitamin D in regulating cervical cell proliferation. Addition of physiological concentrations of vitamin D (50–200 nM) [43] suppresses EGF-dependent proliferation. Growth is half-maximally suppressed at 100 nM and maximally suppressed at 1000 nM 1,25-(OH)2D3. This suggests that vitamin D could play a role as a regulator of proliferation in vivo.

Our previous studies showed that glucocorticoids and retinoids produce opposing responses on normal human cervical epithelial cell differentiation: retinoids suppress and glucocorticoids enhance differentiation [1, 2]. In human papillomavirus-immortalized cells, the retinoid-dependent suppression of differentiation is correlated with reduced cell differentiation [44]. The present studies show that vitamin D suppresses proliferation. Vitamin D, however, does not appear to regulate markers of cervical cell differentiation (i.e., keratin gene expression, etc.) (not shown). Thus, retinoids and vitamin D ligands regulate cell phenotype in distinctly different ways.

Vitamin D Regulation of IGFBP-3 Expression

IGFBP-3 elevation is correlated with slowed cell growth in several cell types [8, 45]. Treatment of cervical cells with D3 results in an increase in IGFBP-3 expression. IGFBP-3 levels are half-maximally increased at 100 nM and maximally increased at 1000 nM. These results are consistent with what would be expected if IGFBP serves as a cervical cell marker of vitamin D responsiveness. It is also possible that IGFBP-3 may have a direct role in the inhibition of cell proliferation. IGFBPs have been shown to inhibit IGF-dependent proliferation as well as proliferation dependent upon other agents [8, 9, 45–48]. IGFBP-3 inhibits the action of IGF in a variety of systems, including ECE16-1 cells. In addition to its effects on the IGF system, IGFBP-3 has also been reported to inhibit non-IGF-dependent proliferation, possibly by binding to a membrane-associated IGFBP-3 binding protein [49–51]. We are presently exploring the possibility that IGFBP-3 may be an agent that directly suppresses ECE16-1 cell proliferation.

Vitamin D Regulates IGFBP-3 mRNA Levels

The present studies show that changes in IGFBP-3 level correlate with changes in IGFBP-3 mRNA level, indicating that the change in IGFBP-3 concentration is not due to stimulation of secretion/release of preexisting IGFBP-3. Thus, vitamin D joins vitamin A and EGF [10–12] as agents that regulate IGFBP-3 protein concentration via regulation of IGFBP-3 mRNA levels.

Vitamin D- and Retinoid-Dependent Growth Inhibition Utilize Distinct Signaling Pathways

The retinoid and vitamin D receptors are members of a family of ligand-activated trans-acting factors [29]. Since both VDRs and retinoic acid receptors (RARs) interact with a common companion receptor, retinoid X receptor (RXR), it is possible that the vitamin D could affect the RAR signaling system and that the vitamin D-dependent growth suppression could be due to effects on RAR function. Data obtained using RXR-specific ligands in ECE16-1 cells suggest that RXR ligands, for example, can influence the activity of RAR-specific ligands [10]. To confirm that the D3-dependent growth suppression does not involve RARs, we treated cells with vitamin D in the presence or absence of AGN193109. AGN193109 is a highly active and specific antagonist of RAR action in ECE16-1 cells [52]. Our results show that AGN193109 can effectively antagonize the action of retinoids in ECE16-1 cells but that it does not antagonize the D3-dependent responses. This suggests that vitamin D is acting through the VDR and that the vitamin D-dependent regulation is not due to VDR interaction with retinoid-signaling molecules.


   FOOTNOTES
 
1 This work was supported by a grant from the American Institute for Cancer Research (R.L.E.) and by Allergan Pharmaceuticals (R.L.E.) and utilized the facilities of the Skin Diseases Research Center of Northeast Ohio (NIH, AR39750). Back

2 Correspondence: Richard L. Eckert, Department of Physiology/Biophysics, Rm E532, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, OH 44106-4970. FAX: 216 368 5586; rle2{at}po.cwru.edu Back

Accepted: October 6, 1998.

Received: August 3, 1998.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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