Abstract
BackgroundOsteoprotegerin (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.MethodsThe 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.Results48 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.ConclusionConstructed pcDNA3.1 (+)/chOPG transfected into CEFs expressed bioactive OPG protein that was able to inhibit osteoclast function.
Highlights
Osteoprotegerin (OPG) has been reported to prevent bone resorption by inhibiting the formation, function, and survival of osteoclasts in a variety of animal models
Expression of chicken OPG (chOPG) in chicken embryo fibroblasts (CEFs) transfected with pcDNA3.1 (+)/chOPG reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that CEFs in the group with pcDNA3.1(+)/chOPG transfection expressed OPG mRNA, but there was no expression of OPG mRNA in the control group and pcDNA3.1(+) group (Figure 1D)
Immunofluorescence studies showed that chOPG protein was distributed in the cytoplasma and CEFs showing green fluorescence were observed in the pcDNA3.1 (+)/chOPG group, but were not present in the other groups (Figure 2A)
Summary
Osteoprotegerin (OPG) has been reported to prevent bone resorption by inhibiting the formation, function, and survival of osteoclasts in a variety of animal models. Osteoporosis in laying hens is a condition that involves a progressive loss of bone resulting in bone fragility and increased risk of fracture. Surveys of laying flocks in Europe have indicated that about 30% of the birds experience one or more bone fractures due to osteoporosis during their lifetime. Medullary bone, an extremely labile source for calcium that develops in specific bones of female birds at the onset of sexual maturity, provides a labile source of calcium for shell formation. Bone formation of osteoblasts switches from structural bone to medullary bone [2]. In the absence of structural bone formation, continued osteoclastic resorption of structural bone will result in a depletion of structural bone, leading to osteoporosis
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