A Class III Semaphorin (Sema3e) Inhibits Mouse Osteoblast Migration and Decreases Osteoclast Formation In Vitro

α-Minimum essential medium (α-MEM), fetal calf serum, (FCS), penicillin, and streptomycin were from Invitrogen (Paisley, UK). Tissue culture plates were from Costar (Cambridge, MA). All other reagents were from Sigma (Poole, UK), unless otherwise indicated.

Osteoblasts were isolated from the calvaria of 4-day-old litters of C57Bl6 mice by sequential collagenase/EDTA digestion. Osteoblasts were cultured in α-MEM (10% FCS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 1 mM glutamine) and grown to ~90% confluence. Cells were harvested in trypsin/EDTA, counted on a Neubauer hemocytometer, seeded at 3 × 10⁴ cells/cm², and allowed to adhere overnight prior to treatment. Mineralization was induced using α-MEM supplemented with 10% (v/v) FCS, 100 U/mL penicillin, 100 mg/mL streptomycin, 1 mM glutamine, 10 mM β-glycerophosphate, and 50 μg/mL l-ascorbic acid 2-phosphate. Medium was replaced every 3 days. Mineralization was visualized by staining with alizarin red on ethanol-fixed monolayers.

Mouse osteoclasts were generated in vitro from macrophage colony-stimulating factor (M-CSF)-dependent bone marrow macrophages. Bone marrow cells were flushed from the tibiae and femora of 3- to 4-week-old C57Bl6 mice and cultured overnight in 75-cm² tissue culture flasks (one flask per mouse; BD Biosciences, San Jose, CA) in α-MEM containing 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM glutamine, 10% FCS, and 25 ng/mL murine M-CSF (R&D Systems, Minneapolis, MN). Nonadherent cells were removed, pelleted, and counted before being seeded at 1 × 10⁵ cells/cm² in 75-cm² tissue culture flasks in the presence of 25 ng/mL M-CSF. Medium was changed and fresh M-CSF added every 2 days until confluence, after which cells were trypsinized, counted, and seeded at 5 × 10⁴ cells/cm² in α-MEM containing 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM glutamine, 10% FCS, 50 ng/mL murine M-CSF, and 10 ng/mL murine RANKL (R&D Systems). Fresh medium and cytokines were added every 2 days until multinucleated osteoclasts formed (approximately 5 days).

Mature osteoclasts were isolated from the limbs of New Zealand white rabbits, 2–4 days old, as described previously [11]. Osteoclasts isolated from the minced long bones of one rabbit were vortexed and resuspended in 25 mL of α-MEM containing 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM glutamine, and 10% FCS. Cells were seeded in 125-uL aliquots onto 5-mm-diamter dentine discs before being allowed to adhere for 2 h. Nonadherent cells were washed away in PBS, before incubation in the presence or absence of recombinant Sema3e for 48 h. Cells were then fixed in 4% formaldehyde, stained with TRITC-phalloidin, and counted for the presence of actin rings by inverted fluorescence microscopy. Discs were then stained for tartrate-resistant acid phosphatase (TRAP) as previously described [12], and TRAP-positive multinucleated cells with three or more nuclei were considered to be osteoclasts. Cells were cleaned from the discs, and the area of resorption pits on the surface of the dentine was quantified by reflected light microscopy and custom image analysis software developed using Aphelion ActiveX objects (ADCIS, Herouville-Saint-Clair, France).

RNA was extracted by phenol/chloroform fractionation using TRIzol^® reagent (Invitrogen) per the manufacturer’s instructions. Isolated RNA was further purified using an RNeasy Mini Kit (Qiagen, Valencia City, CA), including a DNAse I treatment step. RNA quantity was determined by spectrophotometry, and A₂₆₀/A₂₈₀ ratios were used to confirm the absence of contamination. RNA quality was confirmed on an Agilent (Palo Alto, CA) Bioanalyzer to ensure that no degradation occurred during extraction. RNA was reverse-transcribed to cDNA using 2 μg of total RNA, 1 μg of random primer oligonucleotides (Invitrogen), and MMLV reverse transcriptase (SuperScript II, Invitrogen).

Intron-spanning oligonucleotide primers were designed using Primer3 [13] and checked via BLAST for specificity (http://​blast.​ncbi.​nlm.​nih.​gov/​Blast): Sema3e forward, 5′-CCA TAC AAT GCT GCT GGA TG-3′; Sema3e reverse, 5′-GTG TGC CAA TTA TAT CCG GG-3′; PlexinD1 forward, 5′-TCA GAG GAG ATC GTG TGT GC-3′; PlexinD1 reverse, 5′-GCT CTA TGC TCC AGG TCC TG-3′; Gapdh forward, 5′-ACT TTG TCA AGC TCA TTT CC-3′; Gapdh reverse, 5′-TGC AGC GAA CTT TAT TGA TG-3′. PCRs were prepared to a total volume of 25 μL containing the equivalent of 50 ng of reverse-transcribed RNA per reaction and 500 nM of each primer. Amplification was performed using Qiagen Taq DNA polymerase under optimized conditions: 95°C for 10 min, 33 cycles of 95°C for 30 s, 58°C for 60 s, and 72°C for 60 s. Products were resolved on a 1.5% agarose gel and detected using ethidium bromide under UV illumination.

Intron-spanning, FAM-labeled hydrolysis probe assays were designed for genes of interest using the Universal Probe Library (Roche, Mannheim, Germany; see Table 1) and multiplexed with a primer-limited, VIC-labeled Gapdh TaqMan^® assay (Applied Biosystems, Foster City, CA). Assays were performed on a LightCycler^® 480 qPCR system (Roche) using LightCycler Probes Mix and an optimized protocol (20 μL reaction volume; 5 min 95°C, 40 cycles of 10 s 95°C, 30 s 60°C). Relative gene-of-interest expression to Gapdh was calculated using a standard curve of serially diluted cDNA to correct for PCR efficiency, per the method of Pfaffl [14].

Protein lysates were prepared in RIPA buffer (1% [v/v] Nonidet P-40, 0.1% [w/v] sodium dodecyl sulfate, 0.5% [w/v] sodium deoxycholate in PBS, plus 1% [v/v] protease inhibitor cocktail [Sigma]). Cell lysate (40 μg) was electrophoresed on 12% polyacrylamide-SDS gels (Criterion XP system; Bio-Rad, Hemel Hempstead, UK) under reducing conditions. Proteins were transferred to polyvinylidene difluoride membranes and then hybridized with 0.2 μg/mL anti-Sema3E antibody (R&D Systems), followed by 1 μg/mL anti-goat immunoglobulin G-horseradish peroxidise conjugate (Merck, Darmstadt, Germany). Bands were visualized using chemiluminescence (Supersignal West Dura reagent; Pierce, Rockford, IL) and a Bio-Rad Fluor-S Max MultiImager.

Alkaline phosphatase activity was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenyl. Osteoblasts were cultured on 96-well plates and lysed in 150 μL of lysis buffer (1 M diethanolamine [DEA], 1 mM MgCl₂, and 0.05% Triton X-100). Lysate (50 μL) was mixed with 50 μL of substrate solution (20 mM p-nitrophenol phosphate, 1 M DEA, and 1 mM MgCl₂). The amount of p-nitrophenol generated after 30 min was calculated by measuring the absorption of each well at 414 nm in comparison with a standard curve of 1.25–30 nmol/well.

A 0.5-mm wound was made, using a pipette tip, across a confluent monolayer of osteoblasts on 24-well Imagelock plates (Essen Instruments, Ann Arbor, MI). Cell layers were washed three times, treated with 40 nM PTH or 100 ng/mL recombinant Sema3e in medium with or without 10% (v/v) serum, incubated in an IncuCyte microscope (Essen Instruments), and imaged every 30 min. Wound healing was calculated using the IncuCyte Scratch Wound Assay software application. Results were confirmed using replicate plates and images taken on a phase-contrast inverted light microscope.

Using a panel of quantitative PCR (qPCR) assays to measure all seven known members of the class III semaphorins, we examined their expression profile in both proliferating (P, after 48-hour growth in normal medium) or mineralizing (M, after 14 additional days in osteoblast differentiation medium) C57Bl6 calvarial osteoblasts (Fig. 1a). Six of the seven class III semaphorins were detected in osteoblasts (the exception being Sema3f), as was expression of the receptor components PlexinA1 and -D1. Sema3c was the most abundant, with low levels of Sema3b, -3d, and -3 g detected in proliferating osteoblasts. Fourteen days treatment in differentiation medium resulted in mineralization (as confirmed by alizarin red staining) (data not shown) and a significant increase in expression of Sema3a, -3d, and -3 g as well as PlexinD1 (by 2.2-, 14.6-, 6.5-, and 4.6-fold, respectively). Sema3e significantly decreased by 90% in mineralizing cultures, while Sema3c remained unchanged and Sema3f remained undetected. We chose three of the significantly regulated genes for further investigation, choosing Sema3a, -3d, and -3e based on reports of each displaying effects on cells or in tissues unrelated to axonal guidance.

Long culture periods (>1 week) in the presence of differentiation medium are required to produce cultures of mineralizing osteoblasts. In order to discount the effects of culture time or cell density on the expression of key members of the class III semaphorins, we examined expression of Sema3a, -3d, and -3e in osteoblasts seeded at a low density and grown toward confluence in normal, nondifferentiation medium (Fig. 1b). Osteoblasts proliferated until confluence at 96–120 h postseeding and remained viable during the time course. Expression of Sema3a remained unchanged, while 3d significantly increased in expression to 11.2-fold over control by 216 h. Sema3e significantly decreased in expression as cells grew to confluence, reaching a fivefold decrease by 216 h. After reaching confluence, expression of both Sema3d and -3e stabilized. These observations suggest that the changes in expression of Sema3d and -3e observed in cultures of proliferating and mineralizing cultures were related to the effects of cell growth and density and that the increase in Sema3a expression in mineralizing cultures was not related to cell growth or density.

Next, we examined the expression of Sema3a, -3d, and -3e in response to factors known to influence osteoblast differentiation. PTH may act as both an anabolic and a catabolic factor in bone, depending on the timing and duration of exposure [15]. Short-term exposure of calvarial osteoblasts to PTH induced an elevation of alkaline phosphatase activity (data not shown), indicating that osteoblast differentiation had been initiated [16]. Despite this, we did not see an effect on the expression of Sema3a, -3d, or -3e after 24-hour exposure (Fig. 2a–c) or at earlier or later time points (data not shown) following PTH stimulation. Proliferation and differentiation of osteoblasts in vitro can also be induced by 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) treatment [17, 18] and has previously been reported to regulate Sema3b expression in osteoblasts [7]. In contrast to the inert effects of PTH, 1,25(OH)₂D₃ caused a significant increase in expression of Sema3e after 24 h (Fig. 2c), which persisted for at least 48 h posttreatment (data not shown) but had no effect on Sema3a or 3d expression (Fig. 2a, b). Stimulation of the canonical Wnt signaling pathway promotes the translocation of β-catenin to the nucleus and the subsequent regulation of gene transcription via binding to TCF/LEF. Wnt signaling is important in the development and maintenance of mineralized tissues [19] and has previously been implicated in the regulation of Sem3d in zebrafish [20] and Sema3a in the dental mesenchyme [21] but has not been investigated in osteoblasts in vitro. Downstream stimulation of the canonical Wnt pathway can be achieved by pharmacological inhibition of GSKβ (which phosphorylates β-catenin to target it for ubiquitin-mediated degradation [22]) by 6-bromoindirubin-3′-oxime (BIO) [23]. Treatment with MeBIO, the kinase inactive negative control of BIO, had no significant effect on expression of Sema3a, -3d, or -3e, while BIO caused a significant decrease in expression of both Sema3a and -3e (Fig. 2a, c). Cells were also treated with 40 mM LiCl, which inhibits GSKβ but via a different mechanism from BIO. LiCl inhibition of GSKβ also caused a decrease in Sema3a and -3d expression, supporting the data observed with BIO (data not shown). These data demonstrate that there is evidence of differential regulation of class III semaphorins in response to modulation of signaling pathways known to be important for osteoblast function.

As Sema3b has recently been reported to be a 1,25(OH)₂D₃-responsive gene in osteoblasts [7], we decided to focus on the similarly 1,25(OH)₂D₃-responsive Sema3e for further study. Sema3e is a unique class III semaphorin as it does not require the presence of a neuropilin in its receptor complex, instead consisting solely of PlexinD1. Since class III semaphorins can antagonize VEGF signaling by competing for neuropilins, Sema3e will not exhibit non-Plexin-mediated effects associated with neuropilin sequestration, thereby avoiding potential interpretation problems due to the expression of VEGF receptors in osteoblasts (reviewed in [24]). Sema3e expression was strongly detected in osteoblasts by both end-point and qPCR but below the limit of detection in macrophages and osteoclasts (Fig. 3a, b). Sema3e protein was clearly detected in both proliferating and mineralizing osteoblast cultures but not osteoclasts (Fig. 3d). Trace amounts of Sema3e protein were detected in macrophage cultures, possibly representing some heterogeneity within the cell population. Higher protein levels were observed in cultures of mineralizing osteoblasts compared to proliferating osteoblasts, despite the lower transcript expression observed in mineralizing cultures, although this may be due to deposition and accumulation of secreted Sema3e in the extracellular matrix. All cell types examined (osteoblasts, M-CSF-dependent macrophages, and osteoclasts) expressed the Sema3e receptor protein PlexinD1 (Fig. 3a). Expression of PlexinD1 was significantly higher in macrophages and osteoclasts than in either osteoblast culture (Fig. 3c). As expression of PlexinD1 was detected in all cells, this suggests that osteoblast-derived Sema3e may act as both an autocrine signaling molecule and in a paracrine manner upon macrophages and/or osteoclasts. We found no change in expression of PlexinD1 after treatment with 1,25(OH)₂D₃, BIO, or PTH (data not shown) and encountered problems detecting PlexinD1 protein levels in mouse cells using available antibodies.

Since osteoblasts expressed both Sema3e and PlexinD1, we investigated Sema3e as an autocrine regulator of osteoblast function in vitro. Recombinant mouse Sema3e (100 ng/mL) did not affect osteoblast proliferation, measured by alamar blue assay of viable cell number (data not shown), nor did it affect mineralization, measured by alizarin red staining after 7, 14, or 21 days in culture (21-day representative image shown in Fig. 4a). PTH (40 nM) caused differentiation of osteoblasts and a subsequent doubling in alkaline phosphatase activity after 48-hour treatment. Sema3e (100 ng/mL) did not affect alkaline phosphatase activity in the presence or absence of PTH (Fig. 4b) but did inhibit the migration of osteoblasts in a wound-healing assay using a confluent monolayer of osteoblasts. PTH increased wound healing, and serum-free medium resulted in no migration of cells into the wound area (Fig. 4c). As wound closure can occur as a result of both migration and proliferation, it was important to establish that the effects observed in Fig. 4c were predominantly due to migration. By constructing time-lapse videos from the still images taken for calculating wound width, we could confirm that wound closure occurred due to migration of cells across the wounded region and not due to proliferation of cells along the wound edges (data not shown).

As macrophages and osteoclasts express PlexinD1, we next investigated the effect of 200 ng/mL recombinant mouse Sema3e protein on the formation of multinucleated (three or more nuclei per cell), TRAP-positive osteoclasts in M-CSF- and RANKL-treated cultures of mouse bone marrow macrophages. An abundance of TRAP-positive osteoclast-like cells was observed in control cultures after approximately 5 days in RANKL, while Sema3e-treated cultures had fewer osteoclasts and less intense TRAP staining (Fig. 5a), which was confirmed by Western blotting for TRAP protein (Fig. 5b). Quantification of osteoclasts revealed that Sema3e-treated cultures had 62% fewer osteoclasts than control cultures (Fig. 5c), with a dose-dependent decrease in osteoclast number observed from 50 to 200 ng/mL (data not shown). Accordingly, qPCR analysis showed that genes associated with osteoclast formation and differentiation were 25–40% lower in Sema3e-treated cultures compared to control cultures, with significant decreases observed in Acp5, Cd51, Nfatc1, and Calcr expression (Fig. 5d). All markers in Fig. 5d were substantially upregulated in M-CSF- and RANKL-stimulated osteoclast cultures compared to M-CSF-treated cultures alone (from undetectable levels of Calcr to 100 + -fold increases in Alp5, Mmp9, and Ctsk and a twofold increase in Nfatc1 and Cd51), confirming the formation of osteoclast cells in M-CSF and RANKL cultures (data not shown). In order to ascertain if Sema3e could affect osteoclast function as well as formation, we investigated the effects of Sema3e treatment on mature osteoclast cultures. As mature osteoclast cultures from mice formed variably on dentine, we used mature osteoclasts isolated from the minced long bones of rabbit neonates to remove errors associated with varying formation efficiencies or the inhibitory effects of Sema3e on formation. The use of mature rabbit osteoclasts on dentine is an established method for the measurement of resorption, enabling the easy identification of TRAP-positive osteoclasts and functional actin rings. No effect of Sema3e was observed on the number of TRAP-positive multinucleated cells adhered to the dentine surface, the formation of actin rings, or the area of dentine resorbed by mature osteoclasts (Fig. 6a–c).