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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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CD14 (A) and CD34 (B) immunohistochemistry of isolated bone marrow macrophages (BMMs).
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Figure 1: CD14 (A) and CD34 (B) immunohistochemistry of isolated bone marrow macrophages (BMMs).

Mentions: The immunohistochemical procedure and preparation of controls were carried out according to the manufacturing company’s standards and guidelines. The controls were used to assess the specificity of the reaction as follows: pre-incubation of the cells with the peptide in order to obtain the first antibody, incubation of the cells with an irrelevant primary antibody of the same isotype followed by the second antibody. The sections were incubated with the primary antibodies (CD14 (Santa Cruz, CA, USA, sc-73794) [1:250] and CD34 (Santa Cruz, sc-7045) [1:250]) for 30 minutes at room temperature, washed with TBS and incubated with link antibody for 15 minutes each. Then the sections were washed with TBS and incubated with labeled Streptavidin-biotin (LSAB) for 15 minutes at room temperature, washed again with TBS and incubated with diaminobenzidine (DAB) and substrate chromogen system for 5 minutes at room temperature which resulted in brown-coloured precipitates at the antigen site (Figures 1A-B).


Inhibition of osteoclastogenesis by RNA interference targeting RANK.

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

CD14 (A) and CD34 (B) immunohistochemistry of isolated bone marrow macrophages (BMMs).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: CD14 (A) and CD34 (B) immunohistochemistry of isolated bone marrow macrophages (BMMs).
Mentions: The immunohistochemical procedure and preparation of controls were carried out according to the manufacturing company’s standards and guidelines. The controls were used to assess the specificity of the reaction as follows: pre-incubation of the cells with the peptide in order to obtain the first antibody, incubation of the cells with an irrelevant primary antibody of the same isotype followed by the second antibody. The sections were incubated with the primary antibodies (CD14 (Santa Cruz, CA, USA, sc-73794) [1:250] and CD34 (Santa Cruz, sc-7045) [1:250]) for 30 minutes at room temperature, washed with TBS and incubated with link antibody for 15 minutes each. Then the sections were washed with TBS and incubated with labeled Streptavidin-biotin (LSAB) for 15 minutes at room temperature, washed again with TBS and incubated with diaminobenzidine (DAB) and substrate chromogen system for 5 minutes at room temperature which resulted in brown-coloured precipitates at the antigen site (Figures 1A-B).

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