Introduction
Bone fractures are common traumatic injuries of the bone cortex (1). Fracture healing is a regenerative process that recapitulates a number of the ontological events of embryonic skeletal development (1,2). Bone fracture healing is histologically defined in four steps: The initial inflammation-responsive phase within the first several days after fracture, the generation of surrounding soft callus and subsequent hard callus formation, followed by the osteoblast-induced formation of woven bone on the calcified matrix and recreation of the appropriate anatomical shape (2). The mechanisms involved in fracture healing are highly complex and are closely related to the interplay between cells, the extracellular matrix (ECM) and cytokines (1). It is well recognised that multiple cell types, such as chondrocytes, osteoblasts, endothelial cells, osteocytes and even mesenchymal stem cells, are involved in bone regeneration via the secretion of growth factors and the temporal expression of bone healing-related genes, such as bone sialoprotein and osteocalcin (3).
Osteocytes are the most common functional cell type involved in fracture healing (4,5). Osteocytes connect with each other and with other cell types, such as osteoblasts, bone marrow cells and periosteal cells, through an abundant dendritic network, which allows migration of signalling factors among the cells (4). Therefore, the position and status of osteocytes are critical for bone metabolism, remodelling and generation (6).
Extracellular signal-regulated kinase (ERK) is a critical regulator of animal development (7). ERK activation initiates a phosphorylation cascade within cells, affects gene expression and causes changes in cell phenotypes, such as proliferation, differentiation and motility (7). Oxidative stress causes decreased autophagy of osteocytes, during which inhibition of ERK signalling impairs autophagosome formation and promotes osteocyte cell death (8). In addition, activation of ERK signalling in osteocytes can cause changes in the cytoskeleton, remodelling of the ECM, and further alteration of tissue structure and bones (1).
The regulator of G protein signalling (RGS) family contains key cytosolic proteins that are capable of accelerating GTPase activity and regulating downstream signalling under various physiological conditions (9). For example, RGS18 is associated with platelet production and reactivity, and participates in the haemostatic response after injury (10). Plasma levels of RGS18 are a promising biomarker of gastric cancer (11). Furthermore, RGS18 suppresses osteoclastogenesis by negatively regulating OGR1/NFAT signalling in osteoclasts (12). The present study conducted a bioinformatics analysis of acutely injured subjects (AIS) and screened for elevated RGS18 expression. Gene Set Enrichment Analysis (GSEA) and subsequent experimental verification revealed that ERK signalling is involved in osteocyte proliferation and apoptosis. The present study provides a novel target for bone fracture healing.
Materials and methods
Bioinformatics analysis
The GSE93138 and GSE93215 datasets (13) in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) were selected for the present bioinformatics analysis. Differentially expressed genes were screened using the 'limma' package (14). In the two datasets, peripheral blood samples were obtained from acutely injured subjects (AIS) collected over multiple days, and were compared with those obtained from age- and sex-matched healthy volunteers (HVs). Microarrays were then performed to compare changes in gene expression between the AIS and HVs. The AIS were enrolled upon presentation for fracture care. To comprehensively analyse the basic functions and participating pathways of the differentially expressed genes, GSEA was performed using GSEA software (version 4.2.1; https://www.gsea-msigdb.org/gsea/index.jsp) with the c2.cp.kegg.v7.1.symbols.gmt gene set (https://www.gsea-msigdb.org/gsea/index.jsp) (15,16).
Specimen selection
All experiments were performed with the approval of the Ethics Committee of The Fifth Affiliated Hospital Jinan University (approval no. 2022-10.19.1.0; Heyuan, China). Peripheral blood samples were collected from AIS (<7 days after injury) (n=10). In total, samples were collected from five male patients and five female patients, with a median age of 43.4 years (range, 26-54 years). The inclusion criterion was patients aged between 18 and 55 years. The exclusion criteria were: Prior knee injury or surgery in either knee, posterior cruciate ligament injury, posterolateral corner injury, lateral collateral ligament injury, diabetes or other systemic diseases, and a history of inflammatory arthritis or gout. Peripheral blood samples were also collected from age- and sex-matched HVs (n=10), and were used as controls. The peripheral blood samples were collected between November 2022 and March 2023. The expression levels of RGS18 were determined by reverse transcription-quantitative PCR (RT-qPCR). All donors signed an informed consent form.
Cell culture
Mouse osteocyte MLO-Y4 cells (Procell Life Science & Technology Co., Ltd.) were cultured in α-minimal essential medium (α-MEM; Procell Life Science & Technology Co., Ltd.) containing 10% foetal bovine serum (FBS; Shanghai Basal Media Technologies Co., Ltd.) and 1% penicillin-streptomycin. Mouse osteoblast MC3T3-E1 cells (Nanjing Cobioer Biosciences Co., Ltd.) were cultured in α-MEM containing 20% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin-streptomycin. The cells were maintained at 37°C in an incubator containing 5% CO2 and 95% humidity. The ERK activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and MEK1/2 inhibitor PD98059 were purchased from MilliporeSigma.
RT-qPCR
Total RNA was extracted from the collected peripheral blood samples and MLO-Y4 and MC3T3-E1 cells using Trizol reagent (Beyotime Institute of Biotechnology). RNA then underwent cDNA synthesis using a Prime Script RT-PCR kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. TB Green Fast qPCR Mix (Takara Biotechnology Co., Ltd.) was used for qPCR. The following thermocycling conditions were used: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 10 sec. The expression levels of genes were normalised to GAPDH using the 2−ΔΔCq method (17). The primer sequences were designed and synthesised by Shanghai GenePharma Co., Ltd., as follows: Mouse RGS18, forward 5′-GGC CAA AGA AAC AAG ATG GAG T-3′, reverse 5′-ACA CTC TGC TTT GTG CCG TA-3′; mouse GAPDH, forward 5′-CAG GAG AGT GTT TCC TCG TCC-3′, reverse 5′-GAT GGG CTT CCC GTT GAT GA-3′; human RGS18, forward 5′-GCA GAG ACA GAA AGA AAC GCA G-3′, reverse 5′-CTC TTC AGG GGA GAC TCT TGT-3′; and human GAPDH, forward 5′-CCA TGT TGC AAC CGG GAA G-3′ and reverse 5′-GCC CAA TAC GAC CAA ATC AGA G-3′.
Cell transfection
Small interfering RNA (siRNA) targeting RGS18 (si-RGS18) and a plasmid overexpressing RGS18 (pcDNA-RGS18) were synthesised by Shanghai GenePharma Co., Ltd. Scrambled siRNA (si-NC) and empty pcDNA3.1 vector were used as negative controls, respectively. The siRNA sequences were as follows: si-RGS18, sense 5′-GGAGAGACUCAAGCCAGUAGA-3′, antisense 5′-UCUACUGGCUUGAGUCUCUCC-3′; and si-NC, sense 5′-GAGACAGGGCAGCCAAGUAUA-3′ and antisense 5′-UAUACUUGGCUGCCCUGUCUC-3′. For transfection, MLO-Y4 and MC3T3-E1cells were seeded in six-well plates at a density of 5×105 cells/well. A total of 5 μl Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and 2 μg vectors or 100 nM siRNAs was mixed in 100 μl Opti-MEM™ (Gibco; Thermo Fisher Scientific, Inc.) and added to each well. After 48 h of incubation at 37°C, the medium was replaced with fresh medium and the cells were cultured for another 24 h.
Cell counting kit 8 (CCK-8) assay
Post-transfection with si-RGS18 or pcDNA-RGS18, MLO-Y4 and MC3T3-E1 cells were seeded into 96-well plates at a density of 5,000 cells/well. After incubation for 12, 24, 48 and 72 h at 37°C, CCK-8 solution (Thermo Fisher Scientific, Inc.) was added and incubated for a further 2 h. Absorbance was measured at an optical density of 450 nm using a microplate detector (Thermo Fisher Scientific, Inc.).
5-Ethynyl-2′-deoxyuridine (EdU) assay
Following transfection, the proliferation of MLO-Y4 and MC3T3-E1 cells was determined using an EdU assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, MLO-Y4 and MC3T3-E1 cells (5×103 cells/well in 96-well plates) were stained with EdU (10 μM) at room temperature for 1 h and then fixed in 4% formaldehyde for 15 min at room temperature (23±2°C), and permeabilised with 0.5% Triton X-100 for 10 min at room temperature. DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) was used to label the nuclei for 20 min at room temperature. Images were captured using a fluorescence microscope (Leica Microsystems, Inc.).
Flow cytometry
Cell cycle arrest and apoptosis were assessed by flow cytometry. Briefly, MLO-Y4 and MC3T3-E1 cells were transfected with si-RGS18 or pcDNA-RGS18 as indicated, followed by treatment with 20 μM PD98059 or 200 nM TPA for 24 h at 37°C. For cell cycle analysis, 1×106 cells were collected and fixed in ice-cold 70% ethanol for 12 h at 4°C and then stained with a mixture of PI (50 μg/ml), 1% Triton-X100 (1%) and DNase-free RNase (100 μg/ml) at 4°C for 30 min. To assess apoptosis, 1×105 cells were collected and stained using an Annexin V-FITC/PI double staining kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. The samples were then assessed by flow cytometry (Accuri-C6™ plus; BD Biosciences) and the data were analysed using FlowJo™ software (v7.6.5; FlowJo, LLC).
Western blotting
Total protein lysates were extracted from MLO-Y4 and MC3T3-E1 cells following transfection using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) and were quantified using a BCA kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 μg) were separated by SDS-PAGE on 10-12% gels and transferred onto nitrocellulose membranes. After blocking with 5% non-fat milk at room temperature for 1 h, the membranes were probed using primary antibodies against RGS18 (cat. no. 11866-1-AP; Proteintech Group, Inc.), cyclin D (cat. no. ab239794; Abcam), cyclin E (cat. no. ab33911; Abcam), cleaved caspase-3 (cat. no. ab32042; Abcam), cleaved caspase-9 (cat. no. 9509; Cell Signaling Technology, Inc/), ERK1/2 (cat. no. ab17942; Abcam), phosphorylated (p)-ERK1/2 (cat. no. ab201015; Abcam), p38 (cat. no. ab170099; Abcam), p-p38 (cat. no. ab236527; Abcam), JNK1/2 (cat. no. ab112501; Abcam), p-JNK1/2 (cat. no. ab4821; Abcam), ERK5 (cat. no. ab196609; Abcam), p-ERK5 (cat. no. ab5686; Abcam) (all 1:1,000) and GAPDH (cat. no. 60004-1-Ig; 1:50,000; Proteintech Group, Inc.) overnight at 4°C. The membranes were then incubated with the corresponding secondary anti-mouse or anti-rabbit antibodies conjugated to HRP (cat. nos. ab6789 and ab205718; both 1:2,000; Abcam) at room temperature for 1 h. Protein bands of interest were visualised by incubation with an enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.).
Immunostaining
MLO-Y4 and MC3T3-E1 cells were seeded in confocal dishes at a density of 100,000 cells/well, followed by transfection with si-RGS18 or pcDNA-RGS18. The cells were then washed with PBS, fixed in 4% paraformaldehyde at room temperature for 15 min, permeabilised with 0.5% Triton X-100 at room temperature for 10 min, blocked with 5% BSA at room temperature for 1 h and incubated with a primary antibody against p-ERK1/2 (cat. no. ab201015; 1:200; Abcam) at 4°C overnight. Subsequently, the cells were washed with PBS and incubated with a goat anti-rabbit IgG antibody (Alexa Fluor® 555) (cat. no. ab150078; 1:200; Abcam) and with DAPI for 10 min at room temperature. Five random stained areas were observed under a fluorescence microscope (Leica Microsystems, Inc.).
Statistical analysis
All experiments were repeated three times and all data are presented as the mean ± standard deviation and were analysed using GraphPad Prism 9.5.1 software (Dotmatics). Statistical differences between two groups were measured using unpaired Student's t-test, and differences among three or more groups were analysed using one-way or two-way ANOVA followed by the Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
RGS18 expression is increased in AIS
To identify the potential factors associated with bone fracture, gene expression in AIS and HVs from the GSE93138 and GSE93215 datasets of the GEO database was analysed. The overlap analysis identified four genes: KIAA0825, ANXA3, RGS18 and LIPN that were upregulated in AIS compared with in HVs (Fig. 1A). Given that a previous study reported that RGS18 can impair osteoclast formation (12), RGS18 was selected for further investigation. Further experiments confirmed that the expression levels of RGS18 were increased in AIS compared with those in HVs from the GSE93138 dataset (Fig. 1B) and clinical samples (Fig. 1C).
RGS18 promotes osteoclast viability and proliferation
To assess the function of RGS18 in bone fracture, MLO-Y4 and MC3T3-E1 osteocytes were transfected with RGS18 overexpression vector or RGS18 siRNA, and the transfection efficiency was validated by western blot analysis and RT-qPCR (Fig. 2A and B). The viability of MLO-Y4 and MC3T3-E1 cells was promoted by RGS18 overexpression, but was suppressed by RGS18 knockdown (Fig. 2C and D). Furthermore, overexpression of RGS18 increased and silencing of RGS18 decreased the number of EdU-positive MLO-Y4 and MC3T3-E1 cells (Fig. 2E and F), thus indicating that RGS18 contributes to osteocyte proliferation.
RGS18 promotes osteocyte cell cycle progression
The present study then examined the effect of RGS18 on osteocyte cell cycle progression. It was revealed that overexpression of RGS18 reduced the population of MLO-Y4 and MC3T3-E1 cells in G1 phase, but increased the number of cells in S phase (Fig. 3A and B). By contrast, the number of MLO-Y4 and MC3T3-E1 cells in S phase was decreased by RGS18 knockdown (Fig. 3A and B). Furthermore, the expression levels of cell cycle-related proteins cyclin D and cyclin E were increased by RGS18 overexpression but decreased by RGS18 knockdown in MLO-Y4 and MC3T3-E1 cells (Fig. 3C and D), implying that RGS18 contributes to osteocyte cell cycle progression.
RGS18 suppresses osteocyte apoptosis
The present study also evaluated the function of RGS18 in modulating osteocyte apoptosis (early + late) and necrosis. Notably, apoptosis and necrosis of MLO-Y4 and MC3T3-E1 cells was suppressed by RGS18 overexpression, but enhanced by RGS18 knockdown (Fig. 4A and B). In addition, the expression levels of apoptosis-related proteins cleaved caspase-3 and cleaved caspase-9 were inhibited by RGS18 overexpression and enhanced by RGS18 knockdown in MLO-Y4 and MC3T3-E1 cells (Fig. 4C and D), indicating that RGS18 may suppress osteocyte apoptosis.
RGS18 stimulates MAPK signalling
The present study explored the potential mechanisms underlying RGS18-mediated osteocyte function. GSEA of the GSE93138 dataset identified MAPK signalling as one of the RGS18-stimulated signalling pathways (Fig. 5A). Considering MAPK signalling is a critical signalling pathway that is activated due to mechanical stimuli, and results in osteocyte cytoskeletal changes and ECM remodelling (1), MAPK signalling was selected for further analysis. Phosphorylation of ERK1/2, but not of p38, JNK1/2 or ERK5, was induced by RGS18 overexpression and was inhibited by RGS18 knockdown in MLO-Y4 and MC3T3-E1 cells (Fig. 5B-E). Immunofluorescence analysis confirmed that the levels of p-ERK1/2 were enhanced by RGS18 overexpression and reduced by RGS18 knockdown in MLO-Y4 and MC3T3-E1 cells (Fig. 5F and G).
RGS18 promotes osteocyte proliferation through ERK signalling
The present study evaluated the association between RGS18 and ERK signalling in the modulation of osteocyte proliferation. EdU-positive MC3T3-E1 cells were enhanced by RGS18 overexpression, whereas treatment with the MEK1/2 inhibitor PD98059 blocked this effect (Fig. 6A). Furthermore, overexpression of RGS18 attenuated the proportion of MC3T3-E1 cells in G1 phase, but enhanced the number of MC3T3-E1 cells in S phase, whereas PD98059 reversed this effect (Fig. 6B). In addition, overexpression of RGS18 suppressed MC3T3-E1 cell apoptosis and necrosis, whereas treatment with PD98059 reversed this effect (Fig. 6C). Collectively, these data indicated that RGS18 contributes to osteocyte proliferation by activating ERK signalling.
ERK activation reverses RGS18 knockdown-induced osteocyte death
The present study observed that knockdown of RGS18 decreased the number of EdU-positive MC3T3-E1 cells, whereas treatment with the ERK1/2 activator TPA reversed this effect (Fig. 7A). The distribution of MC3T3-E1 cells in G1 phase was enhanced, but the number of MC3T3-E1 cells in S phase was reduced by RGS18 knockdown, whereas TPA treatment reversed these effects (Fig. 7B). In addition, the apoptosis and necrosis of MC3T3-E1 cells was promoted by RGS18 knockdown, whereas treatment with TPA blocked this effect (Fig. 7C), thus indicating that ERK activation reversed RGS18 knockdown-induced osteocyte death.
Discussion
Bone fracture healing is a complex regenerative process involving various cells, such as chondrocytes, osteoblasts, endothelial cells and mesenchymal stem cells, and released factors, such as bone sialoprotein and osteocalcin (3). Osteocytes serve a crucial role in the modulation of metabolism, remodelling and bone generation during fracture healing. In the present study, the role of RGS18 in the regulation of bone fractures and osteocytes was uncovered.
RGS18 is a member of the RGS family, and participates in multiple physiological and pathological processes. It has been reported that RGS18 inhibits platelet activation, and induces platelet production and survival (10). RGS18 also functions as a myeloerythroid lineage-related modulator of G protein signalling in megakaryocytes (18). In addition, RGS18 regulates cilia-related mechanosensory processes (19) and serves as a negative modulator of osteoclastogenesis by regulating NFAT signalling (12). In the present study, it was revealed that RGS18 was more highly expressed in samples from AIS compared with those from HVs. Cell viability and proliferation of osteocytes were promoted by RGS18 overexpression, but were suppressed by RGS18 knockdown. Furthermore, overexpression of RGS18 increased the number of S-phase osteocytes, and RGS18 knockdown resulted in the opposite effect. Osteocyte apoptosis was suppressed by RGS18 overexpression but induced by RGS18 knockdown. These data suggested that RGS18 contributes to osteocyte proliferation, indicating a potential function of RGS18 in the regulation of bone fracture healing. The present findings elucidate a novel function of RGS18 in bone fracture healing and osteocytes. The effects of RGS18 on bone-fracture healing should be confirmed in future in vitro and in vivo studies.
Regarding the underlying mechanism, GSEA of the GSE93138 dataset revealed that RGS18 could stimulate MAPK signalling. Phosphorylation of ERK1/2 was induced by RGS18 overexpression in osteocytes. Furthermore, the present study confirmed that treatment with the MEK1/2 inhibitor PD98059 reversed the RGS18 overexpression-induced osteocyte proliferation and PD98059 reversed the RGS18 overexpression-inhibited osteocyte apoptosis. Moreover, the ERK1/2 activator TPA reversed the RGS18 knockdown-induced suppression of osteocyte proliferation and the RGS18 knockdown-induced osteocyte apoptosis. These data suggested that RGS18 promotes osteocyte proliferation by activating ERK signalling. The present findings provide novel insights into the mechanism by which RGS18 contributes to bone fracture healing via stimulating ERK signalling. ERK signalling participates in the regulation of osteocyte function during bone fracture healing. It has been reported that HMGB1 contributes to bone fracture healing in a rat tibial fracture model by activating ERK signalling (20). High glucose levels inhibit the expression of connexin 43, and suppress hemichannel function and gap junctions in osteocytes by stimulating ERK signalling (21). Overexpression of Lgr5 in mesenchymal stem cells promotes fracture healing by modulating mitochondrial dynamics and ERK signalling (22). Furthermore, inhibition of microRNA-21 can enhance bone fracture healing by activating the ERK pathway (23). These findings suggested that ERK signalling may be one of the mechanisms by which RGS18 mediates bone fracture healing, and other potential mechanisms should be explored to increase the understanding of RGS18-regulated bone fracture healing.
In conclusion, RGS18 may contribute to osteocyte proliferation and bone fracture healing by activating ERK signalling (Fig. 8). RGS18 may be a key factor in promoting osteoblast proliferation, and this study provides a theoretical basis for further development of fracture healing treatments.