Abstract
Background: Osteocytes, which comprise over 90% of all bone cells, communicate with osteoblasts and osteoclasts to regulate each other's physiological function via dendrites, suggesting that dendrite elongation plays a vital role for bone regeneration. We examined the effect of semaphorin 3A (SEMA3A) on dendritic processes of an osteocyte cell line, since in previous work we found it to be essential for promoting osteoblast differentiation. Materials and Methods: Dendrite length was analyzed by Cellomics Array Scan VTI quantitatively in osteocyte-like cell line, MLO-Y4 cells. We performed cell proliferation assay. Gene and protein expression was examined by real-time reverse-transcriptase polymerase chain reaction and western blotting, respectively. Results: Both total and average dendrite length were significantly increased in MLO-Y4 cells stimulated with SEMA3A compared to control. E11 protein was up-regulated upon SEMA3A stimulation. Moreover, cyclin-dependent kinase 6 (CDK6) was down-regulated in a time-dependent manner. Taken together, these results suggest that SEMA3A regulates dendrites of osteocytes in association with down-regulation of CDK6. SEMA3A may be a promising drug to apply for bone tissue engineering.
Bone tissue is constantly being remodeled to maintain its homeostasis by bone cells under control of hormones, growth factors, nervous system, and mechanical loading (1-3). Bone cells comprise of osteoclasts which resorb bone, osteoblasts which build bone, and osteocytes which orchestrate osteoblasts and osteoclasts (4). Osteocytes, which make up over 95% of bone cells in the adult skeleton, occupy the lacunar space and are surrounded by the bone matrix (4, 5). Numerous cellular projections of the embedding osteocytes are extended toward the vascular space or bone surface. Their dendritic processes are regulated by mechanical loading through the hemichannels of osteocytes (6). Furthermore, E11/glycoprotein 38(GP38), which is a transmembrane glycoprotein expressed in early osteocytes, is found to be increased after mechanical loading and small interfering RNA-mediated knockdown of E11 prevented the fluid flow-induced dendritic processes of an osteocyte-like cell line (7). Additionally, the upregulation of E11 plays a vital role for osteocyte maturation (8).
We have shown that semaphorin 3A (SEMA3A), which is a diffusible axonal chemorepellent that has an important role in axon guidance, regulates bone remodeling indirectly by modulating sensory nerve development (9, 10). SEMA3A exerts an osteoprotective effect by both suppressing bone resorption and increasing bone formation. Osteoblast differentiation is stimulated by SEMA3A through activation of the canonical wingless-type MMTV integration site family/β-catenin pathway (11).
A temporal cell-cycle arrest in G1 phase is essential for cell differentiation (12). Cyclin-dependent kinase (CDK) 4, and 6, can form complexes with cyclin D to induce progression of the cell cycle (13). Emerging evidence demonstrates that down-regulation of CDK6 is critical for differentiation of various cells (14-17).
We hypothesized that SEMA3A promotes osteocyte maturation because it exerts a positive effect on osteoblast differentiation. Thus, in this study, we examined the effect of SEMA3A on dendritic processes of osteocytes and the involvement of CDK6.
Materials and Methods
Cell culture and reagents. MLO-Y4 cells, kindly provided by Dr. Lynda F. Bonewald (The University of Missouri), were cultured on collagen-coated (rat tail collagen type I; 0.15 mg/ml) surfaces and grown in α-modified Eagle's medium (WAKO, Osaka, Japan) supplemented with 2.5% fetal bovine serum (GE Healthcare, Chicago, IL, USA) and 2.5% calf serum (GE Healthcare) containing antibiotics (growth medium). Cells were inoculated on plates or dishes at 3×104 cells per cm2. For the serum-starved experiment, after seeding, the cells were allowed to proliferate for 24 h. Their growth was then arrested by incubation for 12 h in growth medium without serum. Recombinant mouse SEMA3A (rmSEMA3A) was purchased from R&D company (Minneapolis, MN, USA). The serum-starved cells were treated with rmSEMA3A (500 ng/ml) for 1, 3, or 6 h.
Crystal violet staining. The cells were fixed with 10% formalin for 5 min to stain with crystal violet (0.05%) after being cultured for 24 h. Images were taken using an inverted microscope (Olympus IX70, Tokyo, Japan) to visualize the morphology of dendrites in MLO-Y4 cells.
Cell proliferation assay. The cells were incubated in conditioned medium for 48 h with rmSEMA3A (15, 31, 62, 125, 250, 500, or 1,000 ng/ml) or vehicle (phosphate-buffered saline, PBS) for 48 h. The sample cells were quantified using a Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Fitchburg, WI, USA), according to the manufacturer's instructions. Measurements were made of six replicates.
Quantitative analysis of dendrite elongation. The cells were inoculated on 8 mm slide chambers (BD Biosciences, Franklin Lakes, NJ, USA) in the presence of rmSEMA3A (500 ng/ml) or vehicle (PBS) for 24 h and subsequently washed twice with PBS and fixed in 4% paraformaldehyde at room temperature for 10 minutes. Fixed cells were then permealized with RIPA lysis buffer (Santa Cruz, Dallas, TX, USA) containing protease inhibitor cocktail for 5 min, then blocked in 1% bovine serum albumin at room temperature for 1 h. After blocking, cells were stained with Alexa-488-conjugated phalloidin and counterstained with Vectashield Mounting Medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Indices of dendrite elongation were quantified using the Cellomics ArrayScan VTI high content imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Mean dendrite average length (μm) and mean dendrite total length (μm) were analyzed. Acquired images were analyzed using a dedicated proprietary algorithm (Thermo Fisher Scientific, Waltham, MA, USA).
Quantitative reverse-transcriptase polymerase chain reaction (qPCR). Total RNA was extracted from treated cells using ISOGEN (Nippon Gene,Tokyo, Japan) and qPCR was conducted as previously described (18). For qPCR of E11, we used SYBR green-based technique using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan). The gene-specific primer pairs used were as follows: E11 sense, 5’-GAGGAACTGTCCACCTCAGC-3’ and antisense, 5’-ATGGCTAACAAGACGCCAAC-3’; β-actin (ACTB) sense, 5’-AGAAGGACTCCTATGTGGGTGA-3’ and antisense, 5’-CATGATCTGGGTCATCTTTTCA-3’. For qPCR analysis, values were normalized to ACTB using the 2_ΔΔCt method.
Western blot analysis. Western blot analysis was performed as follows. Briefly, the treated cells were rinsed with ice-cold PBS and lysed with a lysis buffer (1% Triton-X 100, 150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 10 μg/ml aprotinin, 0.1 M NaF, 2 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride). The lysates were incubated on ice for 20 min and centrifuged at 15,000 × g for 5 min at 4°C. Equal amounts of proteins were separated by sodium dodecyl sulfate -polyacrylamide gel electrophoresis with Mini-Protean Gel TGX, 8-16% and electroblotted for 30 min at 200 V using the Bio-Rad Trans Blot system onto Mini polyvinylidene fluoride transfer membranes (Bio Rad, Redmond, WA, USA). After the blocking of nonspecific binding by soaking the filters in 5% skim milk, the desired proteins were immunodetected with their respective antibodies. The membrane was developed using Clarity Western ECL Substrate (Bio Rad), according to the manufacturer's instructions. The bands were visualized using a Chemi-Doc XRS system (Bio-Rad). We used anti-goat poyclonal antibody for E11 (R&D), anti-mouse monoclonal antibody for CDK6 [DCS-83] (Santa Cruz), and anti-rabbit polyclonal antibody for CDK4 [C-22] (Santa Cruz). We used donkey anti-goat IgG-horseradish peroxidase (HRP) (sc-2020, Santa Cruz), anti-rabbit IgG HRP-linked antibody (Cell Signaling, Danvers, MA, USA), and donkey anti-mouse IgG-HRP (Santa Cruz) as secondary antibody. Anti-β-actin mouse monoclonal antibody (Santa Cruz) was used as a loading control.
Statistical analysis. Comparisons between two groups were analyzed with the Student's t-tests (p<0.05). All values are represented as the mean±S.E.M.
Results
SEMA3A promotes dendrite elongation in MLO-Y4 cells. We examined whether SEMA3A promotes dendrite elongation in the MLO-Y4 osteocyte cell line. SEMA3A was added to the culture medium of MLO-Y4 and cells were stained with crystal violet. After 24 h, dendrite elongation of these cells was observed (Figure 1A). Then we examined dendrite elongation quantitatively. We used Cellomics ArrayScan VTI and found that both the mean average dendrite length and mean total dendrite length of SEMA3A-treated cells were significantly increased compared to the control (Figure 1B). Additionally, we examined whether SEMA3A affects cell proliferation of MLO-Y4 cells. We found that SEMA3A did not have an effect on proliferation of osteocytes (Figure 1C). These results suggest that SEMA3A promotes dendrite elongation in osteocytes.
E11 protein is up-regulated in MLO-Y4 cells stimulated with SEMA3A. To explore the expression of E11, which is a dendrite marker of osteocytes, we conducted quantitative RT-PCR and western blotting analyses. Although mRNA expression of E11 was not up-regulated in MLO-Y4 cells stimulated with SEMA3A (Figure 2A), protein expression of E11 was dramatically up-regulated (Figure 2B). These results suggest that E11 protein expression is up-regulated via post-transcriptional or post-translational modification.
CDK6 expression is down-regulated in serum-starved MLO-Y4 cells by stimulation with SEMA3A. We next investigated whether SEMA3A inhibits CDK6 expression during osteocyte differentiation or not, because CDK6 down-regulation is essential for the differentiation and maturation of osteoblasts (16). To preclude the effect of various cytokines in the serum, we conducted the experiments under depletion of serum. MLO-Y4 cells were arrested in quiescence by serum starvation for 18 h and subsequently incubated with SEMA3A or vehicle. As expected, CDK6 was down-regulated in MLO-Y4 cells stimulated with SEMA3A in a time-dependent manner (Figure 3). In contrast, the protein level of CDK4, which is essential for cell-cycle progression of the G1 phase, also decreased in a time-dependent manner (Figure 3). According to Figure 1, however, SEMA3A did not have an effect on proliferation of osteocytes (Figure 1C). These results suggest that CDK6 down-regulation upon SEMA3A treatment does not affect cell-cycle progression in osteocytes.
Discussion
The cytoplasmic dendrites of osteocytes form a network for the communication between osteocytes and other bone cells. While it is well known that mechanical stimulation of osteocytes through fluid flow leads to increased dendritic processes (7), little has been reported on humoral factors capable of promoting the extension of dendrite in osteocytes. Karagiosis and colleagues showed that lysophosphatidic acid, a water-soluble phospholipid, is a potent stimulator of dendrite outgrowth in osteocytes (19). Our study, for the first time, demonstrates that the protein SEMA3A can promote the extension of dendrite in osteocytes.
Our findings indicate that SEMA3A promotes not only osteoblast differentiation, but also osteocyte maturation. As SEMA3A promotes differentiation of osteoblasts, it seems to be reasonable that osteocyte maturation should be accompanied by dendrite extension upon SEMA3A treatment. During osteocyte maturation, both E11 mRNA and E11 protein levels are increased in osteocyte-like cell line MLO-A5 stimulated with β-glycerol 2-phosphate disodium salt hydrate (8). However, we observed E11 mRNA expression was not changed treated with SEMA3A in spite of the up-regulation of E11 protein in MLO-Y4 cells, suggesting post-transcriptional or post-translational modification. Martín-Villar et al. demonstrated transcriptional (multiple initiation sites, alternative splicing and polyadenylation) and post-translational (proteolytic processing) mechanisms for E11 expression (20). We speculate that SEMA3A may modify the editing of mRNA or inhibit the degradation of protein such as through the ubiquitin proteasome system or sumoylation. Further experiments are needed to elucidate the precise mechanisms.
Intriguingly, we found that SEMA3A stimulation caused CDK6 down-regulation in osteocytes. This result supports the hypothesis that SEMA3A is essential for the maturation of osteocytes because CDK6 is a critical regulator for differentiation of bone cells. Namely, CDK6 is down-regulated in osteoblasts stimulated with bone morphogenetic protein-2, and in osteoclasts stimulated with receptor activator of nuclear factor-κB ligand (15, 16).
In humans, serum SEMA3A was positively associated with osteocalcin in postmenopausal women independently of age, whereas no correlation between serum SEMA3A and bone mineral density have been reported (21). However, Hayashi et al. showed that the local administration of SEMA3A into an injured site accelerated bone regeneration in a bone-regeneration mouse model of cortical bone defects induced by drill-hole injury (11). It is, thus, expected that SEMA3A could have a role in the application of local drug delivery such as of bone morphogenetic protein-2 (22). Further investigation will be needed to determine whether SEMA3A is useful as an osteoinducive drug.
In conclusion, we demonstrated that SEMA3A up-regulates E11 protein expression and regulates dendrites of osteocytes in association with down-regulation of CDK6.
Acknowledgements
This work was supported in part by a Grant-in-Aid (26293432, to Tsuyoshi Sato) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
- Received January 11, 2016.
- Revision received February 24, 2016.
- Accepted February 26, 2016.
- Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved