The Effect Of BMP-2on Calcitonin Gene Related Peptide-induced MG63Cells Proliferation And Differentiation

Numerous researches provide in vivo suggested that such neuropeptide calcitonin generelated peptide （CGRP） is associated with bone development, metabolism and repair. Invitro studies have demonstrated that CGRP stimulates the osteoblastic proliferation,differentiation and maturity in both osteoblastic cell models as well as bone marrowmesenchymal stromal cell models. CGRP and its receptors have been identified in signaltransduction pathways, such as [Ca2+]i（intracellular Ca2+）,the cAMP（cyclic adenosinemonophosphate）, PKC （protein kinase C）, ERK （extracellular signal-regulated proteinkinase） and MAPK （mitogen-activated protein kinase） pathways.Although CGRP is a recognised neuromodulator of osteoblast cell signal pathway, itsmechanism of action in osteogenesis remains elusive. Bone morphogenetic proteins （BMPs）are multi-functional growth factors that belong to TGF-families. The roles of BMPs, inembryonic development and cellular functions in postnatal and adult animals have beenextensively studied in recent years. Among BMPs, BMP-2has very strong osteoinductiveactivity. Both in vivo and in vitro studies have shown that BMP-2was essential regulator ininducing osteogenic differentiation and bone formation. Bone marrow mesenchymalstromal cells （BM-MSCs） effectively supported bone formation via autocrine and paracrinefunctions when selectively facilitating the delivery and bioavailability of BMP-2. BMP-2promoted the proliferation of pre-osteoblastic cells, induced the osteogenic or chondrogenicdifferentiation of mesenchymal cells, improved the osteogenic phynotype and capacity ofstem cells, through increased ALP activity and osteocalcin mRNA expression. Signaltransduction studies have revealed that Smad, P38MAPK pathways are the immediatedownstream molecules of BMP receptors and play a central role in BMP signaltransduction.To date, the researches about the relationship between BMP and CGRP are limited. Previous studies suggested that bone morphogenetic proteins （BMPs）2,4, or6stimulatedCGRP expressions in60%of DRG neurons. While CGRP promoted the production ofBMP-2in pulp cells. BMP-2was also associated with the spatial and temporal regenerationof CGRP-positive nerve fibers in ectopic bone formation. Evidence suggested thatneuropeptides and receptors like CGRP play an important role in the regulation of boneremodeling and CGRP induces the expression of BMP-2in bone tissues. So we hypothesisthat expression of BMP-2is involved in the mechanism of CGRP induced osteogenesis.The experiment is about the effect of BMP signal pathway on CGRP contributing to MG63proliferation and differentiation, and CGRP contributed to BMP-2expression of MG63andits mechanism was studied.Methods1.Cell culture: Low-passage MG63osteogenic human osteosarcoma cells wereobtained from the the Chinese academy of sciences institute of Shanghai life science andplated, grown, and maintained in RPMI1640（with10%FBS）.Cells were normally platedat a density of5×105cells/cm²and cultivated in humidified5%CO2-95%air at37°C. Thesixth to seventh generations were used in the experiments.2. CGRP-induced MG63cells proliferation and differentiation:2.1Proliferation: Dose-effect (10^-10-10^-7M) and time-effect （24-72h） ofCGRP-induced MG63cells viability were determinated by MTT method; dose-effect (10^-10-10^-7M) and time-effect （24-72h） of CGRP on cell cycle were assayed by flow cytometry.2.2Differentiation: Dose-effect (10^-10-10^-7M) and time-effect （24-72h） ofCGRP-induced MG63cells ALP were stained by PNPP method; effect（48h） of CGRP（10-8M） on cell ALP was stained by immunofluorescence staining; dose-effect (10^-10-10^-7M) andtime-effect （24-72h） of CGRP-induced MG63cells expression of ALP、CollagenⅠ、OCNprotein were detected by Western blot.3. Effect of BMP signal pathway on CGRP-induced MG63cells proliferation anddifferentiation:3.1Groups:10-8M CGRP；10-8M CGRP+100ng/ml Noggin；100ng/ml Noggin；control.Time:48h.3.2Effect on Proliferation: Cell cycle was assayed by flow cytometry. 3.3Effect on Differentiation: ALP was stained by immunofluorescence staining;expression of ALP、CollagenⅠ、OCN protein of MG63were detected by Western blot.4. CGRP on expression of BMP-2of MG63:4.1Dose-effect (10^-10-10^-7M) and time-effect （1-48h） of CGRP-induced MG63cellsexpression of BMP-2mRNA were detected by RT-PCR.4.2Effect of BMP signal pathway on CGRP-induced expression of BMP-2in MG63cells:Groups：10-8M CGRP；10-8M CGRP+100ng/ml Noggin；100ng/ml Noggin； control.Time:48h.The expression of BMP-2mRNA and protein of MG63were detected by RT-PCR andWestern blot.5. The mechanism of CGRP-induced expression of BMP-2in MG63cells:5.1Groups:10-8M CGRP、10-8M CGRP+5μM H-89、5μM H-89、control. Time:48h.5.2The expression of cAMP was detected by ELISA; the expression of BMP-2mRNAwas detected by RT-PCR; the expression of BMP-2、 CREB、 pCREB protein weredetected by Western blot.6. Statistic analysis:Data were expressed as means±S.D. Statistical comparisons of the results were madeusing analysis of variance. Significant differences （p<0.05, p<0.01） between the means ofcontrol and test group were analyzed by Dunnett’s test.Results1. CGRP-induced MG63cells proliferation and differentiation:1.1Proliferation: Through MTT assay, the cell,sgrowth rate in10^-10-10^-7M CGRPgroups was higher than that in control group （P<0.05）, especially in10-8M CGRPexperimental group; in flow cytometry assay， the proportion of S+G2phase cells in10^-10-10^-7M CGRP groups was higher than that in control group （P<0.05），especially in10-8M CGRP （48h）experimental group and a downward trend appeared after48h.1.2Differentiation: OD value of ALP stain in10^-10-10^-7M CGRP groups was higherthan that in control group （P<0.05），especially in10-8M CGRP experimental group, and no increasing after48h; in immunofluorescence staining, fluore-scence number of10-8M CGRP （48h） group was more than control group; in westernblot assay, expression of ALP、CollagenⅠ and OCN protein in10^-10-10^-7M CGRPexperimental groups were higher than control group （P<0.05）.2. Effect of BMP signal pathway on CGRP-induced MG63cells proliferation anddifferentiation:2.1Effect on Proliferation: In flow cytometry assay， the proportion of S+G2phasecells in CGRP and CGRP+Noggin experimental groups was higher than that in controlgroup （P<0.05）; while there was no difference between CGRP and CGRP+Nogginexperimental groups.2.2Effect on Differentiation: The expression of ALP、CollagenⅠ、OCN protein inCGRP group was higher than that in control group and CGRP+Noggin group（p<0.05）.3. CGRP on expression of BMP-2in MG63cells:3.1The BMP-2mRNA expression in10-7M CGRP group was higher than that in10-8M group between8-48h（p<0.01）; the BMP-2mRNA expression in10-9M group detecteduntil48h was lower than that in10-8M and10-7M group （p<0.01）.3.2The expression of BMP-2mRNA and protein were increased in both CGRP andCGRP+Noggin groups, with significant difference when compared with the Noggin andcontrol groups （p<0.01）.4. The mechanism of CGRP-induced expression of BMP-2in MG63cells:4.1ELISA of the related fold of cAMP concentrations in four groups, CGRP groupand CGRP+H-89group were higher than control group （P<0.01）, while there was nodifference between CGRP group and CGRP+H-89group.4.2The expression of CREB did not differ in four groups.4.3The expression of pCREB was significantly increased in CGRP treated group andinhibited in H-89pretreated group （P<0.01）.4.4The expression levels of BMP-2mRNA and protein significantly increased in theCGRP treated group compared with the other three groups （p<0.01）.Conclusions1. CGRP up-regulates proliferation and differentiation of MG63in vitro, with time and dose dependence.2. CGRP-induced cell differentiation may operate by a BMP-2-dependent pathway；CGRP-induced cell proliferation may operate by a BMP-2-independent pathway.3. CGRP up-regulates the expression levels of BMP-2of MG63in vitro, with time anddose dependence.4. cAMP/pCREB pathway is involved in the CGRP promotion of BMP-2mRNA andprotein in MG63.