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Cilengitide inhibits osteoclast adhesion through blocking the αvβ3-mediated FAK/Src signaling pathway - PubMed

  • ️Sun Jan 01 2023

Cilengitide inhibits osteoclast adhesion through blocking the αvβ3-mediated FAK/Src signaling pathway

Dan-Yang Guo et al. Heliyon. 2023.

Erratum in

Abstract

The remodeling of actin cytoskeleton of osteoclasts on the bone matrix is essential for osteoclastic resorption activity. A specific regulator of the osteoclast cytoskeleton, integrin αvβ3, is known to provide a key role in the degradation of mineralized bone matrixes. Cilengitide is a potent inhibitor of integrins and is capable of affecting αvβ3 receptors, and has anti-tumor and anti-angiogenic and apoptosis-inducing effects. However, its function on osteoclasts is not fully understood. Here, the cilengitide role on nuclear factor κB ligand-receptor activator (RANKL)-induced osteoclasts was explored. Cells were cultured with varying concentrations of cilengitide (0,0.002,0.2 and 20 μM) for 7 days, followed by detected via Cell Counting Kit-8, staining for tartrate resistant acid phosphatase (TRAP), F-actin ring formation, bone resorption assays, adhesion assays, immunoblotting assays, and real-time fluorescent quantitative PCR. Results demonstrated that cilengitide effectively restrained the functionality and formation of osteoclasts in a concentration-dependent manner, without causing any cytotoxic effects. Mechanistically, cilengitide inhibited osteoclast-relevant genes expression; meanwhile, cilengitide downregulated the expression of key signaling molecules associated with the osteoclast cytoskeleton, including focal adhesion kinase (FAK), integrin αvβ3 and c-Src. Therefore, this results have confirmed that cilengitide regulates osteoclast activity by blocking the integrin αvβ3 signal pathway resulting in diminished adhesion and bone resorption of osteoclasts.

Keywords: Adhesion; Cilengitide; FAK/Src signaling; Integrin αvβ3; Osteoclast.

© 2023 The Authors.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1

Cilengitide inhibits RANKL-induced osteoclastogenesis in vitro. (A) Chemical structure of cilengitide. (B) Cytotoxic effects of cilengitide on BMMs at 24 h, 48 h, and 72 h using the CCK8 assay. (C) BMMs were treated with 30 ng/ml M-CSF and 100 ng/ml RANKL and various concentrations of cilengitide for 7 days, then stained for TRAP. Scale bar: 200 μm. (D–E) Number and area of TRAP+ osteoclasts, defined by ≥ 3 nuclei. The data are expressed as the means ± SD; ****p＜0.0001 relative to the RANKL-induced group.

Fig. 2
Fig. 2

Cilengitide disrupts osteoclast activation but did not affect the early stages of osteoclastogenesis. (A) Schematic diagram of osteoclast formation. (B) BMMs were treated with 20 μM cilengitide for the indicated times and were stained for TRAP. Scale bar: 200 μm. (C) Schematic diagram of BMMs processed with cilengitide at different treatment periods. (D) Number of TRAP+ osteoclasts with ≥3 nuclei. The data are expressed as the means ± SD; ***p＜0.001 relative to the RANKL-induced group.

Fig. 3
Fig. 3

Cilengitide blocks F-actin ring formation and bone-resorption of osteoclasts in vitro. (A) BMMs were treated with different concentrations of cilengitide (0, 0.002, 0.2, and 20 μM) for 7 days and stained for F-actin rings and DAPI staining. Scale bar: 100 μm. (B) Quantification of F-actin ring area per well. (C) Representative images of bone resorption by osteoclasts treated with different concentrations of cilengtide on bovine bone fragments. Scale bar:100 μm. (D) Quantification of bone resorption area per well. The data are expressed as the means ± SD; ***p < 0.001, ****p < 0.0001 relative to the RANKL-induced group.

Fig. 4
Fig. 4

Cilengitide inhibits cell adhesion and the expression of osteoclast activation related markers in osteoclasts. (A–B) BMMs were seeded into 96-well plates coated with osteopontin (0.5 μg/ml) and PDL (50 μg/ml), respectively, in the presence of different concentrations of cilengitide (0, 0.002, 2, and 20 μM). Cell adhesion was quantified by crystalline violet staining and enzymatic labeling. (C–F) The expression of the osteoclast-related genes NFATc1, CTSK, and integrin genes (αv, β3) were analyzed by real-time PCR, and their expression levels were normalized to that of GAPDH. (G) Representative immunoblot images of integrin αvβ3, NFATc1, CTSK and GAPDH. (H–J) Analysis of the intensity of differentially expressed protein bands at different concentrations of cilengitide. The data are expressed as the means ± SD; **p < 0.01,***p＜0.001, ****p＜0.0001 relative to the RANKL-induced group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5
Fig. 5

Cilengitide inhibits FAK/Src signaling. (A) Representative immunoblot images of FAK, p-FAK, c-Src, p-Src and β-actin. (B–C) Analysis of the intensity of differentially expressed protein bands in the different groups. The data are expressed as the means ± SD; **p＜0.01 relative to the RANKL-induced group.

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