RNA interference of the BMPR-IB gene blocks BMP-2-induced osteogenic gene expression in human bone cells

Cell Biology International 32 (2008) 1362e1370 www.elsevier.com/locate/cellbi

RNA interference of the BMPR-IB gene blocks BMP-2-induced osteogenic gene expression in human bone cells W. Singhatanadgit a,b, V. Salih a, I. Olsen a,* a

Division of Biomaterials and Tissue Engineering, Eastman Dental Institute, University College London, 256 Gray’s Inn Road, London WC1X 8LD, UK b Faculty of Dentistry, Thammasat University, 99 Moo 18, Klong Luang, Pathum-Thani 12121, Thailand Received 3 June 2008; accepted 5 August 2008

Abstract We have previously shown that human bone cells express bone morphogenetic protein receptor-IB (BMPR-IB). However, little is known about the precise role of this receptor in the response of osteoblastic genes to the BMP in these cells. To determine BMPR-IB-dependent osteoblastic gene expression, the present study examined the effects of BMPR-IB knockdown on BMP-induced osteoblast-associated genes. BMPR-IB mRNA and protein were markedly suppressed by transfection of cells with BMPR-IB siRNA. Using three different bone cell samples, BMP-2 stimulation of alkaline phosphatase (ALP), osteocalcin (OC), distal-less homeobox-5 (Dlx5) and core binding factor alpha-1 (Cbfa1) was found to be specifically and significantly reduced in the BMPR-IB siRNA-transfected cultures compared with that of control cultures. Our study has provided evidence that BMPR-IB-dependent signaling plays a crucial role in BMP-2 up-regulation of the ALP, OC, Dlx5 and Cbfa1 genes in bone cells, suggesting a pivotal role of this receptor in BMP-2-induced osteoblast differentiation in vitro. These findings thus suggest the possibility that BMPR-IB could be a therapeutic target for enhancing bone regeneration in vivo. Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: BMP receptor-IB; BMP-2; RNA interference; Bone cells

1. Introduction Bone morphogenetic proteins (BMP) were initially isolated and identified as factors that induce ectopic bone formation in vivo when implanted into muscle tissues (Urist, 1965; Wozney et al., 1988). The BMP are members of the transforming growth factor b (TGF-b) superfamily and promote differentiation of both osteoprogenitor and mesenchymal stem cells into Abbreviations: BMP, bone morphogenetic proteins; BMPR, bone morphogenetic protein receptor; TGF-b, transforming growth factor b; ALP, alkaline phosphatase; Col I, type-I collagen; OP, osteopontin; BSP, bone sialoprotein; OC, osteocalcin; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-kB ligand; Smurf1, smad ubiquitin regulatory factor; Dlx5, distal-less homeobox 5; Msx2, muscle segment homeobox 2; Cbfa1, core binding factor alpha-1; siRNA, small interfering RNA; IF, immunofluorescence; RTePCR, reverse transcriptionepolymerase chain reaction; WB, Western blotting. * Corresponding author. Tel./fax: þ44 20 7915 1254. E-mail address: [email protected] (I. Olsen).

osteoblasts (Lieberman et al., 2002). These factors are also active in chemotaxis, mitogenesis and differentiation (Nakashima and Reddi, 2003) and enhance new bone formation in vivo (Bostrom and Camacho, 1998; Yamaguchi et al., 2000; Cheng et al., 2001; Boden et al., 2002). The biological activities of the BMP are mediated through signal transduction via three BMP-specific receptors, BMPRIA, -IB and -II (Chen et al., 2004). Upon binding of the BMP ligand to heteromeric complexes of type-I and type-II BMPR at the cell surface, the type-II receptor phosphorylates the type-I receptor. This activated type-I receptor in turn phosphorylates Smad1/5/8, which then assemble into complexes with Smad4 and translocate into the nucleus where they regulate the expression of target genes, including those involved in osteogenic differentiation of cells of mesenchymal lineage (Akiyama et al., 1997; Namiki et al., 1997; Chen et al., 1998; Korchynskyi et al., 2003; Osyczka et al., 2004). Thus, BMP signaling via the BMPR has been shown to stimulate a wide variety of mediators

1065-6995/$ - see front matter Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.08.005

W. Singhatanadgit et al. / Cell Biology International 32 (2008) 1362e1370

of osteoblast growth and function, including bone matrix proteins (e.g., alkaline phosphatase (ALP), type-I collagen (Col I), osteopontin (OP), bone sialoprotein (BSP), osteocalcin (OC)), osteogenic regulatory genes (e.g., BMP-2, -4, and -7, osteoprotegerin (OPG), receptor activator of nuclear factor-kB ligand (RANKL)), BMP inhibitory factors (e.g., smad ubiquitin regulatory factor (Smurf1), Smad6, noggin) and osteogenic transcription factors (e.g., distal-less homeobox 5 (Dlx5), muscle segment homeobox 2 (Msx2), core binding factor alpha-1 (Cbfa1)) (Ducy et al., 1997; Hofbauer et al., 1998; Takase et al., 1998; de Jong et al., 2002; Diefenderfer et al., 2003; Otsuka et al., 2003; de Jong et al., 2004; Takagi et al., 2004; Celil et al., 2005). However, the importance of BMPR-IB specifically in BMP-2-induced osteogenic gene expression in human bone cells and the precise role of this receptor in their differentiation in response to BMP-2 are not yet well understood. In the present study, bone cells were therefore transfected with a BMPR-IB small interfering RNA (siRNA) to knockdown BMPR-IB expression in order to delineate BMPR-IB function in osteogenic signal transduction. 2. Materials and methods 2.1. Primary human bone cell culture Primary human bone cells were grown from corticolamellar bone of the maxilla of patients undergoing routine extraction of third molar teeth at the Eastman Dental Hospital. The participants signed informed consent to the use of these tissues, in accordance with the protocol approved by the Joint Research and Ethics Committee of the Eastman Dental Institute and Hospital. The tissue was washed extensively with phosphate-buffered saline (PBS) (Gibco Life Technologies Ltd, Paisley, UK) to remove blood and debris, cut into pieces approximately 1e2 mm3 and incubated in 48-well tissue culture plates (Falcon, Becton Dickinson, Cowley, UK) for 15 min at 37  C in a humidified atmosphere of 5% CO2 in air to enable them to adhere to the plate. Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) containing 10% heatinactivated fetal calf serum (FCS) (PAA Laboratories, Yeovil, UK) (10% FCSeDMEM) supplemented with 100 U/ml of penicillin (Gibco), 100 mg/ml of streptomycin (Gibco) and 2.5 mg/ml of amphotericin B (Gibco) was then added and the explants cultured until the outgrowth of adherent cells reached confluence. The cells were harvested by incubating with trypsineEDTA (0.25% trypsin, 1 mM EDTA) (Gibco) for 5 min at 37  C, centrifuged and then re-cultured as adherent monolayers at 37  C in a humidified atmosphere of 5% CO2 in air, in 10% FCSeDMEM with antibiotic supplements. In the present study, bone cell samples were obtained from three different donors and used between passages 3 and 5. 2.2. Treatment of cells Bone cell cultures were transfected with a BMPR-IB siRNA or a non-specific ‘scramble’ siRNA (used as the control siRNA), which has very limited sequence similarity to known

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genes, as described below. Forty-eight hours after transfection, some of the cultures were treated with BMP-2 (50 ng/ml) for another 24 h and the RNA extracted from both the BMP-2treated and BMP-2-untreated siRNA-transfected cultures. 2.3. Preparation of cells and reverse transfection with siRNA Bone cell cultures were transfected with siRNA using a modified reverse transfection method (Amarzguioui, 2004; Ovcharenko et al., 2005). Briefly, exponentially-growing cells were harvested by trypsinization with trypsin-EDTA for 5 min at 37  C and resuspended at 5  105 cells/ml in DMEM without FCS and antibiotic supplements. Transfection complexes were prepared in a final volume of 100 ml of FCS/ antibiotics-free DMEM by mixing 4 ml of lipofectamine (2 mg/ml; Invitrogen, Paisley, UK) with 10 ml of the control siRNA or with 5e10 ml of the BMPR-IB siRNA (10 mM; Ambion, Huntingdon, UK), to a final concentration of 50e 100 nM. After incubating at room temperature for 30 min, 100 ml of the transfection complexes were gently mixed with 900 ml of bone cell suspension, prepared as described above, placed into 6-well plates and incubated for 24 h at 37  C in a humidified atmosphere of 5% CO2 in air. Subsequently, 1 ml of DMEM containing 20% FCS and 2 antibiotic supplements were added to each well and the cells incubated for another 24 h prior to assay. In some experiments, the cells were seeded onto sterile glass coverslips which had been placed in the wells. 2.4. Immunofluorescence analysis (IF) of BMPR-IB Forty-eight hours post-transfection of cells on sterile glass coverslips in a 6-well plate as described above, the cells were washed three times with PBS, fixed with 1% paraformaldehyde (Merck, Poole, UK) in PBS for 30 min. They were then incubated with 0.1% saponin (Sigma) in PBS, for 30 min at room temperature, to permeabilize the cells in order to allow antibody penetration for the detection of intracellular as well as cell surface antigens. The cells were treated with 20% normal goat serum (NGS) in PBS containing 0.1% saponin for 30 min to block non-specific binding of antibody. They were then washed three times with 2% FCS in PBS (FCSePBS) and incubated for 1 h at room temperature with a primary mouse monoclonal antibody (mAb) against human BMPR-IB (R&D systems, Abingdon, UK), diluted 1:100 in PBS containing 2% NGS and 0.1% saponin. After washing with FCS-PBS, the coverslips were incubated with biotinylated goat anti-mouse IgG secondary antibody (Sigma), diluted 1:500 in PBS containing 2% NGS and 0.1% saponin, for 1 h at room temperature. Streptavidin-FITC (DAKO) was added (1:1000) for 30 min at room temperature. The samples were subsequently mounted on glass slides using Citifluor medium (Agar Scientific, Stansted, UK) and the fluorescent signal examined with a BioRad MRC-600 CLSM confocal fluorescence microscope (BioRad Microscience Ltd, Hemel Hempstead, UK).

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2.5. Semi-quantitative reverse transcriptionepolymerase chain reaction (RTePCR) Total RNA was extracted from the bone cells using RNeasyÒ Mini Kit (Qiagen, West Sussex, UK), in accordance with manufacturer’s instructions. For reverse transcription, 1 mg of total RNA was used with 5 ng of oligo-dT (Promega, Madison, WI) in 40 ml of water. After 5 min at 65  C, the first stand of cDNA was synthesized in a total volume of 50 ml, containing 50 U of cloned Moloney murine leukemia virus (M-MuLV) reverse transcriptase, 1 M-MuLV buffer, 40 mM of each dNTP and 40 U of RNase block (all Stratagene, La Jolla, CA). After incubation at 37  C for 60 min, the enzyme was inactivated by incubation for 5 min at 90  C. For each of the genes indicated in Table 1, an aliquot of each cDNA sample was added to a 25 ml reaction mix containing 2 U REDTaq DNA polymerase, 1 REDTaq PCR buffer, 50 mM of each dNTP (all Sigma), and 2 mM of the forward and reverse primer pair sequences (SigmaeGenosys, Pampisford, UK), as shown in Table 1. The PCR reaction was carried out in a thermocycler (GeneAmp PCR System 2400). An initial denaturation step of 2 min at 94  C was followed by the amplification cycles (as indicated in Table 1) and final extension for 7 min at 72  C to ensure the complete generation of a double-stranded DNA. The optimal number of PCR cycles within the linear range of PCR amplification was determined for each gene (data not shown). The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also amplified to assess the quality of the extracted RNA and as a baseline for calculating the relative level of each gene. The PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. To obtain a semi-quantitative estimate of specific transcript expression, the intensity of the band corresponding to each PCR product was measured by densitometry using the Scion Image program (Scion Corporation, Frederick, MD, USA) and normalized to that of GAPDH. 2.6. Measurement of BMPR-IB expression by Western blotting (WB) The cells were lysed for 20 min at 4  C with a lysis buffer containing 50 mM TriseHCl (pH 7.4), 1% Nonidet P-40 (NP40), 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA and the proteinase inhibitors phenyl-methyl-sulfonyl fluoride (PMSF) 1 mM, aprotinin, leupeptin, pepstatin (1 mg/ml each) (all from Sigma). The lysates (150 mg of protein) were then subjected to SDSePAGE on 12% acrylamide gels at 100 V and 30 mA, for 2 h, then transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P Transfermembrane, Sigma), at 100 V and 200 mA for 45 min. These were placed in 100% methanol for 10 s to remove water and then blocked with 5% BSA in PBS (blocking buffer) for 1 h at room temperature. Immunoblotting was carried out by incubating the membranes overnight at 4  C with the following primary mABs diluted 1:1000 in blocking buffer: anti-BMPR-IB (R&D Systems); and

anti-a-tubulin (Insight Biotechnology, Middlesex, UK). The membranes were washed three times with 1% Tween-20 in PBS and a HRP-conjugated secondary goat anti-mouse IgG antibody (DAKO), diluted 1:2500 in blocking buffer, was added for 2 h. After three washes with 1% Tween-20 in PBS, the immunoreactive bands were visualized using the enhanced chemiluminescence ECL Plus kit (Amersham Biosciences, Little Chalfont, UK), with Lumigen PS-3 Acridan as substrate, and the band intensities measured using the Scion Image program. 2.7. Statistical analysis The data are presented as the mean  SE of measurements from three independent bone cell samples, with the experiments being performed in triplicate. Statistical differences between the mean of the test groups and the control group were analyzed by single sample t-test, with p < 0.05 considered significant. The t-test program in the SPSS 11.0 software (SPSS, Chicago, IL) was used for the analyses. 3. Results 3.1. Transfection of bone cells with BMPR-IB siRNA inhibits endogenous BMPR-IB expression Cells transfected with control siRNA and BMPR-IB siRNA were analyzed for the expression of BMPR-IB 48 h posttransfection, as described in Section 2. The results in Fig. 1A show that the expression of BMPR-IB mRNA was inhibited after transfection with BMPR-IB siRNA, with a concentration of 100 nM reducing the BMPR-IB transcript level by approximately 89% compared with that in the control cultures (transfected with the scramble siRNA). WB analysis demonstrated that the level of BMPR-IB protein also markedly decreased 48 h post-transfection with the BMPR-IB siRNA (100 nM), by approximately 76% (Fig. 1B). In addition, IF analysis of BMPR-IB expression clearly revealed the suppressive effect of BMPR-IB siRNA on the expression of the BMPR-IB antigen in the bone cells, as shown by the notably reduced level of BMPR-IB staining (arrows) in the BMPR-IB siRNA-transfected cultures compared with that in the control cultures (Fig. 1C). Similar results were observed in the other two bone cell samples (data not shown). Thus, the expression of endogenous BMPR-IB in the three different bone cell samples used in the present study was significantly inhibited by transfection of the cells with BMPR-IB siRNA. 3.2. Effects of BMP-2 on osteogenic gene expression In order to determine the role of BMPR-IB signaling in BMP-2-induced osteogenic gene expression, the effect of BMP-2 on these genes was first examined by semi-quantitative RTePCR in control cultures treated with BMP-2. Representative electrophoresis gels in Fig. 2A, and a summary of the results obtained from three bone cell samples in Table 2, show that BMP-2 differentially modulated mRNA expression of the

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Table 1 Sequences of primers and conditions used for RTePCR Genes

Sequence (50 e30 ): Forward (F); Reverse (R); [Amplification cycle conditions]

GAPDH (600 bp)

F: CCACCCATGGCAAATTCCCATGGCA R: CTGGACGGCAGGTCAGGTCCACC [94  C 30 s, 55  C 2 min and 72  C 2 min] F: ACTTGCTGTATTGCTGACCTGG R: GGCTTTCTGCAGAGATGCTTAC [94  C 30 s, 61  C 30 s, 72  C 30 s] F: CCCAAAGGCTTCTTCTTG R: CTGGTAGTTGTTGTGAGC [94  C 1 min, 58  C 1 min, 72  C 1 min] F: GGACACAATGGATTGCAAGG R: TAACCACTGCTCCACTCTGG [94  C 1 min, 58  C 1 min, 72  C 1 min] F: CACCTGTGCCATACCAGTTAAAC R: GGTGATGTCCTCGTCTGTAGCATC [94  C 30 s, 55  C 1 min, 72  C 1 min] F: TGCTCAGCATTTTGGGAAT R: TGCATTGGCTCCAGTGACACT [94  C 1 min, 58  C 1 min, 72  C 1 min] F: ATGAGAGCCCTCACACTCCTC R: GCCGTAGAAGCGCCGATAGGC [94  C 1 min, 58  C 1 min, 72  C 1 min] F: GGAATGACTGGATTGTGGCT R: TGAGTTCTGTCGGGACACAG [94  C 1 min, 57  C 1 min, 72  C 1 min] F: TCAGGCAGTCCTTGAGGATA R: AAGCAGTCTGTGTAGTGTGTGG [94  C 1 min, 57  C 1 min, 72  C 1 min] F: GTGGCAGCATCCAATGAAC R: CTGGTAGGCGCTCATAATTACC [94  C 1 min, 57  C 1 min, 72  C 1 min] F: CTGCTTATAACTGGAAATGGCC R: CTGTGGCAAAATTAGTCACTGG [94  C 1 min, 57  C 1 min, 72  C 1 min] F: AGTGTCTAGAGAGGAGGGCTTTGA R: CCGCACTGTGACTAGAACTTCAGA [94  C 1 min, 57  C 1 min, 72  C 1 min] F: CAGTTCCCAAGCATTTCATCC R: TCAATATGGTCGCCAAACAG [94  C 1 min, 58  C 1 min, 72  C 1 min] F: ACATTCCGCTTTTCATGG R: CGCAACTGTGGACACTTTC [94  C 1 min, 57  C 1 min, 72  C 1 min] F: ATGGATGCTTGTTTCAAAGGG R: TACACCAAGAAAAGCAGGGC [94  C 1 min, 57  C 1 min, 72  C 1 min] F: AAAGACGCACTTTGGCTTA R: CGAATACTTTATTATCGAGTGACTG [94  C 1 min, 57  C 1 min, 72  C 1 min] F: TCGTGAGTTTATTGCATATGTAACA R: CTTCCCACTGTTTTTATCACTGA [94  C 1 min, 57  C 1 min, 72  C 1 min] F: GGAGGAAGTTACAGATGTGGCTGT R: CACTCGGAAATGATGGGGTACTG [94  C 1 min, 60  C 1 min, 72  C 1 min]

BMPR-IB (512 bp)

ALP (357 bp)

Col I (461 bp)

OP (532 bp)

BSP (627 bp)

OC (297 bp)

BMP-2 (171 bp)

BMP-4 (106 bp)

BMP-7 (160 bp)

OPG (135 bp)

RANKL (288 bp)

Cbfa1 (443 bp)

Dlx5 (162 bp)

Msx2 (185 bp)

Smad6 (204 bp)

Smurf1 (203 bp)

Noggin (259 bp)

bone matrix components ALP, Col I, OP, BSP and OC in the bone cells, with the most pronounced changes being observed for ALP. Thus, in the control cultures, the expression of ALP transcripts was significantly up-regulated by BMP-2 compared with that in the control cultures without BMP-2 treatment (319%; p < 0.05). Similarly, BMP-2 also had a significant

cDNA (ml)

Cycles

5

22

10

40

10

38

2

20

5

30

5

30

5

30

2

26

2

26

2

26

2

20

5

38

2

28

2

30

2

30

2

26

2

22

5

30

stimulatory effect on the expression of the OP, BSP and OC genes, although it had little effect on the Col I gene. The effect of BMP-2 on the expression of osteoblastic and osteoclastic genes is shown in Fig. 2B and summarized in Table 2b. These results show that exogenously added BMP-2 increased the expression of BMP-2, BMP-4. BMP-7, OPG and

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A

Control siRNA 100

BMPR-IB siRNA 50

75

100

(nM)

BMPR-IB

512 bp

GAPDH

600 bp 100

B

90

Control siRNA

85

11

BMPR-IB siRNA

BMPR-IB

55 kDa

α-tubulin

55 kDa 100

24

C

Control siRNA

BMPR-IB siRNA

Fig. 1. Effect of BMPR-IB siRNA on the expression of BMPR-IB in bone cells. In panel A, cells were transfected with the control siRNA (100 nM) and with the BMPR-IB siRNA (50, 75 and 100 nM) as described in Section 2. At 48 h post-transfection, the expression of BMPR-IB transcripts was analyzed by RTePCR using GAPDH as an internal control. The numbers under the representative PCR gels show the percentage of BMPR-IB transcripts compared with that in the control culture, defined as 100%. Similar results were observed in three separate experiments and in three different bone cell samples. In panel B, the cells were transfected with 100 nM of control and BMPR-IB siRNA and the expression of the BMPR-IB antigen analyzed by WB 48 h after transfection, using a-tubulin as an internal control. The numbers under the representative immunoreactive bands show the percentage of BMPR-IB antigen in the BMPR-IB siRNA-transfected culture compared with that in the control culture, defined as 100%. Similar results were observed in three separate experiments and in three different bone cell samples. In panel C, cells were transfected with the BMPR-IB siRNA (100 nM) and, 48 h posttransfection, the expression of the BMPR-IB antigen examined by confocal fluorescence microscopy. The expression of the BMPR-IB antigen is indicated by arrows. Similar results were observed in three separate experiments and in all the three different bone cell samples examined. Bars ¼ 10 mm.

RANKL transcripts by between 107% and 140%, although none of these changes was found to be statistically significant. In marked contrast, there was a statistically significant effect of BMP-2 on the mRNA expression of Smurf1, Smad6 and noggin, three main inhibitory factors involved in the BMP/ BMPR pathway. The results in Fig. 2C and Table 2c demonstrate that BMP-2 strongly up-regulated all of these three genes by approximately 2.9-, 4.5- and 3.5-fold, respectively. The osteogenic transcription factors Dlx5, Msx2, and Cbfa1 regulate a wide range of osteogenesis-associated genes (Ducy et al., 1997; Shirakabe et al., 2001; Tadic et al., 2002; Barnes et al., 2003; Kim et al., 2004), and their importance has been demonstrated in both skeletal abnormalities in vivo (Korchynskyi and ten Dijke, 2000; ten Dijke et al., 2003) and osteoblast differentiation in vitro (de Jong et al., 2002, 2004; Takagi et al., 2004). The effect of BMP2 on the expression of these transcripts in bone cell cultures is shown in the representative PCR gels in Fig. 2D and the summary of results in Table 2d, which demonstrate, as with the BMPinhibitory genes, that BMP-2 had a pronounced stimulatory effect on all of these three transcription factor transcripts (up-regulation by between 166% and 175%; p < 0.05). 3.3. Effects of BMPR-IB siRNA transfection on BMP-2-induced osteogenic gene expression The specific role of BMPR-IB signaling in BMP-2-induced bone gene expression was examined by determining the effect of BMPR-IB siRNA transfection on BMP-2-induced transcript expression of the four groups of gene described above. Representative electrophoresis gels in Fig. 2A (lane 3) show that transfection of bone cells with BMPR-IB siRNA differentially affected the expression of the BMP-2-induced bone matrix genes. A summary of the results obtained from three different bone cell samples (Table 3a) shows that BMPR-IB knockdown significantly inhibited the BMP-2-induced ALP and OC genes (by approximately 49% and 30%, respectively) compared with the level in control cultures treated with BMP-2. In contrast, BMPR-IB siRNA had little effect on BMP-2-induced Col I, OP and BSP transcripts. These results thus suggest that BMPR-IB siRNA-mediated suppression of BMPR-IB signaling at least partly blocks BMP-2 up-regulation of the ALP and OC genes, but not of the Col I, OP and BSP genes. Notably, BMPR-IB siRNA transfection had no significant effect on all the BMP-2induced osteogenic, osteoclastic and BMP-inhibitory genes (Fig. 2B and C; Table 3a and c), indicating that signaling via BMPR-IB is unlikely to be a major pathway in the regulation of these mediators by BMP-2. In contrast, the representative electrophoresis gels in Fig. 2D and summary in Table 3d show that bone cells transfected with the BMPR-IB siRNA expressed significantly lower levels of Dlx5 and Cbfa1 transcript (71% and 70% of the level detected in control cultures, respectively), whereas Msx2 gene expression was unaffected. 4. Discussion A role for BMPR-IB signaling in osteoblast differentiation has previously been implicated in cells of mesenchymal

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A

B

Bone matrix genes 1

2

3

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Osteogenic regulatory genes 1

2

3

ALP

357 bp

BMP-2

171 bp

Col I

461 bp

BMP-4

106 bp

OP

532 bp

BMP-7

160 bp

BSP

627 bp

OPG

135 bp

OC

297 bp

RANKL

288 bp

GAPDH

600 bp

GAPDH

600 bp

C

BMP-inhibitory factor genes 1

2

D

Transcription factor genes

3

1

2

3

Smurf1

203 bp

Dlx5

162 bp

Smad6

204 bp

Msx2

185 bp

Noggin

259 bp

Cbfa1

443 bp

GAPDH

600 bp

GAPDH

600 bp

Fig. 2. Representative PCR gels showing transcript expression of the genes encoding bone matrix proteins, osteogenic regulatory proteins, BMP-inhibitory factors and osteogenic transcription factors in siRNA-transfected bone cells culture with and without BMP-2. Cultures were transfected with siRNA (control siRNA or BMPR-IB siRNA) and were subsequently treated with or without BMP-2 (50 ng/ml for 24 h). The expression of ALP, Col I, OP, BSP, OC, BMP-2, BMP-4, BMP7, OPG, RANKL, Smurf1, Smad6, noggin, Dlx5, Msx2 and Cbfa1 transcripts was examined by RTePCR, using GAPDH as an internal control. The results show representative gels of the PCR products of each gene. Similar results were observed in three separate experiments and in three different bone cell samples. Lane 1: control cultures without BMP-2; lane 2: control cultures with BMP-2; lane 3: BMPR-IB siRNA-transfected cultures with BMP-2.

lineage (ten Dijke et al., 2003). Although BMPR-IB-mediated signaling has also been reported in skeletal development in vivo (Kawakami et al., 1996; Zou et al., 1997; Yi et al., 2000) and in the osteogenic pathway of the 2T3 murine mesenchymal cell line (Chen et al., 1998), the ROB-C26 rat osteoprogenitor-like cell line (Nishitoh et al., 1996) and in human bone cells (Singhatanadgit et al., 2006), the precise role of this receptor in bone cells is not yet well established. The results of the present study have provided strong evidence of the importance of BMPR-IB in a number of key osteogenic functions of bone cells, using an RNA interference approach to suppress the expression of endogenous BMPR-IB specifically and thus ablate the BMPR-IB signal transduction pathway. At the functional level, this disruption of BMPR-IB signaling significantly suppressed the expression of BMP-2induced ALP and OC transcripts, indicating that BMPR-IB is at least partly necessary for the expression of these two genes and thereby the corresponding proteins, which are generally regarded as characteristic early and late BMP-responsive markers of osteoblast differentiation, respectively (Lecanda et al., 1997; Hay et al., 1999; Ebara and Nakayama, 2002; van der Horst et al., 2002; Noth et al., 2003). BMPR-IB is also required for the BMP-2-mediated induction of these osteoblast marker genes in the precursor cell lines 2T3 and C2C12 (Akiyama et al., 1997; Namiki et al., 1997; Chen et al., 1998) and for the expression of the OC gene in vivo (Zhao et al.,

2002), although other studies in human bone marrow stromal cells have found that BMPR-IB overexpression had no effect on ALP activity (Osyczka et al., 2004), suggesting that the lack of ALP regulation by BMP-2 was not due to lack of BMPR-IB. This discrepancy suggests the possibility that the induction of ALP via BMPR-IB in the bone cells reported here may involve downstream signaling molecules which differ from those in the bone marrow stromal cells. Consistent with previous reports using human bone cells (Fromigue et al., 1998; Hay et al., 1999), knockdown of BMPR-IB was found here to have no significant effect on the expression of the Col I gene at the transcriptional level. However, it is possible that BMPR-IB-mediated signaling could nevertheless be involved in the regulation of Col I by a post-transcriptional mechanism, since it has previously been shown that there is a significant decrease in collagen fibrils in 2T3 cell cultures which lack of functional BMPR-IB (Chen et al., 1998). OP and BSP are also considered to be essential components of the bone matrix (Hunter and Goldberg, 1993; Denhardt and Noda, 1998), and their expression has been shown to be stimulated by BMP-2 in human marrow stromal cells and human osteoblasts (Lecanda et al., 1997; Diefenderfer et al., 2003). However, while the present study has also demonstrated that OP and BSP mRNA levels were upregulated by BMP-2 in the bone cells used here, this was found to be independent of BMPR-IB signaling. This suggests

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Table 2 Relative transcript levels in control cultures treated with BMP-2

Table 3 Relative transcript levels in BMPR-IB siRNA-transfected cultures treated with BMP-2

Genes

BMP-2 treatment

a. Bone matrix proteins ALP Col I OP BSP OC

319  39* 130  23 175  31* 167  22* 170  10*

a. Bone matrix proteins ALP Col I OP BSP OC

b. Osteogenic regulatory proteins BMP-2 BMP-4 BMP-7 OPG RANKL

140  33 107  6 112  11 140  25 132  30

b. Osteogenic regulatory proteins BMP-2 BMP-4 BMP-7 OPG RANKL

c. BMP-inhibitory factors Smurf1 Smad6 Noggin

292  31* 453  45* 349  30*

c. BMP-inhibitory factors Smurf1 Smad6 Noggin

93  1.2 98  0.9 93  1.3

d. Transcription factors Dlx5 Msx2 Cbfa1

174  15* 166  20* 175  10*

d. Transcription factors Dlx5 Msx2 Cbfa1

71  0.3* 92  1.2 70  0.2*

Genes

The results are presented as the mean percent (  SE) of the respective transcript level in control cultures without BMP-2 treatment, defined as 100%, obtained from three different bone cell samples. Numbers in bold indicate statistically significant differences: * p < 0.05 vs. control cultures without BMP-2.

the possibility that other pathways, for example BMPR-IAmediated signaling, might be responsible for the effects of BMP-2 on OP and BSP in the these cells. A number of naturally-occurring mediators associated with bone remodeling, including BMP, OPG and RANKL, have also been shown to be regulated by BMP-2. For example, BMP-2 has been reported to stimulate the expression of its own transcripts in human marrow stromal cells (Diefenderfer et al., 2003), of OPG transcripts in human osteoblastic cells (Hofbauer et al., 1998) and of RANKL transcripts in C2C12 cells (Otsuka et al., 2003). However, the present BMPR-IB knockdown experiments have demonstrated that BMP-2 had no significant effect on the expression of any of these transcripts in the bone cells, possibly because of limited or absent expression of some of the signaling components downstream of BMP-2 that are required for the expression of these genes in the bone cells specifically. Thus, unlike ALP and OC, the results here indicate that the regulation of BMP-2, -4, -7, OPG and RANKL by BMP-2 does not involve BMPR-IB-mediated signaling. BMPR-mediated BMP signal transduction is also known to be negatively controlled by BMP inhibitory molecules, including Smurf1, Smad6 and noggin (Imamura et al., 1997; Gazzerro et al., 1998; Murakami et al., 2003). We have previously shown that the expression of Smurf1 transcripts is up-regulated by BMP-2 (Singhatanadgit et al., 2006), while the genes coding for Smad6 and noggin have also been reported to be transcriptionally activated in response to BMP-2

BMPR-IB siRNA transfection 51  0.7* 96  1.1 94  0.9 80  0.9 70  0.3*

89  1.2 103  0.4 97  0.8 86  1.1 98  0.7

The results are presented as the mean percent (  SE) of the respective transcript level in control cultures treated with BMP-2, defined as 100%, obtained from three different bone cell samples. Numbers in bold indicate statistically significant differences: *p < 0.05 vs. control cultures treated with BMP-2.

treatment in vitro (Takase et al., 1998; Diefenderfer et al., 2003; Li et al., 2003; Osyczka et al., 2004). The present finding that BMP-2 markedly induces the expression of Smurf1 as well as Smad6 and noggin transcripts in the bone cells could thereby lead to down-regulation of the BMP/ BMPR signal transduction pathway. This is consistent with our findings in bone cells that, in response to prolonged BMP-2 treatment, the level of p-Smad1/5/8 decreased (unpublished observation), possibly reflecting increased expression of such BMP inhibitors. However, BMP-2 induction of these BMP inhibitory factor genes has been shown here to be unlikely to be mediated via BMPR-IB signaling in the bone cells, in contrast to a previous study showing induction of noggin by transfection of human marrow stromal cells with constitutively active BMPR-IB (caBMPR-IB) (Osyczka et al., 2004). Although the reason for this discrepancy remains unclear, it is possible that the role of BMPR-IB signaling in the expression of noggin might be due to differences in the type and differentiation stage of these cells. The osteoblast-associated transcription factors Dlx5, Msx2 and Cbfa1 play a pivotal part in osteoblast differentiation and bone formation (Ducy et al., 1997; Komori et al., 1997; Shirakabe et al., 2001; Tadic et al., 2002). However, while Dlx5 and Cbfa1 have been shown to stimulate osteogenesisrelated functions such as ALP activity and the expression of the Col I, OP, BSP and OC genes, the Msx2 gene has been shown to down-regulate these genes (Ducy et al., 1997;

W. Singhatanadgit et al. / Cell Biology International 32 (2008) 1362e1370

Shirakabe et al., 2001; Tadic et al., 2002; Barnes et al., 2003; Kim et al., 2004). Although these transcription factors have previously been reported to be regulated by BMP (Ducy et al., 1997; Miyama et al., 1999; Ashique et al., 2002; Lee et al., 2003), the specific role of the BMPR in mediating these changes is still unclear. In the present study, while BMP-2 was found to up-regulate Dlx5 transcript expression via BMPR-IB signaling, Msx2 expression did not involve the BMPR-IB pathway in the bone cells, as has also been reported in BMPRIB transgenic mice in vivo (Zhang et al., 2000). As with the regulation of OP and BSP noted above, BMPR-IA might provide an alternative signaling pathway for Msx2. The results reported here also showed that, as with Dlx5, the expression of the Cbfa1 gene was up-regulated by BMP-2 and was mediated at least in part by BMPR-IB signaling, consistent with previous studies showing that BMP-2-induced expression of Cbfa1 mRNA was blocked by a truncated inactive BMPR-IB in 2T3 cells (Chen et al., 1998) and in homozygous truncated BMPR-IB transgenic mice (Zhao et al., 2002). The present results have provided evidence that BMPR-IB is required, at least partly, for BMP-2 induction of ALP, OC, Dlx5 and Cbfa1 gene expression. Although the role of BMPRIB in BMP-2-induced mineralized bone matrix formation was not examined here, a previous study has reported that this process was completely blocked by overexpression of truncated BMPR-IB in 2T3 cells (Chen et al., 1998). Thus, while BMPR-IB signaling therefore appears to be a key factor in a number of pivotal BMP-2-induced osteoblast functions including mineralization, our experiments demonstrate that this receptor is unlikely to have a major role in signal transduction pathways which control the response of certain BMP inhibitory genes, e.g., Smurf1, Smad6, noggin and Msx2. In conclusion, we have shown that BMPR-IB-dependent signaling plays a crucial role in BMP-2 stimulation of the expression of the ALP, OC, Dlx5 and Cbfa1 genes in bone cells. These findings thus provide important insight into the role of BMPR-IB in BMP-2-induced osteoblastic functions of these cells in vitro, and highlight the possibility that this receptor could be a therapeutic target for enhancing bone formation in vivo. Acknowledgement This work was supported in part by a scholarship of the Royal Thai Government to W.S. References Akiyama S, Katagiri T, Namiki M, Yamaji N, Yamamoto N, Miyama K, et al. Constitutively active BMP type I receptors transduce BMP-2 signals without the ligand in C2C12 myoblasts. Exp Cell Res 1997;235:362e9. Amarzguioui M. Improved siRNA-mediated silencing in refractory adherent cell lines by detachment and transfection in suspension. Biotechniques 2004;36:766e8. 770. Ashique AM, Fu K, Richman JM. Signalling via type IA and type IB bone morphogenetic protein receptors (BMPR) regulates intramembranous bone formation, chondrogenesis and feather formation in the chicken embryo. Int J Dev Biol 2002;46:243e53.

1369

Barnes GL, Javed A, Waller SM, Kamal MH, Hebert KE, Hassan MQ, et al. Osteoblast-related transcription factors Runx2 (Cbfa1/AML3) and MSX2 mediate the expression of bone sialoprotein in human metastatic breast cancer cells. Cancer Res 2003;63:2631e7. Boden SD, Kang J, Sandhu H, Heller JG. Use of recombinant human bone morphogenetic protein-2 to achieve posterolateral lumbar spine fusion in humans: a prospective, randomized clinical pilot trial: 2002 Volvo Award in clinical studies. Spine 2002;27:2662e73. Bostrom MP, Camacho NP. Potential role of bone morphogenetic proteins in fracture healing. Clin Orthop 1998;(Suppl)::S274e82. 355. Celil AB, Hollinger JO, Campbell PG. Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling. J Cell Biochem 2005;95:518e28. Chen D, Ji X, Harris MA, Feng JQ, Karsenty G, Celeste AJ, et al. Differential roles for bone morphogenetic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages. J Cell Biol 1998;142:295e305. Chen D, Zhao M, Harris SE, Mi Z. Signal transduction and biological functions of bone morphogenetic proteins. Front Biosci 2004;9:349e58. Cheng SL, Lou J, Wright NM, Lai CF, Avioli LV, Riew KD. In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene. Calcif Tissue Int 2001;68:87e94. de Jong DS, van Zoelen EJ, Bauerschmidt S, Olijve W, Steegenga WT. Microarray analysis of bone morphogenetic protein, transforming growth factor beta, and activin early response genes during osteoblastic cell differentiation. J Bone Miner Res 2002;17:2119e29. de Jong DS, Vaes BL, Dechering KJ, Feijen A, Hendriks JM, Wehrens R, et al. Identification of novel regulators associated with early-phase osteoblast differentiation. J Bone Miner Res 2004;19:947e58. Denhardt DT, Noda M. Osteopontin expression and function: role in bone remodeling. J Cell Biochem 1998;(Suppl):92e102, 30e31. Diefenderfer DL, Osyczka AM, Garino JP, Leboy PS. Regulation of BMPinduced transcription in cultured human bone marrow stromal cells. J Bone Joint Surg Am 2003;3(Suppl):19e28. 85-A. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997;89:747e54. Ebara S, Nakayama K. Mechanism for the action of bone morphogenetic proteins and regulation of their activity. Spine 2002;27:S10e5. Fromigue O, Marie PJ, Lomri A. Bone morphogenetic protein-2 and transforming growth factor-beta2 interact to modulate human bone marrow stromal cell proliferation and differentiation. J Cell Biochem 1998;68:411e26. Gazzerro E, Gangji V, Canalis E. Bone morphogenetic proteins induce the expression of noggin, which limits their activity in cultured rat osteoblasts. J Clin Invest 1998;102:2106e14. Hay E, Hott M, Graulet AM, Lomri A, Marie PJ. Effects of bone morphogenetic protein-2 on human neonatal calvaria cell differentiation. J Cell Biochem 1999;72:81e93. Hofbauer LC, Dunstan CR, Spelsberg TC, Riggs BL, Khosla S. Osteoprotegerin production by human osteoblast lineage cells is stimulated by vitamin D, bone morphogenetic protein-2, and cytokines. Biochem Biophys Res Commun 1998;250:776e81. Hunter GK, Goldberg HA. Nucleation of hydroxyapatite by bone sialoprotein. Proc Natl Acad Sci USA 1993;90:8562e5. Imamura T, Takase M, Nishihara A, Oeda E, Hanai J, Kawabata M, et al. Smad6 inhibits signalling by the TGF-beta superfamily. Nature 1997;389: 622e6. Kawakami Y, Ishikawa T, Shimabara M, Tanda N, Enomoto-Iwamoto M, Iwamoto M, et al. BMP signaling during bone pattern determination in the developing limb. Development 1996;122:3557e66. Kim YJ, Lee MH, Wozney JM, Cho JY, Ryoo HM. Bone morphogenetic protein-2-induced alkaline phosphatase expression is stimulated by Dlx5 and repressed by Msx2. J Biol Chem 2004;279:50773e80. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997;89:755e64. Korchynskyi O, Dechering KJ, Sijbers AM, Olijve W, ten Dijke P. Gene array analysis of bone morphogenetic protein type I receptor-induced osteoblast differentiation. J Bone Miner Res 2003;18:1177e85.

1370

W. Singhatanadgit et al. / Cell Biology International 32 (2008) 1362e1370

Korchynskyi O, ten Dijke P. Bone morphogenetic protein receptors and their nuclear effectors in bone formation. In: Vukicevic S, Sampath K, editors. Bone morphogenetic proteins: from laboratory to clinical practice. Boston: Birkhauser; 2000. p. 31e60. Lecanda F, Avioli LV, Cheng SL. Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2. J Cell Biochem 1997;67:386e96. Lee MH, Kim YJ, Kim HJ, Park HD, Kang AR, Kyung HM, et al. BMP-2induced Runx2 expression is mediated by Dlx5, and TGF-beta 1 opposes the BMP-2-induced osteoblast differentiation by suppression of Dlx5 expression. J Biol Chem 2003;278:34387e94. Li X, Ionescu AM, Schwarz EM, Zhang X, Drissi H, Puzas JE, et al. Smad6 is induced by BMP-2 and modulates chondrocyte differentiation. J Orthop Res 2003;21:908e13. Lieberman JR, Daluiski A, Einhorn TA. The role of growth factors in the repair of bone. Biology and clinical applications. J Bone Joint Surg Am 2002:1032e44. 84-A. Miyama K, Yamada G, Yamamoto TS, Takagi C, Miyado K, Sakai M, et al. A BMP-inducible gene, dlx5, regulates osteoblast differentiation and mesoderm induction. Dev Biol 1999;208:123e33. Murakami G, Watabe T, Takaoka K, Miyazono K, Imamura T. Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads. Mol Biol Cell 2003;14:2809e17. Nakashima M, Reddi AH. The application of bone morphogenetic proteins to dental tissue engineering. Nat Biotechnol 2003;21:1025e32. Namiki M, Akiyama S, Katagiri T, Suzuki A, Ueno N, Yamaji N, et al. A kinase domain-truncated type I receptor blocks bone morphogenetic protein-2-induced signal transduction in C2C12 myoblasts. J Biol Chem 1997;272:22046e52. Nishitoh H, Ichijo H, Kimura M, Matsumoto T, Makishima F, Yamaguchi A, et al. Identification of type I and type II serine/threonine kinase receptors for growth/differentiation factor-5. J Biol Chem 1996;271:21345e52. Noth U, Tuli R, Seghatoleslami R, Howard M, Shah A, Hall DJ, et al. Activation of p38 and Smads mediates BMP-2 effects on human trabecular bone-derived osteoblasts. Exp Cell Res 2003;291:201e11. Osyczka AM, Diefenderfer DL, Bhargave G, Leboy PS. Different effects of BMP-2 on marrow stromal cells from human and rat bone. Cells Tissues Organs 2004;176:109e19. Otsuka E, Notoya M, Hagiwara H. Treatment of myoblastic C2C12 cells with BMP-2 stimulates vitamin D-induced formation of osteoclasts. Calcif Tissue Int 2003;73:72e7. Ovcharenko D, Jarvis R, Hunicke-Smith S, Kelnar K, Brown D. Highthroughput RNAi screening in vitro: from cell lines to primary cells. RNA 2005;11:985e93.

Shirakabe K, Terasawa K, Miyama K, Shibuya H, Nishida E. Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5. Genes Cells 2001;6:851e6. Singhatanadgit W, Salih V, Olsen I. Up-regulation of bone morphogenetic protein receptor IB by growth factors enhances BMP-2-induced human bone cell functions. J Cell Physiol 2006;209:912e22. Tadic T, Dodig M, Erceg I, Marijanovic I, Mina M, Kalajzic Z, et al. Overexpression of Dlx5 in chicken calvarial cells accelerates osteoblastic differentiation. J Bone Miner Res 2002;17:1008e14. Takagi M, Kamiya N, Takahashi T, Ito S, Hasegawa M, Suzuki N, et al. Effects of bone morphogenetic protein-2 and transforming growth factor beta1 on gene expression of transcription factors, AJ18 and Runx2 in cultured osteoblastic cells. J Mol Histol 2004;35:81e90. Takase M, Imamura T, Sampath TK, Takeda K, Ichijo H, Miyazono K, et al. Induction of Smad6 mRNA by bone morphogenetic proteins. Biochem Biophys Res Commun 1998;244:26e9. ten Dijke P, Fu J, Schaap P, Roelen BAJ. Signal transduction of bone morphogenetic proteins in osteoblast differentiation. Journal of Bone and Joint Surgery 2003;85:34e8. Urist MR. Bone: formation by autoinduction. Science 1965;150:893e9. van der Horst G, van Bezooijen RL, Deckers MM, Hoogendam J, Visser A, Lowik CW, et al. Differentiation of murine preosteoblastic KS483 cells depends on autocrine bone morphogenetic protein signaling during all phases of osteoblast formation. Bone 2002;31: 661e9. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel regulators of bone formation: molecular clones and activities. Science 1988;242:1528e34. Yamaguchi A, Komori T, Suda T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev 2000;21:393e411. Yi SE, Daluiski A, Pederson R, Rosen V, Lyons KM. The type I BMP receptor BMPRIB is required for chondrogenesis in the mouse limb. Development 2000;127:621e30. Zhang Z, Yu X, Zhang Y, Geronimo B, Lovlie A, Fromm SH, et al. Targeted misexpression of constitutively active BMP receptor-IB causes bifurcation, duplication, and posterior transformation of digit in mouse limb. Dev Biol 2000;220:154e67. Zhao M, Harris SE, Horn D, Geng Z, Nishimura R, Mundy GR, et al. Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation. J Cell Biol 2002;157: 1049e60. Zou H, Wieser R, Massague J, Niswander L. Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage. Genes Dev 1997;11:2191e203.