BMP-2在降钙素基因相关肽调控MG63细胞增殖分化中的作用
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摘要
降钙素基因相关肽(Calcitonin gene-related peptide，CGRP)被证实可以促进成骨细胞的增殖和分化活性，已知的CGRP信号调控通路包括了：胞内钙离子通道(Ca2+)、环磷酸腺苷(cAMP)、蛋白激酶(PKC)、丝裂原活化蛋白激酶(MAPK)、胞外信号调节激酶(ERK)等。大量实验证实，骨形态发生蛋白(BMP)也同样拥有诱导成骨的能力。骨髓基质细胞在有BMP-2参与的基础上，通过旁分泌和自分泌作用，在细胞和细胞间质传导信息，刺激DNA的合成和细胞的复制，促进成骨细胞前体的增殖，诱导未分化间充质细胞不可逆地分化为软骨细胞和成骨细胞，提高干细胞的成骨表型和成骨能力，增强碱性磷酸酶(Alkaline phosphatase，ALP)的活性，促进细胞间的粘附和成骨细胞标记基因的表达。目前已知的BMP对成骨细胞的信号传导主要包括有Smad，P38MAPK通路。
目前的研究发现：BMP-2，4，6可以促进神经分泌CGRP，CGRP在体外促进牙髓细胞分泌BMP-2，BMP-2体内异位诱导成骨组织中CGRP的神经可以再生分布，BMP-2在腹腔注射CGRP的大鼠胫骨骨折愈合的骨痂中高表达，CGRP在体外协同增加BMP-2诱导成骨细胞增殖的能力。这些研究结果提示：CGRP可以诱导骨组织中BMP-2的表达增加，但两者之间相互作用的信号通路机制目前尚不清楚，因此推测CGRP的促成骨作用可能与BMP-2的表达有相关性。本实验将对CGRP促成骨细胞增殖、分化作用中BMP信号通路所发挥的作用；CGRP促成骨细胞BMP-2表达的影响及其机制等进行研究。
方法
1．细胞培养：人骨肉瘤细胞(MG63)细胞株(中国科学院上海生命科学研究院)接种于培养瓶(100ml)中传代培养(5×106个/cm~2)，培养基为RPMI1640(含10%FBS)，放置于5%CO2孵箱中37°C孵育72h传代，传代至6-7代时使用。
2．CGRP对MG63细胞增殖和分化的调控作用：
2.1MG63细胞增殖：MTT法检测CGRP对MG63细胞增殖的调控作用；流式细胞仪检测CGRP对MG63细胞增殖周期的调控。设定量效作用的浓度为(10~(–10)-10~(–7)M)，时效作用为(24-72h)。
2.2MG63细胞分化：PNPP偶氮法检测CGRP对MG63细胞ALP染色的调控作用；Western blot检测CGRP对MG63细胞碱性磷酸酶(ALP)、Ⅰ型胶原(CollagenⅠ)、骨钙素(OCN)表达的调控作用。设定量效作用的浓度为(10~(-10)-10~(-7)M)，时效作用为(24-72h)。免疫荧光染色检测10-8M CGRP对MG63(48h)ALP表达的作用。
3．CGRP对MG63细胞增殖、分化调控中BMP信号通路的作用：
3.1分组：10-8M CGRP；10-8M CGRP+100ng/ml Noggin；100ng/ml Noggin；空白对照组，作用时间为48h。
3.2MG63细胞增殖影响：流式细胞仪检测MG63细胞增殖周期。
3.3MG63细胞分化影响：免疫荧光染色检测MG63细胞的ALP表达；Western blot检测MG63细胞的ALP，ColIaⅠ和OCN蛋白表达。
4．CGRP对MG63细胞表达BMP-2的调控作用：
4.1RT-PCR检测CGRP对MG63细胞表达BMP-2mRNA的调控作用：设定量效作用的浓度为(10~(-10)-10~(-7)M)，时效作用为(1-48h)。
4.2CGRP促进MG63细胞表达BMP-2中Noggin的作用：
分组：10-8M CGRP；10-8M CGRP+100ng/ml Noggin；100ng/ml Noggin；空白对照组，作用时间48h。
RT-PCR检测MG63细胞BMP-2mRNA的表达；Western blot检测MG63细胞BMP-2蛋白的表达。
5．CGRP促进MG63细胞表达BMP-2的机制：
5.1分组：10-8M CGRP、10-8M CGRP+5μM H-89、5μM H-89、空白对照组，作用时间48h。
5.2免疫检测试剂盒检测MG63细胞cAMP表达；RT-PCR检测MG63细胞BMP-2mRNA表达；Western blot检测MG63细胞BMP-2、环磷酸腺苷反应元件结合蛋白(cAMPresponse element binding protein， CREB)、磷酸化环磷酸腺苷反应元件结合蛋白(Phosphorylated Camp Response Element Binding Protein，pCREB)的表达。
6．统计分析：
所有数据均采用均数±标准差(±s)表示，应用Dunnett，s检验中的t检验和方差分析，进行统计分析，分别设定p<0.05以及p<0.01表明有显著性差异和差异非常显著。
结果
1．CGRP对MG63细胞增殖、分化的调控作用：
1.1MG63细胞增殖：MTT法检测发现CGRP加入MG63细胞培养基中24-72h的时间段中，CGRP各浓度组的细胞数均显著高于空白对照组(P<0.05)，其中以10-8MCGRP浓度组最为明显(P<0.05)；流式细胞仪检测细胞增殖周期发现不同浓度的CGRP都加速了细胞周期循环，但是以48h10-8M组最为明显(P<0.05)，48h以后有下降趋势。
1.2MG63细胞分化：PNPP偶氮法检测发现各浓度组的CGRP促进MG63细胞的ALP表达量均高于空白对照组，其中以72h10-8M组最为明显(P<0.05)，时效对比中发现各浓度组细胞的ALP表达量在72h较48h增加不明显(P＞0.05)；蛋白定量分析结果发现各实验组的ALP，ColIaⅠ和OCN蛋白表达量均高于空白对照组(P<0.05)，其中以48h10-8M组最为明显(P<0.05)；免疫荧光染色结果发现48h10-8M组荧光数目较空白对照组多。
2．CGRP对MG63细胞增殖、分化调控中BMP信号通路的作用：
2.1MG63细胞增殖：流式细胞仪检测发现CGRP组和CGRP+Noggin组的S+G2期细胞的数目高于空白对照组(p<0.05)，但是CGRP组和CGRP+Noggin组之间没有明显差异。
2.2MG63细胞分化：Wstern-blot检测CGRP组ALP，ColIaⅠ和OCN蛋白的表达量高于空白对照组(p<0.05)；CGRP+Noggin组低于CGRP组(p<0.05)。
3．CGRP对MG63细胞表达BMP-2的调控作用：
3.1PCR检测发现8-24小时内只有10-8M、10-7M组可以检测到BMP-2mRNA的表达，且10-7M组明显高于10-8M组(p<0.01)；48h10-9M组可检测到BMP-2mRNA表达，但明显低于48h10-8M、48h10-7M两组(p<0.01)。
3.210-8M CGRP作用48h，MG63细胞BMP-2mRNA和蛋白的表达量，CGRP组高于空白对照组和Noggin组(P<0.01)，CGRP+Noggin组与CGRP组无差异。
4．CGRP促进MG63细胞表达BMP-2的机制：
4.1MG63细胞cAMP含量检测发现，CGRP组和CGRP+H-89组均高于空白对照组(P<0.01)，但是前两组之间无显著差异。
4.2CGRP、CGRP+H-89、H-89和空白对照组的CREB表达量无显著差异。
4.3CGRP组的pCREB蛋白表达量高于空白对照组(P<0.01)，但是CGRP+H-89组明显低于CGRP组(P<0.01)。
4.4CGRP组的BMP-2mRNA和蛋白表达量高于空白对照组(P<0.01)，但是CGRP+H-89组明显低于CGRP组(P<0.01)。
结论
1．CGRP促进MG63细胞的增殖与分化，并且具有时间以及剂量依赖性，优势浓度为10-8M，优势时段为48h。
2．CGRP可能通过BMP-2途径促进MG63细胞的分化，而不通过BMP-2途径促进MG63细胞的增殖。
3．CGRP促进MG63细胞表达BMP-2，并且具有时间以及剂量依赖性，优势浓度为10-7M，优势时段为48h。
4．CGRP促进MG63细胞BMP-2表达的可能机制是：通过CGRP促进cAMP合成，从而促进CREB磷酸化，进而促进BMP-2转录。
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.
Methods
1.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/cm~2and 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~(-7)M) and time-effect (24-72h) ofCGRP-induced MG63cells viability were determinated by MTT method; dose-effect (10~(-10)-10~(-7)M) and time-effect (24-72h) of CGRP on cell cycle were assayed by flow cytometry.
2.2Differentiation: Dose-effect (10~(-10)-10~(-7)M) 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~(-7)M) 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~(-7)M) 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.
Results
1. CGRP-induced MG63cells proliferation and differentiation:
1.1Proliferation: Through MTT assay, the cell,sgrowth rate in10~(-10)-10~(-7)M 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~(-7)M 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~(-7)M 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~(-7)M 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).
Conclusions
1. 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.
目前的研究发现：BMP-2，4，6可以促进神经分泌CGRP，CGRP在体外促进牙髓细胞分泌BMP-2，BMP-2体内异位诱导成骨组织中CGRP的神经可以再生分布，BMP-2在腹腔注射CGRP的大鼠胫骨骨折愈合的骨痂中高表达，CGRP在体外协同增加BMP-2诱导成骨细胞增殖的能力。这些研究结果提示：CGRP可以诱导骨组织中BMP-2的表达增加，但两者之间相互作用的信号通路机制目前尚不清楚，因此推测CGRP的促成骨作用可能与BMP-2的表达有相关性。本实验将对CGRP促成骨细胞增殖、分化作用中BMP信号通路所发挥的作用；CGRP促成骨细胞BMP-2表达的影响及其机制等进行研究。
方法
1．细胞培养：人骨肉瘤细胞(MG63)细胞株(中国科学院上海生命科学研究院)接种于培养瓶(100ml)中传代培养(5×106个/cm~2)，培养基为RPMI1640(含10%FBS)，放置于5%CO2孵箱中37°C孵育72h传代，传代至6-7代时使用。
2．CGRP对MG63细胞增殖和分化的调控作用：
2.1MG63细胞增殖：MTT法检测CGRP对MG63细胞增殖的调控作用；流式细胞仪检测CGRP对MG63细胞增殖周期的调控。设定量效作用的浓度为(10~(–10)-10~(–7)M)，时效作用为(24-72h)。
2.2MG63细胞分化：PNPP偶氮法检测CGRP对MG63细胞ALP染色的调控作用；Western blot检测CGRP对MG63细胞碱性磷酸酶(ALP)、Ⅰ型胶原(CollagenⅠ)、骨钙素(OCN)表达的调控作用。设定量效作用的浓度为(10~(-10)-10~(-7)M)，时效作用为(24-72h)。免疫荧光染色检测10-8M CGRP对MG63(48h)ALP表达的作用。
3．CGRP对MG63细胞增殖、分化调控中BMP信号通路的作用：
3.1分组：10-8M CGRP；10-8M CGRP+100ng/ml Noggin；100ng/ml Noggin；空白对照组，作用时间为48h。
3.2MG63细胞增殖影响：流式细胞仪检测MG63细胞增殖周期。
3.3MG63细胞分化影响：免疫荧光染色检测MG63细胞的ALP表达；Western blot检测MG63细胞的ALP，ColIaⅠ和OCN蛋白表达。
4．CGRP对MG63细胞表达BMP-2的调控作用：
4.1RT-PCR检测CGRP对MG63细胞表达BMP-2mRNA的调控作用：设定量效作用的浓度为(10~(-10)-10~(-7)M)，时效作用为(1-48h)。
4.2CGRP促进MG63细胞表达BMP-2中Noggin的作用：
分组：10-8M CGRP；10-8M CGRP+100ng/ml Noggin；100ng/ml Noggin；空白对照组，作用时间48h。
RT-PCR检测MG63细胞BMP-2mRNA的表达；Western blot检测MG63细胞BMP-2蛋白的表达。
5．CGRP促进MG63细胞表达BMP-2的机制：
5.1分组：10-8M CGRP、10-8M CGRP+5μM H-89、5μM H-89、空白对照组，作用时间48h。
5.2免疫检测试剂盒检测MG63细胞cAMP表达；RT-PCR检测MG63细胞BMP-2mRNA表达；Western blot检测MG63细胞BMP-2、环磷酸腺苷反应元件结合蛋白(cAMPresponse element binding protein， CREB)、磷酸化环磷酸腺苷反应元件结合蛋白(Phosphorylated Camp Response Element Binding Protein，pCREB)的表达。
6．统计分析：
所有数据均采用均数±标准差(±s)表示，应用Dunnett，s检验中的t检验和方差分析，进行统计分析，分别设定p<0.05以及p<0.01表明有显著性差异和差异非常显著。
结果
1．CGRP对MG63细胞增殖、分化的调控作用：
1.1MG63细胞增殖：MTT法检测发现CGRP加入MG63细胞培养基中24-72h的时间段中，CGRP各浓度组的细胞数均显著高于空白对照组(P<0.05)，其中以10-8MCGRP浓度组最为明显(P<0.05)；流式细胞仪检测细胞增殖周期发现不同浓度的CGRP都加速了细胞周期循环，但是以48h10-8M组最为明显(P<0.05)，48h以后有下降趋势。
1.2MG63细胞分化：PNPP偶氮法检测发现各浓度组的CGRP促进MG63细胞的ALP表达量均高于空白对照组，其中以72h10-8M组最为明显(P<0.05)，时效对比中发现各浓度组细胞的ALP表达量在72h较48h增加不明显(P＞0.05)；蛋白定量分析结果发现各实验组的ALP，ColIaⅠ和OCN蛋白表达量均高于空白对照组(P<0.05)，其中以48h10-8M组最为明显(P<0.05)；免疫荧光染色结果发现48h10-8M组荧光数目较空白对照组多。
2．CGRP对MG63细胞增殖、分化调控中BMP信号通路的作用：
2.1MG63细胞增殖：流式细胞仪检测发现CGRP组和CGRP+Noggin组的S+G2期细胞的数目高于空白对照组(p<0.05)，但是CGRP组和CGRP+Noggin组之间没有明显差异。
2.2MG63细胞分化：Wstern-blot检测CGRP组ALP，ColIaⅠ和OCN蛋白的表达量高于空白对照组(p<0.05)；CGRP+Noggin组低于CGRP组(p<0.05)。
3．CGRP对MG63细胞表达BMP-2的调控作用：
3.1PCR检测发现8-24小时内只有10-8M、10-7M组可以检测到BMP-2mRNA的表达，且10-7M组明显高于10-8M组(p<0.01)；48h10-9M组可检测到BMP-2mRNA表达，但明显低于48h10-8M、48h10-7M两组(p<0.01)。
3.210-8M CGRP作用48h，MG63细胞BMP-2mRNA和蛋白的表达量，CGRP组高于空白对照组和Noggin组(P<0.01)，CGRP+Noggin组与CGRP组无差异。
4．CGRP促进MG63细胞表达BMP-2的机制：
4.1MG63细胞cAMP含量检测发现，CGRP组和CGRP+H-89组均高于空白对照组(P<0.01)，但是前两组之间无显著差异。
4.2CGRP、CGRP+H-89、H-89和空白对照组的CREB表达量无显著差异。
4.3CGRP组的pCREB蛋白表达量高于空白对照组(P<0.01)，但是CGRP+H-89组明显低于CGRP组(P<0.01)。
4.4CGRP组的BMP-2mRNA和蛋白表达量高于空白对照组(P<0.01)，但是CGRP+H-89组明显低于CGRP组(P<0.01)。
结论
1．CGRP促进MG63细胞的增殖与分化，并且具有时间以及剂量依赖性，优势浓度为10-8M，优势时段为48h。
2．CGRP可能通过BMP-2途径促进MG63细胞的分化，而不通过BMP-2途径促进MG63细胞的增殖。
3．CGRP促进MG63细胞表达BMP-2，并且具有时间以及剂量依赖性，优势浓度为10-7M，优势时段为48h。
4．CGRP促进MG63细胞BMP-2表达的可能机制是：通过CGRP促进cAMP合成，从而促进CREB磷酸化，进而促进BMP-2转录。
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.
Methods
1.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/cm~2and 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~(-7)M) and time-effect (24-72h) ofCGRP-induced MG63cells viability were determinated by MTT method; dose-effect (10~(-10)-10~(-7)M) and time-effect (24-72h) of CGRP on cell cycle were assayed by flow cytometry.
2.2Differentiation: Dose-effect (10~(-10)-10~(-7)M) 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~(-7)M) 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~(-7)M) 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.
Results
1. CGRP-induced MG63cells proliferation and differentiation:
1.1Proliferation: Through MTT assay, the cell,sgrowth rate in10~(-10)-10~(-7)M 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~(-7)M 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~(-7)M 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~(-7)M 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).
Conclusions
1. 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.
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