Regulator of G protein signalling 18 promotes osteocyte proliferation by activating the extracellular signal‑regulated kinase signalling pathway

Abstract

Osteocyte function is critical for metabolism, remodelling and regeneration of bone tissue. In the present study, the roles of regulator of G protein signalling 18 (RGS18) were assessed in the regulation of osteocyte proliferation and bone formation. Target genes and signalling pathways were screened using the Gene Expression Omnibus (GEO) database and Gene Set Enrichment Analysis (GSEA). The function of RGS18 and the associated mechanisms were analysed by Cell Counting Kit 8 assay, 5‑ethynyl‑2'‑deoxyuridine assay, flow cytometry, reverse transcription‑quantitative PCR, western blotting and immunostaining. Overlap analysis of acutely injured subjects (AIS) and healthy volunteers (HVs) from the GSE93138 and GSE93215 datasets of the GEO database identified four genes: KIAA0825, ANXA3, RGS18 and LIPN. Notably, RGS18 was more highly expressed in peripheral blood samples from AIS than in those from HVs. Furthermore, RGS18 overexpression promoted MLO‑Y4 and MC3T3‑E1 cell viability, proliferation and S‑phase arrest, but inhibited apoptosis by suppressing caspase‑3/9 cleavage. Silencing RGS18 exerted the opposite effects. GSEA of GSE93138 revealed that RGS18 has the ability to regulate MAPK signalling. Treatment with the MEK1/2 inhibitor PD98059 reversed the RGS18 overexpression‑induced osteocyte proliferation, and treatment with the ERK1/2 activator 12‑O‑tetradecanoylphorbol‑13‑acetate reversed the effects of RGS18 silencing on osteocyte proliferation. In conclusion, RGS18 may contribute to osteocyte proliferation and bone fracture healing via activation of ERK signalling.

Keywords: bone fracture; extracellular signal‑regulated kinase signalling; osteocytes; proliferation; regulator of G protein signalling 18.

Figure 1

RGS18 expression is increased in samples from AIS. (A) Screening of AIS-related genes using the Gene Expression Omnibus database. (B) RGS18 was upregulated in samples from AIS compared with those from HVs from the GSE93138 dataset. (C) Reverse transcription-quantitative PCR evaluation of RGS18 in collected peripheral blood samples. ^*P<0.05. AIS, acutely injured subjects; HV, healthy volunteer; RGS18, regulator of G protein signalling 18.

Figure 2

RGS18 promotes the proliferation of osteocytes. (A) Western blotting and (B) reverse transcription-quantitative PCR were performed to detect the expression levels of RGS18 in MLO-Y4 and MC3T3-E1 cells after overexpression or knockdown of RGS18. Viability of (C) MLO-Y4 and (D) MC3T3-E1 cells after overexpression or knockdown of RGS18, as determined by Cell Counting Kit 8 assay. Proliferation of (E) MLO-Y4 and (F) MC3T3-E1 cells after overexpression or knockdown of RGS18, as determined by EdU staining (magnification, ×200). ^*P<0.05. EdU, 5-ethynyl-2′-deoxyuridine; NC, negative control; OD, optical density; RGS18, regulator of G protein signalling 18; si, small interfering.

Figure 3

RGS18 promotes the cell cycle progression of osteocytes. Flow cytometry was performed to detect cell cycle progression of (A) MLO-Y4 and (B) MC3T3-E1 cells after overexpression or knockdown of RGS18. Western blotting was conducted to detect the expression levels of cell cycle-associated proteins in (C) MLO-Y4 and (D) MC3T3-E1 cells after overexpression or knockdown of RGS18. ns, not significant; ^*P<0.05. NC, negative control; RGS18, regulator of G protein signalling 18; si, small interfering.

Figure 4

RGS18 suppresses apoptosis of osteocytes. Flow cytometry was performed to detect the proportions of apoptotic and necrotic (A) MLO-Y4 and (B) MC3T3-E1 cells after overexpression or knockdown of RGS18. Western blotting was conducted to detect the expression levels of apoptotic proteins in (C) MLO-Y4 and (D) MC3T3-E1 cells after overexpression or knockdown of RGS18. ^*P<0.05. NC, negative control; RGS18, regulator of G protein signalling 18; si, small interfering.

Figure 5

RGS18 stimulates MAPK signalling. (A) Gene Set Enrichment Analysis of RGS18-activated signalling pathways. Expression levels of ERK signalling pathway-related proteins in (B) MLO-Y4 and (C) MC3T3-E1 cells after overexpression or knockdown of RGS18 were detected by western blotting. Quantitative analysis results of the expression of ERK signalling pathway-related proteins in (D) MLO-Y4 and (E) MC3T3-E1 cells. Immunofluorescence staining of p-ERK in (F) MLO-Y4 and (G) MC3T3-E1 cells (magnification, ×400). ^*P<0.05. ERK, extracellular signal-regulated kinas; NC, negative control; ns, not significant; p-, phosphorylated; RGS18, regulator of G protein signalling 18; si, small interfering.

Figure 6

RGS18 promotes the proliferation of osteocytes through ERK signalling. MC3T3-E1 cells with RGS18 overexpression were treated with or without the ERK inhibitor PD98059 (20 μM). (A) Proliferation of MC3T3-E1 cells was determined by EdU staining (magnification, ×200). (B) Cell cycle progression, and (C) apoptosis and necrosis of MC3T3-E1 cells were detected by flow cytometry. ^*P<0.05. EdU, 5-ethynyl-2′-deoxyuridine; ERK, extracellular signal-regulated kinas; RGS18, regulator of G protein signalling 18.

Figure 7

ERK activation reverses RGS18 knockdown-induced osteocyte death. MC3T3-E1 cells with RGS18 knockdown were treated with or without ERK activator TPA (200 nM). (A) Proliferation of MC3T3-E1 cells was determined by EdU staining (magnification, ×200). (B) Cell cycle progression, and (C) apoptosis and necrosis of MC3T3-E1 cells were detected by flow cytometry. ^*P<0.05. EdU, 5-ethynyl-2′-deoxyuridine; ERK, extracellular signal-regulated kinas; RGS18, regulator of G protein signalling 18; si, small interfering; TPA, 12-O-tetradecanoylphorbol-13-acetate.

Figure 8

A schematic diagram showing the findings of the present study. RGS18 contributed to the proliferation of osteocytes and bone fracture healing through activating ERK signalling. ERK, extracellular signal-regulated kinas; RGS18, regulator of G protein signalling 18.