Substance P stimulates late-stage rat osteoblastic bone formation through neurokinin-1 receptors

2.1. Cell isolation and primary culture of osteogenic cells 

Rat osteogenic cells were isolated essentially as previously described (Bellows et al., 1986). Briefly, calvarias were dissected aseptically from 3-day-old Wistar rats. They were minced and digested in a collagenase-containing enzyme mixture at 37°C for 10, 20, 30, and 50min, yielding populations I through IV. Cells retrieved from each step of the digestion sequence were plated in 60mm culture dishes in α-minimal essential medium (α-MEM) containing 10% heat-inactivated fetal bovine serum (FBS; Wako Pure Chemical Industries, Osaka, Japan), 0.3μg/mL fungizone (Gibco, Grand Island, NY), plus antibiotics: 100μg/mL penicillin G (Wako) and 50μg/mL gentamicin (Gibco). After 2 days, the cultures were washed with phosphate-buffered saline (PBS) to remove nonviable cells and other debris, then incubated with 0.25% trypsin (Gibco), and counted using a hemocytometer. Cells from populations II through IV were pooled and resuspended in α-MEM containing 10% FBS and antibiotics and plated in 35mm culture dishes or on coverslips (22mm diameter; Matsunami Glass Ind., Osaka, Japan) in 35mm culture dishes at 3×104cells/dish. After 24h, the culture medium was changed to the above medium, supplemented with 50μg/mL ascorbic acid (Wako), 10nM dexamethasone (Wako), and 10mM β-glycerophosphate (Nacalai Tesque, Kyoto, Japan). These conditions are optimal for the formation of mineralized osteoid nodules. The culture medium was changed three times each week, and cells were cultured up to 21 days. Fresh SP or compounds were added when the culture medium was changed.

To examine the dose-dependent effects of SP, 10−12–10−6MSP was added and cultured for 21 days. To examine the effects of SP antagonists or SP receptor antagonists, cells were cultured with 10−7M or 10−8MSP, 10−7M or 10−8MSP+10−6M spantide (SP antagonist; Peptide Institute, Osaka, Japan), and 10−7M or 10−8MSP+10−6MFK888 (NK1-R antagonist; Fujisawa Pharmaceutical, Osaka, Japan), as described by Fujii et al. (1992). To examine the time-dependent effects of SP on osteoblastic bone formation, 10−7MSP was added during the first of the three weeks, the first two weeks of the three weeks, or all three weeks when the medium was changed. All experiments for bone formation assay were assessed after 3 weeks culture.

2.2. Bone formation analysis 

To detect minerals in bone-like nodules, the samples were stained with von Kossa reagent. In summary, the specimens were washed three times in PBS and fixed with 3.7% formaldehyde in PBS for 10min, washed in distilled water three times, and then incubated for 1h in 0.5% silver nitrate (Wako). They were then washed three times in distilled water, incubated for 3min in 0.3% sodium thiosulfate pentahydrate (Nacalai Tesque), washed in distilled water, and dried. The stained mineralized bone-like nodules were imaged using a CCD camera, and the images were transferred to a computer-assisted image analysis system (MCID, Image Research, Brock University, Ontario, Canada).

2.3. Immunocytochemistry 

Cultured rat osteoblastic cells were fixed in 90% ethanol for 10min, and then washed in PBS. The fixed cells were pre-incubated in 0.1M PBS with 1% normal goat serum for 30min at room temperature. Then, the cells were incubated with rabbit polyclonal antibodies against NK1-R (1:1000; Molecular Probes, Eugene, OR) for 2h at 37°C, washed in PBS and incubated with goat anti-rabbit IgG conjugated to fluorescein (1:100; Molecular Probes) for 1.5h at 37°C. Finally, the sections were washed in 0.1M PBS and placed under a coverslip.

The samples were examined under a fluorescence microscope (Olympus Optical, Tokyo, Japan) equipped with a CoolSNAP CCD camera (RS Photometrics, Tucson, AZ, USA) or a confocal laser scan microscope (Radiance 2100, Bio-Rad, Herts, UK).

2.4. RT-PCR analysis 

To confirm the expression of NK1-R, rat calvarial osteoblastic cells and ROS osteoblastic cells were cultured for 7 and 14 days without SP in medium containing 10% FCS, dexamethasone, ascorbic acid, and β-glycerophosphate. The RNA in the rat striatum was extracted as a positive control.

To examine the time-dependent effects of SP on the differentiation of osteobalstic clls, semi-quantitative RT-PCR for runX2, Col I, and OCN expression was performed on 7-, 14- and 21-day-cultured calvarial osteoblastic cells with 10−7MSP, 10−7MSP+10−6MSP antagonist (spantide), 10−7MSP+10−6MFK888, and 10−8M calcitonin-gene-related peptide (CGRP; Peptide Institute), which is another neuropeptide in sensory neurons.

Cellular mRNA was extracted using a Total RNA Extraction Miniprep System (Viogene, Sunnyvale, CA), according to the manufacturer’s protocol. To reduce DNA contamination, the RNA samples were treated with RNase-free DNaseI (Takara Bio, Shiga, Japan) for 3h at 37°C; cDNA was synthesized from 2μg total RNA in 30μL of a reaction buffer composed of 500μM dNTPs, 20 U ribonuclease inhibitor (Promega, Madison, WI), and 200 U Superscript II reverse transcriptase (Invitrogen Life Technology, Carlsbad, CA). The reaction was carried out at 70°C for 7min, then at 45°C for 60min, followed by 10min at 70°C, with final cooling to 4°C. The primers used to amplify NK1-R were 5′-AGG ACA GTG ACC AAT TAT TTC CTG G-3′ and 5′-CTG CTG GAT GAA CTT CTT-3′ (GeneBank J05097; 745–769 and 1386–1410). The primers used to amplify runX2 were 5′-GCT TCA TTC GCC TCA CAA ACA-3′ and 5′-TGC TGT CCT CCT GGA GAA AGT T-3′ (GeneBank AF325502; 626–646 and 672–693). The primers used to amplify Col I were 5′-GGC AAC AGT CGA TTC ACC-3′ and 5′-AGG GCC AAT GTC CAT TCC-3′ (GeneBank Z78279; 4171–4188 and 4330–4347). The primers used to amplify OCN were 5′- GTC CCA CAC AGC AAC TCG-3′ and 5′-CCA AAG CTG AAG CTG CCG-3′ (GeneBank M25490; 611–628 and 1464–1481). The primers for the GAPDH internal control were 3′-TGA AGG TCG GTG TCA ACG GAT TTG GC-3′ and 5′-CAT GTA GGC CAT GAG GCT CAC CAC-3′ (GeneBank M17701; 35–60 and 994–1017). Each cycle consisted of denaturation (NK1-R, 94°C for 30s; runX2, 94°C for 1min; Col I, 94°C for 1min; OCN, 94°C for 1min; GAPDH, 94°C for 1min), annealing (NK1-R, 55°C for 30s; runX2, 52°C for 1min; Col I, 60°C for 1min; OCN, 61°C for 40s; GAPDH, 65°C for 1min), and 72°C for 25 cycles of 1min steps for NK1-R, 40 cycles for runX2, 20 cycles for Col I, 24 cycles for OCN, and 38 cycles for GAPDH, with a final extension step (72°C for 9min). To confirm its reproducibility, we replicated the RT-PCR experiment three or more times. Each PCR mixture consisted of cDNA (reverse-transcribed RNA), Taq polymerase buffer and 1μL each of the sense and anti-sense primers, for a total volume of 20μL. The PCR products were electrophoresed in 2% agarose gels and visualized with ethidium bromide. The data were analyzed using NIH Image.

The oligonucleotide RT-PCR primer sequences were designed and their specificity was confirmed using a BLAST-assisted internet search of non-redundant nucleotide sequence databases (National Library of Medicine, Bethesda, MD).

2.5. Statistical analysis 

The data were analyzed using Stat View (Abacus Concepts, Berkeley, CA, USA). An analysis of variance (ANOVA), followed by Fisher’s protected least-squares difference (PLSD) and the Bonferroni correction, were used to analyze the difference in bone formation between the control and each group. The values were expressed as mean±standard deviation (SD).

3.1. Expression of NK1-R in osteoblastic cells 

To examine the expression of NK1-R in osteoblastic cells, RT-PCR was performed using NK1-R specific primers. NK1-R expression was not detected in rat calvarial osteoblastic cells or ROS osteoblastic cells cultured for 7 days in medium containing ascorbic acid and dexamethasone β-glycerophosphate (Fig. 1a). In 14-day cultures, NK1-R was expressed in calvarial osteoblastic cells, but not in ROS osteoblastic cells (Fig. 1b). To confirm our findings regarding NK1-R expression in calvarial osteoblast cultures, we performed immunocytochemistry using the antibody against NK1-R, which showed that the 7-day cultures of calvarial and ROS cells lacked immunopositive reactions for NK1-R (Fig. 1c and d). However, the 14-day cultures showed many punctate immunopositive reactions for NK1-R localized in calvarial cells (Fig. 1e).

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Fig. 1. Expression of NK1-R in osteoblastic cells. (a) Expression of NK1-R mRNA by ROS osteoblastic and primary osteoblastic (pOB) cells cultured for 7 days; and (b) ROS and pOB cells cultured for 14 days. The striatum is a positive control. RT(+) and RT(−): with and without reverse transcription, respectively. (c) Immunostaining for NK1-R in pOB cells cultured for 7 days; and (d) ROS cells, (e) pOB cells cultured for 14 days. Bar=10μm.

3.2. Time- and dose-dependent effect of SP on osteoblastic bone formation 

Rat calvarial osteoblastic cells isolated in medium containing 10% FCS, dexamethasone, ascorbic acid, and β-glycerophosphate formed numerous mineralized bone nodules by day 21 (Fig. 2a). These were recognized as dark brown spots using von Kossa staining under normal light. To examine the dose-dependent effect of SP on osteoblastic bone formation, 10−12–10−6MSP was added to the culture medium from week 1–3 and maintained for 21 days. The addition of ⩾10−8MSP significantly increased the average bone formation by calvarial osteoblastic cells (Fig. 2b).

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Fig. 2. Dose- and time-dependent effects of SP on bone formation by primary osteoblastic (pOB) cells. (a) Bone nodule formation by rat calvarial osteoblastic cells cultured for 21 days without SP (cont) or with 10−12M, 10−9M, and 10−6MSP. (b) Calvarial osteoblastic cells cultured for 3 weeks with 10−12–10−6MSP. Bone formation increased significantly compared to the control (∗p<0.05, ∗∗p<0.01). (c) and (d) Calvarial osteoblastic cells cultured for three weeks with or without SP (cont). 10−7MSP was added during the first of the three weeks (1w), the first two weeks of the three weeks (1,2w), or all three weeks (1–3w) when the medium was changed. After three weeks culture, the formation of bone nodule were assessed. (c) Bone formation increased significantly compared to the control (∗p<0.05, ∗∗p<0.01). Data represent the mean total bone nodule area±SD (n=10). (d) Mean bone nodule area (total bone nodule area/number of bone nodules). The differences were statistically significant (∗p<0.05). Data represent the mean bone nodule are ±SD (n=10).

To establish the time-dependent effects of SP on osteoblastic bone formation, we added 10−7MSP to 3-week osteoblast cultures during the first of the three weeks (week 1), the first two weeks of the three weeks (week 1–2), or all three weeks (week 1–3) when the medium was changed. In week 1, we observed no significant increase in bone formation by calvarial osteoblasts in the presence of 10−7MSP compared to bone formation without SP. However, during weeks 1–2 and 1–3, the addition of 10−7MSP to the culture medium significantly increased bone formation (Fig. 2c). Furthermore, the mean area of bone nodule was significantly increased only when the cells were cultured with SP for 1–3week (Fig. 2d).

3.3. Effects of SP and SP-receptor antagonists 

To examine whether the effect of SP on bone formation was dependent on the interaction of SP with its receptor, we investigated the effects of an SP antagonist and SP-receptor (SP-R) antagonist on the SP-induced increase in bone formation of calvarial osteoblasts. Enhanced bone formation in the presence of 10−7MSP was inhibited by the addition of 10−6Mspantide or 10−6MFK888 (Fig. 3). The increased bone formation with 10−8MSP was inhibited by the adding 10−6Mspantide.

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Fig. 3. Effects of 10−6MSP antagonist (spantide) and 10−6NSP-receptor antagonist (FK888; NK1-R antagonist) on bone formation by calvarial osteoblastic cells stimulated with 10−7M or 10−8MSP. Data are mean±SD (n=10). The differences were statistically significant (∗p<0.05, ∗∗p<0.01).

3.4. Effects of SP on the expression of runX2, Col I, and OCN 

The effects of SP on the early differentiation of calvarial osteoblastic cells were examined by measuring the expressions of runX2 and Col I in the cells cultured for 7 days (Fig. 4). The relative density of runX2 and Col I expression revealed that SP had no effects on the early stage of osteoblastic cell differentiation. However, the relative density of runX2 and Col I expression were increased at day 14. Using semi-quantitative RT-PCR analysis, OCN expression was not enhanced by the addition of 10−7MSP at day 7. However, OCN expression was apparently increased in the presence of 10−7MSP compared with control value at day 14 and 21. Adding 10−6Mspantide or 10−6MFK888 inhibited the stimulated SP expression. We also found that OCN mRNA expression was not enhanced in cells cultured with 10−7MCGRP.

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Fig. 4. Semi-quantitative RT-PCR for runX2, Col I, and OCN expression on 7-, 14- and 21-day-cultured calvarial osteoblastic cells (control; (1)) with 10−7MSP (2), 10−7MSP+10−6MSP antagonist (spantide) (3), 10−7MSP+10−6MFK888 (4), and 10−8M calcitonin-gene-related peptide (CGRP; Peptide Institute) (5). The total RNA was analyzed using RT-PCR with runX2-, Col I-, OCN- and GAPDH-specific primers. MW=molecular weight markers. The bottom lows indicate the relative density of runX2-, Col I-, or OCN expression versus control. NM=molecular marker.