Apoptosis repressor with caspase recruitment domain enhances survival and promotes osteogenic differentiation of human osteoblast cells under Zoledronate treatment

Abstract

Zoledronate is one of the most potent nitrogen-containing bisphosphonates which has been demonstrated to result in osteoblast apoptosis and impact osteogenic differentiation in vitro. This effect of Zoledronate on osteoblasts may partially explain bisphosphonate‑associated osteonecrosis of the jaw, a serious complication associated with treatment with bisphosphonates. Apoptosis repressor with caspase recruitment domain (ARC) is a multifunctional inhibitor of apoptosis that is physiologically expressed predominantly in post‑mitotic cells such as cardiomyocytes, neurons and skeletal muscle cells. However, its effect on human osteoblasts remains unclear. The current study aimed to investigate the effects of ARC on human osteoblasts under the treatment of high concentrations of Zoledronate. ARC‑overexpressed human osteoblasts were established and were exposed to Zoledronate with different concentrations (0, 1 and 5 µM) in vitro. Cell numbers were detected using the MTT assay, and flow cytometry was used to identity cell apoptosis. Alkaline phosphatase staining, quantitative analysis and ectopic osteogenesis in nude mice were used to evaluate the osteogenic differentiation of ARC‑overexpressed osteoblasts. It was observed that ARC is able to reverse the inhibitory effect of Zoldronate on osteoblasts. ARC is additionally able to promote osteogenic differentiation of osteoblasts and inhibit their apoptosis. These observations suggest a critical role for ARC in the regulation of human osteoblasts under Zoledronate treatment.

Figure 1

ARC overexpression in human osteoblasts by stable transduction and it can enhance survival of human osteoblasts. (A) The fluorescence microscopy detection of enhanced green fluorescent protein-positive human osteoblasts on day 3 subsequent to lentiviral infection (magnification, ×100). (B) ARC mRNA and (C) protein expression levels were increased significantly in ARC-overexpressed cells compared with control cells. ^****P<0.0001 vs. control group. (D) Cell numbers [as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] were significantly higher in ARC-overexpressed human osteoblasts compared with empty vector-transfected controls under 0, 1 and 5 µM concentrations of Zoledronate. ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001 vs. empty vector-transfected control groups. ARC, apoptosis repressor with caspase recruitment domain; OB, osteoblast; con, control; nol, Zoledronate.

Figure 2

ARC inhibits apoptosis of human osteoblasts. (A) Cell apoptosis was detected by flow cytometry. One representative image from 3 independently performed experiments is presented. (B) Cell apoptosis was significantly reduced in ARC-overexpressed human osteoblasts compared with empty vector-transfected controls on day 3 under 0, 1 and 5 µM concentrations of Zoledronate treatment. ^*P<0.05, ^**P<0.01 vs. empty vector-transfected controls. (C) Western blot analysis identified reduced expression of cleaved-caspase-3 in ARC-overexpressed human osteoblasts compared with empty vector-transfected controls on day 7 under 0, 1 and 5 µM concentrations of Zoledronate treatment. ARC, apoptosis repressor with caspase recruitment domain; PI, propidium iodide; OB, osteoblast; con, control; nol, Zoledronate.

Figure 3

ARC promotes osteogenic differentiation of human osteoblasts. (A) ALP staining and (B) ALP activity of ARC-overexpressed human osteoblasts and empty vector-transfected controls at day 7. (C) ALP staining and (D) ALP activity of ARC-overexpressed human osteoblasts and empty vector-transfected controls at day 14. The ALP activity was significantly upregulated in ARC-overexpressed human osteoblasts compared with empty vector-transfected controls under 0, 1 and 5 µM concentrations of Zoledronate treatment. (E) Reverse transcription-quantitative polymerase chain reaction analysis of the expression of ALP, BSP, OCN and Runx2 at day 7. The expression levels were significantly increased in ARC-overexpressed human osteoblasts compared with empty vector-transfected controls under 0 and 1 µM concentrations of Zoledronate treatment. While in the 5 µM drug-treated groups, no significant differences between ARC-overexpressed human osteoblasts and empty vector-transfected controls were observed in the ALP, BSP and OCN expression levels. ^*P<0.05, ^**P<0.01 vs. empty vector-transfected controls. (F) Western blot analysis identified that increased expression of OCN in ARC-overexpressed human osteoblasts compared with empty vector-transfected controls on day 7 under 0, 1 and 5 µM concentrations of Zoledronate treatment. ARC, apoptosis repressor with caspase recruitment domain; ALP, alkaline phosphatase; BSP, bone sialoprotein; OCN, osteocalcin; Runx2, runt-related transcription factor 2; OB, osteoblast; con, control; nol, Zoledronate.

Figure 4

(A) Histological observations at 10 weeks subsequent to implantation (magnification, ×100). (a) 0 µM Zoledronate CPC/OB-nol3; (b) 0 µM Zoledronate CPC/OB-con; (c) 1 µM Zoledronate CPC/OB-nol3; (d) 1 µM Zoledronate CPC/OB-con. Newly formed bone was observed in the CPC/OB-nol3 groups (a and c) and the CPC/OB-con groups (b and d). (B) The percentages of new bone area were significantly increased in the CPC/OB-nol3 groups compared with the CPC/OB-con groups by histomorphometric analysis (^**P<0.01 vs. CPC/OB-con groups). CPC, calcium phosphate cement; OB, osteoblast; nol, Zoledronate; con, control.