ATF1/miR-214-5p/ITGA7 axis promotes osteoclastogenesis to alter OVX-induced bone absorption

Abstract

Background: The dynamic balance of osteoblast and osteoclast is critical for bone homeostasis and overactive osteoclastic function may lead to osteoporosis. Activating transcription factor 1 (ATF1) is involved in osteoclastogenesis. However, the detailed mechanisms remain to be explored.

Methods: RAW264.7 cells were used and induced toward osteoclast by RANKL administration. We performed flow cytometry, CCK-8 assay and tartrate-resistant acid phosphatase (TRAP) staining to examine cell apoptosis, proliferation and differentiation of RAW264.7 cells, respectively. Mice were subjected to ovariectomy to induce osteoporosis. Micro CT, HE staining and TRAP staining were performed to evaluate bone loss in the OVX mouse model. Bioinformatics methods, luciferase assays and Chromatin Immunoprecipitation (ChIP) were used to predict and validate the interaction among ATF1, miR-214-5p, and ITGA7.

Results: ATF1 and miR-214-5p were up-regulated while ITGA7 was inhibited in RANKL-induced osteoclasts. MiR-214-5p was transcriptionally activated by ATF1. ATF1 knockdown suppressed osteoclast formation by miR-214-5p inhibition. ITGA7 was the direct target of miR-214-5p. Knockdown of miR-214-5p abolished osteoclastogenesis, which was reversed by ITGA7 knockdown. In OVX model, miR-214-5p knockdown suppressed osteoclast differentiation and prevented bone loss.

Conclusion: ATF1/miR-214-5p/ITGA7 axis regulated osteoclast formation both in vivo and in vitro, thereby affecting OVX-induced bone resorption in mice. Knockdown of ATF1 might be a promising strategy to manage osteoporosis.

Keywords: ATF1; ITGA7; Osteoclastogenesis; Osteoporosis; miR-214-5p.

Fig. 1

ATF1, miR-214-5p are upregulated and ITGA7 is downregulated in RANKL-induced osteoclast. A Expression of osteoclast markers examined by western blotting. B TRAP staining results in control and RANKL treatment group. C Expression of ATF1, miR-214-5p and ITGA7 examined by qRT-PCR. D Protein expression of ITGA7 examined by western blotting. Experiments were performed for at least 3 times. Data are shown as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 2

MiR-214-5p is transcriptionally activated by ATF1. Knockdown or overexpression of ATF1 were performed in RAW264.7 cells, ATF1 (A) and miR-214-5p (B) expression in each group were detected via qRT-PCR. C Two predicted binding sites by JASPAR. D ATF1 and miR-214-5p promoter binding site mutation sequence. E, F Luciferase reporter assay in cells transfected with a plasmid containing miR-214-5p-WT, Site 1-MUT and Site 2-MUT. Sh-ATF1 and ATF1 overexpression vector were administrated, respectively. G ChIP assays was conducted in RAW264.7 cells. qRT-PCR was utilized to examine miR-214-5p promoter expression associated with ATF1. Experiments were performed for at least 3 times. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 3

ATF1 inhibition abolishes osteoclast formation, which is reversed by miR-214-5p upregulation. RAW264.7 cells were administrated with sh ATF1/sh NC with or without miR-214-5p mimic/mimic NC. A Expression of ATF1, miR-214-5p and ITGA7 was detected by qRT-PCR. B Protein level of ITGA7 was detected by western blotting. C Cell viability was evaluated by CCK-8 assay. D Cell apoptosis was assessed by flow cytometry. E Expression of osteoclast markers was evaluated by western blotting. F TRAP staining results in each group. Experiments were performed for at least 3 times. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 4

MiR-214-5p directly interacts with ITGA7. Cells were treated with miR-214-5p mimic/mimic NC or inhibitor NC/ miR-214-5p inhibitor. A Expression of miR-214-5p and ITGA7 after different treatment was detected by qRT-PCR. B Protein level of ITGA7 was detected by western blotting. C The possible binding site of miR-214-5p in the ITGA7 3′-UTR. D Luciferase reporter assay in RAW264.7 cells transfected with a plasmid containing ITGA7 WT, ITGA7 MUT, miR-214-5p mimic/mimic NC. Experiments were performed for at least 3 times. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 5

MiR-214-5p downregulation inhibits osteoclastogenesis and ITGA7 knockdown reverses this trend. RAW264.7 cells were treated with miR-214-5p mimic/mimic NC with or without sh ITGA7/sh NC. A Expression of miR-214-5p and ITGA7 was examined by qRT-PCR. B Protein level of ITGA7 was examined by western blotting. C Cell viability was evaluated by CCK-8 assay in each group. D Cell apoptosis was assessed by flow cytometry. E Expression of osteoclast markers was evaluated by western blotting. F TRAP staining results in each group. Experiments were performed for at least 3 times. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 6

MiR-214-5p inhibition prevents OVX-induced osteoporosis. OVX or sham-operated mice were injected with miR-214-5p antagomir/antagomir NC. A Expression of miR-214-5p and ITGA7 was examined by qRT-PCR. B Protein level of ITGA7 was examined by western blotting. C Bone mineral density (BMD, g/cc), trabecular number (Tb.N, 1/mm) and trabecular thickness (Tb.Th, μm) of each sample were investigated by Micro CT. D Expression of osteoclast markers was evaluated by western blotting. E, F Bone tissue sections were exposed to H&E and TRAP staining. G, H Number of osteoclasts per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS) of mice obtained. N = 6. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001