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. 2011 Mar 24;53(1):21.
doi: 10.1186/1751-0147-53-21.

Effects of osteoprotegerin from transfection of pcDNA3.1(+)/chOPG on bioactivity of chicken osteoclasts

Lele Hou  1 , Jiafa Hou, Jing Yao, Zhenlei Zhou
Affiliations

Effects of osteoprotegerin from transfection of pcDNA3.1(+)/chOPG on bioactivity of chicken osteoclasts

Lele Hou et al. Acta Vet Scand. .

Abstract

Background: Osteoprotegerin (OPG) has been reported to prevent bone resorption by inhibiting the formation, function, and survival of osteoclasts in a variety of animal models. However, the effects of OPG on bone metabolism in avian species have not been described. The objective of this study was to investigate the effects of chicken OPG (chOPG) expressed in chicken embryo fibroblasts (CEFs) on chicken osteoclast function in vitro.

Methods: The chOPG sequence containing the open reading frame (ORF) was amplified from chicken embryo frontal bone and inserted into the pcDNA3.1 (+) vector. PcDNA3.1 (+)/chOPG was transiently transfected into CEFs by lipofectamine 2000. Transcription of OPG mRNA and expression of chOPG recombinant protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence. The level of chOPG recombinant protein was detected by enzyme-linked immunosorbent assay. The suspension of osteoclasts was separated from chicken embryos and divided into three groups (control group, pcDNA3.1 (+) group and pcDNA3.1 (+)/chOPG group). The percentage of osteoclast apoptosis was detected by flow cytometry. The tartrate-resistant acid phosphatase (TRAP) secreted by osteoclasts was measured by the diazol method. The resorbing activity of osteoclasts was evaluated by the area of lacunae on bone flaps and the concentration of calcium in the supernatant liquid of osteoclasts.

Results: 48 h after transfection, the exogenous OPG gene transcription was detected by RT-PCR. After 72 h, the CEFs transfected from pcDNA3.1 (+)/chOPG displayed green fluorescence and the concentration of chOPG protein was 15.78 ± 0.22 ng/mL. After chicken osteoclasts were cultured for 5 d in a medium containing supernatant from transfected CEFs, the percentage of osteoclast apoptosis was increased significantly, the concentration of TRAP, the area of lacunae on bone flaps and calcium concentration were decreased significantly in the pcDNA3.1(+)/OPG group compared to the control group and the pcDNA3.1 (+) group.

Conclusion: Constructed pcDNA3.1 (+)/chOPG transfected into CEFs expressed bioactive OPG protein that was able to inhibit osteoclast function.

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Figures

Figure 1
Figure 1
Gel electrophoresis of chOPG. 1A: Gel electrophoresis of reverse transcription polymerase chain reaction (RT-PCR) product. Total RNA extracted from chicken embryo frontal bone was analyzed using RT-PCR with specific primers. About 1.2 kbp gene of chicken osteoprotegerin (chOPG) was amplified (lane 1); DL2000 marker (lane 2); 1B: Gel electrophoresis of pcDNA3.1 (+)/chOPG PCR product. chOPG fragment was inserted into the eucaryon expression vector pcDNA3.1 (+) between Nhe| and Xho|. Negative plasmid (lane 1) and positive plasmid (lane 2) were chosen using PCR; marker (lane 3); 1C: Gel electrophoresis of pcDNA3.1 (+)/chOPG double restriction enzyme assay. Positive plasmid (lane 3) was identified by Nhe|and Xho|double restriction digestion and showed pcDNA3.1 (+) and OPG (lane 2); marker (lane 1); 1D: Gel electrophoresis of RT-PCR analysis showing the expression of chOPG gene at 48 h. Amplification of chOPG using cDNA from lane 1 (control group) and lane 2 (pcDNA3.1 (+) transfected CEFs group) showing negative result. Amplification of chOPG using cDNA from lane 4 (pcDNA3.1 (+)/chOPG transfected chicken embryo fibroblasts group) showing about 1200 bp gene of chOPG. Lane 3: marker
Figure 2
Figure 2
The expression of chOPG protein and effect on osteoclast morphology, apoptosis and resorption. 2A: Immunofluorescence assay for a possible chicken osteoprotegerin (chOPG) protein. Chicken embryo fibroblasts (CEFs) were grown on coverslips, fixed, and examined by indirect immunofluorescence. Cells were incubated with rabbit anti-chOPG serum. The secondary antibody was fluorescein-conjugated goat anti-rabbit immunoglobulin G (green). The nuclei of the corresponding cells were visualized by DAPI staining (blue). Fluorescence signals were analyzed by Fluoview microscopy (×200). Negative results are shown on card l (control group) and card 2 (pcDNA3.1 (+) transfected CEFs group), positive green fluorescence for CEFs are shown on card 3 (pcDNA3.1 (+)/chOPG transfected CEFs group). 2B: The morphology of osteoclasts was observed by inverted phase contrast microscope (×200). The adherent osteoclasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing supernatant of control group (l), pcDNA3.1 (+) transfected CEF group (2) and pcDNA3.1 (+)/chOPG transfected CEF group (3) for 5 d. 2C: Effect of the supernatant of three groups on the apoptosis of osteoclasts by flow cytometry. 2D: Toluidine blue staining of bone slices showing resorption lacunae (×200). The adherent osteoclasts were cultured in DMEM containing supernatant for 5 d in three groups.
Figure 3
Figure 3
The change of TRAP enzyme activity and concentration of Ca2+ in three groups. 3A: Effect of culture supernatant from chicken embryo fibroblasts transfected on osteoclastic TRAP enzyme activity (Mean ± SD; n = 8). ** indicates P < 0.01 compared with the control group. 3B: The concentration of Ca2+ in the supernatant containing bovine bone slices (Mean ± SD; n = 8). ** indicates P < 0.01 compared with the control group.
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