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Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with Simian Virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, ostenectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.
Bone morphogenetic proteins (Bmps) were initially identified by their ability to induce bone and cartilage formation when implanted subcutaneously or intramuscularly in animals . The Bmps are structurally related to transforming growth factor-β (TGF-β). They contain over 20 different Bmps and are subclassified into at least four groups depending on their structures . Bmp2 and Bmp4 are most similar to Decapentaplegic (Dpp) in Drosophi and belong to the Bmp2/4 subclass . Among Bmp family proteins, Bmp2 is one of the best characterized molecules. Recombinant Bmp2 has been shown to be a potent stimulator of osteogenesis both in vitro and in vivo [4–9]. Overexpression Bmp2 induces expression of osteocalcin and other genes that are important for osteoblast differentiation . Moreover, there is growing evidence indicating that Bmp4 also plays an essential role in osteoblast differentiation, bone formation and fracture healing [11–14]. However, detail understandings of the mechanisms through which Bmp2 and Bmp4 exert their effects on osteoblast functions remain elusive as it has been difficult to decipher the specific roles of Bmp2 or Bmp4 during osteogenesis because of functional redundancy between them . Recently, mice simultaneously lacking Bmp2 and Bmp4 has been produced and displayed severe impairment of osteogenesis as well as exhibited different genotypic and phenotypic characteristics compared to either that of Bmp2 or Bmp4 null mice . Thus, generation of a floxed Bmp2/4 osteoblast cell line would be a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events involved in matrix mineralization in vitro. Such information will help realize the potential of Bmps as therapeutic agents and for the rational targeting of specific Bmps to the appropriate clinical indication.
In the present study, we established an immortalized mouse floxed Bmp2/4 osteoblast cell line using transfection of simian virus 40 T-antigen (SV40). The cells show stable growth and retain the phenotypic and genotypic characteristics similar to primary floxed Bmp2/4 osteoblasts in vitro.
A conditional allele of the mouse Bmp2 gene was created by introducing Cre recombinase recognition sites (loxP), which were integrated to excise the protein-coding region in exon 3 of the Bmp2 gene. Mice carrying floxed Bmp4 allele were kindly provided by Dr. Hogan . The loxP sites were placed upstream and downstream of exons 3 and 4, within intron 2 and 3′ of the poly-adenylation site, and the entire Bmp4 protein coding sequence was excised by the Cre recombinase . For generation of Bmp2 and Bmp4 double conditional mice, Bmp2C/C animals were crossed with Bmp4C/C animals to generate Bmp2C/+/Bmp4C/+ animals. These animals were crossed with Bmp2C/C or Bmp4C/C animals to generate Bmp2C/C/Bmp4C/+ or Bmp2C/+/Bmp4C/C animals, respectively. Bmp2C/C/Bmp4C/+ or Bmp2C/+/Bmp4C/C animals were crossed with each other to generate Bmp2C/C/Bmp4C/C animals. Genotyping of Bmp2C/C/Bmp4C/C animals were done by PCR analyses using floxed Bmp2 and Bmp4 specific primers (Table 1). Genomic DNA was isolated from the mouse tails by DNA purification kit, Wizard® Genomic (Promega, Madison, WI). For Bmp2, the floxed allele was amplified as a 400 bp product, while for Bmp4, the floxed allele amplified as a 184 bp band. Protocols utilized for mouse experiments were approved by the Animal Care and Use for Research of the University of Texas Health Science Center at San Antonio, TX, USA.
The calvarial bones of 1-day floxed Bmp2/4 mice were isolated and washed with phosphate buffered saline (PBS), cut into pieces (about 0.1 cm3), attached to a flask with Minimum Essential Medium Alpha Medium (a-MEM, Invitrogen, San Diego, CA) containing 10% fetal calf serum plus penicillin (100 unit/ml) and streptomycin (100 μg/ml) and cultured at 37°C in a humidified atmosphere of air containing 5% CO2.
Primary floxed Bmp2/4 osteoblast cells in passage 3 were transfected with pSV3neo (ATCC, No. 37150, Manassas, VA), a plasmid containing coding sequences of SV40 T-Ag and a neomycin (G418)-resistance gene by electroporation. One day after transfection, cells were cultured in standard medium supplemented with 600 μg/ml of G418 (Sigma-Aldrich, St. Louis, MO). Several colonies were formed, and well-isolated colonies were removed selectively and replated at low densities to obtain the secondary selection. One of the selected secondary colonies was named iBmp2/4 (immortalized floxed Bmp2/4). iBmp2/4 cells of passage 50 and primary floxed Bmp2/4 osteoblast cells of passage 3 were used for the following characterization.
Morphology of iBmp2/4 and primary cells was observed by a light inverted microscope. Cell proliferation assay was performed by direct cell counting and MTT method. Briefly, cells were seeded into 6-well plates at 5×104 cells per well. The cells were trypsinized and counted daily using a hemocytometer for up to 4 days. For MTT assay, cells were seeded into 96-well plates with 1.5×103 cells per well and detected at day 1, 2, 3, 4 respectively by MTT cell proliferation assay kit (ATCC, No. 30-1010K, Manassas, VA).
Simian virus 40 sequences were accessed in Genbank (Accession No. J02400) and specific primers were synthesized (Table 1). Genomic DNA was isolated from iBmp2/4 and primary cells. pSV3 neo plasmid was used as positive control. Two-hundred nanograms of DNA (for pSV3 neo plasmid DNA 10 ng) were diluted in a 25 μl polymerase chain reaction (PCR) mix (Sigma-Aldrich). Five μl of PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. For detection of SV40 protein expression, iBmp2/4 and primary cells were seeded on coverslips in 6-well plates and cultured for 48 hours in standard a-MEM medium. The coverslips were rinsed with PBS and fixed with cold acetone and methanol (1:1). The cells were blocked with 10% goat serum and incubated with a primary anti-SV40 large T antigen monoclonal antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at 37°C. Then the cells were washed with PBS containing 0.1% goat serum and incubated with the secondary antibody conjugated with Alexa Fluo® 568 red (Molecular Probes, Eugene, OR) for 1 hour at room temperature. For negative control, the primary SV40 antibody was replaced by mouse IgG I (Dakocytomation, Carpinteria, CA). For cell nucleus staining, the cells were treated with Hoechst (Sigma-Aldrich). Images of Alexa Fluo® 568 red staining of the SV40 protein were obtained at the Core Optical Imaging Facility at UTHSCSA under the same parameters in a Nikon inverted microscope.
For detection of ALP activity, cultures of both primary and immortalized osteoblast cells for 3 weeks were fixed with 70% ethanol for 5 min and washed in the buffer (100 mM Tris-HCl, pH 9.5; 100 mM NaCl; 50 mM MgCl2). In situ ALP staining was performed according to the supplier’s instructions (Bio-Rad, Hercules, CA).
iBmp2/4 and primary cells were plated in 6-well plates at a density of 4×105 per well and cultured in calcifying medium (a-MEM supplemented with 10% FBS, penicillin (100 unit/ml) and streptomycin (100 μg/ml), 50 μg/ml ascorbic acid, 10 nM dexamethasone and 10 mM sodium β-glycerophosphate) at 37°C for 3 weeks respectively. The cells were fixed in 10% formaldehyde neutral buffer solution and then stained with Alizarin Red S (Sigma-Aldrich).
Total RNA was extracted from iBmp2/4 and primary cells by RNA STAT-60 kit (Tel-Test, Inc. Friendswood, TX), treated with DNase I (Promega), and purified with the RNeasy Mini Kit (Qiagen Inc., Valencia, CA). RNA concentration was determined at an optical density of OD260. The RNA was transcribed into cDNA by SuperScript II reverse transcriptase (Invitrogen). Specific primers for the RT-PCR were shown in Table 1, and these included Gapdh, Runx2, Dlx3, Osx, ATF4, Dmp1, Bsp, Opn, Oc, Osn, ALP and collagen type I. The PCR reaction was first denatured at 95°C for 10 min, and then carried out at 95°C for 60s, at 50–60°C for 60s and at 72°C for 60s for 35 cycles and with a final 10 min extension at 72°C. Five μl of PCR products were analyzed by agarose gel with ethidium bromide staining. Corrective DNA was verified by DNA sequencing.
iBmp2/4 and primary cells were prepared as described above. For detection of osteoblast-related proteins, fluorescent immunohistochemistry was performed using antibodies directed against mouse Bsp and Dmp1 (gifts from Dr. Larry Fisher, NIDCR), Runx2, Osx, Opn, Oc, and Col1A1 (Santa Cruz Biotechnology Inc.) and Dlx3 (Abcam, Cambridge, MA). Negative control of mouse IgG 1 was purchased from Dakocytomation (Carpinteria, CA). Immunohistochemical assay was performed as described above with corresponding secondary antibodies conjugated with Alexa Fluor 488 green fluorescent labeling (Molecular Probes). Microphotographs were obtained under a Nikon microscope using a Nikon Cool pix 4500 digital camera.
All values were represented as the mean ± standard deviation (S.D.). Statistical significance was determined using the unpaired Student’s t-test with a P-value of < 0.05 being statistical significant.
To establish Bmp2 and Bmp4 conditional mice, Bmp2 conditional mice were crossed with Bmp4 conditional animals. The floxed Bmp2/4 mice were confirmed by PCR using specific Bmp2 and Bmp4 primers (Fig. 1A & Table 1) and Southern blot analysis (data not shown).
To create immortalization of mouse floxed Bmp2/4 osteoblast cell lines, primary cells were transfected with pSV3 neo plasmid containing SV40 T-Ag gene and then selected with G418. The G418 resistant cells were formed after 2–3 week selections. These cells bypassed senescence and grew over 50 population doublings (PDLs) without significant growth retardation and termed as iBmp2/4. On the other hand, non-transfected primary cells entered crisis at about 5 PDLs. The iBmp2/4 cells were passaged at 50 generation and used for detail characterization. PCR analysis shows that SV40 T-Ag gene was detected in iBmp2/4 cells and pSV3 neo plasmid, but not seen in the primary cells (Fig. 1B). Immunohistochemistry further confirmed that simian virus 40 T-Ag was expressed in all of iBmp2/4 cells whereas immunostaining was not present in the primary cells (Fig. 1C–D).
Cell morphology was studied using a light inverted microscopy (Fig. 2A). iBmp2/4 cells display spindle shape similar to that of the primary cells. There was no microscopical finding indicating senescence or neoplastic nature in iBmp2/4 cells. Proliferation of both the primary and iBmp2/4 cells appeared stable. However, the iBmp2/4 cells displayed a more rapid growth rate than the primary cells as revealed by daily cell counting and MTT assay (Fig. 2B).
To assess bone-related gene expressions in iBmp2/4 and primary cells, we studied expression of collagenous and non-collagenous as well as transcription factor genes using RT-PCR assay. Figure 3A shows that both of the transformed and primary cells synthesized collagenous and non-collagenous protein genes such as collagen type I, ALP, Bsp, Dmp1, Oc, Osn, Opn. These cells also expressed osteoblast-specific transcription factors, Runx2, Osx and ATF4 (Fig. 3A). Immunohistochemistry studies further confirmed that these cells expressed those proteins (Fig. 3B). These results indicate that iBmp2/4 cells retain their genotypic characteristics similar to the primary osteoblastic cells.
To evaluate the differentiation and mineralization activities of these cells, we examined ALP activity, one of osteoblast differentiation markers, by in situ ALP histochemistry. Figure 4A shows that the positive expression of ALP in both the primary and iBmp2/4 cells after a culture of 3 weeks in calcifying media. Also, mineralized nodules in both the primary and immortalized osteoblast cells were seen by Alizarin Red S staining (Fig. 4B).
In this study, we established an immortalized mouse floxed Bmp2/4 osteoblast cell line, named iBmp2/4, from primary mouse floxed Bmp2/4 osteoblasts by transfection of SV40 T-Ag, a viral oncogene. iBmp2/4 cells present SV40 positive and retain high ALP activity and strong mineralization ability. Moreover, they express all of the identification markers of osteoblasts. These data suggest that the cell line iBmp2/4 is functionally active and displays genotypic and phenotypic characteristics similar to that of the primary osteoblast cells.
The strategy for this study was to introduce a pSV3 neo, a plasmid containing coding sequences of SV40 T-Ag and a neomycin-resistance gene into primary floxed Bmp2/4 osteoblasts, as pSV3 neo has been demonstrated to be an effective agent for immortalization manipulation in a variety of studies [18–20]. Moreover, numerous studies have shown that the viral oncogene SV-40 large T antigen is capable of immortalizing various cells including bone-derived cell line RCT3 [21–24]. In the present experiment, we transfected SV40 into the mouse primary floxed Bmp2/4 osteoblast cells, and the transfected iBmp2/4 cells express SV40 detected by PCR and immunostaining. The iBmp2/4 cells have been continuously cultured for over one year, indicating a stable genomic integration of SV40 T antigen coding sequences.
Although it was reported that SV40 might alter the nature of cells such as growth with extension and cell morphology , cell morphology of immortalized cells transfected by SV-40 similar to the primary ones was also observed [19, 26–27]. In the present experiment, the growth pattern of the transfected cells is altered with a more rapid proliferation rate. However, the iBmp2/4 cells retain phenotypic and genomic characteristics with no obvious cell morphology changes. Osteoblast cells immortalized with SV-40 large T antigen are capable of maintaining the differentiated features were also reported [28–29]. These characteristics include ALP activity, type I collagen synthesis, presence of osteopontin transcripts which could be stimulated with 1,25 (OH)2D3, and ability of producing mineralizing extracellular matrix. In this study, we examined the ALP activity, mineralized nodule formation and the expression of bone-related genes that act as osteoblast terminal phenotype identification markers [30–31]. Our results show that both the primary and transfected iBmp2/4 cells displayed strong mineralization ability and characteristics of bone-related gene expression.
In summary, we were able to establish a cell line of immortalized mouse floxed Bmp2/4 osteoblast cells with differentiation and mineralization capability. The cell line can provide an excellent resource of large amounts suitable for Bmp2/4 knock out experiments with the addition of Cre recombinase in vitro. Thus, we for the first time established the iBmp2/4 cells that can be a useful cell model for investigating the mechanism of Bmp2/4 effects on osteoblast proliferation, differentiation and mineralization.
This work was supported in part by National Institutes of Health Grants DE019802 (S.C.), AR054616 (S.H.) and the grant from the Natural Science Foundation of China 30801293 (LA Wu) and 2009-10 3M ESPE Preventative Pediatric Dentistry Postdoctoral Research Fellowship of AAPD (LA Wu).
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