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Egg White Ovotransferrin Attenuates RANKL-Induced Osteoclastogenesis and Bone Resorption

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Egg White Ovotransferrin Attenuates RANKL-Induced Osteoclastogenesis and Bone Resorption

Nan Shang et al. Nutrients.

Abstract

Ovotransferrin, a member of the transferrin family, is the second main protein found in egg white. Ovotransferrin was reported to have antimicrobial, antioxidant, and immunomodulating activities. The aim of this work was to characterize the cellular and molecular functions of egg white ovotransferrin on osteoclasts differentiation and function. Osteoclasts were prepared from mouse macrophage RAW 264.7 cells stimulated with receptor activator of nuclear factor κB ligand (RANKL). Ovotransferrin inhibited osteoclasts differentiation and the calcium-phosphate resorptive ability via the suppression of RANKL-induced nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Ovotransferrin induced apoptosis of matured osteoclasts, accompanied by increased expression of Bcl-2-like protein 11 (Bim) and Bcl-2-assoicated death promoter (Bad), but decreased expression of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra-large (Bcl-xl). We established a novel role of egg white ovotransferrin as an inhibitor of osteoclastogenesis, which may be used for the prevention of osteoporosis.

Keywords: MAPK pathway; NF-κB pathway; apoptosis; osteoclastogenesis; osteoclasts.

Conflict of interest statement

The authors have no conflicts to declare.

Figures

Figure 1
Figure 1
Effect of ovotransferrin on RANKL-induced osteoclastogenesis. (A) RAW 264.7 cells were incubated with DMEM containing RANKL (100 ng/mL) and different concentrations of ovotransferrin (OVT) for 6 days before the TRAP staining was performed. All images were captured under 10× magnification. (B) TRAP+ multinucleated cells with at least three nuclei were identified and counted as mature osteoclasts. The results are expressed as means ± SEM with representative of 6 independent experiments. Means with different letter indicate p < 0.05.
Figure 2
Figure 2
Effect of ovotransferrin on RANKL-induced NF-κB activation. RAW 264.7 cells were pretreated with ovotransferrin (1–1000 μg/mL) for 30 min prior to stimulation with RANKL (100 ng/mL). Then, RAW 264.7 cells were treated with both RANKL and ovotransferrin for 4 h. Whole cell lysates were used for Western blot analysis of (A) p-NF-κB p65 (ser 536)/NF-κB p65, (B) p-IκB (ser 32)/IκB, and (C) p-IKKα/β (ser 176/180)/IKKα+IKKβ. (D) All bands are shown. The results are expressed as means ± SEM representative of 4 independent experiments. Means with different letters indicate p < 0.05.
Figure 3
Figure 3
Effect of ovotransferrin on RANKL-induced MAPK activation. RAW 264.7 cells were pretreated with ovotransferrin (1–1000 μg/mL) for 30 min prior to stimulation with RANKL (100 ng/mL). Then, RAW 264.7 cells were treated with both RANKL and ovotransferrin for 4 h. Whole cell lysates were used for Western blot analysis of (A) p-p44/42 Erk1/2 (Thr 202/Tyr 204)/p44/42 Erk1/2, (B) p-p38 (Thr 180/Tyr182)/p38, and (C) p-JNK (Thr 183/Tyr 185)/JNK. (D) All bands are shown. The results are expressed as means ± SEM representative of 4 independent experiments. Means with different letters indicate p < 0.05.
Figure 4
Figure 4
Effect of ovotransferrin on RANKL-induced expression of proteins involved in osteoclastogenesis. RAW 264.7 cells were pretreated with ovotransferrin (1–1000 μg/mL) for 2 h prior to stimulation with RANKL (100 ng/mL). Then, RAW 264.7 cells were co-cultured with RANKL and ovotransferrin for 12 h. Whole cell lysates were used for Western blot analysis of (A) TRAF6, (B) cFos, (C) NFATc1, and (D) cathepsin K. (E) All bands are shown. The results are expressed as means ± SEM representative of 4 independent experiments. Means with different letters indicate p < 0.05.
Figure 5
Figure 5
Effect of ovotransferrin on RANKL-induced bone resorption in vitro. RAW 264.7 was cultured onto fluoresceinated calcium phosphate (CaP)-coated plate and stimulated with RANKL (100 ng/mL) for 6 days. After 1 day for adhesion, cells were treated with ovotransferrin (1–1000 μg/mL) and RANKL together for 6 days. (A) The fluorescence intensity of resorbed CaP during 6-day treatment. * p < 0.05. (B) The total resorbed CaP after 6 days. (C) The total area of resorption pits after 6 days. (D) Image of resorption area. All images were captured under 10× magnification. The results are expressed as means ± SEM representative of 4 independent experiments. Means with different letters indicate p < 0.05.
Figure 6
Figure 6
Effect of ovotransferrin on RANKL-induced osteoclasts apoptosis. Osteoclasts were generated by stimulating RAW 264.7 with RANKL (100 ng/mL) for 4–6 days and multinucleated cells were identified by microscopy. Then cells were treated with ovotransferrin (1–1000 μg/mL) for 12 h. (A) Osteoclasts apoptosis percentage. Means with different letters indicate p < 0.05. (B) Osteoclasts apoptosis measured by flow cytometry. The lower left quadrant: PI-/V-FITC-; upper left quadrant: PI+/V-FITC-; lower right quadrant: PI-/V-FITC+; upper right quadrant: PI+/V-FITC+. (C) The gene expression of the Bcl-2 family (Bcl-2, Bcl-xl, Bim, and Bid). The results are expressed as means ± SEM representative of 4 independent experiments. * p < 0.05; ** p < 0.01.
Figure 7
Figure 7
The mechanism of ovotransferrin attenuating RANKL-induced bone resorption. The mature osteoclast differentiates from its precursor and is responsible for bone resorption. OVT inhibits the activation of NF-κB and MAPK pathways during the differentiation, and then down-regulates several proteins (c-Fos, NFATc1, and cathepsin K) involved in osteoclastogenesis. OVT stimulates cell apoptosis in mature osteoclasts via regulating the expression of Bcl-2 family members. Based on this, OVT may have the ability to protect against bone loss.

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