journal of prosthodontic research 65 ( 2021) 393–399

Journal of Prosthodontic Research

Official Journal of Japan Prosthodontic Society

Original article Effect of aging on bone metabolism: the involvement of complement C1q Fumiko Aonumaa, b, Sen Higashia , Shintaro Tsukab , Tomoko Ohsumia , Chihiro Masakib , Ryuji Hosokawab , Hiroshi Takeuchia* aDivision of Applied Pharmacology, Kyushu Dental University, Kitakyushu, Japan bDepartment of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Japan

Abstract Purpose: Impairment of normal bone remodeling affects the successful osseointegration of dental implants. Recently, it has been reported that complement C1q level increases with age and delays wound healing by modulating Wnt signaling. As Wnt signaling is known to play an essential role in bone remodeling, we hypothesized that aging-dependent increases in C1q affect bone remodeling. In this study, we examined whether C1q affects the differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts, and investigated whether C1q could modify cellular signaling, including the Wnt/β-catenin pathway in these cells. Methods: Osteogenic differentiation of MC3T3-E1 cells was assessed using alkaline phosphatase staining. Differentiation of osteoclasts from mouse bone marrow cells was assessed using tartrate-resistant acid phosphatase staining. Activation of canonical Wnt signaling and protein phosphorylation was monitored using Western blotting. Results: C1q, at 5-15 µg/mL promoted osteoclast fusion, whereas it did not affect the differentiation of osteoblasts. On the other hand, a higher concentration of C1q (50 µg/mL) suppressed both bone morphogenetic protein-2-induced osteogenic differentiation and osteoclast formation. C1q did not induce an obvious activation of Wnt/ β-catenin signaling in either pre-osteoblasts or pre-osteoclasts, contrary to previous reports using other tissues. Instead, C1q upregulated the receptor activator of nuclear factor-kappa B ligand (RANKL)-induced phosphorylation of Akt. Conclusions: C1q could affect cellular signaling and modify the differentiation of osteoblasts and osteoclasts, depending on the concentration. Therefore, an increase in C1q with age could be one of the factors that determine the prognosis of treatment of elderly patients. Keywords: Aging, C1q, Bone, Osteoclast

Received 6 February 2020, Accepted 25 August 2020, Available online 29 October 2020

zirconia crowns are an alternative to metal crowns in posterior regions a candidate biomarker of sarcopenia in elderly individuals [7, 8]. Because 1. Introduction impairment of bone homeostasis naturally occurs with an advancing age and causes osteoporosis [9], an elevated C1q level in the elderly Aging is associated with an increased risk of frailty and related diseases, could play a direct or indirect role in bone homeostasis; therefore, it such as sarcopenia, osteoporosis, and cancer. Chronological age is also could be involved in the development of osteoporosis and be associated considered to be one of the risk factors for successful dental implant with the reduced rate of successful osseointegration of dental implants. treatment, while it is unclear whether aging itself could be an important Recently, C1q was also shown to be increased in the serum of aged factor, rather than the factors related to aging [1]. Therefore, further mice and to activate the canonical Wnt signaling pathway by directly research based on sufficient evidence is needed to assess the survival and binding to the Wnt receptor, (Fzd) 8, followed by the digestion success of dental implants in elderly patients. of low-density lipoprotein (LDL)-related receptor 5 (Lrp5) or Lrp6, which Complement C1q is a component of the , which consists functions as a co-receptor of the Fzd family [10]. It has been shown that of C1q, C1r, and C1s serine proteases, which play critical roles in the chronic activation of Wnt signaling by C1q leads to the development initiating the classical complement pathway [2, 3]. C1q is also known of muscle fibrosis, an aging-related phenomenon. The Wnt receptor is to have various functions that are mediated through a mechanism primarily a heterodimeric complex consisting of a seven-transmembrane independent of its role in the [4-6].It has been receptor, Fzd, and a co-receptor, Lrp5 or Lrp6 [11]. shown that the serum level of C1q increases steadily with age, above There are ten mammalian Fzd proteins that provide primary platforms the 40 years of age in humans, and that it is negatively correlated with for Wnt ligand binding. Wnt binding to the receptor leads to the muscle mass and strength; thus, serum C1q levels are suggested to be inactivation of the constitutively active kinase glycogen synthase kinase 3β (GSK3β), which keeps β-catenin phosphorylated in the absence of Wnt, preventing the continuous degradation of β-catenin via the ubiquitin/ proteasome pathway. Therefore, stimulation of cells with the Wnt ligand * Corresponding author at: Division of Applied Pharmacology, Kyushu Dental results in the accumulation of stabilized β-catenin,that enters the nucleus University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan and promotes the expression of to exert a variety of cellular functions E-mail address: [email protected] (H. Takeuchi). [12, 13]. Wnt/β-catenin signaling is also referred to as the canonical Wnt https://doi.org/10.2186/jpr.JPOR_2019_644 signaling pathway. 1883-1958/© 2020 Japan Prosthodontic Society. All rights reserved. Considering that Wnt signaling plays an important role in the regulation 394 F. Aonuma et al. / journal of prosthodontic research 65 (2021) 393–399 of bone homeostasis [11, 14, 15], we examined the effect of C1q on the Images were acquired using an optical microscope equipped with a digital differentiation of both osteoblasts and osteoclasts, and then tried to clarify camera (BZ9000; KEYENCE, Osaka, Japan). whether C1q could modify any cellular signaling, including the Wnt/ β-catenin signaling pathway, in bone metabolizing cells. The results would 2.5. mRNA expression analysis help understand how aging affects the bone condition in the elderly and serve to assess the success of implant treatment using serum C1q level as a Total RNA was isolated from the cells using the RNA extracting novel factor. reagent ISOGEN II (NIPPON , Tokyo, Japan) or NucleoSpinRNA (TakaraBio, Shiga, Japan). Total RNA was reverse transcribed (RT) using 2. Materials and methods ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). Cellular mRNA expression was analyzed by performing semi-quantitative RT- 2.1. Osteoblast differentiation PCR using OneTaq Quick-Load 2 × Master Mix DNA polymerase (NEB, Ipswich MA, USA). The primers used in this study are listed in Table 1. The mouse pre-osteoblast cell line MC3T3-E1 was obtained from the Riken Cell Bank (Ibaraki, Japan) and maintained in α-modified Eagle’s 2.6. β-catenin stabilization assay and Western blotting minimum essential medium (Fujifilm Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum (Biosera, Boussens, France), 100 units/ Bone marrow cells were prepared and cultured for 3 days and were mL penicillin, 100 µg/mL streptomycin (Nacalai tesque, Kyoto, Japan) subjected to a β-catenin stabilization assay, in which the accumulation of in a humidified incubator at 37ºC and 5% CO2. To induce osteogenic β-catenin in the cytoplasmic fraction of the cells was measured to assess the differentiation of MC3T3-E1 cells, the medium was replaced with activation of Wnt/β-catenin signaling. Cells treated with C1q, C1, Wnt3a, differentiation medium [growth medium containing 10 ng/mL bone or LiCl were washed with ice-cold PBS and lysed in a hypotonic media morphogenetic protein (BMP-2; Proteintech, Rosemont, IL)] after the cells buffer (10 mM Tris-HCl, 10 mM NaCl, 2 mM ethylenediaminetetraacetic reached confluence. To examine the effects of human complement C1q and acid pH 7.5) supplemented with a protease inhibitor cocktail (Nacalai C1 (EMD Millipore, Darmstadt, Germany), Wnt3a (R&D Systems Inc., Tesque). The fraction containing the nuclei and intact cells was pelleted Minneapolis, MN), or LiCl (Nacalai Tesque, Kyoto, Japan), osteogenic using centrifugation at 600 × g for 2 minutes at 4°C. The supernatant differentiation was induced in the presence or absence of C1q, C1, Wnt3a, containing the cytoplasmic and membranous fractions was cleared using or LiCl. After three days, osteogenic differentiation was assessed by centrifugation at 15,000 × g for 30 minutes at 4°C, followed by the staining the cells and measuring the alkaline phosphatase activity in the measurement of the protein concentration of the samples using the Protein cell lysates, as described below. Assay BCA Kit (Fujifilm Wako). Equal protein amounts of the samples were boiled in Laemmli’s sample buffer (1% sodium dodecyl sulfate, 1% 2.2. Staining for alkaline phosphatase activity β-mercaptoethanol, 6% glycerol, 0.01% bromophenol blue, and 0.2 M Tris- HCl), and then subjected to standard Western blot analysis using mouse MC3T3-E1 cells, which were cultured in differentiation medium for anti-β-catenin antibody (BD Bioscience, Franklin Lakes, NJ, USA) and three days, were fixed with a 4% paraformaldehyde solution, and ethanol/ mouse anti-β-actin antibody (Sigma-Aldrich). acetone. To assess cell differentiation, after washing with phosphate- Blots were developed with horseradish peroxidase-coupled secondary buffered saline (PBS), cells were stained for alkaline phosphatase activity antibodies and visualized using the enhanced chemiluminescent substrate using a Nitroblue Tetrazolium (NBT)/ 5-bromo-4-chloro-3-indolyl reagent ImmunoStar LD (Fujifilm Wako) or Immobilon (Merck-Millipore, phosphate (BCIP) stock solution (Sigma-Aldrich), according to the Billerica, MA, USA). Images were obtained using a LAS-3000mini manufacturer’s protocol. Images were obtained using a general digital analyzer (Fujifilm, Tokyo, Japan) and the intensity of the protein bands was camera. measured using ImageJ software (National Institute of Health, Bethesda, MD, USA). 2.3 Assay for alkaline phosphatase activity To analyze the RANKL-induced phosphorylation of Akt, bone marrow cells were incubated for 24 h in medium containing M-CSF with or To measure the alkaline phosphatase activity, we used a LabAssay ALP without C1q, C1, Wnt, or LiCl by replacing the medium on day 3. After 24 (Fujifilm Wako) kit, following the manufacturer’s protocol. Briefly, the h of treatment, the cells were washed with phosphate-buffered saline and cells were washed with PBS and solubilized in 20 mM Tris-HCl, pH 7.4, incubated in serum-free medium containing C1q, C1, Wnt, or LiCl for 3 0.2% Triton-X100. After freezing and thawing to disrupt the cells, the h, followed by stimulation with 100 ng/mL RANKL. Then, the cells were cell lysate was clarified using centrifugation at 15,000 × g for 10 minutes lysed in Laemmli’s sample buffer, followed by boiling for 10 minutes. The at 4°C. The cell lysate was mixed with p-nitrophenylphosphate as a following procedure was performed as described above. The antibodies substrate and incubated for 30 minutes at 37°C, followed by an absorbance used were anti-Akt and anti-phospho-Akt (Ser-473) (Cell Signaling measurement at 405 nm to quantify the alkaline phosphatase activity. Technology, Danvers, MA, USA).

2.4. Osteoclast formation 2.7. Assay for cell viability

Bone marrow cells were isolated from the thigh and shinbones of 6 The proliferation and viability of the cells were evaluated using a WST- – 10-weeks-old male mice (strain ddY, which stands for Deutschland, 8 [2-(2-methoxy-) 4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- Denken, and Yoken) and were cultured in α-modified Eagle’s minimum 2H-tetrazolium]-based colorimetric assay using a Cell Counting Kit- essential medium supplemented with 10% fetal bovine serum (Sigma- 8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan), by following the Aldrich), 100 units/mL penicillin, 100 µg/mL streptomycin, 10,000 units/ manufacturer’s instructions. MC3T3-E1 cells plated at a density of 5 × 103 mL macrophage colony-stimulating factor (M-CSF) (KYOWA KIRIN, cells/well or BMMs in 96-well plates were treated with or without C1q, Tokyo, Japan), and 10 ng/mL receptor activator of NF-kB ligand (RANKL) C1, Wnt3a, or LiCl for 48 - 72 h, and then subjected to the colorimetric

(Fujifilm Wako) in 96-well plates for two days at 37ºC and 5% CO2 to assay. To analyze the number of viable cells, WST-8 solution was directly obtain pre-osteoclast bone marrow-derived macrophages (BMMs). Then, added to the wells and these were incubated at 37°C for 2 h, followed by the medium was replaced with medium containing 10,000 units/mL the measurement of the absorbance at 450 nm. M-CSF and 100 ng/mL RANKL on days 3 and 5 in the presence or absence 2.8. Statistical analysis of C1q, C1, Wnt3a, or LiCl. After seven days of culture, osteoclastogenic differentiation was assessed using staining for tartrate-resistant acid Unless otherwise indicated, data are expressed as mean + SEM. Statistical phosphatase (TRAP) activity of the cells. For this, a TRAP/ALP staining analysis of data was performed using one-way analysis of variance kit (Fujifilm Wako) was used, following the manufacturer’s protocol. (ANOVA) among means, followed by post-hoc comparisons to determine F. Aonuma et al. / journal of prosthodontic research 65 (2021) 393–399 395

Table 1. Primers used for RT-PCR. Target Forward Reverse Lrp5 5’-AAGGGTGCTGTGTACTGGAC-3’ 5’-AGAAGAGAACCTTACGGGACG-3’ Lrp6 5’-TTGTTGCTTTATGCAAACAGACG-3’ 5’-GTTCGTTTAATGGCTTCTTCGC-3’ Fzd1 5’-CAGCAGTACAACGGCGAAC-3’ 5’-GTCCTCCTGATTCGTGTGGC-3’ Fzd2 5’-GCCGTCCTATCTCAGCTATAAGT-3’ 5’-TCTCCTCTTGCGAGAAGAACATA-3’ Fzd3 5’-ATGGCTGTGAGCTGGATTGTC-3’ 5’-GGCACATCCTCAAGGTTATAGGT-3’ Fzd4 5’-TGCCAGAACCTCGGCTACA-3’ 5’-ATGAGCGGCGTGAAAGTTGT-3’ Fzd5 5’-CCAAACCTACGCTCCCAGG-3’ 5’-CGCACCTTGTTGTAGAGTGG-3’ Fzd6 5’-ATGGAAAGGTCCCCGTTTCTG-3’ 5’-GGGAAGAACGTCATGTTGTAAGT-3’ Fzd7 5’-CGGGGCCTCAAGGAGAGAA-3’ 5’-GTCCCCTAAACCGAGCCAG-3’ Fzd8 5’-ATGGAGTGGGGTTACCTGTTG-3’ 5’-CACCGTGATCTCTTGGCAC-3’ Fzd9 5’-GTGCCAAGCGATGGAGATCC-3’ 5’-GCGTAGAGCGAGCAGAAGAA-3’ Fzd10 5’-CATGCCCAACCTGATGGGTC-3’ 5’-GCCACCTGAATTTGAACTGCTC-3’ Actb 5’-GCCAACCGTGAAAAGATGACC-3’ 5’-TGCCAATAGTGATGACCTGGCC-3’

the significant differences between groups using Tukey-Kramer HSD was enhanced by BMP-2, and it was further promoted by activating the (honestly significant difference) test. JMP 15 software (SAS Institute, Cary, canonical Wnt signaling pathway with either Wnt3a or LiCl. However, NC, USA) was used for the statistical analysis. neither C1q nor C1 promoted alkaline phosphatase activity in MC3T3-E1 cells. On the other hand, both C1q and C1 suppressed osteogenic 3. Results differentiation at 50 µg/mL, the highest concentration examined in the study (Fig. 2A and 2B). To confirm the effects of each compound on 3.1. Expression of Wnt receptor genes in bone marrow-derived pre- cell proliferation and viability, the cells were cultured in the presence or osteoclasts and MC3T3-E1 cells absence of various concentrations of C1q, C1, Wnt3a, or LiCl for 3 days and were subjected to a tetrazolium-based colorimetric assay. Neither C1q Expression patterns of all genes encoding Wnt receptor components in nor C1 significantly affected cell viability, even at a concentration of 50 µg/ various mice tissues and cultured osteoblasts and osteoclasts have already mL, the highest concentration examined (Fig. 2C), suggesting that a high been reported by Albers et al. [16]. They showed that Fzd1, 2, 3, 7, 8, concentration of C1q has an inhibitory effect on osteogenic differentiation 9, and Lrp5 and 6 were expressed at levels comparable to those in other of osteoblasts. tissues in osteoblasts, whereas only Fzd8 and Lrp5, and 6 were detected in Then, we performed a β-catenin stabilization assay to test whether C1q osteoclasts. To investigate the direct effects of C1q on the differentiation activates the canonical Wnt signaling pathway in MC3T3-E1 cells. Wnt3a of bone-metabolizing cells with special reference to the Wnt signaling and LiCl were used as positive controls; Wnt3a is a physiological ligand pathway, we first examined the mRNA expression of Wnt receptors in that activates Wnt signaling by binding to the receptor Fzd, and LiCl is an both the pre-osteoblast and pre-osteoclast cells used in this study. Mouse inhibitor of GSK3β, which mimics Wnt/β--catenin signaling by stabilizing BMMs, which are bone marrow cells cultured for two days in the presence β-catenin. MC3T3-E1 cells were incubated with various concentrations of M-CSF and a low concentration of RANKL (10 ng/mL), and a mouse of C1q, C1, Wnt3a, and LiCl for 3 h and analyzed for the amount of calvaria-derived cell-line, MC3T3-E1, were used as precursor cells for cytoplasmic β-catenin using Western blotting. The amount of β-catenin was osteoclasts and osteoblasts, respectively. Total RNA was prepared from robustly increased in cells treated with Wnt3a or LiCl in a dose-dependent these cells, followed by examination of the gene expression of Wnt manner (Fig. 3, bar graph). However, we could scarcely detect cytoplasmic receptors, that is, Lrp5, Lrp6, and Fzd1 - Fzd10. As shown in Fig. 1, the β-catenin accumulation in cells treated with either C1q or C1 (Fig. 3). detected expression of both Lrp5 and Lrp6 was at an equivalent level to that in BMMs and MC3T3-E1 cells. MC3T3-E1 cells expressed a variety 3.3. Effects of C1q on osteoclast formation in BMM culture of Fzd family genes at relatively high levels, whereas the expression level of Fzd was very low in BMMs. Fzd9 was detected in BMMs, but Then, we examined the effects of exogenously added C1q on osteoclast not in MC3T3-E1 cells. We did not detect Fzd2, 7, or 10 at all, even formation in mouse BMMs. Mouse BMMs were induced to differentiate when the PCR amplification was extended to 40 cycles in either BMMs into osteoclasts by RANKL. in the presence or absence of various or MC3T3-E1 cells (data not shown). Although the mRNA expression of concentrations of C1q, C1, Wnt3a, or LiCl. Although the total number of Fzd8, which is the primary receptor of C1q [10], was detected in both cell multinucleated TRAP-positive cells was not affected by 5 to 15 µg/mL C1q types, its expression level was very low in BMMs. No PCR products were and C1, the proportion of large osteoclasts was increased in the presence of detected in the samples in which reverse-transcriptase was omitted during C1q and C1, while the proportion of small osteoclasts decreased, resulting the reverse-transcription treatment, indicating that there was no genomic in an apparent increase in the average cell size (Fig. 4B and 4C). This result DNA contamination in the preparation of the RNA samples. suggested that the cell fusion of osteoclasts was enhanced by C1q and C1. However, C1q and C1 did not promote osteoclast formation, but rather 3.2. Effect of C1q on Wnt signaling and osteogenic differentiation of suppressed osteoclast formation at 50 µg/mL, the highest concentration MC3T3-E1 cells used in this study (Fig. 4B). Treatment of the cells with Wnt3a and LiCl, both of which are known to activate Wnt/β-catenin signaling, did not As a high expression level of the candidate receptor of C1q was significantly affect RANKL-induced osteoclast formation (Fig. 4B and 4C). detected in MC3T3-E1 cells, we first examined the effects of C1q and C1 To examine whether a high concentration of the complement suppressed on the osteogenic differentiation of MC3T3-E1 cells. This was assessed osteoclast formation by inhibiting cell growth, the cells were cultured for by quantifying the increase in alkaline phosphatase activity, which was 48 h, followed by assessment of the viable cell number via a colorimetric compared with the results following Wnt3a and LiCl treatments, both of assay. The number of viable cells was decreased with 50 µg/mL of either which are known to promote osteoblast differentiation [11]. As shown in C1q or C1 but with neither Wnt3a nor LiCl (Fig. 4D), suggesting that Fig. 2A and 2B, the alkaline phosphatase activity associated with the cells exogenously added C1q and C1 at 50 µg/mL are both cytotoxic to BMMs 396 F. Aonuma et al. / journal of prosthodontic research 65 (2021) 393–399

Fig. 1. Expression of Wnt receptors in MC3T3-E1 cells and BMMs Total RNA was extracted from BMMs and MC3T3-E1 cells and subjected to reverse transcription reaction with (+) or without (-) reverse transcriptase (RT), followed by general PCR using primers for the indicated genes. Fig. 2. The effect of C1q on differentiation and proliferation of MC3T3-E1 cells (A) MC3T3-E1 cells were induced to differentiate into osteoblasts with BMP-2 in the presence or absence of increasing concentrations of C1q, C1, Wnt3a, and at similar levels. LiCl. Representative images of cells stained for alkaline phosphatase activity on day 4 are shown. The concentrations indicated as 0, 1, 2, and 3, are 0, 5, 15, and 3.4. Effects of C1q on osteoclast formation in BMM culture 50 μg/ml for C1 and C1q; 0, 10, 30, and 100 ng/ml for Wnt3a; and 0, 1, 3, and 10 mM for LiCl, respectively. (B) MC3T3-E1 cells were induced to differentiate We next examined whether C1q can activate Wnt/β-catenin signaling in into osteoblasts as in (A). The alkaline phosphatase activity of cell lysates from each well is expressed as the percentage of the control (white bar), in which cells osteoclast precursor cells. Freshly prepared mouse bone marrow cells were were cultured in the absence of BMP2. The bar graph shows the mean values + cultured for three days in the presence of M-CSF and then treated with SEM. *P < 0.05 and **P < 0.01 versus cells cultured in the absence of BMP- C1q, C1, Wnt3a, or LiCl with or without RANKL, followed by analysis of 2 (white bar), #P < 0.01 versus control cells cultured in the presence of BMP- canonical Wnt signal activation using a β-catenin stabilization assay. When 2. (C) To assess the cell proliferation of MC3T3-E1 cells, the cells were seeded the cells were treated with Wnt3a or LiCl alone, accumulation of β-catenin at 5,000 cells per well in 96-well plates, and viable cells were estimated by a was observed, starting at 1 h, and the amount did not decrease until 6 h. colorimetric assay. The effect of compounds on cell proliferation and viability was confirmed by incubating the cells for two days in the presence of the In contrast, neither C1q nor C1 induced β-catenin accumulation (Fig. indicated concentrations of C1q, C1, Wnt3a, or LiCl, followed by estimation of 5A), indicating that neither C1q nor C1 could activate the canonical Wnt the viable cell number using a colorimetric assay. The data are expressed as the signaling pathway on their own. means + SEM of three independent experiments performed in triplicate and as Considering that C1q was recently reported to activate phosphoinositide the percentage of the control, which was cultured in the absence of the indicated 3-kinase/Akt signaling by directly binds to a collagen receptor, namely, compounds. No significant differences were observed. discoidin domain receptor 1 (DDR1) [17], we next investigated the effect of C1q on Akt signaling, which is also involved in the modification of osteoclastogenic differentiation [18, 19]. BMMs were pre-incubated with or without C1q, C1, Wnt3a, or LiCl for 24 h, followed by stimulation with RANKL. While pretreatment did not promote Akt phosphorylation without RANKL stimulation (Time 0 min), RANKL-induced phosphorylation of Akt was enhanced by pretreating the cells with C1q or C1 (Fig. 5B). Pretreatment of the cells with Wnt3a or LiCl did not affect the RANKL- induced phosphorylation level of Akt (Fig. 5B).

4. Discussion

The main purpose of this study was to examine the effect of complement C1q on the differentiation of bone-metabolizing cells. Here, we showed that exogenously added C1q at a concentration of up to 15 µg/mL promotes RANKL-induced cell fusion of osteoclasts without affecting the osteogenic differentiation of MC3T3-E1 cells, while at 50 µg/mL, the highest concentration examined in the study, C1q suppressed both osteogenic and osteoclastogenic differentiation. The inhibitory effects Fig. 3. Activation of Wnt/β-catenin signaling by C1q in MC3T3-E1 cells of C1q at 50 µg/mL on the differentiation of osteoclasts can be partially MC3T3-E1 were treated with indicated concentrations of C1q, C1, Wnt3a, or explained by the inhibitory effects of C1q on cell survival, as observed in LiCl for 3h. Next, the cell lysates were subjected to Western blotting to detect the colorimetric assays. However, as the proliferation of MC3T3-E1 cells β-catenin and β-actin. Representative images of the blots are shown in the top panel. The densities of the β-catenin bands were normalized with those of β-actin was not significantly suppressed by C1q alone or by the C1 complex, the and expressed as the percentage of the non-treated control in the graph. Data are mechanisms underlying the strong inhibition of osteoblast and osteoclast shown as the means + SEM from three independent experiments. *P < 0.05 and differentiation by C1q are currently unclear. **P < 0.01 versus control cells (None). Although the concentration of endogenous C1q in both human and murine serum was estimated to be approximately 130 µg/mL [8], there F. Aonuma et al. / journal of prosthodontic research 65 (2021) 393–399 397

Fig. 4. The effect of C1q on osteoclast formation and viability of BMMs Fig. 5. Effect of C1q on Wnt/β-catenin and Akt signaling (A) BMMs were induced to differentiate into osteoclasts in the presence or (A) BMMs were stimulated with C1q (15 μg/ml), C1 (15 μg/ml), Wnt3a (100 absence of the indicated concentrations of C1q, C1, Wnt3a, or LiCl following the ng/ml), or LiCl (10 mM) for the indicated time periods, followed by Western schedule summarized in the materials and methods section. The images represent blot analyses of the amount of β-catenin in the cytosol. The relative amount of cells stained for TRAP activity on day 7. Scale bars, 300 μm. (B) The numbers of β-catenin was estimated from the density of the bands. The data were normalized TRAP-positive multinucleated cells containing more than 3 nuclei in each well with that for β-actin, and they are expressed as the means + SEM of three were counted, and they are presented as the means + SEM of ten wells from two independent experiments. **P < 0.01 versus nontreated control cells. (B) BMMs independent experiments. *P < 0.05 and **P < 0.01 versus control cells. (C) The were pre-incubated with C1q (15 μg/ml), C1 (15 μg/ml), Wnt3a (30 ng/ml), or area of individual TRAP-positive multinucleated cells were measured using the LiCl (3 mM) for 24 h and then serum-starved for 3 h, followed by stimulation ImageJ software, and the total area was divided by the cell number to obtain the with 100 ng/ml of RANKL for the indicated time periods. Total cell lysates were average OC size in each well. The data represents the mean + SEM of ten wells subjected to Western blotting, and the amount of phosphorylated Akt and total from two independent experiments. *P < 0.05 and **P < 0.01 versus control Akt were estimated by the density of the bands. The level of phosphorylated Akt cells. (D) BMMs in 96-well plates were cultured in the presence of the indicated was normalized with that of total Akt, and it is expressed as the means + SEM concentrations of C1q, C1, Wnt3a, or LiCl for 48 h. Next, WST-8 solution of more than three independent experiments. *P < 0.05 and **P < 0.01 versus was added to each well and incubated for 2 h, followed by measurement of the control cells with each pre-treatment but without RANKL-stimulation. #P < 0.01 absorbance versus cells without any pretreatment (None) but stimulated with RANKL for the at 450 nm to assess the viable cell number. The data represents the means + SEM corresponding time period. of three independent experiments performed in triplicate, and they are expressed as the percentage of control cells. *P < 0.05 and **P < 0.01 versus control cells. called “coupling factor,” which mediates the coupling of bone resorption to its subsequent formation during the bone remodeling process [22]. are various C1q-binding molecules that buffer cells from the toxic effects Studies have shown that the expression level of C3 increases during of high concentrations of C1q in the body unlike in vitro tissue culture osteoclastogenesis and that mature osteoclasts release , the cleavage conditions [20]. Endogenous C1q-binding molecules, such as C1 inhibitor, product of C3, which stimulates osteoblastogenesis by binding to the are known to regulate C1q functions in the body, that is, the concentration C3a receptor on the cell surface of osteoblasts. Sato et al. reported of C1q that is available to modify bone homeostasis is determined by the that C3 was also produced in osteoblasts [23]; it potentiates M-CSF- balance between the levels of C1q and C1q-binding molecules. Yonemasu et dependent proliferation of bone marrow cells and induction of osteoclast al. reported that the serum level of C1q increased steadily to approximately differentiation [24]. Furthermore, it was shown that C3 deficiency 160 µg/mL with aging after the 40 years of age [7, 8], indicating that reduced bone loss in ovariectomized mice [25]. Another complement an excess of up to 30 µg/mL C1q might exist in people above 40 years component, C5, was reported to elicit osteoblast chemotaxis to the of age. Therefore, the concentration of C1q that promoted osteoclast fracture callus [26]. Although complement components, other than fusion in this study, 5 - 15 µg/mL, would be physiologically reasonable. C1q, are involved in bone homeostasis, the serum levels of these In addition to aging, previous studies have investigated the relationship components do not change as dramatically with aging as that of C1q between the complements and bone metabolism. Teo et al. [21] reported [8].culture; however, the underlying mechanisms remain unresolved. that C1q is expressed in osteoclasts and enhanced osteoclastogenesis Another component, C3a, was also identified as a candidate for the so- inculture; however, the underlying mechanisms remain unresolved. called “coupling factor,” which mediates the coupling of bone resorption Another component, C3a, was also identified as a candidate for the so- to its subsequent formation during the bone remodeling process [22]. 398 F. Aonuma et al. / journal of prosthodontic research 65 (2021) 393–399

Studies have shown that the expression level of C3 increases during osteoclastogenesis and that mature osteoclasts release C3a, the cleavage product of C3, which stimulates osteoblastogenesis by binding to the C3a receptor on the cell surface of osteoblasts. Sato et al. reported that C3 was also produced in osteoblasts [23]; it potentiates M-CSF- dependent proliferation of bone marrow cells and induction of osteoclast differentiation [24]. Furthermore, it was shown that C3 deficiency reduced bone loss in ovariectomized mice [25]. Another complement component, C5, was reported to elicit osteoblast chemotaxis to the fracture callus [26]. Although complement components, other than C1q, are involved in bone homeostasis, the serum levels of these components do not change as dramatically with aging as that of C1q [8]. Despite the high expression level of multiple Fzd genes, we did not Fig. 6. Effect of aging-dependent increase in serum C1q level on cellular observe a significant activation of Wnt/β-catenin signaling by C1q in both signaling and the success of dental implant treatment, as supposed by the results osteoblast and osteoclast precursor cells. Wnt3a, a physiological ligand for from this study the receptors, and LiCl, which bypasses the Wnt receptor to activate the (A) Moderately increased C1q with age upregulates bone resorption by canonical Wnt signaling pathway, showed robust accumulation of β-catenin accelerating osteoclast fusion. Next, considerable increase in C1q with further in this study, indicating that the Wnt/β-catenin pathway was functional in aging leads to impairment of bone remodeling by inhibiting the differentiation both MC3T3-E1 and BMMs. of both osteoblasts and osteoclasts. Therefore, aging-dependent increase in C1q level may affect the prognosis of dental implant treatment. (B) When Wnt Naito et al. used 50 and 100 µg/mL C1q to activate Wnt signaling signaling is in inactive state, β-catenin is phosphorylated by constitutively active in several types of cells and in subsequent experiments, including the GSK3β followed by continuous degradation through the ubiquitin/proteasomal measurement of the regenerative capacity of skeletal muscles [10]. In pathway. Upon binding of Wnt ligand to a component of its receptor, Frizzled, the present study, although 50 µg/mL of C1q strongly inhibited the canonical Wnt signaling is activated leading to inactivation of GSK3β followed differentiation of both osteoblasts and osteoclasts in our study, we could by releasing β-catenin from the continuous degradation. Then the stabilized not detect the activation of the canonical Wnt signaling pathway by C1q. β-catenin is accumulated in the cell and enters the nucleus to promote gene 23 expression. While it was shown that C1q increases with age and activates Wnt/ As the enzymatic activity of another component, C1s, could be required for β-catenin signaling by directly binding to Frizzled in cells of the skeletal muscle C1q to activate Wnt signaling [10], we additionally used the C1 complex, and vascular smooth muscle cells, activation of canonical Wnt signaling was which consists of C1q, C1r, and C1s, in parallel with C1q alone. However, not observed in precursors of osteoblasts and osteoclasts in this study. Instead, there were no differences between C1q alone and the C1 complex C1q upregulated RANKLinduced Akt activation that could be one candidate regarding the activation of Wnt/β-catenin signaling and the effects on the mechanism how C1q promoted osteoclast differentiation. differentiation of osteoblasts and osteoclasts, ensuring that C1q could not activate Wnt/β-catenin signaling in either osteoblast or osteoclast precursor cells. Currently, the reasons for the inability of C1q to activate canonical osseointegration of dental implants in elderly patients. Wnt signaling in these bone-metabolizing cells is completely obscure. Although we did not detect an obvious activation of canonical Wnt 5. Conclusion signaling pathway, C1q could modify RANKL-induced osteoclast differentiation. Therefore, we investigated whether C1q affects any cellular The results of the present study suggest that complement component signaling pathway involved in osteoclast differentiation. We found that the C1q is known to increase with age after 40 years of age, affecting cellular treatment of pre-osteoclast cells with C1q enhanced the RANKL-induced signaling and the differentiation of osteoclasts and osteoblasts, leading to a phosphorylation of Akt, which is known to promote osteoclast formation reduced bone remodeling. The serum level of C1q in the elderly could be [18, 19]. We treated the cells with a specific inhibitor of Akt (MK-2206) considered as a factor that influences osseointegration of dental implants. or with its upstream molecule, phosphoinositide 3-kinase (LY294002), in the presence or absence of C1q to clarify whether Akt is involved in Acknowledgments the function of C1q, but this experiment failed because RANKL-induced osteoclast differentiation was impaired by these inhibitors, even in the The present study was supported by Japan Society for the Promotion control cells (data not shown). Thus, the involvement of Akt in the effect of of Science (KAKENHI grants 18K17154 to F.A., 18H02995 to R.H. and C1q on RANKL-induced osteoclast differentiation remains to be clarified. 17K11649 to H.T.); and the grant-in-aid from Kyushu Dental University Taken together, the results of this study suggest that a moderate increase Internal Grants (to HT). in C1q with age suppressed the normal bone remodeling by enhancing bone resorption by osteoclasts; a further increase in C1q inhibits the Conflict of interest statement differentiation of both osteoblasts and osteoclasts, leading again to an impaired bone remodeling (Fig. 6A). Although C1q has been shown to The authors reported no conflicts of interest related to this study. activate canonical Wnt signaling in other tissues, neither C1q nor C1 complex activated the canonical Wnt signaling pathway in osteoblasts Animal Studies or osteoclasts in this study. Therefore, the molecular mechanisms by which C1q affects bone-metabolizing cells and aging-dependent changes This study was approved by and performed in accordance with in muscles [10] would be different, meaning that the precise mechanism guidelines of the institutional animal research ethics committee of Kyushu by which C1q affects osteoclast fusion remains unclear and needs to Dental University (Approval number, 16-013). be clarified in the future by, for example, performing a comprehensive analysis of gene expression and post-translational modification of proteins, References in addition to verifying a possible mechanism in which the RANKL- induced activation of Akt is promoted by C1q, as observed in this study [1] Ikebe K, Wada M, Kagawa R, Maeda Y. Is old age a risk factor for dental implants? J Dent Sci Rev 2009;45:59-64. (Fig. 6B). The effect of C1q on osteoclasts and osteoblasts observed in this [2] Reid KB. Activation and control of the complement system. Essays Biochem study should be separated from the role of C1q in the complementsystem, 1986;22:27-68. because the complement components included in the serum were heat- [3] Walport MJ. Complement. First of two parts. N Engl J Med 2001;344:1058-66. inactivated, and both C1q alone and the C1 complex showed comparable [4] Nayak A, Pednekar L, Reid KB, Kishore U. Complement and non-complement activating functions of C1q: a prototypical innate immune molecule. Innate effects on bone-metabolizing cells. Understanding the precise role of Immun 2012;18:350-63. C1q in bone remodeling may provide new strategies to improve the F. Aonuma et al. / journal of prosthodontic research 65 (2021) 393–399 399

[5] Bohlson SS, O'Conner SD, Hulsebus HJ, Ho MM, Fraser DA. Complement, [18] Moon JB, Kim JH, Kim K, Youn BU, Ko A, Lee SY, et al. Akt induces c1q, and c1q-related molecules regulate macrophage polarization. Front osteoclast differentiation through regulating the GSK3beta/NFATc1 signaling Immunol 2014;5:402. cascade. J Immunol 2012;188:163-9. [6] Ghiran I, Tyagi SR, Klickstein LB, Nicholson-Weller A. Expression and [19] Wu M, Chen W, Lu Y, Zhu G, Hao L, Li YP. Galpha13 negatively controls function of C1q receptors and C1q binding proteins at the cell surface. osteoclastogenesis through inhibition of the Akt-GSK3beta-NFATc1 signalling Immunobiology 2002;205:407-20. pathway. Nat Commun 2017;8:13700. [7] Watanabe S, Sato K, Hasegawa N, Kurihara T, Matsutani K, Sanada K, et [20] Nicholson-Weller A, Klickstein LB. C1q-binding proteins and C1q receptors. al. Serum C1q as a novel biomarker of sarcopenia in older adults. FASEB J Curr Opin Immunol 1999;11:42-6. 2015;29:1003-10. [21] Teo BH, Bobryshev YV, Teh BK, Wong SH, Lu J. Complement C1q production [8] Yonemasu K, Kitajima H, Tanabe S, Ochi T, Shinkai H. Effect of age on C1q by osteoclasts and its regulation of osteoclast development. Biochem J and C3 levels in human serum and their presence in colostrum. Immunology 2012;447:229-37. 1978;35:523-30. [22] Matsuoka K, Park KA, Ito M, Ikeda K, Takeshita S. Osteoclast-derived [9] Eastell R, O'Neill TW, Hofbauer LC, Langdahl B, Reid IR, Gold DT, et al. complement component 3a stimulates osteoblast differentiation. J Bone Miner Postmenopausal osteoporosis. Nat Rev Dis Primers 2016;2:16069. Res 2014;29:1522-30 [10] Naito AT, Sumida T, Nomura S, Liu ML, Higo T, Nakagawa A, et al. [23] Sato T, Hong MH, Jin CH, Ishimi Y, Udagawa N, Shinki T, et al. The specific Complement C1q activates canonical Wnt signaling and promotes aging-related production of the third component of complement by osteoblastic cells treated phenotypes. Cell 2012;149:1298-313. with 1 alpha,25-dihydroxyvitamin D3. FEBS Lett 1991;285:21-4. [11] Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell [24] Sato T, Abe E, Jin CH, Hong MH, Katagiri T, Kinoshita T, et al. The 2012;149:1192-205 biological roles of the third component of complement in osteoclast formation. [12] Huang HC, Klein PS. The Frizzled family: receptors for multiple signal Endocrinology 1993;133:397-404. transduction pathways. Genome Biol 2004;5:234. [25] MacKay DL, Kean TJ, Bernardi KG, Haeberle HS, Ambrose CG, Lin F, et al. [13] MacDonald BT, He X. Frizzled and LRP5/6 receptors for Wnt/beta-catenin Reduced bone loss in a murine model of postmenopausal osteoporosis lacking signaling. Cold Spring Harb Perspect Biol 2012;4. . J Orthop Res 2018;36:118-28. [14] Baron R, Kneissel M. WNT signaling in bone homeostasis and disease: from [26] Ignatius A, Ehrnthaller C, Brenner RE, Kreja L, Schoengraf P, Lisson P, et al. human mutations to treatments. Nat Med 2013;19:179-92. The anaphylatoxin receptor C5aR is present during fracture healing in rats and [15] Nusse R, Clevers H. Wnt/beta-Catenin Signaling, Disease, and Emerging mediates osteoblast migration in vitro. J Trauma 2011;71:952-60. Therapeutic Modalities. Cell 2017;169:985-99. [16] Albers J, Keller J, Baranowsky A, Beil FT, Catala-Lehnen P, Schulze J, et al. Canonical Wnt signaling inhibits osteoclastogenesis independent of osteoprotegerin. J Cell Biol 2013;200:537-49. [17] Lee JH, Poudel B, Ki HH, Nepali S, Lee YM, Shin JS, et al. Complement C1q stimulates the progression of hepatocellular tumor through the activation of discoidin domain receptor 1. Sci Rep 2018;8:4908.