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Patients with inflammatory bone disease or cancer exhibit an increased risk of fractures and delayed bone healing. The S100A4 protein is a member of the calcium-binding S100 protein family, which is abundantly expressed in inflammatory diseases and cancers. We investigated the effects of extracellular S100A4 on osteoblasts, which are cells responsible for bone formation. Treating primary calvarial osteoblasts with recombinant S100A4 resulted in matrix mineralization reductions. The expression of osteoblast marker genes including osteocalcin and osterix was also suppressed. Interestingly, S100A4 stimulated the nuclear factor-kappaB (NF-κB) signaling pathway in osteoblasts. More importantly, the ex vivo organ culture of mouse calvariae with recombinant S100A4 decreased the expression levels of osteocalcin, supporting the results of our in vitro experiments. This suggests that extracellular S100A4 is important for the regulation of bone formation by activating the NF-κB signaling pathway in osteoblasts.
S100 proteins are calcium-binding proteins comprised of more than 20 members and they play important roles in inflammation, cytoskeleton dynamics, enzyme activity, as well as cell growth and differentiation (1–3). Many studies have revealed that S100 proteins are involved in pathological conditions, especially with regard to tumor progression and arthritis (3). For instance, S100A2, S100A4, S100A6, S100A7, and S100B were found to be differentially expressed in cancer cells, while S100A12 was detected at a high level in synovial tissues of arthritis patients (4). However, each kind of the S100 proteins is thought to play a specific part or an, as yet, unidentified critical role under certain circumstances. Notably, S100A8 has been shown to activate osteoclasts, cells responsible for bone resorption, by interacting with the toll-like receptor 4 (TLR4) (5).
Over the last decade, S100A4 has been linked to rheumatoid arthritis pathogenesis. Klingelhofer and colleagues reported that synovial tissues of rheumatoid arthritis patients express S100A4 (6). In addition, S100A4 was reported to stimulate matrix metalloproteinase (MMP)-13 secretion via the receptor for advanced glycation end products (RAGE) in chondrocytes, resulting in cartilage degradation (7). S100A4 also plays important roles in tumor progression by increasing tumor cell migration and invasion as well as MMP secretion (8). Both intracellular and extracellular functions of S100A4 have been studied. Intracellularly, S100A4 binds to the tumor suppressor protein p53, where it aids in tumor survival; tumor cell migration is regulated through interactions with non-muscle myosin II. Extracellularly, S100A4 binds to cell surface receptors such as RAGE, activating nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase pathways for tumor cell invasion and survival (9).
Recently, the NF-κB signaling pathway was reported to be a negative regulator of osteogenesis (10, 11). NF-κB activation by proinflammatory cytokines such as the tumor necrosis factor (TNF)-α in osteoblast precursors inhibits osteogenic differentiation (12). RAGE, a receptor of S100A4, was shown to mediate activation of NF-κB in endothelial cells, macrophages, and lymphocytes (13). A study by Ogawa et al. reported that osteoblastic cells expressed RAGE and that the addition of high glucose with AGE, one of the ligands of RAGE, inhibited mineralization (14). Therefore, we postulated that S100A4 signals through RAGE to activate the NF-κB pathway and to suppress osteoblast function.
In this study, we demonstrated that extracellular S100A4 did not affect early osteoblast differentiation. However, it inhibited mineralization activity due to activation of the NF-κB signaling pathway in osteoblasts. Also, the ex vivo culture of neonatal mice calvariae with recombinant S100A4 decreased the expression of osteocalcin.
To test the effect of S100A4 on bone formation, we first investigated its influence on an in vitro culture of primary osteoblasts. Preosteoblasts derived from mouse calvarial tissues were treated with recombinant mouse S100A4 protein or an control vehicle of an osteogenic differentiation medium. S100A4 protein did not affect the viability of calvarial preosteoblasts at concentrations up to 2 μg/ml (Fig. 1A). Cells became positive for ALP, an early stage marker of osteoblast differentiation, within three days of culture in osteogenic media (Fig. 1B). S100A4 treatment did not alter the extent of ALP staining compared to the vehicle-treated culture (Fig. 1B). Consistent differences in ALP activity by S100A4 treatment at the second or fourth day of the culture could not be observed (Fig. 1C). We next assessed mRNA and protein levels of Runx2, the key transcription factor of early osteoblast differentiation. The mRNA expression of Runx2 in S100A4-treated cultures was comparable to that in the vehicle-treated group (Fig. 1D, upper part). On the other hand, a slight decrease in Runx2 protein level by S100A4 treatment at day 3 of the culture could be observed (Fig. 1D, lower part). Taken together, these results indicate that S100A4 does hardly influences the early stage of osteoblast differentiation.
We examined the effect of S100A4 on matrix mineralization, a crucial function of osteoblasts during bone formation. Calvarial preosteoblasts were cultured in osteogenic media for ten days and the assessment of mineralization was performed by Alizarin-red staining. The cell proliferation and survival was not affected by the presence of S100A4 at concentrations up to 2 μg/ml until day 6 of the culture and only slightly decreased at 2 μg/ml in 9-day cultures (Fig. 2A). Interestingly, the addition of S100A4 at 1 μg/ml in osteogenic media significantly delayed matrix mineralization by calvarial osteoblasts (Figs. 2B and 2C). The expression level of osteocalcin (Bglap), a critical component of the bone matrix, was also lowered by the S100A4 treatment (Fig. 2D). Induction of the osterix protein level, a transcription factor necessary for mineralization, was also suppressed (Fig. 2E). In overall, our results demonstrate that an excessive amount of extracellular S100A4 impairs the mineralization activity of osteoblasts and delays induction of late osteoblast markers.
A number of studies have reported that activation of the NF-κB pathway in osteoblast precursors impairs osteogenic differentiation (10, 11). To investigate the mechanism of mineralization inhibition by S100A4, we tested whether S100A4 activated the NF-κB pathway in calvarial osteoblasts. Stimulating them with recombinant mouse S100A4-increased IKKα/β, IκB, and p65 phosphorylation within 60 minutes (Fig. 3A). We also assessed the protein level of p65 in the nucleus by fractionating the cytosol and nuclear proteins. Increased levels of p65 were observed in the nuclei of S100A4-treated cells (Fig. 3B). Consistent with our Western blotting results, immunofluorescence confocal microscopy also demonstrated localization of p65 in the nuclei after S100A4 stimulation (Fig. 3C). Finally, the decreases in mineralization caused by S100A4 were attenuated by treatment with BAY11-7085, a NF-κB inhibitor (Fig. 3D). These data suggest that S100A4 negatively regulates osteogenic differentiation by activating the NF-κB signaling pathway.
To obtain further evidence supporting the regulatory role of S100A4 on the bone mineralization activity of osteoblasts, we next utilized an ex vivo organ culture. Calvariae from four-day-old mice were cultured for nine days in osteogenic media with or without S100A4 protein. The osteogenic medium was changed in 2-day intervals and total RNAs were isolated for cDNA synthesis (Fig. 4A). Real-time PCR analyses revealed a strong suppression of Bglap (osteocalcin) gene expression by S100A4 treatment (Fig. 4B).
In this study, we investigated the role of extracellular S100A4 on the inhibition of bone formation by activating the NF-κB pathway in osteoblasts. The early differentiation of osteoblast was not affected (Fig. 1). However, both late-stage differentiation of osteoblasts and the mineralization activity were reduced by S100A4 treatment (Fig. 2). Consistent with the results of our in vitro experiments, S100A4 addition to ex vivo culture of neonatal calvariae decreased the expression levels of osteocalcin (Fig. 4). The NF-κB activation in osteoblasts by S100A4 seems to be responsible for the inhibition of bone formation (Fig. 3).
Bone-loss under inflammatory conditions largely depends on enhanced bone resorption by osteoclasts and delayed bone formation by osteoblasts. Among the inflammatory cytokines, TNF-α was shown to directly inhibit the mineralized nodule formation and osteocalcin secretion from osteoblasts (18). Moreover, the expression of Runx2 was decreased after TNF-α treatment (19) as well as a TNF-α induced expression of the Smad ubiquitination regulatory factor 1/2 (Smurf 1/2), which ubiquitylates Runx2 for proteasomal degradation (20). Yamazaki et al. demonstrated that bone morphogenetic protein 2 (BMP2)-mediated activation of Smads, crucial transcription factors of osteoblast differentiation, abrogated by TNF-α via NF-κB activation (12). Moreover, the activation of NF-κB by TNF-α induced a cAMP response element-binding protein H (CREBH), which - in turn - up-regulated Smurf 1 to inactivate the Smad/Runx2 regulatory system (21). Mice that specifically overexpressed the dominant negative form of IKK in osteoblasts displayed increased bone mass (10). Such studies are in accordance with our finding of S100A4 mediating inhibition of mineralization through NF-κB activation and strongly support a negative relationship between bone anabolism and the activation of NF-κB in osteoblasts.
The NF-κB signaling pathway is also critical to the differentiation of osteoclasts by RANKL (receptor activator of NF-κB ligand) (22). Interestingly, mice lacking S100A4 showed a decrease in the number of osteoclasts (23). Therefore, it is reasonable to postulate that S100A4 also mediates the activation of NF-κB in osteoclasts, accelerating bone catabolism. We are currently investigating whether S100A4 directly regulates osteoclastogenesis via the NF-κB pathway and if this could be a useful therapeutic target to treat bone metabolic disorders.
It was previously demonstrated that the synovial fluids of rheumatoid arthritis patients had a mean concentration of 1.98 μg/ml of S100A4, significantly higher than 0.247 μg/ml in osteoarthritis patients (6). Since rheumatoid arthritis promotes and maintains a more inflammated condition near the articular joints than osteoarthritis, the effect of extracellular S100A4 on bone cells might be more pronounced in rheumatoid arthritis. In one of our previous studies, we determined that S100A4 was secreted from lipopolysaccharide-stimulated human periodontal ligament cells, supporting the possibility that S100A4 regulates bone cells in inflammatory diseases (24). The concentrations of recombinant S100A4 used in this study for the in vitro tests were 1 or 2 μg/ml, similar to the level found in arthritis patients.
We described the effect of extracellular S100A4 with respect to the NF-κB pathway’s activation. Detecting receptors that mediate activation in osteoblasts is an appealing subject for further studies. RAGE and TLR4 are among the two best characterized receptors for S100A4 in a variety of cell types (25–28). NF-κB signaling in peripheral blood mononuclear cells was shown to be activated by S100A4 via TLR4 (25), while a series of studies demonstrated that S100A4 mediates signals via RAGE, especially in cancer cells (26–28). Both receptors are critical components of the NF-κB signaling pathway.
In conclusion, our study demonstrates that extracellular S100A4 interrupts the mineralization function of osteoblasts and causes an imbalance in bone homeostasis by inhibiting new bone formation. We demonstrated for the first time that this phenomenon is mediated through NF-κB activation. Further investigations of S100A4 may open new avenues for developing novel therapies against inflammatory bone-destructive conditions.
All animals were kept in a specific pathogen-free animal facility with a consistent temperature (22°C) and humidity (55%), as well as a 12-hour light/dark cycle. The facility was operated by experienced animal handlers who were responsible for changing bedding material as well as for the provision of food and sterilized water under the supervision of a veterinarian. One-day-old ICR mice were purchased from OrientBio (Sungnam, Korea) and used for both the in vitro calvarial osteoblast preparation and the ex vivo organ culture experiments. Animal experiments were approved by the Committee on the Care and Use of Animals in Research at Seoul National University (SNU-130311-4-3).
Recombinant mouse S100A4 was purchased from Prospec (East Brunswick, NJ, USA). Phosphospecific antibodies against IκB kinase (IKK)α/β (Ser176/180), IκB (Ser32), p65 (Ser536), IKKα/β, IκB, and p65 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against osterix, laminB, and p65 were acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-β-actin (AC-74) was purchased from Sigma-Aldrich (St. Louis, MO, USA), anti-Runx2 from MBL International (Woburn, MA, USA), the alkaline phosphatase (ALP) assay kit from Takara Bio Inc. (Fukui, Japan). β-Glycerophosphate, cetylpyridium chloride, and ascorbic acid from Sigma-Aldrich, Bay11-7082 from Alexis Biochemicals (Grunberg, Germany), and the Cell Counting Kit-8 (CCK-8) from Dojindo (Kumamoto, Japan).
Calvarial osteoblasts were seeded in 96-well plates at the density of 1 × 104 cells per well. Cells were cultured in the presence of 10 mM β-glycerophosphate and 100 μM ascorbic acid together with vehicle or recombinant mouse S100A4 (0.1–2 μg/ml) for 3–9 days. Cells at days 0, 3, 6, and 9 were subjected to cytotoxicity assay using the CCK-8 assay kit. After incubating cells with the reagents for one hour, the optical density was measured at 450 nm with an iMark Microplate Absorbance Reader (Biorad, Hercules, CA, USA).
Quantification of the mRNA level by real-time PCR analysis was performed as previously described (15). Primers for real-time PCR analysis were as follows: Runx2 forward, 5′-CGCACGACAACCGCACCA-3′; Runx2 reverse, 5′-CAGCACG GAGCACAGGAAGTT-3′; Bglap forward, 5′-CCGGGAGCAG TGTGAGCTTA-3′; Bglap reverse, 5′-TAGATGCGTTTGTAGG CGGTC-3′; Hprt (hypoxanthine-guanine phosphoribosyltransferase) forward, 5′-CCTAAGATGAGCGCAAGTTGAA-3′; Hprt reverse, 5′-CCACAGGGACTAGAACACCTGCTAA-3′.
Calvarial preosteoblasts were prepared from 1-day-old mice as previously described (16). These were seeded onto 48-well or 6-well cell culture plates in alpha minimum essential medium (WELGENE, Daegu, Korea), supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Osteogenic differentiation was induced by supplementing the culture media with 10 mM β-glycerophosphate and 100 μM ascorbic acid. To evaluate the osteoblast differentiation, ALP activity was measured using the ALP assay kit, following the manufacturer’s protocol. Mineralization was assessed after 10 days of culture by Alizarin-red staining as described before (17) and the mean intensity was assessed using the ImageJ program. Alternatively, Alizarin-red stained wells were incubated with 100 mM cetylpyridium chloride for two hours at 37°C and the optical density of eluates was measured at 415 nm.
Cells were washed with cold PBS and lysed with RIPA buffer (10 mM Tris pH 7.2, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1% sodium deoxycholate, 5 mM ethylenediaminetetraacetic acid). Whole cell lysates or nuclear lysates were subjected to Western blot analysis as previously described (16).
Calvariae were dissected from 4-day-old ICR mice and cultured in 60 mm center well culture dishes (Corning, NY, USA). Osteogenic differentiation was induced by supplementing BGJb medium (Invitrogen) with 10 mM β-glycerophosphate and 100 μM ascorbic acid. The culture medium containing mouse recombinant S100A4 (1 or 5 μg/ml) or vehicle was changed every other day during nine days. For real-time PCR analysis, calvariae were snap-frozen in liquid nitrogen and ground to powder for total RNA isolation using TRIzol (Invitrogen).
Calvarial osteoblasts seeded on 12-mm cover glass were fixed with 3.7% formaldehyde and made permeable with 0.1% Triton X-100 for 15 minutes. After blocking non-specific sites with 1% bovine serum albumin in PBS for 1.5 hours, the cells were incubated with anti-p65 (1/200 dilution) and anti-laminB (1/200 dilution) antibodies for two hours, followed by a 1-hour incubation with secondary mouse-FITC and goat-Cy3 (1/300 dilution) antibody. The prepared slides were viewed under a Zeiss LSM 700 laser-scanning microscope with the following requirements: Objective lenses; C-Apochromat 40x/1.20 W, detectors; PMT, filter model; green (BP 490–555), red (560 IF), and lasers; 488 nm, 555 nm. Pictures were taken and analyzed using the ZEN2010 program (version 126.96.36.1990).
All experiments, except the mice calvariae organ culture studies, were performed at least three times. All quantitative measurements had 3 or 4 replicates. Two-way ANOVA followed by a Bonferroni test and Student’s t test were performed to define differences between multiple and 2 groups, respectively. Quantitative data are presented as the mean ± SD. A P value < .05 was considered statistically significant.
This work was supported by a grant from the National Research Foundation of Korea (NRF-2014R1A2A1A10050406) to H-HK.
CONFLICTS OF INTEREST
The authors have no conflicting financial interests.