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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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Related in: MedlinePlus

RANK silencing in BMMs by stable transduction by retroviruses. BMMs stably transduced with shRNA against the RANK were analyzed for RANK protein levels by Western blot (A, B); β-actin served as a loading control.
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Figure 4: RANK silencing in BMMs by stable transduction by retroviruses. BMMs stably transduced with shRNA against the RANK were analyzed for RANK protein levels by Western blot (A, B); β-actin served as a loading control.

Mentions: Subsequently, the effect of shRANK-3 on infected BMMs was studied to determine the biological influence of RANK on osteoclastogenesis in BMMs. Western blot was used to evaluate inhibition of RANK expression in stable cells. Expression was reduced by about 80.7% (p < 0.01), as compared with uninfected BMM cells (Figures 4). Meanwhile, the levels of RANK protein were significantly reduced in the 3rd-passage cells, which were used in all experiments in this study. The expression of GFP was continuously monitored with fluorescence microscopy to test whether the cells stably express GFP-retro-puro retrovirus. The 10th-passage cells were detected to stably express GFP protein.


Inhibition of osteoclastogenesis by RNA interference targeting RANK.

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

RANK silencing in BMMs by stable transduction by retroviruses. BMMs stably transduced with shRNA against the RANK were analyzed for RANK protein levels by Western blot (A, B); β-actin served as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: RANK silencing in BMMs by stable transduction by retroviruses. BMMs stably transduced with shRNA against the RANK were analyzed for RANK protein levels by Western blot (A, B); β-actin served as a loading control.
Mentions: Subsequently, the effect of shRANK-3 on infected BMMs was studied to determine the biological influence of RANK on osteoclastogenesis in BMMs. Western blot was used to evaluate inhibition of RANK expression in stable cells. Expression was reduced by about 80.7% (p < 0.01), as compared with uninfected BMM cells (Figures 4). Meanwhile, the levels of RANK protein were significantly reduced in the 3rd-passage cells, which were used in all experiments in this study. The expression of GFP was continuously monitored with fluorescence microscopy to test whether the cells stably express GFP-retro-puro retrovirus. The 10th-passage cells were detected to stably express GFP protein.

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