OBJECTIVETo explore the effect of zoledronate (ZOL) on the osteoclast adhesion and expression of integrin α(v) and β3 in vitro.

METHODSMice RAW264.7 cells were used for osteoclast differentiation in vitro, and osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining and dentin resorption lacunae examination. The cells were then divided into 2 groups, the control group and ZOL treatment group (treated with 1 x 10(-6) mol · L(-1) ZOL for 2 d). The adhesion ability of osteoclasts and mRNA and the protein expressions of integrin α(v) and β3 were examined by crystal violet staining, real-time fluorescence quantitative polymerase chain reaction, Western blot analysis, and immunofluorescent chemistry.

RESULTSTRAP staining and dentin resorption lacunae examination revealed the formation of multi-nuclear osteoclasts. ZOL treatment significantly decreased the adhesion ability of osteoclasts (P < 0.01). In the ZOL-treated group, the mRNA levels of integrin α(v) and β3 were 0.66 ± 0.05 and 0.59 ± 0.08, respectively. In the control group, the mRNA levels of integrin α(v) and β3, were 1.01 ± 0.01 and 1.01 ± 0.02, respectively; these values were higher than those in the ZOL-treated group (P < 0.01). The protein level of integrin α(v) and β3 in the ZOL-treated group (31,934.84 ± 112.91 and 18,812.79 ± 194.13) was downregulated by approximately 39.19% and 40.17%, respectively, compared with those in the control group (52,517.81 ± 211.72 and 31,441.93 ± 456.87) (P < 0.01). Immunofluorescent examination showed that the fluorescent intensities of integrin α(v) and β3 in the ZOL-treated group (9.491 ± 0.748 and 4.744 ± 0.759) were also significantly decreased compared with those in the control group (15.159 ± 1.143 and 11.418 ± 1.095) (P < 0.01).

CONCLUSIONZOL significantly inhibits osteoclast adhesion and downregulates integrin α(v) and β3, expression, thus contributing to the ZOL-induced inhibition of osteoclast- mediated bone resorption.

Animals ; Bone Resorption ; Diphosphonates ; Gene Expression ; Imidazoles ; Integrin alphaV ; Mice ; Osteoclasts ; RNA, Messenger

OBJECTIVETo explore the effect of zoledronate (ZOL) on osteoclast differentiation and expressions of transient receptor potential vanilloid 5 channel (TRPV5) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1).

METHODSRAW264.7 cells were divided into two groups for treatment with RANKL for 5 days (group A) or with additional ZOL treatment in the last 2 days of RANKL treatment (group B). Osteoclastogenesis of the cells and the mRNA and protein expressions of TRPV5 and NFATc1 after the treatments were examined.

RESULTSIn group B, the number of newly generated osteoclasts (≥ 3 nuclei), number and size of dentin resorption lacunaes were 29.0 ± 2.4, 24.8 ± 1.1, and 2 030.0 ± 165.7 µm², respectively, which were significantly lower than those in group A (56.5 ± 4.5, 49.3 ± 0.9, and 3 946.7 ± 367.5 µm², respectively, P<0.01). Fluorescent intensity of TRPV5 and NFATc1 were also significantly decreased in group B (P<0.01). Compared with those in group A, TRPV5 mRNA and protein expressions in group B were down-regulated by 50.4% and 37.8%, and those of NFATc1 by 68.0% and 48.4%, respectively (P<0.01).

CONCLUSIONZOL can significantly inhibit osteoclastogenesis and bone resorption, which may be attributed, at least partly, to ZOL-induced inhibition of TRPV5 and NFATc1 expressions.

Animals ; Bone Resorption ; Calcium Channels ; metabolism ; Cell Differentiation ; drug effects ; Cell Line ; Diphosphonates ; pharmacology ; Down-Regulation ; Imidazoles ; pharmacology ; Mice ; NFATC Transcription Factors ; metabolism ; Osteoclasts ; drug effects ; RANK Ligand ; pharmacology ; RNA, Messenger ; TRPV Cation Channels ; metabolism

Objective To explore the effect of zoledronate (ZOL) on the osteoclast adhesion and expression of integrin αv and β3 in vitro. Methods Mice RAW264.7 cells were used for osteoclast differentiation in vitro, and osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining and dentin resorption lacunae examination. The cells were then divided into 2 groups, the control group and ZOL treatment group (treated with 1×10-6 mol·L-1 ZOL for 2 d). The adhesion ability of osteoclasts and mRNA and the protein expressions of integrin αv and β3 were examined by crystal violet staining, real-time fluorescence quantitative polymerase chain reaction, Western blot analysis, and immunofluorescent chemistry. Results TRAP staining and dentin resorption lacunae examination revealed the formation of multi-nuclear osteoclasts. ZOL treatment significantly decreased the adhesion ability of osteoclasts (P<0.01). In the ZOL-treated group, the mRNA levels of integrin αv and β3 were 0.66±0.05 and 0.59±0.08, respectively. In the control group, the mRNA levels of integrin αv and β3 were 1.01±0.01 and 1.01±0.02, respectively; these values were higher than those in the ZOL-treated group (P<0.01). The protein level of integrin αv and β3 in the ZOL-treated group (31 934.84±112.91 and 18 812.79±194.13) was downregulated by approximately 39.19% and 40.17%, respectively, compared with those in the control group (52 517.81± 211.72 and 31 441.93±456.87) (P<0.01). Immunofluorescent examination showed that the fluorescent intensities of integrin αv and β3 in the ZOL-treated group (9.491±0.748 and 4.744±0.759) were also significantly decreased compared with those in the control group (15.159±1.143 and 11.418±1.095) (P< 0.01). Conclusion ZOL significantly inhibits osteoclast adhesion and downregulates integrin αv and β3 expression, thus contributing to the ZOL-induced inhibition of osteoclastmediated bone resorption.