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Introduction

It is evident that mandibular prognathism (Online Mendelian Inheritance in Man #176700) is a multifactorial phenotype (1,2). To identify the susceptibility loci of mandibular prognathism, the first genome-wide association study (GWAS) was performed using microsatellites in Japanese patients including 240 individuals with mandibular prognathism and 360 individuals who were healthy (3,4). This previous GWAS (4) suggested that six loci [1p22.3, 1q32.2 (Table I), 3q23, 6q23.2, 7q11.22 and 15q22.22] were identified as susceptibility regions of mandibular prognathism. The mutations of synovial sarcoma, X breakpoint 2 interacting protein, plexin A2 (PLXNA2) (Table II), Ras p21 protein activator 2, transcription factor 21, calneuron 1 and RAR (retinoic acid receptor)-related orphan receptor α genes were suggested as candidate genes, respectively.

Table I. Results of a previous genome-wide association of mandibular prognathism using microsatellite markers.

Table I.

Results of a previous genome-wide association of mandibular prognathism using microsatellite markers.

				Allele frequency		Fishers exact test P-value
Microsatellite	Cytobands	Alleles (n)	Significant allelea	Cases	Controls	2×2	2×m	Odds ratio (95% confidence interval)
D1S1358i	1q32.2	7	92	0.362	0.465	0.000422	0.007	0.65 (0.51–0.83)

a Significant allele presents the size of the amplicon. m, Number of alleles for the marker.

Table II. Information on nearest candidate gene of susceptibility locus obtained from previous genome-wide association study of mandibular prognathism using microsatellites.

Table II.

Information on nearest candidate gene of susceptibility locus obtained from previous genome-wide association study of mandibular prognathism using microsatellites.

		Physical position of amplicona
Microsatellite	Cytobands	From	To	Nearest gene	Gene locationa
D1S1358i	1q32.2	208365606	208365706	PLXNA2	(chr1: 208200588 - 208391267)

a Physical position of amplicon was referred to GRCh build 37/hg19. PLXNA2, plexin A2.

PLXNA2 gene is located on chromosome 1q32.2 and encodes plexin A2, a member of the plexin-A family of semaphorin co-receptors (5). Semaphorins are a large family of secreted or membrane-bound proteins that mediate repulsive effects on axon path finding during nervous system development (5). Semaphorin 3A (sema3A) is a secreted protein and expressed by osteoblasts, whereas sema3A is not detected in osteoclasts (6). Sema3A exerts an osteoprotective effect by suppressing osteoclastic bone resorption and promoting osteoblastic bone formation. Plexin A2 is expressed in osteoblasts, and it is suggested that sema3A and plexin A2 binding stimulates osteoblast differentiation (6). Yoshida et al (7) reported that sema3A may induce cell migration, proliferation and the odontoblastic differentiation of human dental pulp stem cells. However, human mandibular growth consists of a periosteal growth of cortical bone and an endochondral growth of the mandibular condyle (8). Active mandibular growth occurs in an endochondral growth of the condyle (8). Gomez et al (9) reported that sema3A and plexin A2 mRNA are expressed in mouse chondrogenic cell line MC 615. However, to the best of our knowledge, there have been no previous studies on the role of sema3A or plexin A2 in human chondrocytes.

The objectives of the present study were to examine the function of sema3A and its receptor, plexin A2, in human chondrocytes.

Materials and methods

Cell culture

Normal human chondrocytes (PromoCell GmbH, Heidelberg, Germany) were seeded into a T25 culture vessel (10,000–20,000 cells/cm2) and grown in commercial medium (Chondrocyte Growth Medium; PromoCell GmbH) containing 10% fetal calf serum (PromoCell GmbH). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air, and the medium was changed every 2–3 days. Subcultures were obtained by removing the cells from the dish using 0.025% trypsin in 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) buffered saline solution containing 0.01% ethylenediaminetetraacetic acid (EDTA) (PromoCell GmbH).

Cells were seeded into 100 mm diameter culture plates. After 1 day, they were exposed to new media with either: i) A 100 ng/ml concentration, the concentration determined in a previous study (7), of recombinant human sema3A (R&D Systems, Inc., Minneapolis, MN, USA; high); ii) A 1 ng/ml concentration of sema3A (low); or iii) without sema3A (control). The effects of sema3A were analyzed at days 7 and 14 of culturing, and representative data were obtained from five culture plates of each group.

Protein assay and type II collagen and human parathyroid hormone-related peptide (PTHrP) receptor 1 (PTH-R1) concentration determination

Cells and extracellular matrices were assayed for concentrations of PTH-R1 on days 7 and 14 of culturing using an Enzyme-linked Immunosorbent assay kit (cat no. SEC743Hu; Cloud-Clone Corp., Houston, TX, USA) as previously described (10). Cells and extracellular matrices were detached with 0.025% trypsin in HEPES buffered saline solution containing 0.01% EDTA, collected by centrifugation (220 × g for 3 min at 4°C), washed three times, resuspended in 1× PBS, subject to ultrasonication, and centrifuged (1,500 × g for 10 min at 4°C) to remove cellular debris in accordance with the manufacturer's protocol.

The cells and extracellular matrices were also assayed for concentrations of type II collagen using an enzyme immunoassay kit (Type II Collagen Detection kit; Chondrex, Inc., Redmond, WA, USA) as previously described (11,12). In accordance with the manufacturer's protocol, the cells and extracellular matrices were solubilized by pepsin digestion (at 4°C for 24 h) and elastase digestion (at 4°C for 24 h) prior to with the assay. Culture media were also assayed for concentrations of type II collagen. Protein content of the cells and extracellular matrices were measured using a bicinchoninic acid assay reagent kit (Thermo Fisher Scientific, Waltham, MA, USA) as previously described (13,14). Bovine serum albumin was used as the control. All assays were performed according to the manufacturer's protocol.

Statistical methods

The data were represented as the mean ± standard deviation. Representative data were obtained from five culture plates of each group. Parametric tests were used in the present study as the distribution of the concentrations of type II collagen, PTH-R1 and the protein expression levels of control, low and high sema3A groups were normally distributed. One-way analysis of variance was used to compare the protein content and concentrations of type II collagen and PTH-R1 among three groups. Post hoc multiple comparisons were performed using Bonferroni's test. Statistical analyses were performed using SPSS version 23.0 statistical package (IBM Corporation, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Evaluation of human chondrocytes response to sema3A treatment

Fig. 1 reveals the effect of recombinant human sema3A on the morphology of human chondrocytes. At culture day 7, the cells were polygonal, and low and high concentrations of exogenous sema3A appeared to promote the growth of chondrocytes compared with the control. At culture day 14, the cells remained polygonal in the three groups.

Figure 1. Effect of exogenous sema3A on the morphology of human chondrocytes. Cells cultured for 1 day were exposed to 1 ng/ml (low) or 100 ng/ml (high) sema3A for 7 or 14 days in 100 mm diameter culture plates. Upper lane, day 7; lower lane, day 14. Scale bars, 100 µm. Sema3A, semaphorin 3A.

Fig. 2 demonstrates the mean ± standard deviation of the protein content, in accordance with cell growth and accumulation of extracellular matrices, of human chondrocytes. At culture day 7, high and low concentrations of exogenous sema3A significantly increased the protein content of chondrocytes (0.044±0.010 and 0.051±0.006 mg/ml, respectively) compared with the control (0.017±0.005 mg/ml) (P=0.0008 and 0.00002, respectively). At culture day 14, a high concentration of sema3A significantly increased the protein content of chondrocytes (0.174±0.020 mg/ml) compared with the control (0.124±0.014 mg/ml) (P=0.002).

Figure 2. Protein content in human chondrocytes and matrices in the absence or presence of exogenous sema3A. Cells cultured for 1 day were grown without any addition (control), with 1 ng/ml sema3A (low) or 100 ng/ml sema3A (high). Left, day 7; right, day 14. The data represent the mean ± standard deviation of five samples. ***P<0.001 and **P<0.01 with comparisons shown by lines. Sema3A, semaphorin 3A.

Effect of sema3A on type II collagen expression in human chondrocytes

Fig. 3 presents the mean ± standard deviation of concentrations of type II collagen in the culture medium of human chondrocytes. At culture day 7, exogenous sema3A dose-dependently decreased the concentrations of type II collagen in the culture medium of chondrocytes, although the differences were not statistically significant among the three groups. At culture day 14, the concentration of type II collagen in the culture medium of chondrocytes was not detected in the three groups. Concentrations of type II collagen in cells and extracellular matrices were not also detected in the three groups.

Figure 3. Concentration of type II collagen in culture medium of human chondrocytes at culture day 7 in the absence or presence of exogenous sema3A. Cells cultured for 1 day were grown without any addition (control), with 1 ng/ml sema3A (low) or 100 ng/ml sema3A (high). The data represent the mean ± standard deviation of five samples. Sema3A, semaphorin 3A.

Effects of sema3A on PTH-R1 expression in human chondrocytes

Fig. 4 presents the mean ± standard deviation of concentrations of PTH-R1 in human chondrocytes. At culture day 7, neither high nor low concentrations of exogenous sema3A significantly influenced the concentration of PTH-R1 of chondrocytes (0.241±0.036 and 0.242±0.029 ng/ml, respectively) compared with the control (0.253±0.084 ng/ml). At culture day 14, a high concentration of sema3A significantly decreased the concentration of PTH-R1 in chondrocytes (0.211±0.017 ng/ml) when compared with the control (0.310±0.053 ng/ml) (P=0.004).

Figure 4. Concentration of PTH-R1 in human chondrocytes and matrices in the absence or presence of exogenous sema3A. Cells cultured for 1 day were grown without any addition (control), with 1 ng/ml sema3A (low) or 100 ng/ml sema3A (high). Left, day 7; right, day 14. The data represent the mean ± standard deviation of five samples. **P<0.01 with comparisons shown by lines. PTH-R1, parathyroid hormone-related peptide receptor 1; sema3A, semaphorin 3A.

Discussion

To investigate the effects of sema3A on the various functions of human dental pulp stem cells, Yoshida et al (7) added a 10 ng/ml concentration of recombinant human sema3A in medium for human dental pulp stem cells. To examine the function of sema3A in the regulation of osteoclast differentiation, Hayashi et al (6) added a 500 ng/ml concentration of recombinant sema3A in medium for osteoblasts. The concentrations of recombinant human sema3A used in the present study were selected according to these previous studies.

Type II collagen produced by cells is incorporated into the extracellular matrix (11,12). However, in the present study, type II collagen was not detected in cells and matrix, nevertheless type II collagen was digested by pepsin and elastase for solubilizing collagen. Instead of type II collagen in extracellular matrix, concentrations of type II collagen in culture medium were measured out, which was already soluble.

Sema3A is expressed in osteoblasts (6). Plexin A2, which is a semaphorin receptor, is also expressed in osteoblasts, and it has been suggested that sema3A and plexin A2 binding stimulates osteoblast differentiation (6). Gomez et al (9) reported that sema3A and plexin A2 mRNA were expressed in a mouse chondrogenic cell line. In the present study, the addition of recombinant human sema3A significantly (P<0.001) increased the protein content of human chondrocytes (Fig. 2). Protein content is thought to indicate the degree of cell growth and accumulation of extracellular matrices (13,14). The results of the present study and a previous study (9) therefore suggest that plexin A2 may be expressed in not only mouse but also human chondrocytes, and that exogenous sema3A may bind plexin A2 on proliferative human chondrocytes and induce the acceleration of cell growth and/or accumulation of extracellular matrices of chondrocytes, although the present study did not assert whether the expression of plexin A2 is constant or regulated by exogenous sema3A.

Active mandibular growth occurs in an endochondral growth of the condyle of the temporomandibular joint (8). In the temporomandibular joint, endogenous cytokines are reportedly produced by non-inflammatory cells including osteoblasts and chondrocytes and interact with their receptors (13). The PTHrP gene is located on chromosome 12p12.1-p11.2 and encodes a member of the parathyroid hormone family. The protein PTHrP, via its receptor, PTH-R1, regulates endochondral bone development. The PTHrP ligand is expressed in mouse undifferentiated (proliferative) chondrocytes in the growth plate of developing bones (15,16). PTH-R1 is expressed in mouse chondrocytes located mainly in the proliferative zone and the hypertrophic zone of the growth plate (15–17). Expression of the PTH-R1 gene coincides with the type II collagen gene (16). Activation of PTH-R1 interferes with the early stages of chondrogenesis through the cyclic AMP/protein kinase A signaling pathway (18). Namely, PTH-R1 functions to maintain proliferative chondrocytes and delays the production of hypertrophic chondrocytes in mice (18). Indian hedgehog is expressed in pre-hypertrophic chondrocytes and stimulates the production of PTHrP in mice (18,19). Experiments using chimaeric mice (19) revealed that a negative feedback loop functions in chondrogenesis. In the present study, to examine whether sema3A is associated with the endochondral growth of the condyle of the temporomandibular joint, effects of exogenous sema3A on PTH-R1 in human chondrocytes were preliminarily investigated.

In the present study, the addition of a high concentration (100 ng/ml) of recombinant human sema3A significantly (P=0.002) decreased the expression of PTH-R1 in human chondrocytes at culture day 14 (Fig. 4). The novel results of the present study suggested that exogenous sema3A may function on plexin A2 in proliferative human chondrocytes and suppresses PTH-R1 and/or PTHrP expression in proliferative human chondrocytes, although the mechanism remains unknown. Suppression of PTH-R1 and/or PTHrP expression potentially accelerates the differentiation of hypertrophic chondrocytes and the subsequent endochondral ossification at puberty (in other words, early termination of condylar endochondral growth). The mutation of the PLXNA2 gene may inhibit this action of sema3A on chondrocytes and delay this early termination of condylar endochondral growth. Therefore, the mutation of PLXNA2 gene encoding plexin A2 may be a candidate gene of mandibular prognathism, even though the results of the present study were limited.

In the present study, there was no data of expression of sema3A, plexin A2 and PTH-R1 when the calcium concentration was changed. In general, PTH enhances the release of calcium from the large reservoir contained in the bones. PTH and PTH-R1 binding stimulates osteoblasts to increase their expression of receptor activator of nuclear factor κ-B (RANKL). The binding of RANKL to RANK stimulates these osteoclast precursors to fuse, forming novel osteoclasts, which ultimately enhances bone resorption (6). Interestingly, Ohta et al (20) reported that plexin A2 mediates cell-cell adhesion via a homophilic binding mechanism under the presence of calcium ions. Therefore, the association between the expression of plexin A2 and calcium concentration will be analyzed in the future.

Another limitation in the present study lies in the ‘dedifferentiation’ of chondrocytes. Chondrocytes grow and express the phenotypes of chondrocytes in monolayer cultures (21,22). However, in monolayer cultures cultured over two weeks in a previous study, the growing chondrocytes appeared to be dedifferentiated (21,22). Chondrocytes dedifferentiated by monolayer cultures transform into a fibroblast-like morphology and express type I collagen accompanied with a fibroblast-like phenotype (23,24), whereas proliferative chondrocytes are polygonal and express type II collagen accompanied with a chondrocyte phenotype. In the present study, the increased protein content and the decreased type II collagen and PTH-R1 expression in human chondrocytes at culture day 14 may result from the dedifferentiation accelerated by exogenous sema3A.

To facilitate the ‘redifferentiation’ of the dedifferentiated chondrocytes incubated by monolayer cultures over two weeks, high cell density three-dimensional cultures performed by assembling chondrocyte in alginate beads, synthetic polymer gels or compressing into pellets (spheroid formation) have been attempted (25,26). In future studies, three-dimensional cultures for human chondrocytes will be utilized.

In conclusion, a previous GWAS had revealed PLXNA2 encoding a member of the plexin-A family of semaphorin co-receptors as one of the candidate genes for mandibular prognathism. The present preliminary study proposed that sema3A functions on not only murine but also human chondrocytes and revealed the novel result that exogenous sema3A suppresses the expression of PTH-R1 in human proliferative chondrocytes. Exogenous sema3A may function on human chondrocytes by binding plexin A2. Suppression of PTH-R1 and/or PTHrP expression potentially accelerates the early termination of condylar growth. The mutation of the PLXNA2 gene may delay the early termination of the condylar growth. Therefore, the mutation of the PLXNA2 gene encoding plexin A2 may be a candidate gene of mandibular prognathism.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Japanese Society for the Promotion of Science KAKENHI (Grants-in-Aid for Scientific Research; grant no. JP16K20630) and National Institutes of Health/National Institute if Dental and Craniofacial Research (grant no. R01DE023538).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

TK conceived and designed the study, acquired the data, analyzed and interpreted the data and drafted the article. AO conceived and designed the study and revised the article for important intellectual content. MH acquired the data, analyzed and interpreted the data and revised the article for important intellectual content. JYamaz analyzed and interpreted the data and revised the article for important intellectual content. JYamas analyzed and interpreted the data and revised the article for important intellectual content. JI conceived and designed the study and revised the article for important intellectual content.

Ethics approval and consent to participate

Not applicable.

Patients consent for publication

Not applicable.

Competing interest

The authors declare that they have no competing interests.

References