| Neuropeptide which is secreted by peripheral nerve plays an important role in fracture healing and bone metabolism. Calcitonin gene-related peptide （CGRP）, a 37-amino acid peptide generated by tissue-specific alternative splicing of the calcitonin gene, has been shown to be expressed in nerve fibers during bone development and regeneration, and CGRP-knockout mice as well as functional studies have demonstrated that CGRP is an important factor during bone formation and repair. CGRP receptors have been identified both in vitro osteoblast-like cell models as well as downstream signal transduction pathways, such as the cyclic adenosine monophosphate （cAMP）, intracellular Ca2+ and mitogen-activated protein kinase （MAPK） pathways.Nitric oxide （NO） has been reported to have biphasic effects on osteoblastic bone regeneration. These biological characteristics of NO are determined by the mechanisms of NO action. NO secreted from constitutive NOS （cNOS, including eNOS and nNOS）, 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 intracellular Ca2+ concentration ([Ca2+]i). In osteoblasts, eNOS is widely and constitutively expressed; iNOS is only induced by stimulation with pro-inflammatory cytokines, and nNOS is hardly expressed at all. Low NO concentrations, constitutively produced by osteoblasts, have been shown to act as an autocrine stimulator of osteoblast growth and cytokine production, while high NO concentrations, which are stimulated by pro-inflammatory cytokines, have been shown to have inhibitory effects on osteoblast growth and differentiation. In contrast, NO is secreted from iNOS in larger quantities and for prolonged time periods, resulting in cytotoxicity due to oxygen-derived free radicals and lipid peroxidation.This study was designed to verify the effect of CGRP on the osteoblasts’proliferation and differentiation in vitro; to clear the effect of CGRP on cell NO prodcution, NOS activity and expression; to explore the the regulation mechanism of CGRP on NO production; at last, to find the biological meaning of CGRP-NO signaling pathway in osteoblasts.Methods1. Cell culture: MG-63 cell （ATCC） were cultured in Dulbecco’s modified Eagle’s Medium supplemented with 10% fetal calf serum. Cells were subcultured every 72 h in a humidified 5% CO2 incubator. Cells from passages 6–8 were used in the experiments.2. Effect of CGRP on proliferation and differentiation of MG-63:2.1 Proliferation: Time-effect （24-96 h） and dose-effect (10–10 -10–7mol/L) of CGRP on cell viability of MG-63 were determinated by MTT method; time-effect （0-24 h） of CGRP on cell cycle was assayed using flow cytometry; time-effect （0-80 m） of CGRP on expression of c-fos/c-jun mRNA in cell was detected by RT-PCR.2.2 Differentiation: Dose-effect (10–10 -10–7mol/L) of CGRP on cell ALP was stained by reforming Kaplow method; dose-effect (10–10 -10–7mol/L) of CGRP on cell calcifacaiton was stained by alizarin bordeaux; dose-effect (10–10 -10–7mol/L) of CGRP on expression of CollagenⅠmRNA in cell was detected by RT-PCR; dose-effect (10–10 -10–7mol/L) of CGRP on expression of OC in cell culture supernatant was detected by ELISA.3. Effect of CGRP on NO production in MG-63: Time-effect （0-48 h） of 10–8 mol/L CGRP on NO production in cell culture supernatants was detected by Griess reaction; time-effect （0-4 h） of 10–8 mol/L CGRP on NO concentration in cell was detected by DAF-FM DA fluorescence detection; effects of 10–8 mol/L CGRP on eNOS, nNOS and iNOS activity in cell were detected by NOS enzyme kit; time-effects （0-48 h） of 10–8 mol/L CGRP on expression of eNOS, nNOS and iNOS were stained and detected by immunoflorescence.4. Regulation of CGRP on NO production in MG-63:4.1 Regulation of CGRP on activity of eNOS via [Ca2+]i : Time-effect （0-15 m） of 10–8 mol/L CGRP on [Ca2+]i in MG-63 was dectected by Fluo-3/AM fluorescence detection; depressive effect of verpamil on CGRP induced NO concentration in MG-63 was detected by DAF-FM DA fluorescence detection; depressive effect of verpamil on CGRP induced NO production in cell culture supernatants was detected by Griess reaction.4.2 Regulation of CGRP on expression of iNOS mRNA : Time-effect （24-96 h） of 10–8 mol/L CGRP on expression of iNOS mRNA in cell was detected by Real time PCR; after inducted by TNF-αand INF-γ, effect of CGRP on expression of iNOS mRNA in MG-63 was detected by Real time PCR.5. Biological meaning of CGRP-ON regulation in MG-63:5.1 Proliferation: Time-effect （24-96 h） of L-NAME, inhibitor of eNOS on CGRP induced cell proliferation was detected by MTT method; depresive effect of L-NAME on CGRP induced cell cycle acceleration was assayed using flow cytometry; depresive effect of L-NAME on CGRP induced up-expression of c-fos/c-jun mRNA in cell was detected by Real time PCR.5.2 Differentiation: Depresive effect of L-NAME on CGRP induced up-expression of CollagenⅠmRNA in cell was detected by Real time PCR; depresive effect of L-NAME on CGRP induced up-expression of OC in cell culture supernatant was detected by ELISA. Results:1. Effect of CGRP on proliferation and differentiation of MG-63:1.1 Proliferation: In MTT assay, growth rate of cell in 10–10 -10–7mol/L CGRP experimental groups were significantly higher than control group （P<0.05 or P<0.01）; IP and the proportion of S phase cells in CGRP experimental groups were high at 8 h, but no statistical difference at 24 h; expression of c-fos mRNA in experimental groups’cell were significant higher than control group at 20 m （P<0.01）,expression of c-jun mRNA were lower than control group at 60m （P<0.01）.1.2 Differentiation: OD value of ALP stain in 10–10 -10–7mol/L CGRP experimental groups were high than control group （P<0.05 or P<0.01）,number of alizarin bordeaux stain in 10–10 -10–7mol/L CGRP experimental groups were higher than control group （P<0.01）,expression of CollagenⅠmRNA in 10–9 -10–7mol/L CGRP experimental groups were higher than control group （P<0.05 or P<0.01）,expression of OC in cell culture supernatant in 10–10 -10–7mol/L CGRP experimental groups were high than control group （P<0.05 or P<0.01）.2. Effect of CGRP on NO production in MG-63: After induced by CGRP, production in cell culture supernatant in CGRP experimental groups were higher than control group （P<0.01） at 12 and 24 h,NO concentration in cell in CGRP experimental groups were higher than control group （P<0.05 or P<0.01） at 1, 2 and 3 h,activity of NOS in inhibited eNOS group was significant lower than CGRP reduced group （P<0.01）, but no statistical difference in inhibited nNOS and iNOS groups. After CGRP indeced 0-48 h, except for expression of eNOS was higher than control （P<0.05） at 36 h, there was no stastical difference in eNOS, nNOS and iNOS experission.3. Regulation of CGRP on NO production in MG-63:3.1 Regulation of CGRP on activity of eNOS via [Ca2+]i : After induced by CGRP, [Ca2+]i in experimental groups’cells increased from 250-600 s contrast with control; NO concentration in [Ca2+]i inhibited group was lower than CGRP induced group （P<0.01）, NO production in culture supernatant in [Ca2+]i inhibited group was lower than CGRP induced group （P<0.01）.3.2 Regulation of CGRP on expression of iNOS mRNA : After induced by CGRP for 48 h, there was no difference of iNOS mRNA expression between experimental group and control group; after inducted by TNF-αand INF-γfor 48 h, expression of iNOS mRNA in experimental group cells was significant higher than control （P<0.01）, but no difference between TNF-αand INF-γinduced group and CGRP+ TNF-α+INF-γgroup.4. Biological meaning of CGRP-ON regulation in MG-63:4.1 Proliferation: Growth rate of cell in L-NAME experimental groups were significantly lower than control group at 48, 72 and 96 h（P<0.01）, but no difference at 24 h; growth rate of cell in L-NAME inhibited group was lower than CGRP induced group at 24 h （P<0.01）; proportion of G2 phase cells in L-NAME inhibited group were lower than CGRP induced group （P<0.01）, but no statistical difference in IP; expression of c-fos mRNA in L-NAME inhibited group were lower than CGRP induced group at 20 m （P<0.01）,and no difference in c-jun mRNA. 4.2 Differentiation: After induced by CGRP for 24 h, there was no difference of CollagenⅠmRNA and OC expression between L-NAME inhibited group and CGRP induced group .Conclusion:1. CGRP up-regulates proliferation of MG-63 in vitro, and 10–8mol/L is the most effective concentration; effect of CGRP on MG-63 proliferation reflected in the activation of AP-1, expression of c-fos mRNA, and accelerates cell into mitosis to raise the proliferation of cell; CGRP up-regulates activity of ALP, mineralized nodule formation, expression of CollagenⅠmRNA and OC to simulate differentiation of MG-63.2. CGRP stimulates activity of eNOS in MG-63 cell, increases concentration of intracellular NO, and increases production of NO in culture medium; CGRP dose not regulate expression of NOS isoforms in MG-63.3. CGRP increases intracellular NO concentration via up-regulating [Ca2+]i to stimulate activity of eNOS in MG-63, but not regulating expression of iNOS mRNA; even stimulated by inflammatory cytokines, expression of iNOS mRNA can not be effected by CGRP.4. CGRP induced c-fos mRNA up-regulation can be inhibited via inhibiting eNOS activity; this inhibiting effect also be found in CGRP induced proliferation and cell cycle change; but it not be found in expression of CollagenⅠmRNA and OC, which reflect differentiation of MG-63. |
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