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Inhibition of osteoclastogenesis by RNA interference targeting RANK.

Ma R, Xu J, Dong B, Kauther MD, Jäger M, Wedemeyer C - BMC Musculoskelet Disord (2012)

Bottom Line: Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK) ligand (RANKL) as well as the macrophage colony-stimulating factor (M-CSF).The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines.The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedics, University of Duisburg-Essen, Essen, Germany.

ABSTRACT

Background: Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK) ligand (RANKL) as well as the macrophage colony-stimulating factor (M-CSF). The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption.

Methods: Three pairs of short hairpin RNAs (shRNA) targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs) using the optimal shRNA by targeting RANK.

Results: Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p < 0.01). shRANK-3 also brought about a marked inhibition of osteoclast formation and bone resorption as demonstrated by tartrate-resistant acid phosphatase (TRAP) staining and osteoclast resorption assay. The results of our study show that retrovirus-mediated shRANK-3 suppresses osteoclast differentiation and osteolysis of BMMs.

Conclusions: These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.

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Inhibition of osteoclastogenesis by RANK silencing. (A, B) Osteoclast differentiation of BMMs as identified by TRAP staining. (C) Quantification of the data. The multinucleated TRAP-positive cells (>3 nuclei) in eight random view areas (at 200× magnification) in each of the five replicates were counted. Osteoclast resorption was quantified using toluidine blue staining (at 400× magnification) (D,E,F). Comparison of the resorption area of bone slices between untreated and treated BMM cells was evaluated by Scanning Electron Microscopy (at 400× magnification) (G, H).
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Figure 5: Inhibition of osteoclastogenesis by RANK silencing. (A, B) Osteoclast differentiation of BMMs as identified by TRAP staining. (C) Quantification of the data. The multinucleated TRAP-positive cells (>3 nuclei) in eight random view areas (at 200× magnification) in each of the five replicates were counted. Osteoclast resorption was quantified using toluidine blue staining (at 400× magnification) (D,E,F). Comparison of the resorption area of bone slices between untreated and treated BMM cells was evaluated by Scanning Electron Microscopy (at 400× magnification) (G, H).

Mentions: We performed functional assays to determine the effect of RANK inhibition on osteoclast differentiation and osteolysis. Infected BMM cells or BMM cells were treated with RANKL (100 ng/ml) and M-CSF (30 ng/ml) for an additional nine days and subjected to TRAP staining and osteoclast resorption assay. TRAP+ cells with more than three nuclei were counted and the area of osteoclast resorption was measured with a scanning electron microscope (SEM). We noted a reduction in the number of TRAP+ multinucleated cells in infected BMMs (6.8 ± 1.64) compared with BMMs (82 ± 4.64) (p < 0.05) (Figures 5 A-C). Furthermore, a significant reduction in resorption areas on the bone slices (Figures 5 D-F) in infected BMMs (16.6% ± 2.70%) compared with BMMs (2.8% ± 0.84%) (p < 0.05) was observed, indicating profound inhibition of osteoclastic bone resorption. Meanwhile, the resorption area of bone slices between untreated and treated BMM cells was evaluated by Scanning Electron Microscopy (Figures 5 G, H). The difference is obvious.


Inhibition of osteoclastogenesis by RNA interference targeting RANK.

Ma R, Xu J, Dong B, Kauther MD, Jäger M, Wedemeyer C - BMC Musculoskelet Disord (2012)

Inhibition of osteoclastogenesis by RANK silencing. (A, B) Osteoclast differentiation of BMMs as identified by TRAP staining. (C) Quantification of the data. The multinucleated TRAP-positive cells (>3 nuclei) in eight random view areas (at 200× magnification) in each of the five replicates were counted. Osteoclast resorption was quantified using toluidine blue staining (at 400× magnification) (D,E,F). Comparison of the resorption area of bone slices between untreated and treated BMM cells was evaluated by Scanning Electron Microscopy (at 400× magnification) (G, H).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475138&req=5

Figure 5: Inhibition of osteoclastogenesis by RANK silencing. (A, B) Osteoclast differentiation of BMMs as identified by TRAP staining. (C) Quantification of the data. The multinucleated TRAP-positive cells (>3 nuclei) in eight random view areas (at 200× magnification) in each of the five replicates were counted. Osteoclast resorption was quantified using toluidine blue staining (at 400× magnification) (D,E,F). Comparison of the resorption area of bone slices between untreated and treated BMM cells was evaluated by Scanning Electron Microscopy (at 400× magnification) (G, H).
Mentions: We performed functional assays to determine the effect of RANK inhibition on osteoclast differentiation and osteolysis. Infected BMM cells or BMM cells were treated with RANKL (100 ng/ml) and M-CSF (30 ng/ml) for an additional nine days and subjected to TRAP staining and osteoclast resorption assay. TRAP+ cells with more than three nuclei were counted and the area of osteoclast resorption was measured with a scanning electron microscope (SEM). We noted a reduction in the number of TRAP+ multinucleated cells in infected BMMs (6.8 ± 1.64) compared with BMMs (82 ± 4.64) (p < 0.05) (Figures 5 A-C). Furthermore, a significant reduction in resorption areas on the bone slices (Figures 5 D-F) in infected BMMs (16.6% ± 2.70%) compared with BMMs (2.8% ± 0.84%) (p < 0.05) was observed, indicating profound inhibition of osteoclastic bone resorption. Meanwhile, the resorption area of bone slices between untreated and treated BMM cells was evaluated by Scanning Electron Microscopy (Figures 5 G, H). The difference is obvious.

Bottom Line: Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK) ligand (RANKL) as well as the macrophage colony-stimulating factor (M-CSF).The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines.The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedics, University of Duisburg-Essen, Essen, Germany.

ABSTRACT

Background: Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK) ligand (RANKL) as well as the macrophage colony-stimulating factor (M-CSF). The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption.

Methods: Three pairs of short hairpin RNAs (shRNA) targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs) using the optimal shRNA by targeting RANK.

Results: Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p < 0.01). shRANK-3 also brought about a marked inhibition of osteoclast formation and bone resorption as demonstrated by tartrate-resistant acid phosphatase (TRAP) staining and osteoclast resorption assay. The results of our study show that retrovirus-mediated shRANK-3 suppresses osteoclast differentiation and osteolysis of BMMs.

Conclusions: These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.

Show MeSH
Related in: MedlinePlus