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. Author manuscript; available in PMC: 2014 Sep 4.
Published in final edited form as: Cancer Biol Ther. 2007 Aug;6(8):1313–1317. doi: 10.4161/cbt.6.8.4507

BMP Suppresses PTEN Expression via RAS/ERK Signaling

Stayce E Beck 1,3, John M Carethers 1,2,3,4,*
PMCID: PMC4154563  NIHMSID: NIHMS209555  PMID: 18059158

Abstract

Bone morphogenetic protein (BMP), a member of the transforming growth factor β family, classically utilizes the SMAD signaling pathway for its growth suppressive effects, and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

Keywords: bone morphogenetic protein, PTEN, RAS, ERK, TGFβ, colon cancer

INTRODUCTION

Bone Morphogenetic Protein (BMP) is a member of the TGFβ family of signaling ligands known to regulate cell proliferation, apoptosis, and differentiation, and participates in the mesenchymal development of most tissues and organs in vertebrates. However, its role in epithelial growth regulation is not well understood. As part of the TGFβ family, BMP utilizes a similar signaling cascade to that of TGFβ. BMP ligands bind and activate serine-threonine kinase receptors, type IA (BMPRIA), type IB (BMPRIB) and type II (BMPRII), to transmit their signal into the cell.1–4 Upon BMP ligand binding, BMPRII phosphorylates BMPRI, which in turn phosphorylates intracellular mothers against decapentaplegic, Drosophila (SMAD) 1, 5, or 8 at their C-termini.5 The phosphorylated SMADs associate with SMAD4, and the complex translocates to the nucleus as a transcription factor to regulate the expression of various genes that control cell proliferation, cell differentiation, and apoptosis.6

Classically, BMP ligands utilize the SMAD signaling pathway to transmit signals to the nucleus, but when SMAD4 is not present as is often seen in later stages of colon cancer development, BMP-SMAD signaling is impaired. Juvenile polyposis (JP), an autosomal dominant gastrointestinal hamartomatous polyposis syndrome that increases the afflicted patient’s risk for developing colon cancer ~12-fold over the general population, occurs in patients with germline mutations in the tumor suppressors SMAD4 or BMPRIA.7,8 Moreover, a small percentage of JP kindreds may have germline mutations in PTEN, although this is controversial when patients are young and may not manifest certain phenotypic features of other hamartomatous syndromes.9 Because mutations in BMP signaling pathway components and possibly PTEN cause JP, a very important question that is still not understood is whether the BMP signaling pathway interacts or regulates PTEN expression. Waite and Eng suggested that cells treated with BMP increased PTEN protein levels by decreasing the association of PTEN with ubiquitin degradation proteins.10 Qiao et al used a Cre-loxP approach to disrupt SMAD4 in skin to study epidermal tumorigenesis, and showed that absence of SMAD4 blocked TGFβ and BMP-SMAD signaling, and that the mice developed malignant skin tumors.11 Interestingly, these authors found that tumorigenesis was accompanied by inactivation of PTEN with subsequent activation of Akt.11 These studies suggest that there may be an interaction between the BMP and PTEN pathways. Additionally, BMPs sister ligand, TGFβ, has been shown to regulate PTEN expression in keratinocytes,12 and PTEN mRNA levels were also reduced in a model of TGFβ1 overexpressing transgenic mice that develop pancreatic fibrosis.13 Transcriptional or translational regulation of PTEN by BMP has not been examined.

The utilization of SMAD-independent pathways by TGFβ family members has been studied extensively to understand the observed phenomenon of reversal of the growth suppression role for TGFβ.14–22 Canonical SMAD signaling is fairly well understood, although the regulation of SMAD signaling, as well as pathways that constitute non-SMAD signaling, are not. Traditionally, SMAD signaling is thought of as growth suppressive for cancer development. However, there are cellular ways to modulate SMAD signaling,23 and/or there is the existence of non-SMAD pathways that normally balance the SMAD suppressive signaling.24 Loss of SMAD signaling would create an imbalance that could lead to growth proliferation.

Here, we hypothesized that BMP might regulate PTEN at the transcriptional level, which might be unmasked when SMAD-signaling is impaired. We find that BMP downregulates PTEN and this coincides with the onset of growth stimulation when SMAD4 is absent, and is mediated by RAS/ERK mitogenic signaling.

MATERIALS AND METHODS

Cell culture

SW480 cells, which are null for SMAD4, were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) (Invitrogen Corporation, Carlsbad, CA) with 10% fetal bovine serum and penicillin G/streptomycin (Invitrogen Corporation, Carlsbad, CA. BMP2 (R&D systems, Minneapolis, MN) ligand was added into the media at 100 ng/ml where indicated. PD98059 (Chemicon, Temecula, CA), the MEK1/2 pharmacological inhibitor, was used at 25 μM thirty minutes prior to BMP treatment where indicated.

Transfections

Dominant negative K-RAS (DN KRAS; a generous gift from Dr. Rik Derynck, University of California, San Francisco), which inhibits the function of activated RAS, and mock vectors (pcDNA3.0) were transiently delivered by Transfectin (Promega, Madison, WI) at a ratio of 3:1 of vector to transfection reagent in OPTI-MEM reduced serum free media (GIBCO Carlsbad, CA). After 2–3 hours, IMDM with FBS and penicillin G/streptomycin was added to the transfected cells. Two hours post-transfection, complete media was added, and 84 hours later the cells were used in the experiments.

Total cell lysis and western blotting

Cells were lysed using total lysis buffer (12 mM Tris HCl pH 8.3, 100 mM NaCl, 1% SDS, 1% DCA, 1% Triton X-100, 2 mM EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 50 μM DTT, and 2 mM PMSF). The protein was denatured at 100°C for 5 min. and then loaded onto a 15% polyacrylamide gel. After electrophoresis, the proteins were transferred onto a nylon membrane, blocked for 1 hr with 5% milk, and probed overnight with primary antibody at 4°C. Blotting was done with antibodies to PTEN at a 1:200 dilution (SC7974, Santa Cruz Biotechnology, Santa Cruz, CA), pAkt (1:1000 dilution, #SC16646-R, Santa Cruz Biotechnology, Santa Cruz, CA), total Akt (1:1000 dilution, #SC8312, Santa Cruz Biotechnology, Santa Cruz, CA), pERK (1:600 diution, cat #9101, Cell Signal, Danvers, MA), total ERK (1:600 dilution, cat #9102, Cell Signal, Danvers, MA), and GAPDH 1:2000 dilution, #AM4300, Ambion, Austin, TX). The following day, several PBS-Tween 0.1% washes were performed along with appropriate secondary antibody incubation. Blotted proteins were detected with horseradish peroxidase-linked secondary antibodies (Sigma, St. Louis, MO) followed by ECL detection (Amersham, Little Chalfont, UK).

MTT assay

The effect of BMP treatment on cell growth was assessed by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based metabolic assay. Cells were seeded in 48-well plates at a density of 10 to 20,000 cells/well in 0.4 ml of culture medium supplemented with BMP2 [100 ng/ml]. Metabolic activity, corresponding to growth, was assayed for up to 8 days of incubation at fixed intervals for MTT-dependent absorbency. For this, cells were stained 3 hrs with MTT dye, the reaction product released by lysis with SDS, and absorbency detected at 570λ using a Beckman-Coulter DU640B spectrophotometer (Beckman-Coulter, Fullerton, CA). Experiments were performed at least three times.

Cell growth assay

Cells were seeded at a density of 10,000 cells per well, and 24 hours later were treated with 100 ng/ml BMP2, or 200 ng/ml noggin in the presence of FBS. Media with growth factors and inhibitors were replaced every 48 hrs. At the indicated time points, cells were removed from the tissue culture plate with 0.05% trypsin and 10 μl of trypan blue (Gibco, Grand Island, NY) was added to 100 μl of the cell suspension and 10 μl of this mix was counted using a hemocytometer. Briefly, all four quadrants were counted and the number was divided by 4 and multiplied by 104 to get the number of cells/ ml of suspension, this was then multiplied by the dilution factor to give the total number of cells per well. Experiments were replicated at least three times.

Total RNA extraction and semi-quantitative reverse transcriptase-PCR

Total RNA extraction was performed using Trizol reagent (Invitrogen Corporation, Carlsbad, CA). Cells grown on 6-well plates were lysed with 1 ml/well of trizol. After lysis, the cell-trizol mix was combined with chloroform and mixed. Supernatants were then precipitated with isopropanol, and the RNA pellets were washed with 75% ethanol and air-dried, and resuspended in water. Two micrograms of total RNA was converted into cDNA by reverse transcriptase and amplified for PTEN transcripts (SuperScript II, Invitrogen Corporation). Briefly, following inactivation at 65°C for 10 min, 1 μL of the reaction mixture was incubated in buffer containing 0.2 mM concentrations of dATP, dCTP, dGTP, dTTP, 0.2 μM concentrations each of oligonucleotide primers, 3 mM MgCl2 and a 10X buffer consisting of 200 mM Tris-HCl (pH 8.0), 500 mM KCl, and 1 Taq polymerase. The following primers were designed for PTEN: forward, 5′-GGACGAACTGGTGTAATGATATG-3′, and reverse, 5′-TCTACTGTTTTTGTGAAGTACAGC-3′. GAPDH served as a loading control (forward 5′-ACCACAGTCCATGCC ATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′). PCR was performed as follows: denaturation at 95°C for 3 min and 35 cycles of 94°C for 30 s, 55°C for 30 s, and 74°C for 4 min for GAPDH; for PTEN: denaturation at 95°C for 3 min. Experiments were replicated at least three times.

Luciferase assays

Transient transfection of colon cancer cells with the PTEN-luc plasmid was used to assess the effects of BMP2 on PTEN transactivation. Reporter vectors (0.75mg/ml) and the pRL-TK vector (20 ng/well) were transiently delivered by Transfectin (Promega, Madison, WI) in 12-wells plates with a ratio of 3:1 of vector to transfection reagent in OPTI-MEM reduced serum free media (GIBCO Carlsbad, CA). Two hours post-transfection, 1 ml of complete media was added per well and 12 to 16 hours post-transfection, cells were treated with 100 ng/ml of BMP2 or 200 ng/ml noggin. Luciferase activity was measured by a dual-luciferase kit (Promega, Madison, WI) 36 hours after the treatment; normalization was performed using the Renilla luciferase activity expressed by the cotransfected pRL-TK vector. Experiments were replicated at least three times.

Statistical analysis

Statistical significance was determined using either the student’s t-test or two-factor without replication ANOVA. Probability values less than 0.05 were considered to be significant.

RESULTS

BMPs growth effects are biphasic, converting from initial growth suppression to stimulation

The growth of SMAD4-null SW480 cells is initially suppressed through 48 hrs, as we previously reported.24 However, we found that when we extended the length of time that SW480 cells were treated with BMP to 8 days, the number of SMAD4-null SW480 cells is significantly increased over untreated cells as assayed by MTT assay or direct cell counting (Fig. 1).

Figure 1.

Figure 1

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BMP treatment induces a biphasic growth pattern in SW480 cells over eight days of culture. Cells were treated with 100 ng/mL of BMP2 with or without PD98059 (25μM), and cells were counted. Results are expressed as percent change in growth with BMP2 treatment compared to untreated controls. The MEK1/2 inhibitor PD980059 blocked the growth proliferative capability of BMP2 by day eight of growth. Results are representative of three independent experiments.

BMP-induced growth promotion corresponds to decreases in PTEN expression

To assess the mechanism of BMP-induced growth stimulation, we hypothesized that the BMP ligand may cause downregulation of growth suppressive genes. We examined the tumor suppressor gene PTEN due to its potential involvement in juvenile polyposis, and its observed regulation by BMPs sister molecule, TGFβ. BMP2 ligand treatment resulted in a reduction in levels of PTEN at the transcription, mRNA and protein levels. The decrease in PTEN mRNA and transcription occurred after 36 hours of BMP2 treatment in the SMAD4-null SW480 cells (Figs. 2 and 3), followed by a decrease in PTEN protein levels that occurred after 84 hours of BMP2 treatment (Fig. 4). Simultaneously with the PTEN protein decrease, a corresponding increase in phospho-Akt levels occurred, indicating that the BMP-induced PTEN decrease had a functional effect on the phosphatidylinositol-3-kinase (PI3K) pathway (Fig. 4). The decrease in PTEN coupled by the increase in phospho-Akt corresponds to the growth stimulatory phase of the SMAD4-null SW480 cells that is significant after eight days of BMP2 treatment. Additionally, treatment with noggin, a BMP ligand inhibitor, prevents the BMP-induced decrease in PTEN mRNA levels, and is indicative of the direct effects of the BMP ligand upon PTEN regulation (Fig. 2).

Figure 2.

Figure 2

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BMP2 reduces PTEN mRNA in SW480 cells at 36 hours after treatment, which is blocked by noggin or ERK inhibition. (A) Semi-quantitative RT-PCR of PTEN after 36 and 48 hours of BMP2 treatment, with relative amounts of PTEN:GAPDH shown in the bar graph. (B) Semi-quantitative RT-PCR of PTEN was performed in the presence or absence of the BMP ligand inhibitor noggin or RAS/ERK inhibition with PD98059 (25 μM). GAPDH was used as a loading control. Results are representative of three independent experiments.

Figure 3.

Figure 3

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BMP2 reduces the transcription of PTEN in SW480 cells at 36 hours post-treatment, which is reversed with ERK inhibition. The luciferase construct, PTEN-luc, was transfected into SW480 cells, and cells were treated with or without BMP2 and/or PD98059 for 36 hours, and compared to untreated controls. Luciferase activity was normalized to Renilla luciferase activity as detected by the cotransfected pRL-TK vector. Results are representative of three independent experiments.

Figure 4.

Figure 4

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BMP suppresses PTEN protein in SW480 cells after 84 hours of treatment, which is blocked with ERK inhibition. (A) Representative Western blots of PTEN, phosphor-Akt, total Akt, and GAPDH, which was used as a loading control. Cells were treated with increasing dosages of BMP2 (0 to 200 ng/mL), with or without PD98059. (B) Densitometry of PTEN from Western blot without PD98059 treatment. (C) Densitometry of phosphor-Akt from Western blot without PD98059 treatment. Results are representative of 3 independent experiments.

BMP-induced PTEN downregulation and growth stimulation are mediated through the RAS/ERK pathway

Although it is clear that PTEN levels decrease over time with BMP treatment of the SMAD4-null SW480 cells, we wanted to understand the regulatory mechanism for this downregulation. We treated cells with BMP2 and with PD98059, a MEK1/2 kinase inhibitor. With semi-quantitative RT-PCR for PTEN (Fig. 2), PTEN luciferase assay (Fig. 3), and Western blotting of PTEN (Fig. 4), we demonstrate that when ERK signaling is inhibited, BMP-induced PTEN suppression is blocked at the transcriptional, mRNA, and protein levels. Correspondingly, there was no increase in phospho-Akt levels with MEK inhibition after BMP2 treatment (Fig. 4). Prevention of ERK activation with PD98059 blocked the BMP-induced growth stimulation (Fig. 1). Additionally, when SMAD4-null SW480 cells are transfected with dominant negative K-RAS to inactivate RAS, which is upstream of ERK in its signaling cascade, the decrease in PTEN protein levels was blocked, as was any increase in phospho-Akt protein levels (Fig. 5). Indeed, PTEN protein levels appeared to increase with BMP2 treatment and RAS/ERK inhibition by DN K-RAS transfection (Fig. 5) with no increase in the phospho-Akt levels, suggesting a relative increase in balance of PTEN over PI3K function. These results suggest that it is through the RAS/ERK pathway that BMP2 switches from being growth suppressive to growth stimulatory via a decrease in PTEN levels and increases in phospho-Akt.

Figure 5.

Figure 5

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RAS inhibition blocks BMP-induced PTEN suppression in SW480 cells after 84 hours of treatment. Representative Western blots of PTEN, phospho Akt, total Akt, phosphor-ERK, total ERK, and GAPDH (used as a loading control) from SW480 cells treated with or without BMP2, and with transfection of mock or DN K-RAS vector. Note the lack of PTEN suppression by BMP when DN K-RAS is present, and the corresponding lack of phosphor-Akt changes that are observed when the mock vector is transfected. Results are representative of 3 independent experiments.

Discussion

We sought to understand the mechanism of BMP-induced growth stimulation in cells in which SMAD4 is inactivated. Loss of SMAD4 is common in the germline of JP patients (with simultaneous somatic allele inactivation within polyps), and is the key pathway co-SMAD for TGFβ, activin, and BMP signaling. These pathways are commonly inactivated at many levels in sporadic colorectal and pancreatic cancers. TGFβ has been demonstrated to be able to switch from growth suppression to growth stimulation in certain cell types, although the mechanisms are not fully understood.14–22 Here, we uncovered evidence for growth stimulation induced by BMP in a SMAD4-null environment, suggesting that relative loss of BMP-SMAD signaling (thought generally to be growth suppressive in epithelial cells) can be overcome, or imbalanced, by a BMP-SMAD4-independent pathway that is growth promoting.

SMAD4 mutations have been observed in approximately 10% of colon adenomas and nonmetastatic carcinomas,25 and in 30% of invasive metastatic carcinomas and in colon cancer metastases,26 suggesting that loss of SMAD signaling is important for metastasis of advanced colon cancers. Previous work by our lab has shown that upon transient reconstitution of SMAD4 in SMAD4-null SW480 cells, a strong transcriptional activation of BMP-regulated SMAD signaling occurs in the absence of exogenous BMP ligands, indicating high autocrine stimulation of the BMP receptors.24 The autocrine stimulation by BMP was verified by treating the SMAD4-transfected cells with noggin (which binds free BMP ligand) thus preventing binding of the endogenous BMP2 ligand to the receptor and subsequent BMP-induced transcriptional activity.24 Taken together with our current findings of growth promotion after prolonged BMP exposure, autocrine BMP ligand production might be a stimulus for growth stimulation when the BMP-SMAD cascade is not operative.

The SMAD4-null SW480 cells exhibited significant decreases in growth at early time points when treated with BMP2, and BMP2-induced growth suppression was reversed with transfection of a dominant negative BMPR1A vector, indicating the importance of this receptor for transducing BMPs effects.24 However, in this SMAD4-null cell, prolonged BMP treatment revealed a biphasic growth pattern that switched from an initial growth suppressive to a growth promoting pattern. This finding suggests that BMP can induce SMAD4-independent growth effects in SW480 cells. Indeed, levels of the tumor suppressor PTEN decreased with long-term BMP2 treatment (36 hours for the mRNA and 84 hours for the protein), which corresponds to the switch from growth suppression to growth promotion. The simultaneous increase in phospho-Akt levels parallels the decrease in PTEN levels, indicating the downstream effects of the PI3K pathway was coupled with the BMP-induced decreases in PTEN levels. These changes could be mechanistic for the BMP-induced growth promotion.

A mediator for the BMP-induced PTEN downregulation is the RAS/ERK pathway. RAS/ERK signaling has been shown to slow or inhibit BMP-SMAD signaling when SMAD signaling is intact.23 In a SMAD4-null environment, inhibition of the activated RAS/ERK pathway27 with PD98059 or DN K-RAS reversed BMP-induced decreases in PTEN levels and increases in phospho-Akt. Additionally, we no longer observed the biphasic pattern or growth promotion with BMP treatment when the RAS/ERK pathway was inhibited. Thus BMP2 utilizes the RAS/ERK pathway in a SMAD4-independent manner to decrease PTEN levels with subsequent increases in phospho-AKT levels, and mediates the growth stimulatory phase in the SMAD4-null SW480 cells. Overall, BMP-induced PTEN suppression induces growth promotion, but also indicates the existence of SMAD4-independent pathways that are important in mediating the growth effects of the colon cancer cell.

Acknowledgments

Supported by the U.S. Public Health Service (T32-HL07212 for SEB; CA90231 and DK067287 to JMC) and the VA Research Service. A portion of this work was presented in abstract form at the May 2006 annual meeting of the American Gastroenterological Association in Los Angeles, California. We thank Dr. Rik Derynck, University of California, San Francisco for the DN-KRAS construct.

ABBREVIATIONS

BMP

Bone Morphogenetic Protein

TGFβ

Transforming growth factor beta

ERK

extracellular signal-related kinase

GAPDH

glyceraldehydes-3-phosphate dehydrogenase

RT-PCR

reverse transcriptase polymerase chain reaction

DN

dominant negative

JP

juvenile polyposis syndrome

PTEN

phosphatase and tensin homolog deleted on chromosome ten

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