Bone Morphogenetic Protein-4 Inhibits Corticotroph Tumor Cells: Involvement in the Retinoic Acid Inhibitory Action
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《内分泌学杂志》
Laboratorio de Fisiología y Biología Molecular (D.G., D.R., J.G., E.A.), Departamento de Fisiología, Biología Molecular y Celular, Facultad Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina
Max-Planck Institute of Psychiatry (M.P.-P., M.T., M.L., U.R., G.K.S.), 80804 Munich, Germany
Hospital Santa Lucía (A.C., S.B.), 1232 Buenos Aires, Argentina
Department of Neurosurgery (M.L.), Ospedale San Raffaele Instituto di Ricovero e Cura a Carattere Scientifico, 20132 Milan, Italy
Department of Neurosurgery (M.B.), University of Gottingen Medical School, 37075 Gottingen, Germany
Affectis Pharmaceuticals (M.P.-P.), 80804 Munich, Germany
Abstract
The molecular mechanisms governing the pathogenesis of ACTH-secreting pituitary adenomas are still obscure. Furthermore, the pharmacological treatment of these tumors is limited. In this study, we report that bone morphogenetic protein-4 (BMP-4) is expressed in the corticotrophs of human normal adenohypophysis and its expression is reduced in corticotrophinomas obtained from Cushing’s patients compared with the normal pituitary. BMP-4 treatment of AtT-20 mouse corticotrophinoma cells has an inhibitory effect on ACTH secretion and cell proliferation. AtT-20 cells stably transfected with a dominant-negative form of the BMP-4 signal cotransducer Smad-4 or the BMP-4 inhibitor noggin have increased tumorigenicity in nude mice, showing that BMP-4 has an inhibitory role on corticotroph tumorigenesis in vivo. Because the activation of the retinoic acid receptor has an inhibitory action on Cushing’s disease progression, we analyzed the putative interaction of these two pathways. Indeed, retinoic acid induces both BMP-4 transcription and expression and its antiproliferative action is blocked in Smad-4dn- and noggin-transfected Att-20 cells that do not respond to BMP-4. Therefore, retinoic acid induces BMP-4, which participates in the antiproliferative effects of retinoic acid. This new mechanism is a potential target for therapeutic approaches for Cushing’s disease.
Introduction
CORTICOTROPH ADENOMAS are usually small microadenomas at the time of diagnosis, accounting for about 10–15% of all clinically recognized pituitary adenomas (1, 2, 3). ACTH-secreting pituitary tumors generate a glucocorticoid hypersecretion disorder, called Cushing’s disease, which produces adrenal hyperplasia, abnormal fat deposition, thinning of the skin, hypertension, osteoporosis, diabetes, and psychological disturbances (4, 5, 6). Monoclonal expansion of transformed corticotroph cells of the anterior pituitary is one of the mechanisms that leads to ACTH-secreting pituitary tumors development (7), although pluriclonality has also been suggested (8). Although transsphenoidal surgical removal of ACTH-secreting pituitary microadenomas (<1 cm diameter) is an effective approach with an overall cure rate of about 70%, it is only effective in 30% of macroadenomas (>1 cm diameter) (9, 10, 11). Moreover, some inhibitors of ACTH production have been described but they are not effective in Cushing’s disease treatment (12).
Much work has been done to find out not only the molecular pathogenetic mechanism of ACTH-secreting pituitary tumors development but also an effective drug therapy for these pituitary tumors. In a previous study, we demonstrated that retinoic acid treatment reduces proopiomelanocortin (POMC) gene transcription and ACTH production by inhibiting the AP-1 and Nur77/Nurr1 transcriptional activities in pituitary ACTH-secreting tumor cells (13). Expression in ACTH-secreting AtT-20 cells of the orphan receptor COUP-TFI, a negative regulator of the retinoic acid response pathway (14), blocked the inhibitory action of retinoic acid (13). In vivo experiments in nude mice have shown that retinoic acid administration completely blocks corticotroph tumor growth and reverses the endocrine alterations and symptoms of Cushing’s disease (13). All these data support the notion that retinoic acid might be an interesting drug for Cushing’s disease treatment (13). Although the inhibitory mechanism of retinoic acid on ACTH biosynthesis at the level of AP-1 and Nur77/Nurr1 transcriptional activities and POMC transcription in ACTH-secreting tumor cells has been demonstrated, the inhibitory mechanism over cell proliferation is still not clear. Furthermore, the related receptor, peroxisome proliferator-activated receptor- has also been shown as an important target for drug therapies for Cushing’s disease. Peroxisome proliferator-activated receptor--activating ligands, induce cell-cycle arrest and apoptosis in corticotrophinoma cells, induce tumor growth arrest in vivo, and inhibit ACTH and corticosterone secretion from tumoral cells (15).
Several hypothalamic factors and hormones as well as growth factors such as TGF-, platelet-derived growth factor, and cytokines are involved in the molecular and cellular mechanisms that lead to pituitary tumor development (2, 5, 16, 17, 18). Especially estrogens and members of the TGF- family are involved in the control of lactotroph cell proliferation and promote prolactinomas formation (19, 20, 21, 22, 23). Bone morphogenetic protein-4 (BMP-4), a member of the TGF- family that plays a central role during pituitary organogenesis (24, 25), is over-expressed in different prolactinomas models including dopamine receptor 2 knockout (D2R–/–) mice, Fisher rats treated with estradiol and in human tumors, compared with normal tissue and other pituitary adenoma types. Furthermore, GH3 rat lactosomatotrophinoma cells stably transfected with plasmids that express proteins that block BMP-4 action had reduced tumorigenicity in nude mice, providing evidence that the BMP-4 stimulatory pathway plays a role in prolactinoma development in vivo (26).
BMPs, as well as other members of the TGF- family, mediate their effects by binding to a family of membrane tyrosine kinases receptors and subsequently activating a family of receptor substrates, the Smad proteins, which act as transcription factors in the nucleus (27, 28, 29). The Smad-4 protein functions as a signal cotransducer for the different Smad proteins and is required for active transcriptional complexes to assemble. The resulting Smad complex is then free to move into the nucleus and competent to bind Smad-binding elements in association with transcriptional coactivators or corepressors. Proteins such as noggin, gremlin, and caronte bind BMP proteins and prevent their association with signaling tyrosine kinase receptors, thus controlling the extracellular activating phase of BMP pathway (27, 28, 29).
Taking into consideration the crucial role of BMP-4 during pituitary organogenesis and prolactinoma tumorigenesis, we decided to study its potential action in corticotrophinoma growth. In this study, we report the differential BMP-4 expression in normal and adenomatous corticotrophs and its inhibitory action on corticotrophinoma cell proliferation. Furthermore, we show that BMP-4 mediates the antiproliferative action of retinoic acid in these cells. These results point to a novel mechanism by which BMP-4 and retinoic acid may prevent Cushing’s disease development.
Materials and Methods
Cell culture
AtT-20 pituitary corticotroph tumor cells were obtained from the American Type Culture Collection (Rockville, MD) (30). AtT-20 cells were cultured in DMEM (pH 7.3) supplemented with 10% fetal calf serum (FCS), 2.2 g/liter NaHCO3, 10 mM HEPES, and 2 mM glutamine until they were confluent under 5% CO2 atmosphere at 37 C.
Cells were treated with 10, 50, 100, or 200 ng/ml BMP-4, 0.001–1 μM noggin, 10 ng/ml TGF- (all three from R&D Systems, Minneapolis, MN), or 10 or 100 nM all-trans retinoic acid (Sigma, St. Louis, MO) that was dissolved in ethanol-DMSO as a 10 mM stock solution and handled in the dark. The diluting mixture was used in all the in vitro experiments as control.
Patients and tissues
Patients with pituitary adenomas and clinical symptoms of Cushing’s disease were diagnosed by hormonal testing and magnetic resonance imaging as previously described (31). All tumors were benign and graded according to the modified Hardy’s classification (32). Tumor tissue was obtained by transsphenoidal surgery and was transported in sterile medium. Samples from five human pituitary glands were obtained from autopsy cases without any evidence of endocrinological disease, with postmortem delay between 8 and 12 h. The tissues were snap-frozen and stored at –80 C. For immunohistochemistry, 8-μm sections of shock-frozen ACTH-secreting tumor or normal tissue were thaw-mounted onto SuperFrost Plus slides (Menzel-Glaser, Hamburg, Germany), fixed in 4% paraformaldehyde in PBS, and stored in 96% ethanol at 4 C until used.
Cell proliferation and viability
A WST-1 assay (Roche Molecular Biochemicals, Basel, Switzerland) was used to measure cell proliferation and cell viability following the manufacturer’s instructions as previously described (33). The method was validated by total cell count. Acridine orange-ethidium bromide staining was used to rule out toxic effects.
Plasmids
The Smad-4 dominant-negative mutant (Smad-4dn) expression vector consists of a truncated DPC-4 (Smad-4) cDNA (1–514) fused to FLAG expressed under the control of the cytomegalovirus promoter (34). The noggin expression vector contains 1 kb of the mouse noggin cDNA driven by the EF1 promoter (35). The mouse BMP-4 1A promoter contains the –2372/+258 fragment from the 5' flanking region and includes all the necessary sequences for the expression and regulation of BMP-4 in pituitary cell cultures (36). COUP-TFI expression vector contains the 1.5-kb segment of the mouse COUP-TFI cDNA under the control of a cytomegalovirus promoter, and COUP-TFI control vector contains an incomplete segment of the mouse COUP-TFI cDNA that codifies for a truncated inactive protein (37). The human c-myc promoter construction contains the –2329/+510 fragment from the 5' flanking region upstream from the luciferase gene of the pGL3-Basic vector (38).
AtT-20 stable clones
AtT-20 cells were stably transfected with noggin or Smad-4dn expression vectors or the corresponding control vector using lipofectamine as described below, and after 48 h, were selected in culture medium with 600 ng/ml G418 (Invitrogen, Eggenstein, Germany). The selected AtT-20 noggin and AtT-20 Smad-4dn clones were tested for noggin and Smad-4dn expression by Northern blot and Western blot, respectively as described below. Three additional stable clones for each construct had similar results as the one that is shown.
Transfection and reporter assays
Cell transfection was performed with lipofectamine using standard procedures, as previously described (39). After plating in six-well plates, the cells were transfected for 6 h in OPTIMEM (Invitrogen) using 8 μl of lipofectamine (Invitrogen) per well and 1 μg total plasmid DNA. Cells were then washed and left overnight in serum-free DMEM. The following day, cells were washed and treated for 6 or 24 h in serum-free DMEM with the indicated compounds. An additional 200 ng of the RSV--gal construction was cotransfected in all the experiments to correct for variations in transfection efficiency as previously described (39). Luciferase activity was measured with a Junior luminometer (Berthold, Bad Wildbad, Germany). Results are ratios of luciferase and -galactosidase activity. Protein content was measured by a Bradford assay as a further control for the reproducibility of the sampling procedure.
Western blot analysis
Western blot was performed as previously described (26). Briefly AtT-20 cells were washed once with PBS (pH 7.0), and then cell lysates were prepared in standard cracking buffer and boiled for 3 min. Equal levels of protein (30 μg) were electrophoresed by 12% SDS-PAGE. Proteins were blotted onto nitrocellulose western blotting membranes (Sigma) using standard procedures and anti-BMP-4 (1:1,000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-GADPH (1:50,000) (Santa Cruz Biotechnology Inc.), or anti-FLAG antibody (Sigma) were added. The primary antibodies were detected with HRP-conjugated antigoat IgG secondary antibodies (Santa Cruz Biotechnology Inc.).
Northern blot analysis
Total RNA extraction and Northern blot analysis were performed as described previously (31). Briefly, total RNA was isolated by the guanidine isothiocyanate phenol-chloroform extraction method, denatured with glyoxal, electrophoresed, and transferred to a nylon membrane. Filters were prehybridized for 1 h at 60 C and then the probe was added and hybridized for 12 h. Blots were washed at increasing salt and temperature stringency and then exposed to Kodak XAR5 film at –70 C with intensifying screens for 6 d. A 0.5-kb noggin cDNA fragment (35) and a 6.6-kb EcoRI fragment of a 28s and part of the 18s mouse cDNA (31) were labeled with a random-priming kit using [-32P]dCTP (specific activity, 2–4 x 108 cpm/μg). The loading control with the fragment of 18s cDNA as probe was performed in each blot.
Immunohistochemistry
To detect BMP-4 immunoreactivity in normal pituitary and in corticotrophinomas, a goat polyclonal BMP-4 antibody (1:100) (Santa Cruz Biotechnology Inc.) was used in combination with antigoat biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) and avidin-biotin-peroxidase complex (Vector Laboratories). Immunoreactivity was visualized with diaminobenzidine (Sigma) and 0.01% hydrogen peroxide. ACTH was detected with a monoclonal antibody (1:800) (Dako Diagnostika, Hamburg, Germany) in combination with an antimouse IgG (Sigma) and mouse alkaline phosphatase-anti-alkaline phosphatase (Sigma), using vector red (Vector Laboratories). Specific antibodies for prolactin (PRL), FSH, LH, GH, and TSH were also used (1:500) (all from Immunotech, Karlsruhe, Germany). Negative controls were performed by omitting the primary antibody.
In vivo tumor formation
ACTH-secreting experimental tumors were formed by injecting AtT-20 control, AtT20 noggin, or AtT20 Smad-4dn clone cells into a subdermal pouch in BALB/cAnN Crl-nu BR nude mice as previously described (26, 30). To evaluate BMP-4 role on tumor formation, suboptimal amounts of clone cells were injected in nude mice to distinguish differences in tumor growth. All the cells were controlled for viability. Tumor formation was monitored every 2 d during 3 wk. The tumor volume was measured as previously described (26). Briefly a vernier was used to measure the short and long axis of the tumor and the following formula was used: tumoral volume = [(short axis)2 x long axis x 0.4]. Blood samples were collected by heart puncture from halothane-anesthetized animals. Histological examination was performed by hematoxylin-eosin staining of shock-frozen tissues. All animal experiments were performed in accordance with institutional regulations of animal care.
Hormone measurements
ACTH was determined by radioimmunoassay as previously described (31, 33).
Statistical analysis
Statistics were performed using one-factor ANOVA in combination with Scheffe’s test.
Results
BMP-4 expression in pituitary corticotroph tumors
In the normal human adenohypophysis, BMP-4 was present in 40% of endocrine cells, which were mostly found in clusters and rarely scattered (Fig. 1A). Double immunohistochemistry revealed that 30% of the somatotroph, corticotroph, and thyrotroph population were immunopositive for BMP-4 (Fig. 1B). On the other hand, PRL and FSH/LH immunopositive cells rarely stained for BMP-4. Immunohistochemistry in 15 corticotrophinomas revealed variable BMP-4 expression (Fig. 1C and Table 1). Seven of 15 cases had very low BMP-4 immunoreactivity and five of 15 cases did not express BMP-4. Only three of 15 cases had a number of BMP-4-expressing cells higher than 10%. No correlation with tumor grade was observed. The overall expression in corticotrophinomas was lower than in prolactinomas, as we have already described (26). An overall reduced BMP-4 expression is observed in Cushing’s disease adenomas compared with normal corticotroph cells (Fig. 1 and Table 1). Based on this differential BMP-4 expression pattern between normal and tumor corticotroph cells, we further studied a possible role of BMP-4 in corticotroph tumor function and growth.
BMP-4 inhibits murine corticotroph tumor cells ACTH-secretion and cell proliferation
We examined the effect of BMP-4 on ACTH secretion by AtT-20 corticotroph tumor cells. BMP-4 treatment inhibited both the endogenous and forskolin-stimulated ACTH induction in a dose-dependent manner (Fig. 2A). The specific BMP-4 antagonist noggin blocked dose dependently this inhibitory effect (Fig. 2B). The inhibitory effect of BMP-4 on ACTH production was not affected by dexamethasone, because cotreatment with both factors did not shown an increment in BMP-4 inhibitory effect (Fig. 2C).
To further establish the actions of BMP-4 on corticotroph cells, we studied cell proliferation. We observed that BMP-4 inhibited AtT-20 cell proliferation after 3 d of BMP-4 treatment, and the effect is still observed after 5 d (Fig. 3A). On the contrary, TGF- treatment did not affect cell proliferation (Fig. 3A). Similar results were obtained by total cell count, and no cell death was observed by acridine orange-ethidium staining, ruling out toxic effects (data not shown).
The c-myc oncogene is a critical regulator of cell cycle progression and it was recently described that BMP-4 induces c-myc expression in GH3 lactosomatotrophinoma cells and in human prolactinomas (26). Thus, we determined whether BMP-4 exerts its antiproliferative action in AtT-20 cells by altering c-myc activity. Indeed, as shown in Fig. 3B, BMP-4 inhibited c-myc promoter activity.
BMP-4 effects on murine corticotrophs tumor cells in vivo
Smad-4 protein functions as a signal cotransducer for the different TGF- superfamily factors and is necessary for active transcriptional complexes to assemble. Preventing dimerization of active complexes can block BMP-4 signaling, and BMP-4 extracellular binding can be prevented by noggin, which is a BMP-4-specific antagonist.
To test whether BMP-4 antiproliferative effect on AtT-20 cells also affects the ability of ACTH-secreting cells to promote tumors in vivo, we produced AtT-20 clones stably transfected with the Smad-4dn form or noggin to block BMP-4 signaling (Fig. 4, A and B, inset). Expression of Smad-4dn or noggin blocked the antiproliferative effect of BMP-4 (Fig. 4, A and B). Taking into account the BMP-4 inhibitory action in vitro, we hypothesized that blocking BMP-4 inhibitory growth control would result in enhanced tumor growth. Thus, to observe differences in tumor growth, nude mice were injected with a suboptimal concentration of AtT-20 cells stably transfected with an empty vector, Smad-4dn, or with noggin. When a suboptimal concentration of AtT-20 cells stably transfected with the empty vector (control cells) were injected into nude mice no tumors, were observed even after 8 wk (Fig. 5, A and B). In contrast, after 2 wk, tumors developed in all the mice injected with AtT-20 Smad-4dn or AtT-20 noggin clone cells (Fig. 5, A and B). Moreover, the phenotype of the three mice groups was different; whereas the control group did not show any change, the Smad-4dn and noggin groups presented a characteristic Cushing’s disease appearance with noticeably thinner skin and sc fat tissue increase.
Retinoic acid inhibits corticotroph cells by inducing BMP-4
Given the reduced expression of BMP-4 and its inhibitory action in tumor corticotroph growth, we studied its putative involvement in retinoic acid antiproliferative action. We analyzed whether retinoic acid induces BMP-4 in AtT-20 cells. After 24 h of treatment with 10–100 nM retinoic acid, an increase in BMP-4 promoter transcriptional activity was observed (4-fold induction) (Fig. 6A). An almost complete inhibition of the BMP-4 promoter responsiveness to retinoic acid was observed when cotransfecting the BMP-4-LUC promoter construct with a COUP-TFI expression vector (Fig. 6A). Moreover, the expression of COUP-TFI enhances the transcription of BMP-4 (3-fold induction) (Fig. 6A). To control the ability of BMP-4 promoter to be further activated in the presence of COUP-TFI, we treated the cells with cAMP and observed the induction of BMP-4 promoter (Fig. 6A). We further evaluated BMP-4 expression by Western blot. Retinoic acid increased BMP-4 protein levels and this stimulatory effect diminished after 24 h of treatment (Fig. 6B).
To test the hypothesis that the inhibitory effect of retinoic acid in AtT-20 cell proliferation could be mediated by BMP-4, we examined whether BMP-4 signaling is necessary for retinoic acid effects. In both AtT-20 Smad-4dn and AtT-20 noggin cells that do not respond to BMP-4, retinoic acid treatment was not able to inhibit cell proliferation (Fig. 7, A and B).
AtT-20 control cells responded to both BMP-4 and retinoic acid treatment. However, combined administration of the two compounds did not result in a further antiproliferative effect, supporting the notion that BMP-4 and retinoic acid might interact (Fig. 7C).
Discussion
The pathogenic mechanisms as well as the treatment of Cushing’s disease remains still an open question. In this study, we demonstrate for the first time that BMP-4 expression is reduced in corticotrophinoma cells and that BMP-4 regulates not only corticotroph cell physiology but also inhibits its tumorigenic potential. This inhibitory effect of BMP-4 constitutes a novel mechanism for pituitary corticotroph pathogenesis. We also found that retinoic acid, used as a pharmacological agent in different types of cancer (40, 41, 42) and which inhibits corticotrophinoma cells growth (13), may act through BMP-4 expression in corticotroph cells. Therefore, BMP-4 induction by retinoic acid may be an interesting mechanistic target for retinoic acid as a therapeutic option for Cushing’s disease treatment.
We demonstrate in this study that BMP-4 inhibits both the endogenous ACTH production and forskolin-induced ACTH stimulation in a dose-dependent manner, and this inhibitory effect was blocked by noggin, which is a specific antagonist of BMP-4. These results are in agreement with recently published data showing that BMP-4 has an inhibitory effect on the transcriptional activity of POMC promoter (43). BMP-4 inhibits not only AtT-20 cell proliferation but also c-myc promoter transcriptional activity. We have demonstrated that, in lactosomatotrophinoma GH3 cell line and human prolactinomas in which BMP-4 has a promoting role during tumor development, BMP-4 induces c-myc expression. This differential effect on c-myc depending on the cell lineage can be explained by the association of Smad-1 (BMP-4 signaling transducer protein) with different cofactors presented in each cell lineage. For example in GH3 cells BMP-4 stimulation triggers Smad-1/ER physical interaction that may have stimulatory effects over transforming gene promoters as c-myc. In corticotrophs, Smad-1 may possibly be interacting with other proteins resulting in negative effects over c-myc expression. Based on these results and the different studies that place TGF- family as important regulating factors in pituitary tumor development (23, 26), we studied a possible role for BMP-4 in corticotroph cells tumorigenicity in vivo. At low cell concentration doses AtT-20 cells present no tumorigenic capacity when injected in nude mice. In contrast, AtT-20 Smad-4dn, and AtT-20 noggin clones in which the BMP-4 signaling pathway is blocked developed visible tumors starting at 1 wk after injection as well as symptoms of Cushing’s disease. Therefore, inhibition of the BMP-4 signaling results in an increase of the AtT-20 cells tumorigenic potential, demonstrating an inhibitory role of BMP-4 in the development of corticotroph tumors in vivo.
BMP-2 and –4 play a central role during the initial steps of the anterior pituitary development (25, 44, 45, 46). BMP-4 is required during the first stage of pituitary organogenesis for the proliferation of the Rathke’s pouch placode, which gives rise to Pit-1 lineage cells, which includes the PRL-secreting cells, among others. Moreover, overexpression of noggin or a dominant-negative BMP-receptor (BMPR1A) in the anterior pituitary leads to the arrest of the development of the pit-1-expressing lineage. During the second pituitary organogenesis stage an inhibition of BMP-2 ventrodorsal gradient by an FGF8 dorsoventral gradient, leads to corticotroph differentiation (25, 44, 45, 46). Overexpression of FGF8 in the developmental pituitary results in Pit-1 lineage cells absence and corticotroph enhanced differentiation, suggesting that it is necessary to inhibit BMP signaling for normal corticotroph development (47). We have shown that BMP-4 has a strong stimulatory effect on prolactinoma cell proliferation (26). Moreover the activin/BMP system has also been described in the development of human pituitary gonadotropinomas and nonfunctioning adenomas (48). In the present study, we show BMP-4 expression in 40% of endocrine cells in the normal adenohypophysis and most of the BMP-4 immunopositive cells were somatotrophs and corticotrophs but not lactotrophs. On the other hand, in pituitary adenomas, BMP-4 is expressed at low variable levels in corticotrophinoma cells but at high levels in prolactinoma cells. Thus BMP-4, a corticotroph growth inhibitor, is expressed in the normal pituitary but is reduced in corticotroph tumors. In analogy to the situation during pituitary organogenesis, this distinctive expression may allow corticotroph tumoral growth promotion. BMP-4 not only is crucial during pituitary organogenesis but has an opposite role in the different pituitary lineages. BMP-4 promotes pituitary prolactinoma pathogenesis through a Smad/estrogen receptor cross-talk but inhibits corticotroph pathogenesis and Cushing’s disease. It is not clear at this stage, which is the trigger for BMP-4 action (enhancement of proliferation in prolactinomas and loss of inhibition control in Cushing’s corticotroph) in adult pituitary tumor cells. Thus, BMP-4 acts as a regulator no only of pituitary cells undergoing organogenesis differentiation, but also of transformed, not normal, adult pituitary cells, and it has a pituitary linage cell specificity during the tumorigenic process.
Retinoic acid treatment in AtT-20 corticotroph cells induces BMP-4 transcription and BMP-4 expression. Similar actions of retinoic acid have been described in human and murine oesteoblastic cell lines (49). Inhibiting retinoic acid signaling with COUP-TFI (14, 50, 51, 52) blocks the stimulatory effect of retinoic acid on BMP-4, although the promoter may still be stimulated by other pathways. These results are in agreement with previous ones that establish that COUP-TFI can block retinoic acid inhibitory effects on POMC-LUC reporter activity. Also COUP-TFI expression only in normal corticotroph cells but not in pituitary Cushing tumors might be the explanation for the effects of retinoic acid in transformed but not in normal corticotroph cells (13). Moreover, we show that overexpression of COUP-TFI enhances, by a mechanism independent of retinoic acid, the transcription of BMP-4 in AtT-20 cells, suggesting that the differential expression of BMP-4 in normal but not tumor cells may be under the control of COUP-TFI, as it has been described in an osteoblast model (36).
Both BMP-4 and retinoic acid inhibit AtT-20 cell proliferation, but there was no further inhibition in the presence of both factors, even at low doses, suggesting that they may share an overlapping mechanism (which is saturated at low doses). BMP and steroid receptor cross-talk takes place in several cells. A cross-talk mechanism between BMP-4 and estrogens that regulates cell proliferation has been described and a physical interaction between steroid receptors and TGF--induced Smad proteins has been recently shown (26, 53, 54). Several studies have also established a different interaction between retinoic acid and BMPs (55, 56, 57). In embryonal carcinoma cells, retinoic acid controls BMP-2 and BMP-4 regulation (58, 59, 60), and the combination of retinoic acid and BMP-2 or BMP-4 synergistically induces apoptosis of these cells inhibiting neurons and glial cells but leading to smooth muscle cell differentiation (61, 62). To investigate whether retinoic acid transduces its antiproliferative effect through BMP-4 induction, its effect was tested in cells in which the BMP-4 pathway was blocked. In AtT-20 noggin and AtT-20 Smad-4dn cells, the inhibitory action of retinoic acid is abolished, showing that, besides the possibility of a further cross-talk with BMP-4 signaling pathway, retinoic acid is acting through the induction of BMP-4 expression, which in turn has a central role in preventing corticotroph cell proliferation and tumor cell growth.
The present study highlights a role of BMP-4 in the inhibition of corticotrophinoma cell growth and suggests a novel mechanism for the pathogenesis of Cushing’s-associated pituitary adenomas. Furthermore, the fact that BMP-4 is induced by and mediates some of the effects of retinoic acid highlights a novel mechanism of retinoic acid action in corticotrophinoma cells. The knowledge of the cross-talk between retinoic acid and BMP-4 may be a useful tool for the design of new—based on retinoic acid—therapeutic approaches for Cushing’s disease treatment.
Acknowledgments
We thank J. Massague for the Smad-4dn expression vector, K. Monzen for the noggin expression vector, M. Tsai for the COUP-TFI expression vector, S. E. Harris for the mouse BMP-4 luciferase reporter vector, and K. Mitsuyasu for the human c-Myc luciferase reporter vector. We also thank Esther Jelen for technical assistance.
Footnotes
This work was supported by grants from the University of Buenos Aires, the Argentine National Research Council (CONICET), and Agencia Nacional de Promocion Científica y Tecnologica-Argentina.
D.G., D.R., and E.A. are members of the Instituto de Fisiología, Biología Molecular y Neuro Ciencias-Argentine National Research Council (CONICET).
First Published Online September 29, 2005
1 D.G. and M.P.-P. have contributed equally.
Abbreviations: BMP-4, Bone morphogenetic protein-4; FCS, fetal calf serum; POMC, proopiomelanocortin; PRL, prolactin.
Accepted for publication September 17, 2005.
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Max-Planck Institute of Psychiatry (M.P.-P., M.T., M.L., U.R., G.K.S.), 80804 Munich, Germany
Hospital Santa Lucía (A.C., S.B.), 1232 Buenos Aires, Argentina
Department of Neurosurgery (M.L.), Ospedale San Raffaele Instituto di Ricovero e Cura a Carattere Scientifico, 20132 Milan, Italy
Department of Neurosurgery (M.B.), University of Gottingen Medical School, 37075 Gottingen, Germany
Affectis Pharmaceuticals (M.P.-P.), 80804 Munich, Germany
Abstract
The molecular mechanisms governing the pathogenesis of ACTH-secreting pituitary adenomas are still obscure. Furthermore, the pharmacological treatment of these tumors is limited. In this study, we report that bone morphogenetic protein-4 (BMP-4) is expressed in the corticotrophs of human normal adenohypophysis and its expression is reduced in corticotrophinomas obtained from Cushing’s patients compared with the normal pituitary. BMP-4 treatment of AtT-20 mouse corticotrophinoma cells has an inhibitory effect on ACTH secretion and cell proliferation. AtT-20 cells stably transfected with a dominant-negative form of the BMP-4 signal cotransducer Smad-4 or the BMP-4 inhibitor noggin have increased tumorigenicity in nude mice, showing that BMP-4 has an inhibitory role on corticotroph tumorigenesis in vivo. Because the activation of the retinoic acid receptor has an inhibitory action on Cushing’s disease progression, we analyzed the putative interaction of these two pathways. Indeed, retinoic acid induces both BMP-4 transcription and expression and its antiproliferative action is blocked in Smad-4dn- and noggin-transfected Att-20 cells that do not respond to BMP-4. Therefore, retinoic acid induces BMP-4, which participates in the antiproliferative effects of retinoic acid. This new mechanism is a potential target for therapeutic approaches for Cushing’s disease.
Introduction
CORTICOTROPH ADENOMAS are usually small microadenomas at the time of diagnosis, accounting for about 10–15% of all clinically recognized pituitary adenomas (1, 2, 3). ACTH-secreting pituitary tumors generate a glucocorticoid hypersecretion disorder, called Cushing’s disease, which produces adrenal hyperplasia, abnormal fat deposition, thinning of the skin, hypertension, osteoporosis, diabetes, and psychological disturbances (4, 5, 6). Monoclonal expansion of transformed corticotroph cells of the anterior pituitary is one of the mechanisms that leads to ACTH-secreting pituitary tumors development (7), although pluriclonality has also been suggested (8). Although transsphenoidal surgical removal of ACTH-secreting pituitary microadenomas (<1 cm diameter) is an effective approach with an overall cure rate of about 70%, it is only effective in 30% of macroadenomas (>1 cm diameter) (9, 10, 11). Moreover, some inhibitors of ACTH production have been described but they are not effective in Cushing’s disease treatment (12).
Much work has been done to find out not only the molecular pathogenetic mechanism of ACTH-secreting pituitary tumors development but also an effective drug therapy for these pituitary tumors. In a previous study, we demonstrated that retinoic acid treatment reduces proopiomelanocortin (POMC) gene transcription and ACTH production by inhibiting the AP-1 and Nur77/Nurr1 transcriptional activities in pituitary ACTH-secreting tumor cells (13). Expression in ACTH-secreting AtT-20 cells of the orphan receptor COUP-TFI, a negative regulator of the retinoic acid response pathway (14), blocked the inhibitory action of retinoic acid (13). In vivo experiments in nude mice have shown that retinoic acid administration completely blocks corticotroph tumor growth and reverses the endocrine alterations and symptoms of Cushing’s disease (13). All these data support the notion that retinoic acid might be an interesting drug for Cushing’s disease treatment (13). Although the inhibitory mechanism of retinoic acid on ACTH biosynthesis at the level of AP-1 and Nur77/Nurr1 transcriptional activities and POMC transcription in ACTH-secreting tumor cells has been demonstrated, the inhibitory mechanism over cell proliferation is still not clear. Furthermore, the related receptor, peroxisome proliferator-activated receptor- has also been shown as an important target for drug therapies for Cushing’s disease. Peroxisome proliferator-activated receptor--activating ligands, induce cell-cycle arrest and apoptosis in corticotrophinoma cells, induce tumor growth arrest in vivo, and inhibit ACTH and corticosterone secretion from tumoral cells (15).
Several hypothalamic factors and hormones as well as growth factors such as TGF-, platelet-derived growth factor, and cytokines are involved in the molecular and cellular mechanisms that lead to pituitary tumor development (2, 5, 16, 17, 18). Especially estrogens and members of the TGF- family are involved in the control of lactotroph cell proliferation and promote prolactinomas formation (19, 20, 21, 22, 23). Bone morphogenetic protein-4 (BMP-4), a member of the TGF- family that plays a central role during pituitary organogenesis (24, 25), is over-expressed in different prolactinomas models including dopamine receptor 2 knockout (D2R–/–) mice, Fisher rats treated with estradiol and in human tumors, compared with normal tissue and other pituitary adenoma types. Furthermore, GH3 rat lactosomatotrophinoma cells stably transfected with plasmids that express proteins that block BMP-4 action had reduced tumorigenicity in nude mice, providing evidence that the BMP-4 stimulatory pathway plays a role in prolactinoma development in vivo (26).
BMPs, as well as other members of the TGF- family, mediate their effects by binding to a family of membrane tyrosine kinases receptors and subsequently activating a family of receptor substrates, the Smad proteins, which act as transcription factors in the nucleus (27, 28, 29). The Smad-4 protein functions as a signal cotransducer for the different Smad proteins and is required for active transcriptional complexes to assemble. The resulting Smad complex is then free to move into the nucleus and competent to bind Smad-binding elements in association with transcriptional coactivators or corepressors. Proteins such as noggin, gremlin, and caronte bind BMP proteins and prevent their association with signaling tyrosine kinase receptors, thus controlling the extracellular activating phase of BMP pathway (27, 28, 29).
Taking into consideration the crucial role of BMP-4 during pituitary organogenesis and prolactinoma tumorigenesis, we decided to study its potential action in corticotrophinoma growth. In this study, we report the differential BMP-4 expression in normal and adenomatous corticotrophs and its inhibitory action on corticotrophinoma cell proliferation. Furthermore, we show that BMP-4 mediates the antiproliferative action of retinoic acid in these cells. These results point to a novel mechanism by which BMP-4 and retinoic acid may prevent Cushing’s disease development.
Materials and Methods
Cell culture
AtT-20 pituitary corticotroph tumor cells were obtained from the American Type Culture Collection (Rockville, MD) (30). AtT-20 cells were cultured in DMEM (pH 7.3) supplemented with 10% fetal calf serum (FCS), 2.2 g/liter NaHCO3, 10 mM HEPES, and 2 mM glutamine until they were confluent under 5% CO2 atmosphere at 37 C.
Cells were treated with 10, 50, 100, or 200 ng/ml BMP-4, 0.001–1 μM noggin, 10 ng/ml TGF- (all three from R&D Systems, Minneapolis, MN), or 10 or 100 nM all-trans retinoic acid (Sigma, St. Louis, MO) that was dissolved in ethanol-DMSO as a 10 mM stock solution and handled in the dark. The diluting mixture was used in all the in vitro experiments as control.
Patients and tissues
Patients with pituitary adenomas and clinical symptoms of Cushing’s disease were diagnosed by hormonal testing and magnetic resonance imaging as previously described (31). All tumors were benign and graded according to the modified Hardy’s classification (32). Tumor tissue was obtained by transsphenoidal surgery and was transported in sterile medium. Samples from five human pituitary glands were obtained from autopsy cases without any evidence of endocrinological disease, with postmortem delay between 8 and 12 h. The tissues were snap-frozen and stored at –80 C. For immunohistochemistry, 8-μm sections of shock-frozen ACTH-secreting tumor or normal tissue were thaw-mounted onto SuperFrost Plus slides (Menzel-Glaser, Hamburg, Germany), fixed in 4% paraformaldehyde in PBS, and stored in 96% ethanol at 4 C until used.
Cell proliferation and viability
A WST-1 assay (Roche Molecular Biochemicals, Basel, Switzerland) was used to measure cell proliferation and cell viability following the manufacturer’s instructions as previously described (33). The method was validated by total cell count. Acridine orange-ethidium bromide staining was used to rule out toxic effects.
Plasmids
The Smad-4 dominant-negative mutant (Smad-4dn) expression vector consists of a truncated DPC-4 (Smad-4) cDNA (1–514) fused to FLAG expressed under the control of the cytomegalovirus promoter (34). The noggin expression vector contains 1 kb of the mouse noggin cDNA driven by the EF1 promoter (35). The mouse BMP-4 1A promoter contains the –2372/+258 fragment from the 5' flanking region and includes all the necessary sequences for the expression and regulation of BMP-4 in pituitary cell cultures (36). COUP-TFI expression vector contains the 1.5-kb segment of the mouse COUP-TFI cDNA under the control of a cytomegalovirus promoter, and COUP-TFI control vector contains an incomplete segment of the mouse COUP-TFI cDNA that codifies for a truncated inactive protein (37). The human c-myc promoter construction contains the –2329/+510 fragment from the 5' flanking region upstream from the luciferase gene of the pGL3-Basic vector (38).
AtT-20 stable clones
AtT-20 cells were stably transfected with noggin or Smad-4dn expression vectors or the corresponding control vector using lipofectamine as described below, and after 48 h, were selected in culture medium with 600 ng/ml G418 (Invitrogen, Eggenstein, Germany). The selected AtT-20 noggin and AtT-20 Smad-4dn clones were tested for noggin and Smad-4dn expression by Northern blot and Western blot, respectively as described below. Three additional stable clones for each construct had similar results as the one that is shown.
Transfection and reporter assays
Cell transfection was performed with lipofectamine using standard procedures, as previously described (39). After plating in six-well plates, the cells were transfected for 6 h in OPTIMEM (Invitrogen) using 8 μl of lipofectamine (Invitrogen) per well and 1 μg total plasmid DNA. Cells were then washed and left overnight in serum-free DMEM. The following day, cells were washed and treated for 6 or 24 h in serum-free DMEM with the indicated compounds. An additional 200 ng of the RSV--gal construction was cotransfected in all the experiments to correct for variations in transfection efficiency as previously described (39). Luciferase activity was measured with a Junior luminometer (Berthold, Bad Wildbad, Germany). Results are ratios of luciferase and -galactosidase activity. Protein content was measured by a Bradford assay as a further control for the reproducibility of the sampling procedure.
Western blot analysis
Western blot was performed as previously described (26). Briefly AtT-20 cells were washed once with PBS (pH 7.0), and then cell lysates were prepared in standard cracking buffer and boiled for 3 min. Equal levels of protein (30 μg) were electrophoresed by 12% SDS-PAGE. Proteins were blotted onto nitrocellulose western blotting membranes (Sigma) using standard procedures and anti-BMP-4 (1:1,000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-GADPH (1:50,000) (Santa Cruz Biotechnology Inc.), or anti-FLAG antibody (Sigma) were added. The primary antibodies were detected with HRP-conjugated antigoat IgG secondary antibodies (Santa Cruz Biotechnology Inc.).
Northern blot analysis
Total RNA extraction and Northern blot analysis were performed as described previously (31). Briefly, total RNA was isolated by the guanidine isothiocyanate phenol-chloroform extraction method, denatured with glyoxal, electrophoresed, and transferred to a nylon membrane. Filters were prehybridized for 1 h at 60 C and then the probe was added and hybridized for 12 h. Blots were washed at increasing salt and temperature stringency and then exposed to Kodak XAR5 film at –70 C with intensifying screens for 6 d. A 0.5-kb noggin cDNA fragment (35) and a 6.6-kb EcoRI fragment of a 28s and part of the 18s mouse cDNA (31) were labeled with a random-priming kit using [-32P]dCTP (specific activity, 2–4 x 108 cpm/μg). The loading control with the fragment of 18s cDNA as probe was performed in each blot.
Immunohistochemistry
To detect BMP-4 immunoreactivity in normal pituitary and in corticotrophinomas, a goat polyclonal BMP-4 antibody (1:100) (Santa Cruz Biotechnology Inc.) was used in combination with antigoat biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) and avidin-biotin-peroxidase complex (Vector Laboratories). Immunoreactivity was visualized with diaminobenzidine (Sigma) and 0.01% hydrogen peroxide. ACTH was detected with a monoclonal antibody (1:800) (Dako Diagnostika, Hamburg, Germany) in combination with an antimouse IgG (Sigma) and mouse alkaline phosphatase-anti-alkaline phosphatase (Sigma), using vector red (Vector Laboratories). Specific antibodies for prolactin (PRL), FSH, LH, GH, and TSH were also used (1:500) (all from Immunotech, Karlsruhe, Germany). Negative controls were performed by omitting the primary antibody.
In vivo tumor formation
ACTH-secreting experimental tumors were formed by injecting AtT-20 control, AtT20 noggin, or AtT20 Smad-4dn clone cells into a subdermal pouch in BALB/cAnN Crl-nu BR nude mice as previously described (26, 30). To evaluate BMP-4 role on tumor formation, suboptimal amounts of clone cells were injected in nude mice to distinguish differences in tumor growth. All the cells were controlled for viability. Tumor formation was monitored every 2 d during 3 wk. The tumor volume was measured as previously described (26). Briefly a vernier was used to measure the short and long axis of the tumor and the following formula was used: tumoral volume = [(short axis)2 x long axis x 0.4]. Blood samples were collected by heart puncture from halothane-anesthetized animals. Histological examination was performed by hematoxylin-eosin staining of shock-frozen tissues. All animal experiments were performed in accordance with institutional regulations of animal care.
Hormone measurements
ACTH was determined by radioimmunoassay as previously described (31, 33).
Statistical analysis
Statistics were performed using one-factor ANOVA in combination with Scheffe’s test.
Results
BMP-4 expression in pituitary corticotroph tumors
In the normal human adenohypophysis, BMP-4 was present in 40% of endocrine cells, which were mostly found in clusters and rarely scattered (Fig. 1A). Double immunohistochemistry revealed that 30% of the somatotroph, corticotroph, and thyrotroph population were immunopositive for BMP-4 (Fig. 1B). On the other hand, PRL and FSH/LH immunopositive cells rarely stained for BMP-4. Immunohistochemistry in 15 corticotrophinomas revealed variable BMP-4 expression (Fig. 1C and Table 1). Seven of 15 cases had very low BMP-4 immunoreactivity and five of 15 cases did not express BMP-4. Only three of 15 cases had a number of BMP-4-expressing cells higher than 10%. No correlation with tumor grade was observed. The overall expression in corticotrophinomas was lower than in prolactinomas, as we have already described (26). An overall reduced BMP-4 expression is observed in Cushing’s disease adenomas compared with normal corticotroph cells (Fig. 1 and Table 1). Based on this differential BMP-4 expression pattern between normal and tumor corticotroph cells, we further studied a possible role of BMP-4 in corticotroph tumor function and growth.
BMP-4 inhibits murine corticotroph tumor cells ACTH-secretion and cell proliferation
We examined the effect of BMP-4 on ACTH secretion by AtT-20 corticotroph tumor cells. BMP-4 treatment inhibited both the endogenous and forskolin-stimulated ACTH induction in a dose-dependent manner (Fig. 2A). The specific BMP-4 antagonist noggin blocked dose dependently this inhibitory effect (Fig. 2B). The inhibitory effect of BMP-4 on ACTH production was not affected by dexamethasone, because cotreatment with both factors did not shown an increment in BMP-4 inhibitory effect (Fig. 2C).
To further establish the actions of BMP-4 on corticotroph cells, we studied cell proliferation. We observed that BMP-4 inhibited AtT-20 cell proliferation after 3 d of BMP-4 treatment, and the effect is still observed after 5 d (Fig. 3A). On the contrary, TGF- treatment did not affect cell proliferation (Fig. 3A). Similar results were obtained by total cell count, and no cell death was observed by acridine orange-ethidium staining, ruling out toxic effects (data not shown).
The c-myc oncogene is a critical regulator of cell cycle progression and it was recently described that BMP-4 induces c-myc expression in GH3 lactosomatotrophinoma cells and in human prolactinomas (26). Thus, we determined whether BMP-4 exerts its antiproliferative action in AtT-20 cells by altering c-myc activity. Indeed, as shown in Fig. 3B, BMP-4 inhibited c-myc promoter activity.
BMP-4 effects on murine corticotrophs tumor cells in vivo
Smad-4 protein functions as a signal cotransducer for the different TGF- superfamily factors and is necessary for active transcriptional complexes to assemble. Preventing dimerization of active complexes can block BMP-4 signaling, and BMP-4 extracellular binding can be prevented by noggin, which is a BMP-4-specific antagonist.
To test whether BMP-4 antiproliferative effect on AtT-20 cells also affects the ability of ACTH-secreting cells to promote tumors in vivo, we produced AtT-20 clones stably transfected with the Smad-4dn form or noggin to block BMP-4 signaling (Fig. 4, A and B, inset). Expression of Smad-4dn or noggin blocked the antiproliferative effect of BMP-4 (Fig. 4, A and B). Taking into account the BMP-4 inhibitory action in vitro, we hypothesized that blocking BMP-4 inhibitory growth control would result in enhanced tumor growth. Thus, to observe differences in tumor growth, nude mice were injected with a suboptimal concentration of AtT-20 cells stably transfected with an empty vector, Smad-4dn, or with noggin. When a suboptimal concentration of AtT-20 cells stably transfected with the empty vector (control cells) were injected into nude mice no tumors, were observed even after 8 wk (Fig. 5, A and B). In contrast, after 2 wk, tumors developed in all the mice injected with AtT-20 Smad-4dn or AtT-20 noggin clone cells (Fig. 5, A and B). Moreover, the phenotype of the three mice groups was different; whereas the control group did not show any change, the Smad-4dn and noggin groups presented a characteristic Cushing’s disease appearance with noticeably thinner skin and sc fat tissue increase.
Retinoic acid inhibits corticotroph cells by inducing BMP-4
Given the reduced expression of BMP-4 and its inhibitory action in tumor corticotroph growth, we studied its putative involvement in retinoic acid antiproliferative action. We analyzed whether retinoic acid induces BMP-4 in AtT-20 cells. After 24 h of treatment with 10–100 nM retinoic acid, an increase in BMP-4 promoter transcriptional activity was observed (4-fold induction) (Fig. 6A). An almost complete inhibition of the BMP-4 promoter responsiveness to retinoic acid was observed when cotransfecting the BMP-4-LUC promoter construct with a COUP-TFI expression vector (Fig. 6A). Moreover, the expression of COUP-TFI enhances the transcription of BMP-4 (3-fold induction) (Fig. 6A). To control the ability of BMP-4 promoter to be further activated in the presence of COUP-TFI, we treated the cells with cAMP and observed the induction of BMP-4 promoter (Fig. 6A). We further evaluated BMP-4 expression by Western blot. Retinoic acid increased BMP-4 protein levels and this stimulatory effect diminished after 24 h of treatment (Fig. 6B).
To test the hypothesis that the inhibitory effect of retinoic acid in AtT-20 cell proliferation could be mediated by BMP-4, we examined whether BMP-4 signaling is necessary for retinoic acid effects. In both AtT-20 Smad-4dn and AtT-20 noggin cells that do not respond to BMP-4, retinoic acid treatment was not able to inhibit cell proliferation (Fig. 7, A and B).
AtT-20 control cells responded to both BMP-4 and retinoic acid treatment. However, combined administration of the two compounds did not result in a further antiproliferative effect, supporting the notion that BMP-4 and retinoic acid might interact (Fig. 7C).
Discussion
The pathogenic mechanisms as well as the treatment of Cushing’s disease remains still an open question. In this study, we demonstrate for the first time that BMP-4 expression is reduced in corticotrophinoma cells and that BMP-4 regulates not only corticotroph cell physiology but also inhibits its tumorigenic potential. This inhibitory effect of BMP-4 constitutes a novel mechanism for pituitary corticotroph pathogenesis. We also found that retinoic acid, used as a pharmacological agent in different types of cancer (40, 41, 42) and which inhibits corticotrophinoma cells growth (13), may act through BMP-4 expression in corticotroph cells. Therefore, BMP-4 induction by retinoic acid may be an interesting mechanistic target for retinoic acid as a therapeutic option for Cushing’s disease treatment.
We demonstrate in this study that BMP-4 inhibits both the endogenous ACTH production and forskolin-induced ACTH stimulation in a dose-dependent manner, and this inhibitory effect was blocked by noggin, which is a specific antagonist of BMP-4. These results are in agreement with recently published data showing that BMP-4 has an inhibitory effect on the transcriptional activity of POMC promoter (43). BMP-4 inhibits not only AtT-20 cell proliferation but also c-myc promoter transcriptional activity. We have demonstrated that, in lactosomatotrophinoma GH3 cell line and human prolactinomas in which BMP-4 has a promoting role during tumor development, BMP-4 induces c-myc expression. This differential effect on c-myc depending on the cell lineage can be explained by the association of Smad-1 (BMP-4 signaling transducer protein) with different cofactors presented in each cell lineage. For example in GH3 cells BMP-4 stimulation triggers Smad-1/ER physical interaction that may have stimulatory effects over transforming gene promoters as c-myc. In corticotrophs, Smad-1 may possibly be interacting with other proteins resulting in negative effects over c-myc expression. Based on these results and the different studies that place TGF- family as important regulating factors in pituitary tumor development (23, 26), we studied a possible role for BMP-4 in corticotroph cells tumorigenicity in vivo. At low cell concentration doses AtT-20 cells present no tumorigenic capacity when injected in nude mice. In contrast, AtT-20 Smad-4dn, and AtT-20 noggin clones in which the BMP-4 signaling pathway is blocked developed visible tumors starting at 1 wk after injection as well as symptoms of Cushing’s disease. Therefore, inhibition of the BMP-4 signaling results in an increase of the AtT-20 cells tumorigenic potential, demonstrating an inhibitory role of BMP-4 in the development of corticotroph tumors in vivo.
BMP-2 and –4 play a central role during the initial steps of the anterior pituitary development (25, 44, 45, 46). BMP-4 is required during the first stage of pituitary organogenesis for the proliferation of the Rathke’s pouch placode, which gives rise to Pit-1 lineage cells, which includes the PRL-secreting cells, among others. Moreover, overexpression of noggin or a dominant-negative BMP-receptor (BMPR1A) in the anterior pituitary leads to the arrest of the development of the pit-1-expressing lineage. During the second pituitary organogenesis stage an inhibition of BMP-2 ventrodorsal gradient by an FGF8 dorsoventral gradient, leads to corticotroph differentiation (25, 44, 45, 46). Overexpression of FGF8 in the developmental pituitary results in Pit-1 lineage cells absence and corticotroph enhanced differentiation, suggesting that it is necessary to inhibit BMP signaling for normal corticotroph development (47). We have shown that BMP-4 has a strong stimulatory effect on prolactinoma cell proliferation (26). Moreover the activin/BMP system has also been described in the development of human pituitary gonadotropinomas and nonfunctioning adenomas (48). In the present study, we show BMP-4 expression in 40% of endocrine cells in the normal adenohypophysis and most of the BMP-4 immunopositive cells were somatotrophs and corticotrophs but not lactotrophs. On the other hand, in pituitary adenomas, BMP-4 is expressed at low variable levels in corticotrophinoma cells but at high levels in prolactinoma cells. Thus BMP-4, a corticotroph growth inhibitor, is expressed in the normal pituitary but is reduced in corticotroph tumors. In analogy to the situation during pituitary organogenesis, this distinctive expression may allow corticotroph tumoral growth promotion. BMP-4 not only is crucial during pituitary organogenesis but has an opposite role in the different pituitary lineages. BMP-4 promotes pituitary prolactinoma pathogenesis through a Smad/estrogen receptor cross-talk but inhibits corticotroph pathogenesis and Cushing’s disease. It is not clear at this stage, which is the trigger for BMP-4 action (enhancement of proliferation in prolactinomas and loss of inhibition control in Cushing’s corticotroph) in adult pituitary tumor cells. Thus, BMP-4 acts as a regulator no only of pituitary cells undergoing organogenesis differentiation, but also of transformed, not normal, adult pituitary cells, and it has a pituitary linage cell specificity during the tumorigenic process.
Retinoic acid treatment in AtT-20 corticotroph cells induces BMP-4 transcription and BMP-4 expression. Similar actions of retinoic acid have been described in human and murine oesteoblastic cell lines (49). Inhibiting retinoic acid signaling with COUP-TFI (14, 50, 51, 52) blocks the stimulatory effect of retinoic acid on BMP-4, although the promoter may still be stimulated by other pathways. These results are in agreement with previous ones that establish that COUP-TFI can block retinoic acid inhibitory effects on POMC-LUC reporter activity. Also COUP-TFI expression only in normal corticotroph cells but not in pituitary Cushing tumors might be the explanation for the effects of retinoic acid in transformed but not in normal corticotroph cells (13). Moreover, we show that overexpression of COUP-TFI enhances, by a mechanism independent of retinoic acid, the transcription of BMP-4 in AtT-20 cells, suggesting that the differential expression of BMP-4 in normal but not tumor cells may be under the control of COUP-TFI, as it has been described in an osteoblast model (36).
Both BMP-4 and retinoic acid inhibit AtT-20 cell proliferation, but there was no further inhibition in the presence of both factors, even at low doses, suggesting that they may share an overlapping mechanism (which is saturated at low doses). BMP and steroid receptor cross-talk takes place in several cells. A cross-talk mechanism between BMP-4 and estrogens that regulates cell proliferation has been described and a physical interaction between steroid receptors and TGF--induced Smad proteins has been recently shown (26, 53, 54). Several studies have also established a different interaction between retinoic acid and BMPs (55, 56, 57). In embryonal carcinoma cells, retinoic acid controls BMP-2 and BMP-4 regulation (58, 59, 60), and the combination of retinoic acid and BMP-2 or BMP-4 synergistically induces apoptosis of these cells inhibiting neurons and glial cells but leading to smooth muscle cell differentiation (61, 62). To investigate whether retinoic acid transduces its antiproliferative effect through BMP-4 induction, its effect was tested in cells in which the BMP-4 pathway was blocked. In AtT-20 noggin and AtT-20 Smad-4dn cells, the inhibitory action of retinoic acid is abolished, showing that, besides the possibility of a further cross-talk with BMP-4 signaling pathway, retinoic acid is acting through the induction of BMP-4 expression, which in turn has a central role in preventing corticotroph cell proliferation and tumor cell growth.
The present study highlights a role of BMP-4 in the inhibition of corticotrophinoma cell growth and suggests a novel mechanism for the pathogenesis of Cushing’s-associated pituitary adenomas. Furthermore, the fact that BMP-4 is induced by and mediates some of the effects of retinoic acid highlights a novel mechanism of retinoic acid action in corticotrophinoma cells. The knowledge of the cross-talk between retinoic acid and BMP-4 may be a useful tool for the design of new—based on retinoic acid—therapeutic approaches for Cushing’s disease treatment.
Acknowledgments
We thank J. Massague for the Smad-4dn expression vector, K. Monzen for the noggin expression vector, M. Tsai for the COUP-TFI expression vector, S. E. Harris for the mouse BMP-4 luciferase reporter vector, and K. Mitsuyasu for the human c-Myc luciferase reporter vector. We also thank Esther Jelen for technical assistance.
Footnotes
This work was supported by grants from the University of Buenos Aires, the Argentine National Research Council (CONICET), and Agencia Nacional de Promocion Científica y Tecnologica-Argentina.
D.G., D.R., and E.A. are members of the Instituto de Fisiología, Biología Molecular y Neuro Ciencias-Argentine National Research Council (CONICET).
First Published Online September 29, 2005
1 D.G. and M.P.-P. have contributed equally.
Abbreviations: BMP-4, Bone morphogenetic protein-4; FCS, fetal calf serum; POMC, proopiomelanocortin; PRL, prolactin.
Accepted for publication September 17, 2005.
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