Identification of a Putative Autocrine Bone Morphogenetic Protein ...

0013-7227/03/$15.00/0 Printed in U.S.A.

Endocrinology 144(8):3306 –3314 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2003-0185

Identification of a Putative Autocrine Bone Morphogenetic Protein-Signaling Pathway in Human Ovarian Surface Epithelium and Ovarian Cancer Cells TREVOR G. SHEPHERD

AND

MARK W. NACHTIGAL

Dalhousie University, Department of Pharmacology, Halifax, Nova Scotia, Canada B3H 1X5 Bone morphogenetic proteins (BMPs) are members of the TGF␤ superfamily of cytokines that are involved in development, differentiation, and disease. In an analysis of normal ovarian surface epithelium (OSE) and ovarian cancer (OC) cells, we observed BMP4 mRNA expression and found that primary OC cells produce mature BMP4. In addition, each member of the downstream signaling pathway was expressed in primary OSE and OC cells. Smad1 was phosphorylated and underwent nuclear translocation in normal OSE and OC cells upon treatment with BMP4. Interestingly, the BMP target genes ID1 and ID3 were up-regulated 10- to 15-fold in primary

B

OC cells, compared with a 2- to 3-fold increase in normal OSE. The growth of several primary OC cells was relatively unaltered by BMP4 treatment; however, long-term BMP4 treatment of primary OC cells resulted in decreased cell density as well as increased cell spreading and adherence. These data demonstrate the existence and putative function of BMP signaling in normal OSE and OC cells, and thus the continued examination of BMP4 signaling in the regulation of these two processes will be critical to further our current understanding of the role of BMP biology in OC pathogenesis. (Endocrinology 144: 3306 –3314, 2003)

ONE MORPHOGENETIC PROTEINS (BMPs) are members of the TGF␤ superfamily of secreted ligands (1). Approximately 30 different BMPs have been identified in both invertebrates and vertebrates. BMPs function as extracellular dimeric ligands that initiate cellular signaling by binding to and activating BMP receptor complexes at the surface of target cells. Intracellular BMP signaling is mediated by the phosphorylation of receptor-activated Smad (RSmad) proteins, specifically Smads 1, 5, and 8. Activated R-Smads homo- and heterodimers complex with the common partner Smad4 and translocate to the nucleus in which they impart BMP-stimulated alterations in target gene expression. BMP signaling, and the resultant changes in gene expression function in diverse processes such as mesoderm formation at gastrulation, left-right asymmetry, cell-fate determination, vasculogenesis, and apoptosis. BMP signaling is also required for normal ovarian function. BMP4 and BMP7 are expressed by follicular theca cells and appear to modulate FSH-induced hormone production in neighboring granulosa cells (2). BMP15 is expressed by the oocyte, and its requirement is evident in anovulatory female mice lacking the Bmp15 gene, which have defects in folliculogenesis (3). In addition, several strains of Australian Merlino sheep are known to have mutations in the BMP15 and BMPR1B genes (4, 5). Females heterozygous for these mutant alleles have increased ovulation rates leading to increased number of offspring per pregnancy; whereas homozygous mutants are infertile. The surface epithelial cells of the rat

ovary have also been shown to express low but detectable amounts of BMP4; however, the functional significance of this expression remains unknown (2). The majority (90 –95%) of human ovarian cancer (OC) arises from the ovarian surface epithelium (OSE) (6). BMP signaling is growth inhibitory and can induce apoptosis in several different cancer cells (7–12), but in others it has been implicated in their increased metastatic potential (13–16). We therefore screened normal human OSE and human OC cells by RT-PCR for expression of BMP ligands and signaling molecules. We determined that these cells produce BMP4 as well as the molecules required to transmit the BMP4 signal, suggesting that the OSE and OC cells maintain an autocrine signaling loop. Further analysis demonstrated that OSE and OC cells possess an intact BMP signaling pathway, are capable of up-regulating known BMP target genes, and implicate BMP signaling in the regulation of OC cell adhesion. In addition, our results show a differential response in the fold increase of the BMP target genes ID1 and ID3 between normal OSE and primary OC cell cultures. Inhibitor of DNA binding (Id) proteins are negative regulators of basic helix loop helix (bHLH) and non-bHLH transcription factors (17). We hypothesized that an autocrine BMP signaling loop may play a role in coordinating postovulatory OSE repair and OC metastasis by regulating cell adhesion and also contribute to the reduced expression of important regulatory genes via up-regulation of Id molecules leading to ovarian tumorigenesis.

Abbreviations: bHLH, Basic helix loop helix; BMP, bone morphogenetic protein; ECM, extracellular matrix; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Id, inhibitor of DNA binding; IP, immunoprecipitation; nt, nucleotide; OC, ovarian cancer; OSE, ovarian surface epithelium; rh, recombinant human; RSmad, receptor-activated Smad.

Materials and Methods Primary human OSE and OC cells Institutional approval for research with human materials was received before the initiation of these studies (QEII Health Sciences Centre, Research Ethics Committee, QE-RS-99-016, IWK/Grace Hospital Re-

3306

Shepherd and Nachtigal • BMP4 Signaling in Ovarian Cancer Cells

search Ethics). Primary human OC cells were isolated from ascites fluid obtained from chemotherapeutically naive patients with stage III or IV ovarian cancer as described previously (18, 19). Briefly, ascitic fluid containing cells was mixed 1:1 with growth medium [MCDB105 (Sigma, St. Louis, MO)/M199 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Cansera, Etobicoke, Ontario, Canada) and 100 U/ml penicillin, 100 ␮g/ml streptomycin]. After 3 d the supernatant was removed, and attached cells were refed growth medium. All experiments using primary OC cells were performed at culture passages 2 to 6. OSE cells from human ovarian tissue samples were isolated as described previously (19). The surface of the ovary was treated with Dispase II (Roche Molecular Biochemicals, Indianapolis, IN) for 30 min at 37 C with occasional agitation. The OSE cells were gently scraped directly into complete growth medium (MCDB105/M199 supplemented with 10% FBS and 100 U/ml penicillin, 100 ␮g/ml streptomycin) and allowed to attach and grow for 4 d. Homogeneous populations of primary human OSE cells were identified by their characteristic morphology. All experiments using normal primary OSE were performed at culture passages 2 to 4.

Cell lines Three established OC cell lines (CaOV3, SkOV3, and HeyC2) and two immortalized normal ovarian surface epithelial cell lines (IOSE80 and IOSE144; gift of Dr. Nelly Auersperg, UBC) were used in this study. CaOV3 and SkOV3 cells were grown as monolayer cultures and maintained in DMEM (Invitrogen) supplemented with 5% FBS. HeyC2 cells were grown in RPMI 1640 medium (Invitrogen) supplemented with 5% FBS, and IOSE80 and IOSE144 cells were maintained in MCDB105/M199 supplemented with 10% FBS.

RNA isolation and RT-PCR Total cytoplasmic RNA was isolated from cultured cells using the GenElute mammalian total RNA isolation kit (Sigma). For RT-PCR analyses, cDNA was generated with 2 ␮g DNase-treated total RNA using Superscript II reverse transcriptase (Invitrogen) as per the manufacturer’s instructions. Subsequent PCR was used to detect expression of BMP signaling components (30 cycles: 35 sec at 94 C, 35 sec at 59.5 C, 35 sec at 72 C) using primers specific for each human cDNA sequence. The cDNA fragments of human BMP2 [nucleotides (nt) 560-1034], BMP4 (nt 296 –771), BMP7 (nt 793-1293), BMPR1A (nt 145–594), BMPR1B (nt 9071256), BMPR2 (nt 2561–3010), SMAD1 (nt 1038 –1291), SMAD4 (nt 6011050), SMAD5 (nt 202– 660), and SMAD8 (nt 518 – 858) were subcloned into pCRII.TOPO (Invitrogen), and the identity of the amplified fragment was verified by manual sequencing. Sequences of the primers used can be made available upon request.

Northern analysis Primary OC cells and OC cell lines were serum starved in medium containing 0.2% FBS for 24 h before treatment for 90 min with 10 ng/ml recombinant human (rh) BMP4 (R&D Systems, Minneapolis, MN). This dose was chosen because it was the lowest dose observed to produce consistent BMP responses in Smad phosphorylation, BMP target gene expression, and alterations in cell phenotype. Primary normal OSE cells were not serum starved before BMP4 treatment. Total RNA was isolated as described above, and 10 ␮g RNA were separated on a 1.5% agarose formaldehyde gel and transferred to BrightStar Plus membrane (Ambion Inc., Austin, TX). Blots were incubated overnight at 42 C in ULTRAhyb buffer (Ambion Inc.) with 1 ⫻ 106 cpm/ml 32P-labeled cDNA probe generated using StripEZ DNA probe synthesis kit (Ambion Inc.). Fulllength SMAD6 and SMAD7 (gift of Carl-Henrik Heldin, Ludwig Institute for Cancer Research), ID1 (nt 189 –549), and ID3 (nt 356 –750) cDNAs were used to generate the radiolabeled cDNA probes. Northern blots were subsequently washed at room temperature in low-stringency wash solution (2⫻ saline sodium citrate/0.1% SDS) followed by washes at 42– 60 C in high-stringency wash solution (0.1⫻ saline sodium citrate/ 0.1% SDS). Signals were visualized by autoradiography and quantified using a molecular imaging screen and molecular analyst software (BioRad Laboratories, Hercules, CA). Northern blots were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe as

Endocrinology, August 2003, 144(8):3306 –3314 3307

a loading control. The data shown are representative of at least two independent experiments.

Transient transfection and luciferase assays CaOV3 and SkOV3 cells were plated in 12-well plates at 2 ⫻ 104 cells/well the day before transfection. Cells in triplicate wells were transfected with 100 ng pGL2.basic (Promega) or 100 ng pSmad6.lux (contains ⬃8.5 kbp of the mouse Smad6 promoter, gift of Mitsuyasu Kato, Japanese Foundation for Cancer Research) (20) using FuGENE6 transfection reagent (Roche Molecular Biochemicals) in normal growth medium. Total DNA was brought up to 500 ng using pBluescript II KS plasmid (Stratagene, La Jolla, CA). Cells were serum starved (growth medium with 0.2% FBS) for 8 h during the subsequent day and then treated overnight with 10 ng/ml BMP4 in growth medium containing 0.2% FBS. Twenty hours after addition of BMP4, cells were harvested and luciferase activity was determined using the enhanced luciferase assay kit (BD PharMingen, San Diego, CA). Alternatively, cells were cotransfected with 100 ng pCMV5.BMP4 expression vector or the pCMV5 empty vector. The pCMV5.BMP4 is a BMP2/4 chimeric expression vector comprising the BMP2 amino-terminal prodomain and cleavage site fused to the carboxyl-terminal BMP4 mature peptide domain (chimeric cDNA was a gift from Dr. Rik Derynck, University of California, San Francisco). The results shown are the mean data from three independent experiments.

Immunoprecipitation Primary OC cells and OC cell lines grown in 100-mm dishes were radiolabeled for approximately 20 h with 50 ␮Ci per ml 35S-Met/Cys (Promix, Amersham Biosciences, Piscataway, NJ) in 2 ml Cys/Met-free DMEM containing 10% FBS. Culture supernatants were collected and concentrated to a volume of 500 ␮l using 10,000 MWCO Centricon filter devices (Millipore, Bedford, MA) and washed twice with immunoprecipitation (IP) buffer [50 mm HEPES (pH 7.4), 100 mm NaCl, 1% Nonidet P-40, 5 mm EDTA, 1 mm Na pyrophosphate, 1 mm Na orthovanadate, 1⫻ protease inhibitor cocktail (Amersham Biosciences), 10 ␮g/ml phenylmethylsulfonyl fluoride]. Cells were washed with ice-cold 1⫻ PBS and lysed in 500 ␮l IP buffer for 15 min on ice and then clarified by centrifugation. Concentrated supernatants and cell lysates were first precleared with protein A-Sepharose CL-4B (Amersham Biosciences), followed by incubation with 3 ␮g anti-BMP4 antibody (R&D Systems) overnight at 4 C. Immune complexes were precipitated with protein A Sepharose CL-4B for 1 h at 4 C, washed three times with IP buffer and then resuspended in 2⫻ SDS loading buffer containing ␤-mercaptoethanol for SDS-PAGE. Resultant 12% polyacrylamide gels were fixed for 30 min in 30% ethanol/10% acetic acid, incubated in Amplify reagent (Amersham Biosciences), and then dried and visualized by autoradiography. To immunoprecipitate myc epitope-tagged BMP4, SkOV3 cells were plated at 1 ⫻ 105 cells per 35-mm well and transiently transfected with 1 ␮g pMT-BMP4myc (gift of Elizabeth J. Robertson, Harvard University, Boston, MA). Cellular proteins were radiolabeled and immunoprecipitated as described above using anti-c-myc (CLONTECH, Palo Alto, CA) or anti-BMP4 antibodies.

Western analysis Total cellular protein was isolated from normal OSE and OC cells grown to 70 – 80% confluence on 60-mm plates. Cells were washed two times in ice-cold PBS, dissolved in lysis buffer [50 mm Tris-Cl (pH 7.4), 150 mm NaCl, 1 mm EGTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm Na orthovanadate, 1 mm NaF, 1⫻ protease inhibitor mix], clarified by centrifugation (10 min at 15,000 ⫻ g), and quantified by Bradford analysis (Bio-Rad Laboratories). Twenty micrograms of protein extract per lane were separated by SDS-PAGE in the presence of 1% ␤-mercaptoethanol using 10% gels. Rabbit polyclonal anti-Smad1 or anti-phospho-Smad1 (Upstate Biotechnology, Lake Placid, NY) and rabbit polyclonal anti-␤ actin (Sigma) primary antibodies followed by sheep antirabbit IgG-HRP conjugated secondary antibody (Chemicon International, Temeculah, CA) were used to detect protein expression with enhanced chemiluminescence (Perkin-Elmer, Norwalk, CT).

3308

Endocrinology, August 2003, 144(8):3306 –3314


Indirect immunofluorescence Normal OSE and primary OC cells were seeded onto cover slips in 12-well dishes at 4 ⫻ 104 cells/well in complete growth medium and the next day were serum starved for 24 h in medium containing 0.2% FBS. Cells were treated for 1 h with 50 ng/ml rhBMP4 (to ensure maximal signaling activity) and then fixed with 4% paraformaldehyde, followed by treatment with 100% methanol at ⫺20 C for 10 min. Samples were blocked using 5% horse serum and then incubated overnight at 4 C with rabbit anti-Smad1 polyclonal antibody diluted at 1:100 in 5% BSA. Samples were washed and incubated for 1 h at room temperature with Alexa Fluor 488 goat antirabbit IgG secondary antibody (Molecular Probes, Eugene, OR) diluted at 1:2000 in 3% BSA. After the final washes the cover slips were mounted onto slides and cells were visualized by fluorescence microscopy at ⫻200 magnification using an Axiovert 200 (Carl Zeiss, Gottingen, Germany), and images were captured using an Axiocam and Axiovision 3.0 software. Images were subsequently cropped and adjusted for brightness/contrast using Adobe Photoshop 5.5 software. To detect ␤-actin and ␤-catenin primary OC cells were seeded at 1.6 ⫻ 104 cells/well on glass coverslips in complete growth medium, and the next day and then every third day, cells were fed medium and treated with 10 ng/ml BMP4. After 6 d, indirect immunofluorescence was performed as described above.

Growth curves Primary human OC cells were plated on d 0 at 4 ⫻ 104 cells/well in growth medium in six-well plates. On d 1 and then every third day, cells were fed medium and treated with 10 ng/ml BMP4 or 0.1 ng/ml rhTGF␤1 (Sigma-Aldrich, St. Louis, MO). Cells from duplicate wells were counted using a hemacytometer over a 7-d period. The data are representative of three independent experiments. Cells at the end of the experiment were photographed using a camera (Nikon) on an inverted microscope, and the resultant photos were cropped and adjusted for brightness and contrast using Adobe Photoshop 5.5.

Cell adhesion assays Primary OC cells were cultured in complete growth medium without or with 10 ng/ml BMP4 for 48 h. Cells were detached first using 0.06% trypsin in 1⫻ PBS/0.5 mm EDTA and counted using a hemacytometer. The remaining cells were completely detached from the culture dishes using 0.25% trypsin and counted, and cell adhesion was scored as the proportion of cells detached with 0.06% trypsin/total number of detached cells. The results shown are the mean data from at least two independent experiments.

Results BMP signaling components are expressed in OSE and OC cells

To address whether normal OSE and OC cells possess BMP signaling components, RT-PCR experiments were performed using total RNA isolated from early passage primary cultures of normal human OSE and several primary OC cells and established OC cell lines. Previous work by Shimasaki et al. (2) stated that rat OSE produce BMP4 but not BMP7/OP1. We observed that BMP4 mRNA was readily detectable in all samples analyzed (Fig. 1); however, the closely related BMP, BMP2, was expressed in fewer samples. BMP7/OP1 was not detected by RT-PCR in any of the RNA samples. The two type I BMP receptors, BMPR1A and BMPR1B, were detected; however, BMPR1B appears to have a lower level of expression in primary cells and the immortalized OSE cells. In addition the type II receptor BMPR2 was detected, as were the mRNA for the downstream signaling effectors, Smad1, 5, and 8, and the co-Smad, Smad4. These experiments showed that both normal OSE and OC cells possessed the mRNA

FIG. 1. Components of the BMP signaling pathway are expressed in OSE and OC cells. RT-PCR analysis was performed using primers specific for each human gene on total RNA isolated from cultured cells as indicated. Lane 1, negative control (ddH2O); lane 2, OSE3; lane 3, IOSE80; lane 4, IOSE144; lane 5, OC13; lane 6, OC7; lane 7, OC22; lane 8, CaOV3; lane 9, SkOV3; and lane 10, HeyC2. IOSE, Immortalized OSE cells.

required to generate the components required for BMP signaling. We examined whether primary OC cells and established OC cell lines express and secrete mature BMP4 protein. 35SMet/Cys radiolabeled proteins secreted into the cell culture medium were subjected to immunoprecipitation using an anti-BMP4 antibody. All primary OC cell cultures (3/3) express and secrete mature BMP4 (Fig. 2). Similar experiments were not conducted in normal OSE because of the limited


availability of normal samples. Although established OC cell lines produce BMP4 mRNA, the secreted protein was undetectable in conditioned medium. We sought to determine whether OC cell lines transiently transfected with a BMP4 expression vector were capable of proteolytically processing and secreting BMP4 protein. SkOV3 cells were transiently transfected with a myc epitope-tagged BMP4 expression vector, proteins were metabolically labeled and secreted pro-


teins immunoprecipitated using anti-myc or anti-BMP4 antibody. Transfected SkOV3 cells were capable of synthesizing and secreting mature BMP4 (Fig. 2), as were transfected CaOV3 cells (data not shown). Thus, although established OC cell lines do not secrete endogenous BMP4 protein or produce extremely low levels, they have the proteolytic machinery required for BMP4 processing and secretion. BMP signaling is functional in OSE and OC cells

FIG. 2. Primary OC cells secrete mature BMP4 protein. Primary OC cells and established OC cell lines were incubated overnight with 35 S-Cys/Met and radiolabeled BMP4 protein was immunoprecipitated from concentrated culture medium. Monomeric mature BMP4 was readily detected in medium from primary OC cells but was undetectable in the established OC cell lines tested. SkOV3 cells were transfected with a myc epitope-tagged BMP4 expression vector or empty vector control (mock), protein was radiolabeled, and BMP4 protein was immunoprecipitated. Secreted myc epitope-tagged BMP4 was detected using either the anti-BMP4 or anti-Myc monoclonal antibodies.

FIG. 3. Smad1 is phosphorylated and translocates to the nucleus in response to BMP stimulation in primary human OSE, OC, and SkOV3 cells. A, Smad1 protein is phosphorylated in response to BMP4 stimulation in primary OSE (OSE4), OC (OC16, 24, 32), and SkOV3 cells. Western blotting was performed using total cellular protein (20 ␮g) extracts isolated from untreated cells or cells treated with 10 ng/ml rhBMP4 for 30 min. BMP4 signaling was assessed using anti-phosphoSmad1; loading was controlled by detection of total Smad1 and ␤-actin. B, Enhanced nuclear localization of Smad1 in normal OSE (OSE21) and primary OC (OC16) cells after treatment with BMP4. Cells were untreated (UT) or stimulated with 50 ng/ml rhBMP4 (BMP4) for 1 h, and then Smad1 localization was determined by indirect immunofluorescence using anti-Smad1 antibody and fluorescein isothiocyanate-conjugated antirabbit secondary antibody.

One of the first events following BMP receptor activation is the phosphorylation of the R-Smads Smad1, Smad5, and/or Smad8. Cell protein extracts were prepared from serum-starved primary normal OSE cells, OC cells, or established OC cell lines treated with 10 ng/ml rhBMP4 for 30 min. BMP4 treatment led to increased Smad1 phosphorylation in normal (OSE4) and cancer cells (OC16, OC24, OC32, SkOV3; Fig. 3A). Similar results were observed in OSE10, OSE11, OC13, OC14, and OC30. Although the fold increase in Smad1 phosphorylation for OSE4 was 1.7, the mean fold increase for all OSE samples analyzed was 2.4 ⫾ 0.6 (sem). This was similar to the observed mean fold increase in Smad1 phosphorylation for all OC cells (2.0 ⫾ 0.5). The rationale for using 10 ng/ml (⬃250 pm) BMP4 was based on preliminary experiments examining Smad1 phosphorylation in which we found that there was little difference in the level of phosphorylated Smad1 when OC cells were treated with either 10 ng/ml or 50 ng/ml (data not shown). This concentration is very close to the Kd for BMP4 binding to NIH 3T3 cells shown to be 254 pm (21).

3310



Phosphorylation of R-Smads leads to their enhanced nuclear localization. In serum-starved unstimulated primary OSE (OSE21) and OC (OC16) cells, Smad1 is diffusely distributed throughout the cytoplasm and nucleus; however, the majority of the cells possess enhanced nuclear Smad1 following 1 h of treatment with 50 ng/ml rhBMP4 (Fig. 3B). Similar results were obtained for OSE20, OC13, and OC14. The times chosen to examine Smad1 phosphorylation and nuclear translocation were based on previously published work (22). The anti-Smad1 antibody is reported to cross-react with Smad5; however, we were unable to detect endogenous levels of Smad5 with this antibody. BMP receptor activation and nuclear translocation of Smad proteins leads to modulation of target gene transcription. The inhibitory Smad, SMAD6, is regulated by BMPinduced Smad signaling (20, 23). To examine BMP-induced Smad6 promoter activity, CaOV3 and SkOV3 OC cell lines were transfected with a mouse Smad6 luciferase reporter (mSmad6.lux) containing approximately 8.5 kb of the 5⬘ flanking DNA. Luciferase activity increased approximately 1.5- to 3-fold in cells treated with 10 ng/ml rhBMP4, compared with untreated cells (Fig. 4A). A similar increase in luciferase activity was achieved when a BMP4 expression vector (pCMV5.BMP4) was cotransfected with the mSmad6FIG. 5. SMAD6, SMAD7, ID1, and ID3 expression is induced by BMP4 in OSE and OC cells. A, Northern analysis of total RNA isolated from untreated (⫺) cells or cells treated (⫹) with rhBMP4 for 90 min. Probes specific to human SMAD6, SMAD7, ID1, and ID3 were hybridized successively, and GAPDH was used as an RNA loading control. B, BMP4 treatment leads to rapid up-regulation of ID1 and ID3 gene expression in OC cells. Northern analysis was performed using total RNA isolated from cells treated with rhBMP4 for the indicated time points, and GAPDH was used as an RNA loading control.

FIG. 4. BMP4 induces SMAD6 promoter activity in OC cell lines. A and B, BMP4 induces pSmad6-luciferase reporter gene expression in OC cell lines. Cells were transfected with the reporter and treated with 10 ng/ml rhBMP4 (A) or cotransfected with the pCMV5.BMP4 expression vector (B). Luciferase activity was normalized to that of empty expression vector or untreated cells, respectively (set to 1).

lux reporter (Fig. 4B). Luciferase activity was normalized to the pGL2.Basic reporter plasmid, which was unaffected by BMP4 in either type of experiment. Several BMP-induced target genes have been identified in different cell types, and were analyzed in the present study. Northern analyses of total RNA isolated from serum-starved, untreated, or rhBMP4-treated (10 ng/ml) CaOV3 and SkOV3 cells were performed using specific probes for the human BMP target genes SMAD6, SMAD7, ID1, and ID3. Analogous to the transfection experiments described above, BMP4 induced the expression of the endogenous SMAD6 gene in CaOV3 and SkOV3 cells (Fig. 5A). Similar to SMAD6 gene induction, SMAD7 and ID1 genes were stimulated approximately 2-fold by rhBMP4 in CaOV3 and SkOV3 cell (Fig. 5A). Strikingly, the ID3 gene was induced more than 10-fold by BMP4 treatment. Time-course experiments revealed that BMP4-stimulated ID3 gene expression was induced by 30 min and peaked at 90 min in CaOV3 and SkOV3 cells (Fig. 5B). We also tested endogenous gene responses in SkOV3 cells treated with 0.1, 1, or 10 ng/ml BMP4 and observed greater increases in target gene mRNA comparing 0.1–1 or 10 ng/ml BMP4; however, we did not see any difference in mRNA expression levels comparing 1–10 ng/ml. To address whether normal OSE and primary OC cells possess the capacity to induce BMP regulated target genes, Northern analyses were performed on RNA isolated from four normal OSE and five primary OC cell samples either


with or without 10 ng/ml rhBMP4 treatment for 90 min. A representative Northern blot shows that several, but not all, normal OSE and primary OC cells showed elevated SMAD6 and SMAD7 mRNA after rhBMP4 treatment (Fig. 5A). Thus, it appeared that the ability of BMP4 to induce the SMAD6 and SMAD7 genes was variable among patient samples. Despite similarly low basal levels of ID1 expression in normal OSE and primary OC cells, ID1 mRNA was induced to higher levels in primary OC cultures (10- to 15-fold) than normal OSE (2- to 3-fold). Similarly, BMP4 treatment led to a 2- to 3-fold induction of ID3 mRNA in normal OSE, compared with a 10- to 15-fold increase in primary OC cells. The peak level of both ID1 and ID3 expression was at 90 min in a primary OC sample, similar to the results observed for the OC cell lines (Fig. 5B). Thus, BMP4 is able to up-regulate the expression of several BMP target genes in OSE and OC cells, indicating they possess a functional BMP4 signaling pathway. BMP4 effects on OC cell proliferation, morphology, and adherence

BMP treatment of various cancer cells has been documented to induce growth arrest, or apoptosis (9, 24). To address whether BMP4 treatment affects OC cell growth, five primary OC cell samples and the CaOV3 and SkOV3 cell lines were treated with 10 ng/ml rhBMP4 for 6 d (Fig. 6). Treatment of the primary OC cells with 0.1 ng/ml TGF␤1 was used as a control to induce growth arrest. The dose of TGF␤1 was selected based on our previously published work

FIG. 6. BMP4 effects on OC cell proliferation. Primary OC cells and SkOV3 cells were treated with 10 ng/ml rhBMP4 or left untreated for 6 d in complete media (10% FBS). Primary OC cells were also treated with 0.1 ng/ml TGF␤1 to serve as a control for cell growth arrest. SkOV3 cells are not growth inhibited by TGF␤1 (18); therefore, these cells were not treated with TGF␤1. Cells were counted every 2 d, and media were changed on d 3. The y-axis represents cell number (⫻104). The data represent duplicate wells of cells from at least two independent experiments.


showing that 0.1 ng/ml TGF␤1 effectively blocks primary human ovarian cancer cell proliferation (18). We have conducted dose-response curves ranging from 0.1 to 10 ng/ml TGF␤1 and found no difference in the efficacy of growth inhibition in these cells. CaOV3 and SkOV3 cells are not growth inhibited by TGF␤1 (18). Although there was no difference in the overall growth rate among the cell samples tested, the cell density at d 6 of several BMP4-treated primary OC cells (OC13 and OC14 but not OC22) and the SkOV3 cell line was slightly reduced, compared with untreated cultures (Fig. 6). This difference was manifested by altered cell morphology (Fig. 7, A and B). Treated cells were more flattened in appearance, compared with the characteristic cobblestone epithelial morphology of untreated primary OC cells. These alterations were identified in eight different primary OC cultures. However, BMP4 treatment did not change the general cytoskeletal architecture as visualized using anti-␤ actin antibody (Fig. 7, C and D), and it did not grossly alter the formation of adherens junctions of primary OC cells, indicated by membrane localization of ␤-catenin (Fig. 7, E and F). Because BMP4 treatment led to altered OC cell morphology resembling cell spreading, we therefore sought to determine whether BMP4-stimulated OC cells had altered cell attachment to the cell culture substratum. Untreated primary OC cell cultures were readily released on weak trypsin incubation, whereas a greater proportion of BMP4-treated cells remained attached and continued to display the more flattened cell morphology described above (Fig. 8).

3312



FIG. 8. BMP4-treated cells have increased adhesion. Primary OC cells at 80 –90% confluence were treated with 10 ng/ml rhBMP4 (filled bars) or left untreated (open bars) for 48 h in complete medium (10% FBS). Cells were isolated after treatment with 0.06% trypsin, and any adherent cells remaining were lifted with 0.25% trypsin. Cells from each pool were then counted. The proportion of cells lifted by treatment with 0.06% trypsin is shown. Approximately 60 –90% of untreated cells detached from the tissue culture dishes after weak trypsinization, whereas all BMP4-treated cells studied had enhanced adherence. The data represent the mean and SD from at least two independent experiments.

FIG. 7. BMP4 induces morphological change of primary human OC cells. Primary OC cells were left untreated (A, C, E) or treated with 10 ng/ml rhBMP4 (B, D, F) for 6 d in complete medium (10% FBS). Cells (OC16) were photographed on d 6 of the representative experiment. BMP4-treated cells (B) do not display the cobblestone morphology characteristic of confluent OC cells (A) but rather have a reduced cell density and a more flattened and irregular shape. BMP4treated cells do not have altered cytoskeletal characteristics (D), compared with untreated control cultures (C) as visualized using anti␤-actin antibody. Cell-cell contacts via adherens junctions as detected using the anti-␤-catenin antibody were not grossly different between untreated (E) and BMP4-treated OC cells (F).

Discussion

Our discovery that human OSE and OC cells express the components for BMP signaling and maintain a functional BMP pathway provides a rationale to further examine the role that BMP signaling may play in normal OSE biology and ovarian tumorigenesis. The OSE forms a continuous cell monolayer attached to the ovarian surface by a basement membrane composed primarily of fibronectin, type IV collagen, and laminin. Cultured OSE cells express the integrins that are capable of binding these components of the extracellular matrix (ECM) (25). OSE cells also express vitronectin and its associated integrin, ␣v␤3, both of which enhance OSE cell adhesion (26). Ruptures in the OSE after ovulation are quickly repaired by a process involving extracellular matrix production, integrin expression, and reestablishment of adhesion to the underlying basal lamina. Coordinated expression of integrins and ECM substrates during postovulatory

repair is likely critical for maintenance of the OSE (6). Cultured OC cells express many of the same integrins as normal OSE, and their adhesion is dependent on interaction with their respective ECM substrates. OC cells also express ␤1 integrins (27) that may function to promote OC cell adhesion to peritoneal mesothelial surfaces during metastasis (28). The coordinated expression of vitronectin and ␣v␤3 integrin partially mediates OC cell adhesion and regulates the expression of genes involved in ECM degradation (29). Treatment of OC cells with interfering antibodies to ␣v integrin does not interfere with cell attachment; however, it does lead to disruption of OC intercellular contacts and decreased ability of the cells to spread on the cell substratum (30). BMP signaling is known to affect cell adhesion, and expression of BMP signaling components has been implicated in the metastatic potential of some human cancers (14, 15, 31, 32). Osteogenic sarcoma-derived cell lines and human fetal chondrocytes treated with BMP2 have enhanced cell adhesion to types I and IV collagens, fibronectin, and vitronectin as well as down-regulated expression of ␣3␤1 integrin and reduced adhesion to laminin-5 (33). Treatment of primary OC cells with BMP4-enhanced cell adhesion and increased cell spreading. We propose that autocrine BMP4 signaling regulates the expression of ECM components and/or integrin receptors, such as vitronectin and ␣v␤3 integrins, in OSE and OC cells. Confirmation of this concept will rely on abrogation of the BMP signal using neutralizing antibodies or the BMP2/4 inhibitor noggin to establish that autocrine BMP4 signaling may play a role in coordinating postovulatory OSE repair or contribute to OC metastasis. BMP signaling leads to the up-regulation of several distinct target genes, including members of the ID gene family (34, 35). We found that BMP4 signaling leads to the rapid induction of ID1 and ID3 gene expression in both normal OSE and OC cells. Id proteins interact with a variety of bHLH (e.g. E12, E47) and non-bHLH (ETS, paired-domain homeobox) transcription factors that contribute to normal growth regulation, and thus dysregulated Id expression may


contribute to abnormal cell biology (17). Indeed, Id proteins have oncogenic properties (36 –38), and their expression is increased in a variety of human tumors including pancreatic cancer (39), invasive breast carcinoma (40), cervical cancer (41), colorectal adenocarcinoma, multiple myeloma (17), and ovarian cancer (42). BMP2-stimulated mouse embryonic stem cells, as well as BMP4-treated MCF-7 human breast cancer cells, up-regulate the expression of ID1, ID2, and ID3 (34, 35). Strikingly, BMP4 treatment of primary OC cells led to a greater induction of ID1 and ID3 gene expression (⬃10to 15-fold) vs. the induction observed in normal OSE cultures (⬃2- to 3-fold). This observation does not correlate with alterations in mean fold increases in Smad1 phosphorylation levels, which were similar for normal OSE (2.4 ⫾ 0.6), compared with OC cells (2.0 ⫾ 0.5). We hypothesize that elevated levels of Id proteins induced by BMP4 signaling may contribute to abnormal transcriptional regulation of target genes critical for OSE growth control. Recently Schindl et al. (42) determined that Id1 expression in human ovarian cancer in vivo was associated with more aggressive tumor cells and poor patient outcome. Thus, developing in vitro and in vivo models of Id protein overexpression in OSE and OC cells, and identifying the basis for the differential ID1 and ID3 gene expression, will add to our knowledge of ovarian pathogenesis. Ovarian cancer has the highest mortality rate of the gynecological malignancies primarily because of the lack of early detection of the disease. The identification of novel signaling pathways and/or altered gene expression involved in OC progression is critical to the development of better diagnostics and more efficacious therapeutics. Our data are the first to illustrate both the presence and putative function of BMP signaling in OSE and OC cells. In addition, the heightened level of BMP4 regulated ID1 and ID3 gene expression in OC cells has the potential to be exploited in the early detection and treatment of ovarian carcinogenesis. Acknowledgments The authors acknowledge Drs. R. Grimshaw and J. Bentley (Gynecologic Oncology, QEII Health Science Centre) for providing human ovarian tumor samples and Dr. T. F. Baskett and the staff of the QEII Ob/Gyn Unit for providing normal human ovarian tissue. The authors also thank Drs. C.-H. Heldin, M. Kato, and E. J. Robertson for plasmids; Dr. N. Auersperg for immortalized OSE cell lines; and Dr. Y. Fu, E. J. Campbell, and D. C. Lagace for critical reading of the manuscript. Received February 7, 2003. Accepted April 18, 2003. Address all correspondence and requests for reprints to: Mark W. Nachtigal, Dalhousie University, Department of Pharmacology, 5850 College Street, Halifax Nova Scotia, Canada B3H 1X5. E-mail: mark. [email protected]. This work was supported in part by the National Cancer Institute of Canada with funds from the Canadian Cancer Society (13631), the Nova Scotia Health Research Foundation, and CaRE Nova Scotia with funding from the Faculty of Medicine, Dalhousie University. T.G.S. is supported by a CaRE Fellowship with funding from the Canadian Cancer Society, and M.W.N. is a Canadian Institutes of Health Research Scholar.


3.

4.

5.

6. 7. 8. 9.

10. 11. 12.

13.

14. 15.

16. 17. 18. 19. 20.

21.

22. 23.

24.

References 1. Balemans W, Van Hul W 2002 Extracellular regulation of BMP signaling in vertebrates: a cocktail of modulators. Dev Biol 250:231–250 2. Shimasaki S, Zachow RJ, Li D, Kim H, Iemura S, Ueno N, Sampath K, Chang

25.

RJ, Erickson GF 1999 A functional bone morphogenetic protein system in the ovary. Proc Natl Acad Sci USA 96:7282–7287 Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, Prasad SV, Skinner SS, Dunbar BS, Dube JL, Celeste AJ, Matzuk MM 2001 Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol Endocrinol 15:854 – 866 Galloway SM, McNatty KP, Cambridge LM, Laitinen MP, Juengel JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG, Montgomery GW, Beattie AE, Davis GH, Ritvos O 2000 Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosagesensitive manner. Nat Genet 25:279 –283 Wilson T, Wu XY, Juengel JL, Ross IK, Lumsden JM, Lord EA, Dodds KG, Walling GA, McEwan JC, O’Connell AR, McNatty KP, Montgomery GW 2001 Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells. Biol Reprod 64:1225–1235 Auersperg N, Wong AS, Choi KC, Kang SK, Leung PC 2001 Ovarian surface epithelium: biology, endocrinology, and pathology. Endocr Rev 22:255–288 Yamada N, Kato M, ten Dijke P, Yamashita H, Sampath TK, Heldin CH, Miyazono K, Funa K 1996 Bone morphogenetic protein type IB receptor is progressively expressed in malignant glioma tumours. Br J Cancer 73:624 – 629 Ide H, Yoshida T, Matsumoto N, Aoki K, Osada Y, Sugimura T, Terada M 1997 Growth regulation of human prostate cancer cells by bone morphogenetic protein-2. Cancer Res 57:5022–5027 Soda H, Raymond E, Sharma S, Lawrence R, Cerna C, Gomez L, Timony GA, Von Hoff DD, Izbicka E 1998 Antiproliferative effects of recombinant human bone morphogenetic protein-2 on human tumor colony-forming units. Anticancer Drugs 9:327–331 Kawamura C, Kizaki M, Yamato K, Uchida H, Fukuchi Y, Hattori Y, Koseki T, Nishihara T, Ikeda Y 2000 Bone morphogenetic protein-2 induces apoptosis in human myeloma cells with modulation of STAT3. Blood 96:2005–2011 Hjertner O, Hjorth-Hansen H, Borset M, Seidel C, Waage A, Sundan A 2001 Bone morphogenetic protein-4 inhibits proliferation and induces apoptosis of multiple myeloma cells. Blood 97:516 –522 Wach S, Schirmacher P, Protschka M, Blessing M 2001 Overexpression of bone morphogenetic protein-6 (BMP-6) in murine epidermis suppresses skin tumor formation by induction of apoptosis and downregulation of fos/jun family members. Oncogene 20:7761–7769 Hamdy FC, Autzen P, Robinson MC, Horne CH, Neal DE, Robson CN 1997 Immunolocalization and messenger RNA expression of bone morphogenetic protein-6 in human benign and malignant prostatic tissue. Cancer Res 57: 4427– 4431 Autzen P, Robson CN, Bjartell A, Malcolm AJ, Johnson MI, Neal DE, Hamdy FC 1998 Bone morphogenetic protein 6 in skeletal metastases from prostate cancer and other common human malignancies. Br J Cancer 78:1219 –1223 Kim IY, Lee DH, Ahn HJ, Tokunaga H, Song W, Devereaux LM, Jin D, Sampath TK, Morton RA 2000 Expression of bone morphogenetic protein receptors type-IA, -IB and -II correlates with tumor grade in human prostate cancer tissues. Cancer Res 60:2840 –2844 Jin Y, Tipoe GL, Liong EC, Lau TY, Fung PC, Leung KM 2001 Overexpression of BMP-2/4, -5 and BMPR-IA associated with malignancy of oral epithelium. Oral Oncol 37:225–233 Norton JD 2000 ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis. J Cell Sci 113:3897–3905 Dunfield LD, Campbell Dwyer EJ, Nachtigal MW 2002 TGF ␤-induced Smad signaling remains intact in primary human ovarian cancer cells. Endocrinology 143:1174 –1181 Dunfield LD, Shepherd TG, Nachtigal MW 2002 Primary culture and mRNA analysis of human ovarian cells. Biol Proc Online 4:55– 61 Ishida W, Hamamoto T, Kusanagi K, Yagi K, Kawabata M, Takehara K, Sampath TK, Kato M, Miyazono K 2000 Smad6 is a Smad1/5-induced smad inhibitor. Characterization of bone morphogenetic protein-responsive element in the mouse Smad6 promoter. J Biol Chem 275:6075– 6079 Koenig BB, Cook JS, Wolsing DH, Ting J, Tiesman JP, Correa PE, Olson CA, Pecquet AL, Ventura F, Grant RA, Chen G-X, Wrana JL, Massague J, Rosenbaum JS 1994 Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells. Mol Cell Biol 14:5961–5974 Kretzschmar M, Liu F, Hata A, Doody J, Massague J 1997 The TGF-␤ family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev 11:984 –985 Afrakhte M, Moren A, Jossan S, Itoh S, Sampath K, Westermark B, Heldin CH, Heldin NE, ten Dijke P 1998 Induction of inhibitory Smad6 and Smad7 mRNA by TGF-␤ family members. Biochem Biophys Res Commun 249: 505–511 Ghosh-Choudhury N, Ghosh-Choudhury G, Celeste A, Ghosh PM, Moyer M, Abboud SL, Kreisberg J 2000 Bone morphogenetic protein-2 induces cyclin kinase inhibitor p21 and hypophosphorylation of retinoblastoma protein in estradiol-treated MCF-7 human breast cancer cells. Biochim Biophys Acta 1497:186 –196 Kruk PA, Uitto VJ, Firth JD, Dedhar S, Auersperg N 1994 Reciprocal interactions between human ovarian surface epithelial cells and adjacent extracellular matrix. Exp Cell Res 215:97–108

3314


26. Cruet S, Salamanca C, Mitchell GW, Auersperg N 1999 ␣v␤3 and vitronectin expression by normal ovarian surface epithelial cells: role in cell adhesion and cell proliferation. Gynecol Oncol 75:254 –260 27. Cannistra SA, Ottensmeier C, Niloff J, Orta B, DiCarlo J 1995 Expression and function of ␤ 1 and ␣ v ␤ 3 integrins in ovarian cancer. Gynecol Oncol 58: 216 –225 28. Strobel T, Cannistra SA 1999 ␤1-integrins partly mediate binding of ovarian cancer cells to peritoneal mesothelium in vitro. Gynecol Oncol 73:362–367 29. Hapke S, Kessler H, Arroyo de Prada N, Benge A, Schmitt M, Lengyel E, Reuning U 2001 Integrin ␣(v)␤(3)/vitronectin interaction affects expression of the urokinase system in human ovarian cancer cells. J Biol Chem 276:26340 –26348 30. Carreiras F, Cruet S, Staedel C, Sichel F, Gauduchon P 1999 Human ovarian adenocarcinoma cells synthesize vitronectin and use it to organize their adhesion. Gynecol Oncol 72:312–322 31. Heikinheimo KA, Laine MA, Ritvos OV, Voutilainen RJ, Hogan BL, Leivo IV, Heikinheimo AK 1999 Bone morphogenetic protein-6 is a marker of serous acinar cell differentiation in normal and neoplastic human salivary gland. Cancer Res 59:5815–5821 32. Howe JR, Bair JL, Sayed MG, Anderson ME, Mitros FA, Petersen GM, Velculescu VE, Traverso G, Vogelstein B 2001 Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis. Nat Genet 28:184 –187 33. Nissinen L, Pirila L, Heino J 1997 Bone morphogenetic protein-2 is a regulator of cell adhesion. Exp Cell Res 230:377–385 34. Hollnagel A, Oehlmann V, Heymer J, Ruther U, Nordheim A 1999 Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. J Biol Chem 274:19838 –19845


35. Clement JH, Marr N, Meissner A, Schwalbe M, Sebald W, Kliche KO, Hoffken K, Wolfl S 2000 Bone morphogenetic protein 2 (BMP-2) induces sequential changes of Id gene expression in the breast cancer cell line MCF-7. J Cancer Res Clin Oncol 126:271–279 36. Desprez P-Y, Lin CQ, Thomasset N, Sympson CJ, Bissell MJ, Campisi J 1998 A novel pathway for mammary epithelial cell invasion induced by the helixloop-helix protein Id-1. Mol Cell Biol 18:4577– 4588 37. Norton JD, Atherton GT 1998 Coupling of cell growth control and apoptosis functions of Id proteins. Mol Cell Biol 18:2371–2381 38. Wice BM, Gordon JI 1998 Forced expression of Id-1 in the adult mouse small intestinal epithelium is associated with development of adenomas. J Biol Chem 273:25310 –25319 39. Kleeff J, Ishiwata T, Friess H, Buchler MW, Israel MA, Korc M 1998 The helix-loop-helix protein Id2 is overexpressed in human pancreatic cancer. Cancer Res 58:3769 –3772 40. Lin CQ, Singh J, Murata K, Itahana Y, Parrinello S, Liang SH, Gillett CE, Campisi J, Desprez P-Y 2000 A role for Id-1 in the aggressive phenotype and steroid hormone response of human breast cancer cells. Cancer Res 60:1332– 1340 41. Schindl M, Oberhuber G, Obermair A, Schoppmann SF, Karner B, Birner P 2001 Overexpression of Id-1 protein is a marker for unfavorable prognosis in early-stage cervical cancer. Cancer Res 61:5703–5706 42. Schindl M, Schoppmann SF, Stro¨ bel T, Heinzl H, Leisser C, Horvat R, Birner P 2003 Level of Id-1 protein expression correlates with poor differentiation, enhanced malignant potential, and more aggressive clinical behavior of epithelial ovarian tumors. Clin Cancer Res 9:779 –785