|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Effect of calcitonin gene-related peptide on nitric oxide production in osteoblasts: an experimental study
Li Yan*†, Tan Yinghui*1, Yang Bo*, Zhang Gang*, Xiao Xian‡ and Zheng Lu§
*Department of Oral and Maxillofacial Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing, Peoples Republic of China, †Department of Oral and Maxillofacial Surgery, Chengdu Army General Hospital, Chengdu, Peoples Republic of China, ‡The First Outpatient Department of Chengdu Military District Institution, Chengdu Army General Hospital, Chengdu, Peoples Republic of China, and §Department of Hepatobiliary Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing, Peoples Republic of China
The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2′,7′-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.
Key words: calcitonin gene-related peptide, nitric oxide production, osteoblast, signalling pathway
Abbreviations: ALP, alkaline phosphatase, [Ca2+]i, intracellular Ca2+, cAMP, cyclic adenosine monophosphate, CGRP, calcitonin gene-related peptide, cNOS, constitutive nitric oxide synthase, DA, diacetate, DAF-FM, 3-amino, 4-aminomethyl-2′,7′-difluorescein, DMEM, Dulbecco's modified Eagle's medium, eNOS, endothelial nitric oxide synthase, FCS, fetal calf serum, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, IFN-γ, interferon-gamma, IL, interleukin-1, iNOS, inducible nitric oxide synthase, l-NAME, l-arginine, NG-nitro-l-arginine methyl ester, MAPK, mitogen-activated protein kinase, MOB, primary human mandibular osteoblasts, nNOS, neuronal nitric oxide synthase, NO, nitric oxide, NO2, nitrite, NO3, nitrate, RQ%, relative quantity, TNF-α, tumour necrosis factor-alpha
1To whom correspondence should be addressed (email firstname.lastname@example.org).
CGRP (calcitonin gene-related peptide), a 37-amino acid peptide generated by tissue-specific alternative splicing of the calcitonin gene, has been proposed to play a key role in promoting osteoblast recruitment and osteogenic activity (Irie et al., 2002; Kawase et al., 2005). CGRP has been shown to be expressed in nerve fibres during bone development and regeneration (Imai et al., 1997), and CGRP-knockout mice as well as functional studies have demonstrated that CGRP is an important factor during bone formation and repair (McDonald et al., 2004; Ma et al., 2009). CGRP receptors have been identified both in in vitro osteoblast-like cell models as well as downstream signal transduction pathways, such as the cAMP (cyclic adenosine monophosphate) (Villa et al., 2006), [Ca2+]i (intracellular Ca2+) (Burns et al., 2004) and MAPK (mitogen-activated protein kinase) (Kawase et al., 2003, 2005) pathways. In our previous study, we found that the expression and activity of NOS (nitric oxide synthase) was positively correlated with CGRP expression during bone healing, suggesting that NO (nitric oxide) may act downstream of CGRP to promote and regulate fracture healing (Li et al., 2009). However, the regulatory mechanism of CGRP on NO production has not been systematically studied in osteoblasts.
NO has recently been reported to have important effects on osteoblast function and biphasic effects on osteoblastic bone regeneration (van't Hof and Ralston, 2001; Kreja et al., 2008). Low NO concentrations, constitutively produced by osteoblasts, have been shown to act as an autocrine stimulator of osteoblast growth and cytokine production (Riancho et al., 1995; Lin et al., 2008), while high NO concentrations, which are stimulated by proinflammatory cytokines, such as IL-1 (interleukin-1), TNF-α (tumour necrosis factor-alpha) and IFN-γ (interferon-gamma) (Chen et al., 2005; Park et al., 2009), have been shown to have inhibitory effects on osteoblast growth and differentiation (Chen et al., 2005; Fukada et al., 2008). These biological characteristics of NO are determined by the mechanisms of NO action. NO secreted from constitutive NOS [cNOS (constitutive nitric oxide synthase), including eNOS (endothelial nitric oxide synthase) and nNOS (neuronal nitric oxide synthase)], which is constitutively expressed at low levels in its tissues of origin and is regulated at the level of enzymatic activity by changes in free [Ca2+]i (intracellular Ca2+ concentration), results in elevated cGMP levels and causes activation of cGMP-dependent protein kinases (Feelisch and Stamler, 1996). In contrast, NO is secreted from iNOS (inducible nitric oxide synthase) in larger quantities and for prolonged time periods, resulting in cytotoxicity due to oxygen-derived free radicals and lipid peroxidation (Chen et al., 2005). In osteoblasts, eNOS is widely and constitutively expressed; iNOS is only induced by stimulation with proinflammatory cytokines, and nNOS is hardly expressed at all (van't Hof and Ralston, 2001). We propose that CGRP causes changes in NO production in osteoblasts and further explore whether stimulation with CGRP leads to changes in the expression and activity of NOS. We also identify the regulatory mechanism of CGRP action.
2. Materials and methods
Human CGRP and verapamil were purchased from Sigma–Aldrich. CGRP was dissolved in distilled water to a stock concentration of 100 μM and stored in 100 μl aliquots at −70°C. The following reagents were purchased from Beyotime: DAF-FM (3-amino, 4-aminomethyl-2′,7′-difluorescein), DA (diacetate); Griess Reagent; l-NAME (l-arginine, NG-nitro-l-arginine methyl ester); l-canavanine and spermidine. RNAiso Plus, the PrimeScript RT reagent Kit and SYBRR Premix Ex Taq II were purchased from TaKaRa.
2.2. Cell culture
MG-63, an osteogenic human osteosarcoma cell line, was obtained from the ATCC (America Type Culture Collection). Cells were cultured in DMEM (Dulbecco's modified Eagle's medium; Invitrogen) supplemented with 10% FCS (fetal calf serum), 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were subcultured every 72 h in a humidified 5% CO2 incubator. Cells from passages 4–6 were used in the experiments. MOBs (primary cultured human mandibular osteoblasts) were isolated from the mandible of a 24-year-old patient undergoing rebuilding surgery. Following guidelines from the local ethics committee, the patient was informed of the study prior to any surgical procedure and provided consent for use of the materials in this study. Fragments (2 mm×2 mm) of mandibular bone were mechanically isolated and washed several times in PBS to remove blood cells. The fragments were then placed in tissue culture flasks under the same medium and culture conditions as MG-63 cells (Figure 1A). After approximately 2 weeks in culture, bone fragments were discarded, and cells were harvested by trypsinization (Figure 1B). MOBs were identified by ALP (alkaline phosphatase) staining (Figure 1C).
2.3. Determination of NO concentration in cells
Intracellular NO concentrations were measured using a membrane-permeable indicator dye, DAF-FM, which reacts with NO to form a green fluorescent product (Kojima et al., 1999). Cells were seeded on round glass coverslips for 24 h in DMEM containing 10% FCS. After simulating the cells with 10 nM CGRP for 0, 1, 2, 3 or 4 h, individual cultures were incubated for 30 min at 37°C in 10 μM DAF-FM, washed with DMEM and incubated for an additional 30 min in medium without the dye. Cells were rinsed with D-Hanks (Hyclone), and coverslips were affixed to the flow chamber and mounted on a confocal scanning laser microscopy system (LSCM) coupled to an upright microscope (IX-70, ×60 objective; Leica, Germany). Fluorescence intensity was recorded at the excitation wavelength of 488 nm, and the intensity at 0 h was set to 1. The single-cell intracellular NO concentration was analysed as the average intensity of DAF-FM fluorescence, and relative increases in the intracellular NO levels were calculated. Only cells not physically touching adjacent cells were selected for analysis with at least 15 cells counted in each view (magnification, ×200).
2.4. Determination of NO production in cell culture supernatants
MG-63 cells (104/ml) or MOBs (104/ml) were plated in 12-well plates for 24 h and then starved in medium supplemented with 0.1% FCS for 24 h. Cells were incubated with 10 nM of CGRP for 0, 1, 2, 3, 4, 6, 12, 18 or 24 h, and cell culture supernatants were collected and centrifuged at 2000 rev./min for 5 min. NO production was assessed by the Griess reaction (Marzinzig et al., 1997), which measures the stable end product of NO, NO2 (nitrite)/NO3 (nitrate). Briefly, 400 ml samples of conditioned media or NO2 standards (0–100 mM) were mixed with 400 ml of Griess reagent, 1% sulfanilamide and 0.1% naphthylethylene-diamine in 5% phosphoric acid. Samples were measured using a microplate reader, and the absorbance at 530 nm was measured against a blank prepared with non-conditioned medium.
To identify which NOS isoforms were affected, we used l-NAME (50 μM), spermidine (100 μM) and l-canavanine (500 μM) to inhibit eNOS, nNOS and iNOS, respectively. Cells were treated with 10 nM of CGRP and the various inhibitors for 4 h. To determine the effect of [Ca2+]i in CGRP regulation of cNOS, cells were treated with a voltage-dependent L-type Ca2+ channel blocker, verapamil (5 μM), for 4 min. To exclude the influence of any NO synthesis substrates, we co-treated cells with the amino acid l-arginine (20 μM) as a substrate-rich group.
2.5. Assays of eNOS and iNOS mRNA expression
Cells were seeded, starved and treated with 10 nM of CGRP for 0, 6, 12, 18 or 24 h. eNOS and iNOS mRNA expression levels were detected by quantitative RT-PCR. To assay for the regulation of CGRP on iNOS mRNA expression (Park et al., 2009), cytokine-induced groups were used as positive controls, which included stimulation with TNF-α (25 ng/ml), IFN-γ (100 U/ml) and both TNF-α (25 ng/ml)+IFN-γ (100 U/ml) at 24 h; negative control groups were treated with DMEM medium. For each time point and loading treatment, six mRNA samples from each group were extracted using RNAiso Plus and reverse transcribed using a PrimeScript RT reagent Kit (TaKaRa).
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and β-actin were used as internal controls. Primers were designed with the Primer Express software using published human sequences in GenBank and synthesized by TaKaRa: eNOS forward, 5′-CCTCTAAGGTGATCCCTGTCA-3′, reverse, 5′-GCATCCTGCCAGTTTGAGGT-3′ (223 bp); iNOS forward, 5′-TGTTGGGTTCTCTCTGCCATA-3′, reverse, 5′-ACGATTCCAGCCTGATACGC-3′ (157 bp); β-actin forward, 5′-CATGTACGTTGCTATCCAGGC-3′, reverse, 5′-CTCCTTAATGTCACGCACGAT-3′ (250 bp); GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′, reverse, 5′-ATGGTGGTGAAGACGCCAGT-3′ (142 bp).
Quantitative PCR was performed using SYBRR Premix Ex Taq II. Thermal cycling conditions consisted of an initial denaturation at 95°C for 5 min, followed by 40 cycles of 30 s denaturation at 95°C, 30 s annealing at 61°C and 45 s elongation at 72°C and a terminal elongation for 5 min at 72°C. All samples and standards were run in triplicate. Gene expression was quantified using the CT (comparative threshold cycle) method and reported as the fold difference relative to GAPDH and β-actin; the obtained RQ% (relative quantity) of target mRNA was calculated relative to a control sample set to 1.
2.6. Statistical analysis
All results are expressed as the mean±S.E.M. The data were analysed using a one-way analysis of variance (ANOVA) and Newman–Keuls–Student's t test. P<0.05 or P<0.01 was considered significant.
3.1. Effect of CGRP on the NO concentration in osteoblasts
To accurately measure temporal NO concentrations in cells simulated by CGRP, the membrane-permeable indicator DAF-FM, DA was used. This probe is converted by intracellular esterase into DAF-FM, which reacts with NO to form a green fluorescent product. The absorbance of this fluorescent product was then imaged and measured by laser scanning confocal microscopy. As shown, CGRP exhibited a dose-dependent effect (0.1–100 nM) with an effective concentration of 10 nM in both MG-63 cells (Figure 2A) and MOBs (Figure 2B); we subsequently only used 10 nM in all our experiments. The most effective CGRP treatment time point was 1 h in both cell types. Stimulation of MG-63 and MOBs with 10 nM CGRP at 1 h caused peak increases of 323% and 224% in NO concentration, respectively. The NO concentration returned to basal levels after cells were treated for 3 h, but this stimulatory effect was 1 h longer in MG-63 cells than in MOBs.
3.2. Time-dependent effect of CGRP on NO production in cell supernatants
To evaluate long-term NO production in cells, we measured NO2/NO3 concentrations (stable NO end products) in the cell supernatants. As shown, NO concentrations increased linearly in the control groups during the first 24 h with little difference in NO production between MG-63 cells (Figure 3A) and MOBs (Figure 3B) at 24 h, NO production was, respectively, 5.95 and 5.35 μM. CGRP, however, induced a significant increase in NO production between 1 and 6 h, causing increases of 67.5% in MG-63 cells (P<0.01) and 87.6% in MOBs (P<0.01) at the 4-h time point. This maximum difference point of 4 h was designed as the critical observation point in all subsequent experiments. After inclubation for 12 h, CGRP-stimulated NO production in experimental groups coincided with the linear increase in control groups.
3.3. Effect of CGRP on the activity of NOS isoforms
To further explore which NOS isoforms are simulated by CGRP in osteoblasts, we treated cells with inhibitors of eNOS (l-NAME), iNOS (l-canavanine) and nNOS (spermidine) to block CGRP induction. At the same time, we added a NO substrate, L-arginine, to find the CGRP induction change in substrate sufficient conditions. In both MG-63 cells (Figure 4A) and MOBs (Figure 4B), NO production decreased when eNOS was blocked, but not when either iNOS or nNOS was blocked (numbers 1 to 4), suggesting that eNOS is responsible for NO production in osteoblasts. CGRP-induced NO production is effectively blocked 48.3% (P<0.01) and 52.3% (P<0.01) by the eNOS inhibitor l-NAME in both MG-63 and MOBs, respectively (numbers 5 to 8). In substrate-sufficient conditions (numbers 9–13), CGRP-induced NO production was not significant when compared with the conditional control groups (number 9). Interestingly, the eNOS inhibitor l-NAME was still able to block CGRP induction under this condition.
3.4. Effect of [Ca2+]i in CGRP-induced NO production
Given that eNOS is the NOS isoform responsible for CGRP-induced NO production in osteoblasts, we wanted to explore the underlying regulatory mechanism. As Ca2+ is a key factor in eNOS activity and can be induced by CGRP in osteoblasts, we blocked Ca2+ using verapamil, a voltage-dependent L-type Ca2+ channel blocker. Verapamil decreased basal levels of NO both in MG-63 cells by 56.1% (P<0.01, Figure 5A) and in MOBs by 45.0% (P<0.01, Figure 5B), respectively, after stimulation for 4 h. NO levels returned to normal by blocking L-type Ca2+ channels, but CGRP-induced NO production decreased 58.9% (P<0.01) in MG-63 cells and 36.4% (P<0.05) in MOBs. This result suggests that Ca2+ is a necessary signal for CGRP-dependent regulation of eNOS activity.
3.5. Time-dependent effect of CGRP on eNOS and iNOS mRNA expression
Except for its regulatory role on the activity of NOS, CGRP may up-regulate NOS expression to induce further increases in NO production. We thus performed quantitative RT-PCR to determine the effect of CGRP on eNOS and iNOS mRNA expression levels, although it has not been verified that nNOS is expressed in osteoblasts. As shown, we did not observe any statistically significant difference in either eNOS or iNOS mRNA levels between 0 and 24 h after treatment (Figure 6). In addition, we only observed a mild increase of eNOS expression in MG-63 cells at 12 h compared with control groups (P<0.05, Figure 6A); eNOS mRNA expression did not increase in MOBs (Figure 6B).
3.6. Effect of CGRP on cytokine-induced iNOS mRNA expression
Although CGRP does not affect eNOS and iNOS mRNA expression in the first 24 h, we wanted to determine the effect, if any, of CGRP on cytokine-induced increases in iNOS mRNA expression. Regulation of iNOS mRNA levels is an important and effective step in NO production. We used IFN and TNF, previously identified in other osteoblast cell lines, to induce a proinflammatory increase in iNOS mRNA levels. As shown, treating with either cytokine alone had no effect (Figure 7), although TNF treatment did lead to an increase in iNOS mRNA expression in MBOs (Figure 7B). However, co-treating with both IFN and TNF led to a 661% (P<0.01) up-regulation of iNOS mRNA in MG-63 cells (Figure 7A) and a 521% (P<0.01) increase in MBOs (Figure 7B). Finally, when we used CGRP to stimulate cytokine-induced groups, we also did not observe any statistically significant differences compared with the cytokine-induced groups, suggesting that CGRP does not affect cytokine-induced iNOS mRNA expression, at least in vitro.
We demonstrated that incubation of osteoblast-like cells with CGRP leads to a transient time-dependent increase in NO production, primarily through eNOS present in cells. In addition, we elucidated the mechanism of eNOS-mediated NO production via stimulation of CGRP on intracellular Ca2+ levels.
CGRP has been considered to be an endogenous mitogen activator in osteogenic cells because of its effects on cell proliferation (Huebner et al., 2008). Villa et al. (2006) reported that CGRP treatment caused an increase in osteoblast proliferation through the cAMP-dependent signalling pathway, and we have found that CGRP also increases MG-63 cell proliferation through regulation of AP-1 (activator protein-1) (L. Yan, T. Yinghui, Y. Bo, Z. Gang, W. Xuefei and L. Min, unpublished work). Although the major effect of NO appears to be inhibition of cell proliferation in many cell types (Chen, et al., 2005; Tamashiro et al., 2010), there are several reports indicating that the NO produced constitutively by constitutive NOS may have a stimulatory action and may act as a stimulator of osteoblast growth and cytokine production. Because of the positive correlation reported between NO and CGRP expression in bone callus (Li et al., 2009), we hypothesized that NO may also mediate CGRP-induced osteoblast proliferation or differentiation in vitro. In this study, we chose MG-63 and MOBs to investigate this hypothesis. MG-63 express low basal levels of alkaline phosphatase, osteocalcin, synthesize types I and III collagen as immature marrow stromal cells (Ryu et al., 2009) and have been reported to be affected by CGRP in studies (Kawase et al., 2003, 2005; Bums et al., 2004). MOBs are more representative and similar to our experiment in vivo (Li et al., 2009).
NO present in cell supernatants rapidly reacts with oxygen to yield the stable metabolites NO3 and NO2, which can easily be measured by the Griess reaction (Feelisch and Stamler, 1996). In our study, we found that treatment with 10 nM of CGRP induced a significant increase in NO production between 1 to 12 h both in MG-63 cells and MOBs, which recovered after 12 h (comparing between CGRP-stimulated groups and control groups). Therefore, we initially concluded that CGRP promoted NO production in osteoblasts, at least in vitro. However, the amount of accumulated NO3 and NO2 did not accurately reflect the amount of NO present in the cell. We thus used DAF-FM DA to quantitatively image the concentration of NO in the cell by assaying fluorescence photodensity (Kojima et al., 1999). Owing to fluorescence attenuation, we had to limit the time of observation to 4 h. Using this method, we found significant increases in fluorescence intensity within 2 h of CGRP simulation. The most effective concentration that induced NO production was 10 nM, the same concentration that effectively promotes cell proliferation (Villa et al., 2006). It appeared that MG-63 cells were more sensitive than MOBs to secrete NO upon CGRP treatment. We could not detect any statistically significant differences among all of the groups after 2 h of treatment, suggesting that any increases in NO production observed in cell supernatants up to 12 h occurs during the first 2 h. We thus assumed that CGRP-induced increases in NO concentration are directly due to transient and temporal production from cNOS. At the same time, we found that increases in NO were temporal and low in concentration, suggesting that NO may be a downstream messenger to mediate CGRP effect. Despite having cytotoxic effects, NO is involved in many signalling transduction pathways, including cGMP-dependent, EGFR (epidermal growth factor receptor) and MAPK signalling pathways (Chen et al., 2005; Lo and Hung, 2006; Ptasinska et al., 2007). Once NO is demonstrated to be the second messenger of CGRP signalling, we can further explore the effects of CGRP on these other signalling pathways.
Three isoforms of NOS have been identified thus far, and both eNOS and iNOS are constitutively expressed in osteoblasts (Helfrich et al., 1997). Although little is known about the expression of nNOS in bone and cultured bone-derived cells, nNOS mRNA has been detected by RT-PCR in bone (Schmidt et al., 1992; Chen et al., 2005). Here, we applied specific inhibitors of these three NOS isoforms to identify which isoform mediates increases in NO production in osteoblasts. We found that inhibiting the activity of eNOS leads to statistically significant decreases in basal NO levels compared with control groups and was able to recover CGRP-induced NO production. It was demonstrated that eNOS is the isoform responsible for maintaining basal NO levels, and CGRP induces increases in NO levels. To exclude the influence of deficiency of NO donors, we co-incubated cells with 0.1 nM CGRP and 20 μM l-arginine. With sufficient NO donor, CGRP does not induce a higher NO production, but the NO production level can be blocked by inhibiting eNOS. This attenuation of NO may be related with the autoregulatory feedback loop, in which increased NO levels limit additional NO production (Rogers and Ignarro, 1992). CGRP-induced NO production is a normal biological activity of the cell, but increases in NO production lead to cell apoptosis. After blocking intracellular Ca2+ using verapamil, a voltage-dependent L-type Ca2+ channel blocker, CGRP-induced NO production was also recovered by inhibiting eNOS. Burns et al. (2004) reported that in MG-63 cells, CGRP stimulates Ca2+ discharge from intracellular stores through a cAMP-independent mechanism and subsequently stimulates Ca2+ influx through L-type voltage-dependent channels, explaining the effect of CGRP on NO production in osteoblasts. Additionally, we observed a gradual rise of eNOS mRNA expression at 12 h in MG-63 cells treated with CGRP, but otherwise did not observe any statistically significant difference between MG-63 cells and MOBs. Because it only took place at one observation point and in just one kind of cell, we cannot say that CGRP regulates eNOS mRNA expression in osteoblasts. We thus conclude that CGRP causes increases in NO production by regulating eNOS activity, which is dependent on increases in intracellular Ca2+ levels in osteoblasts.
Unlike cNOS, iNOS is primarily regulated at the level of transcription and is activated by proinflammatory cytokines, such as IL-1, TNF-α and IFN-γ (Chen et al., 2005; Park et al., 2009). To identify whether CGRP can regulate the expression of different iNOS isoforms and affect cytokine-induced iNOS activity, we compared iNOS mRNA levels in osteoblasts stimulated with CGRP and those stimulated with TNF, IFN and TNF/IFN as a positive control. We observed iNOS mRNA up-regulation in both MG-63 cells and MBOs with TNF/IFN stimulation and in MBOs with TNF stimulation. However, we did not observe any up-regulation of iNOS mRNA expression or any effect of CGRP on cytokine-induced iNOS mRNA expression. It may be possible that in vivo, CGRP indirectly regulates iNOS expression by recruiting inflammatory cells and stimulating growth or inflammatory factors, although CGRP is not known to regulate the iNOS signalling pathway in osteoblasts in vitro.
In summary, CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro within the first 2 h of treatment. Increased NO levels and extended incubation time appear to not be cytotoxic to osteoblasts; on the contrary, our results were consistent with a role for CGRP in promoting cell proliferation in either a time- or concentration-dependent manner. This temporal, CGRP-dependent induction of NO could play a role in the mitogenic effects of CGRP on osteoblast growth. Future work will aim to identify the components up-regulated by NO during osteoblast proliferation and differentiation.
Li Yan designed the experiments and was in charge of the cytological experiments, data statistics and paper writing. Tan Yinghui was responsible for the financial support, experimental design guidelines and article review. Yang Bo was in charge of the molecular biology experiments. Zhang Gang was in charge of literature review and article review. Xiao Xian was responsible for the molecular biological experiments. Zheng Lu was in charge of the cytological experiments.
This work was funded by the
Burns, DM, Stehno-Bittel, L and Kawase, T (2004) Calcitonin gene-related peptide elevates calcium and polarizes membrane potential in MG-63 cells by both cAMP-independent and -dependent mechanisms. Am J Physiol-Cell Physiol 287, C457-67
Fukada, SY, Silva, TA, Saconato, IF, Garlet, GP, Avila-Campos, MJ and Silva, JS (2008) iNOS-derived nitric oxide modulates infection-stimulated bone loss. J Dent Res 87, 1155-9
Helfrich, MH, Evans, DE, Grabowski, PS, Pollock, JS, Ohshima, H and Ralston, SH (1997) Expression of nitric oxide synthase isoforms in bone and bone cell cultures. J Bone Miner Res 12, 1108-15
Huebner, AK, Keller, J, Catala-Lehnen, P, Perkovic, S, Streichert, T and Emeson, RB (2008) The role of calcitonin and [alpha]-calcitonin gene-related peptide in bone formation. Arch Biochem Biophys 473, 210-7
Imai, S, Rauvala, H, Konttinen, YT, Tokunaga, T, Maeda, T and Hukuda, S (1997) Efferent targets of osseous CGRP-immunoreactive nerve fiber before and after bone destruction in adjuvant arthritic rat: an ultramorphological study on their terminal-target relations. J Bone Miner Res 12, 1018-27
Irie, K, Hara-Irie, F, Ozawa, H and Yajima, T (2002) Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling. Microsc Res Tech 58, 85-90
Kawase, T, Okuda, K and Burns, DM (2003) Immature human osteoblastic MG63 cells predominantly express a subtype 1-like CGRP receptor that inactivates extracellular signal response kinase by a cAMP-dependent mechanism. Eur J Pharmacol 470, 125-37
Kawase, T, Okuda, K and Burns, DM (2005) Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys (Acm) 2, 7] calcitonin gene-related peptide and CGRP8-37. Am J Physiol-Cell Physiol 289, C811-8
Kojima, H, Urano, Y, Kikuchi, K, Higuchi, T, Hirata, Y and Nagano, T (1999) Fluorescent indicators for imaging nitric oxide production. Angew Chem Int Ed Engl 38, 3209-12
Li, Y, Tan, Y, Zhang, G, Yang, B and Zhang, J (2009) Effects of calcitonin gene-related peptide on the expression and activity of nitric oxide synthase during mandibular bone healing in rabbits: an experimental study. J Oral Maxillofac Surg 67, 273-9
Lin, IC, Smartt, JM Jr, Nah, HD, Ischiropoulos, H and Kirschner, RE (2008) Nitric oxide stimulates proliferation and differentiation of fetal calvarial osteoblasts and dural cells. Plast Reconstr Surg 121, 1554-66
Lo, HW and Hung, MC (2006) Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. Br J Cancer 94, 184-8
Ma, H, Young, J and Cherng, S (2009) Calcitonin gene-related peptide and substance P significantly influence coronary flow rate in gene knockout mice. J Bacteriol Res 1, 019-25
Marzinzig, M, Nussler, AK, Stadler, J, Marzinzig, E, Barthlen, W and Nussler, NC (1997) Improved methods to measure end products of nitric oxide in biological fluids: nitrite, nitrate, and S-nitrosothiols. Nitric Oxide 1, 177-89
McDonald, KR, Fudge, NJ, Woodrow, JP, Friel, JK, Hoff, AO and Gagel, RF (2004) Ablation of calcitonin/calcitonin gene-related peptide-α impairs fetal magnesium but not calcium homeostasis. Am J Physiol Endocrinol Metab 287, E218
Park, YG, Kim, KW, Song, KH, Lee, JM, Hong, JJ and Moon, SK (2009) Combinatory responses of proinflamamtory cytokines on nitric oxide-mediated function in mouse calvarial osteoblasts. Cell Biol Int 33, 92-9
Ptasinska, A, Wang, S, Zhang, J, Wesley, RA and Danner, RL (2007) Nitric oxide activation of peroxisome proliferator-activated receptor gamma through a p38 MAPK signaling pathway. FASEB J 21, 950-61
Riancho, JA, Salas, E, Zarrabeitia, MT, Olmos, JM, Amado, JA and Fernández-Luna, JL (1995) Expression and functional role of nitric oxide synthase in osteoblast-like cells. J Bone Miner Res 10, 439-46
Rogers, NE and Ignarro, LJ (1992) Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from l-arginine. Biochem Biophys Res Commun 189, 242-9
Ryu, BM, Li, Y, Qian, ZJ, Kim, MM and Kim, SK (2009) Differentiation of human osteosarcoma cells by isolated phlorotannins is subtly linked to COX-2, iNOS, MMPs, and MAPK signaling: implication for chronic articular disease. Chem Biol Interact 179, 192-201
Schmidt, HH, Gagne, GD, Nakane, M, Pollock, JS, Miller, MF and Murad, F (1992) Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction. J Histochem Cytochem 40, 1439-56
Tamashiro, E, Banks, CA and Cohen, NA (2010) New areas for investigation: nitric oxide. Nasal Polyposis 175-83
Villa, I, Mrak, E, Rubinacci, A, Ravasi, F and Guidobono, F (2006) CGRP inhibits osteoprotegerin production in human osteoblast-like cells via cAMP/PKA-dependent pathway. Am J Physiol-Cell Physiol 291, C529-37
Received 23 November 2010/19 January 2011; accepted 11 March 2011
Published as Cell Biology International Immediate Publication 11 March 2011, doi:10.1042/CBI20100832
© The Author(s)
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 4 Effect of inhibitors of NOS isoforms on CGRP-induced NO production in MG-63 cells (A) and MOBs (B)
Figure 5 Blocking effect of verapamil on the CGRP-induced NO concentrations in MG-63 cells (A) and MOBs (B)
Figure 6 Time-dependent expression of eNOS and iNOS mRNA in MG-63 cells (A) and MOBs (B) after CGRP treatment