The treatment of osteoporosis typically inhibits the activity of osteoclasts, which subsequently results in the suppression of bone formation and maintenance, however the underlying mechanism remains to be elucidated. The receptor activator of nuclear factor κ-B ligand (RANKL)-receptor activator of nuclear factor κ-B (RANK) signaling axis is important in the osteoblast regulation of osteoclasts. RANKL surface-bound molecules expressed on T cells stimulate a reverse signaling transduction in order to regulate the T cells, therefore the present study hypothesized that RANKL expressed on osteoblasts may transfer reverse signals to regulate osteoblasts. A series of experiments were designed to test the hypothesis, using MTT, stealth RNA interference, reverse transcription-quantitative polymerase chain reaction, western blot analysis, alkaline phosphatase activity assay and alizarin red staining. The present study observed the role of RANK-RANKL reverse signaling on osteoblasts, regulated by osteoclasts. Osteoblasts were treated with recombinant RANK proteins. The soluble RANK enhanced the mineralization of osteoblasts. When the RANKL was knocked down in the osteoblast, RANK demonstrated a weak osteogenic effect on the RANKL-deficient osteoblast compared with the wild-type osteoblast which served as a control. Addition of soluble RANK activated the p38 mitogen activated protein kinase (MAPK) signaling pathway in the osteoblast and blocking this same pathway in E1 cells reduced the effect of RANK. In the co-culture system of osteoblasts and osteoclasts, p38 MAPK in E1 cells was phosphorylated a short time following co-culture and the phosphorylation then blocked by abundant soluble RANKL. The findings suggested that RANKL expressed on osteoblasts transferred reverse signals from the exterior of the cell to the interior, which regulated the osteoblasts.
The functional activity of osteoblasts and osteoclasts is synergistic and an imbalance may result in a variety of diseases, including osteoporosis. Osteoblasts regulate osteoclasts via the receptor activator of nuclear factor κ-B ligand (RANKL)-receptor activator of nuclear factor κ-B (RANK) signaling pathway. RANKL is expressed on osteoblasts and T cells. It binds the receptor RANK, which is produced on osteoclasts and their progenitors. The interaction of RANK with RANKL is required for osteoclast formation, differentiation, activation and survival. RANKL-RANK signaling is important in the osteoblast regulation of osteoclasts (
RANKL is a member of the tumor necrosis factor (TNF) super-family and is produced on osteoblasts and T cells. It has previously been suggested that this membrane-integrated TNF ligand may receive signals, subsequently acting as a receptor, to transmit positive and negative feedback signals into the ligand-bearing cell (
It has additionally been reported that bone formation may be regulated by osteoclast activity. The bone remodeling process involves a balance between bone resorption and formation, therefore this equilibrium may be controlled via a concerted action between the two cell types. However, the mechanism by which osteoclast feedback regulates osteoblasts remains to be elucidated. Previous research has indicated that the trans-membrane RANKL expressed on T cells regulates their activity via reverse signaling (
Recombinant soluble RANK (sRANK) and recombinant soluble RANKL (sRANKL) were prepared by
The MC3T3-E1 (E1) and RAW246.7 mouse pre-osteoblastic cells were provided by The Cell Bank Type Culture Collection, Chinese Academy of Sciences (Shanghai China). The cells were incubated with α-Minimal Essential Medium (MEM) (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA) at 37°C in a humidified atmosphere containing 5% CO2. At ~80% confluence, the E1 cells were separated and cultured in complete medium containing osteogenic factors (0.05 mM L-ascorbic acid, 100 nM Dex and 10 mM β-glycerophosphate). Co-culture was initiated when the RAW246.7 cells were plated at a density of 3×105 cells per well on the formed E1 cell layer, following osteogenic differentiation.
Stealth RNAs targeting the 25-nucleotide sequence of RANKL and the control were obtained from Thermo Fisher Scientific, Inc. The primers sequences were as follows: Forward, 5′-GCGCAGAUGGAUCCUAACAdTdT-3′ and reverse, 5′-UGUUAGGAUCCAUCUGCGCdTdT-3′ for siRNA and forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′ for negative control. Before transfection, cells were plated in six-well culture dishes at a density of 1c105 cells per dish. When the confluence reached 70%, E1 cells were transfected with 10 nM stealth RNAs using Lipofectamine™ (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.
The study consisted of four sub-investigations on differing groups: i) E1 cells maintained with 10−6, 10−5 and 10−4 g/l RANK protein (
To investigate the effects of sRANK on cell growth, E1 cells were inoculated at 5×103 cells per well in 96-well culture plates. Following a 24 h culture period, cells were then treated with 10−6, 10−5 and 10−4 g/l of sRANK or without sRANK as a control for 24, 48 and 72 h. The cells were washed twice with PBS. A total of 200 µl complete medium with supplement 20 µl 5 mg/ml MTT solution (Sigma-Aldrich; Merck KGaA) was added to each well. Following incubation at 37°C for 4 h, the medium was replaced with 200 µl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) and vibrated for 15 min. Finally, the absorbance was measured at a wavelength of 490 nm by a microplate spectrophotometer. All experiments were performed in triplicate.
The ALP activity assay was conducted using an ALP kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's protocol. Alizarin red staining was performed to detect calcium deposits. The cultured cells were fixed by 10% neutral buffered formalin and stained with 1% alizarin red S solution for 10 min at room temperature. The calcified nodules were observed under a light microscope (Olympus Corporation, Tokyo, Japan).
Cells were lysed on ice for 5 min using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Beijing, China), and were then sonicated briefly and centrifuged for 10 min at 14,000 × g in a cold microfuge. Protein concentrations were determined using a bicinchoninic acis assay (Beyotime Institute of Biotechnology). Total protein samples (40 µg per lane) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% dried skim milk in TBS/0.05% Tween-20 (TBST) for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against osteocalcin (catalog no. sc-30045; 1:1,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p38 (catalog no. sc-7149; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), phospho-p38 (catalog no. 4092; 1:1,000 dilution; Cell Signaling Technology, Inc. Danvers, MA, USA), and β-actin (catalog no. NB600-503; 1:5,000 dilution; Novus Biologicals, LLC. Littleton, CO, USA) in TBST. Following 3 washes with TBST, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Ig)G antibody (catalog no. A0208; 1:5,000 dilution; Beyotime Institute of Biotechnology) for 1 h at room temperature. Protein expression signals were detected with Enhanced Chemiluminescence-plus substrate (Applygen Technologies, Inc., Beijing, China) using the ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Inc. Hercules. CA, USA), according to the manufacturer's protocol.
Total RNA of the cells was isolated using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.). To carry out qPCR, cDNAs were synthesized from 1 µg of the RNA using Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer's protocol. qPCR analysis of RANKL and GAPDH was conducted using the ABI Prism 7300 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and PowerUp™ SYBR™ Green Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). Each reaction mixture contained 10 µl SYBR Green master mix, 7 µl RNase-free H2O, 1 µl 10 mM forward primer, 1 µl 10 mM reverse primer, and 1 µl cDNA in a final reaction volume of 20 µl. Cycling conditions were as follows: An initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 55°C for 15 sec and elongation at 72°C for 15 sec. Gene expression was normalized to the internal reference gene GAPDH. The 2−∆∆Cq method (
Statistical analysis was performed using SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA). The data are expressed as the mean ± standard deviation from all experiments. Significant differences were determined using one-way ANOVA with Dunnett's post-hoc test or unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
The proliferation activity of E1 cells was examined via MTT assay following sRANK (10−6, 10−5 and 10−4 g/l) treatment for 24, 48 and 72 h. As presented in
RANKL is the only RANK-binding protein described in previous reports, therefore the present study investigated if the interaction of RANK with membrane-bound RANKL on osteoblasts leads to osteoblastogenesis. RANKL expression in E1 cells was knocked down by RNAi. The cells transfected with Stealth™ RNAi oligonucleotide of RANKL for 48 h demonstrated a significant downregulation in gene expression (
In order to understand the mechanism of the reverse signaling, the present study analyzed the signaling pathways of RANK-induced osteoblastogenesis. Activation of p38 MAPK has been revealed to be required for osteoblast differentiation (
The osteoblast and osteoclast co-culture system is unable to be continuously maintained, therefore only the phosphorylation of p38 MAPK signaling factor was examined, a short period following induction of the co-culture (30, 60, 90 and 120 min).
The results indicated that p38 MAPK in co-culture cells was observed to undergo phosphorylation a short time period following osteoblast and osteoclast interaction (
It has previously been demonstrated that osteoblasts regulate osteoclasts via the RANKL-RANK-OPG signaling pathway and are able to exhibit bidirectional signaling (
It has previously been suggested that recombinant murine RANK protein effectively inhibits the activity of osteoclasts and the resulting bone resorption (
RANKL, which is a member of the TNF family, is a type II trans-membrane protein containing a C-terminal TNF homology domain. Numerous TNF family proteins may be expressed in a soluble form or released from the cell surface via specific protease cleavage (
Soluble RANK may bind to RANKL and block the RANKL-RANK forward signal to inhibit osteoclastogenesis. However, the present study suggested that soluble RANK bound to trans-membrane RANKL and activated the RANK-RANKL reverse signal to promote osteoblastogenesis, a pathway reversal which has been confirmed to occur in other TNF family members. RANK is an artificial synthetic peptide that binds RANKL, therefore it was hypothesized that it would act as an endogenous ligand to RANKL. The interaction of RANKL and RANK exhibits the potential to transmit a bidirectional signal to activate osteoclasts and osteoblasts via forward and reverse pathways, respectively.
The crystal structure of RANK-RANKL binding interaction was identified. The present study constructed several variations of RANK and RANKL sequences, each with one amino acid mutation introduced. The affinity of RANK and RANKL was measured using a BIAcore 3000 system as previously described (
Consistent with a previous report that activation of p38 MAPK was involved in RANKL signaling in T cells (
Co-cultures are of primary interest for
In conclusion, it was demonstrated that RANKL present on osteoblasts exhibited a reverse signal transduction ability. Dysfunction of the reverse signaling pathway may act as an underlying mechanism of osteoporosis. The findings may aid identification of the mechanism regulating the coupling between bone formation and resorption. A reverse signaling via RANKL is involved in part in RANK-induced osteoblast differentiation. The RANK-RANKL reverse signaling interaction may act as a novel and potential mechanism to explain and fully elucidate osteoclast regulation of osteoblasts and provide a novel perspective for the treatment of osteoporosis. RANK-RANKL reverse signaling was studied in the present study and other possible reverse signaling interactions require investigation in future studies.
The present study was supported by the National Nature Science Foundation of China (grant no. 81300711).
Signaling via RANKL is involved, in part, in RANK-induced osteoblast differentiation. (A) Expression of RANKL was measured by reverse transcription-polymerase chain reaction. RANKL gene expression was suppressed by small interfering RNA-mediated knockdown with 10 nM stealth RNAi in E1 cells. (B) Treated cells were further cultured with 10−4 g/l sRANK protein and ALP activity was measured. Data was expressed as the mean ± standard deviation. ***P<0.001 vs. each control. (C) Mineralization of E1 cells was evaluated by alizarin red staining. RANK, receptor activator of nuclear factor κ-B; RANKL, receptor activator of nuclear factor κ-B ligand; ALP, alkaline phosphatase activity assay; sRANK, recombinant soluble RANK; Mu-RANK, mutant RANK; KD, Knock down.
Signaling pathways involved in RANK stimulation in E1 cells. Cells were cultured in the presence of 10−4 g/l sRANK or Mu-RANK protein with 0–10 µM p38 inhibitor (SB203580). (A) Post-confluent E1 cells were stimulated with 10−4 g/l RANK protein for 10–240 min. Phosphorylated p38, total p38 and β-actin were detected by western blotting. The osteogenic effects of sRANK on E1 cells in absence or presence of SB203580 were measured by (B) ALP activity on day 7 and examined by (C) alizarin red staining on day 14. **P<0.01 ***P<0.001 vs. 10−4 g/l sRANK protein with no SB203580. RANK, receptor activator of nuclear factor κ-B; ALP, alkaline phosphatase activity assay; sRANK, recombinant soluble RANK; Mu-RANK, mutant RANK; p, phosphorylated.
RANK-RANKL interaction in osteoblast and osteoclast co-culture system. (A) Light microscopy images of the osteoblast and osteoclast co-culture following 30 min. (Magnification ×200). (B) The phosphorylation of p38 was detected by western blotting within a short time period following co-culture (30, 60, 90 and 120 min). (C) A total of 5 nM sRANKL was added to the co-culture system and Mu-RANKL as control. The phosphorylation of p38 was detected by western blotting. RANK, receptor activator of nuclear factor κ-B; RANKL, receptor activator of nuclear factor κ-B ligand; sRANKL, recombinant soluble RANKL; Mu-RANKL, mutant RANKL; p, phosphorylated.