Original Article Effect of Calcitonin-Gene-Related Peptide on Proliferation and Osteogenesis in Rat Bone Mesenchymal Stem Cells

Int J Clin Exp Pathol 2017;10(2):1074-1083 www.ijcep.com /ISSN:1936-2625/IJCEP0040397

Original Article Effect of calcitonin-gene-related peptide on proliferation and osteogenesis in rat bone mesenchymal stem cells

Ting Zhang1,2, Xijiao Yu1,3, Shuang Liu1, Linlin Lv1, Yanzhi Wang1,3, Shu Li1

1Department of Periodontology, School of Stomatology Shandong University; Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan, Shandong, PR China; 2Department of Gerodontics, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, Jiangsu, PR China; 3Department of Gerodontics, Jinan Stomatological Hospital, Jinan, Shandong, PR China Received September 21, 2016; Accepted September 29, 2016; Epub February 1, 2017; Published February 15, 2017

Abstract: Calcitonin-gene-related peptide (CGRP) is an important neurotransmitter in signaling pathways of osteogenesis in mature osteoblasts. The aim of this study was to investigate the function of CGRP in rat bone mesenchymal stem cells (rBMSCs) during the process of osteogenetic differentiation. CCK-8 method was adopted to test the effect of different concentrations of CGRP on rBMSCs proliferation and identify the optimal concentration of

CGRP8-37 which was the antagonist of CGRP. PCR and Western blot were conducted to detect the mRNA and protein expression level of Ramp1 and CRLR. After osteogenic induction, alkaline phosphatase assay, Western blot and real-time qPCR were conducted to detect the protein of alkaline phosphatase (ALP), bone sialoprotein (BSP) and Runx2. Furthermore, Alizarin red staining and calciumion concentration measurement were used to test the differentiation and osteogenesis ability of rBMSCs in the later ossification differentiation period. Consequently, we found that receptors Ramp1 and CRLR of CGRP could be detected in rBMSCs. After osteogenic inducing culture, the expression levels of Runx2, ALP and BSP in rBMSCs were increased in CGRP groups compared with control groups, but the increase was attenuated in CGRP+CGRP8-37 and CGRP8-37 groups at both day 1 and 5. More mineral nodules and concentration of calcium ion were found in CGRP groups than other groups. Collectively, our findings suggest

CGRP promotes the proliferation and osteogenic differentiation of BMSCs, but its receptor antagonist CGRP8-37 at- tenuates the effect.

Keywords: CGRP, neuropeptide, cell differentiation, proliferation, osteogenesis

Introduction ogy view, the physiological growth of every tissues and organs is invariably accompanied by Periodontal disease (PD) is one of the most the synchronous neural growth. Nerve is an commonly known human infectious diseases important factor during individual growth and that affect more than half of population all over development, which not only can serve as a the world. It is usually a progressively destru- nerval conductor, but also can secrete some ctive pathological change of periodontal tis- neurotransmitters to regulate growth of the sues around the teeth which may eventually relevant tissues and cells [2, 3]. Numerous lead to teeth loss. studies have demonstrated that bone nerve In contrast to the conventional approaches of fibers not only can conduct different kinds of periodontal anti-inflammatory therapy, the re- stimulation included temperature, pain, mech- generative therapeutic procedures aim to re- anical and chemical stimulus which lead to pair lost periodontal tissues, including alveo- the damage of the tissues, but also can re- lar bone, periodontal ligament and root ce- lease a variety of neurotransmitters around, mentum, which may face more challenges [1]. such as substance P (SP), calcitonin-gene-re- At present, the clinical and basic science related peptide (CGRP), neurokinin A (NKA) and search methods of periodontal tissue regener- neuropeptide Y (NPY). They play important roles ation only stay in the level of regeneration of in maintaining immune homeostasis, promot- the “structure”, but they ignore “function” re- ing fracture coalescence and facilitating tis- generation. In the point of developmental biol- sue regeneration. Effect of CGRP on proliferation and osteogenesis in rBMSCs

The major manifestation of periodontitis is pro- portant neurotransmitter in signaling pathways gressive destruction of the alveolar bone which of osteogenesis. But the function of CGRP dur- leads to loose and missing of teeth. Alveolar ing stem cell osteogenetic differentiation has bone is the most active bone tissue. The func- few been elucidated. tion of neural factors in the process of alveolar bone reconstruction cannot be overlooked. Our Bone mesenchymal stem cells are frequently preliminary experiments found that the regen- used as seed cells in tissue engineering, which erated alveolar was relatively less dense and have good proliferative capacity, multi-lineage more porous after cutting off inferior alveolar differentiative ability, directional migration abil- nerve. Furthermore, CGRP was the important ity and easy to obtain. So in this experiment factors that can affect the quality of regener- we choose BMSC as the research object cell ated alveolar bone by reduce the bone density model. during mineralization process [4]. Based on this principle, adding CGRP as mediators in On the basis of above-mentioned research the process of periodontal tissue regeneration background, this experiment explored the func- to promote the functional regeneration of peri- tion of CGRP in rat-BMSCs during the process odontal tissue should be an important topic of osteogenetic differentiation in order to pro- which is worthy of continuous exploration and vide theoretical support to further study about research. the role of neural factors in periodontal tissue regeneration. Calcitonin gene related peptide is a 37 ami- no acid neuropeptide generated by alternative Materials and methods splicing of the calcitonin gene which produ- Cell culture ced by nervous system. CGRP comprises two subtypes, which are commonly referred to as Primary bone mesenchymal stem cells (BMSCs) the alpha and beta CGRP [5]. Alpha-CGRP can from 4 to 6-week-old Wistar rats (n=5) were been found in blood vessels, bone marrow, harvested using established technique [14]. periodontium [6], and neuroendocrine cells in Briefly, rats were sacrificed by euthanasia, both the normal lung [7]. On the other hand, beta- femur and tibia were excised aseptically, the CGRP only exists in the sensory nerves of bo- soft tissue was removed, and then, the ends of wel. Alpha-CGRP is an important neuropeptide the femora and tibiae were excised immediate- during the process of bone development and ly. Minimum Essential Medium Alpha (α-MEM, repair [8-10]. While many investigators have Invitrogen) supplemented with 15% fetal bo- identified CGRP promote bone formation. First- vine serum (FBS, HyClone Laboratories Inc.), ly, Many nerve fibers immunostaining experi- 100 IU/mL penicillin, and 100 mg/mL strepto- ments prove that CGRP-immunoreactive nerve mycin was used to flush bone marrow out. The fibers can be found in the bone marrow, perios- cell suspension was obtained by repeatedly teum, and in the epiphyseal trabecular bone aspirating the cells through the 20- and 23- [11]. In addition, the most important CGRP re- gauge needle. The bone marrow cells were then ceptor is a dimer complex of two molecules, seeded into Petri dish with 4.0×105 cells/cm2. activity-modifying protein (RAMP1) and G-pro- All the cells were incubated within a humidifi- tein coupled calcitonin receptor-like receptor ed environment of 5% CO2 at 37°C. Cells were (CRLR), both of which are required for physio- passaged using 0.25% trypsin-EDTA (Invitro- logic activation by CGRP. RAMP1 and CRLR regen, CA, USA). Only cells between passages 2- ceptors are expressed in mature osteoblasts 5 were subsequently used for experiments. [12]. Furthermore, Haegerstrand et al report- ed osteoblasts of transgenic mice which over- The CGRP used in this study was αCGRP (Sig- expressed CGRP have increased bone forma- ma), and CGRP8-37 (Tocris) as the role of CGRP tion activity and trabecular bone mass [13]. receptor antagonists for antagonism. The os-

CGRP8-37 is a C terminal fragment of CGRP, teogenic medium consisting of α-MEM supple- which can be combined with CGRP1 recep- mented with 10% FBS, 10 mmol/l β-glycerol tors. It is one of the CGRP receptor antago- phosphate (Sigma), 50 mg/l ascorbic acid (Sig- nists, which can combat the biological effects ma), 10-8 mol/l dexamethasone, and 1% peni- of CGRP. Lots of studies report CGRP is an im- cillin and streptomycin.

1075 Int J Clin Exp Pathol 2017;10(2):1074-1083 Effect of CGRP on proliferation and osteogenesis in rBMSCs

Table 1. Primer sequences used for real-time PCR Polymerase chain reaction Primer Genbank Sequence (5’-3’) Length (bp) PCR was used to deter- GAPDH NM_017008.4 F: 5’-TGATGGGTGTGAACCACGAG-3’ 125 mine the mRNA transcrip- R: 3’-CCCTTCCACGATGCCAAAGT-5’ tion levels of CGRP rece- RAMP1 NM_031645.1 F: 5’-ACTCACTGCACCAAACTCGT-3’ 180 ptors (calcitonin receptor- R: 3’-GACCGTAATGGGGAGCACAA-5’ like receptor, CLR and re- CRLR NM_012717.1 F: 5’-TATGGCTCTCATCGCAGCAG-3’ 177 ceptor activity modifying R: 3’-TTCCAGCATAGCCATCCGTCT-5’ protein1, RAMP1). Briefly, ALP NM_013059.1 F: 5’-GGAGATGGATGAGGCCATCG-3’ 218 total RNA was extracted R: 3’-CGTCCACCACCTTGTAACCA-5’ from the cells with RNAiso BSP NM_012587.2 F: 5’-GCCACACTCTAGGGGTAAC-3’ 104 Plus (Takara Bio, Shiga, Ja- R: 3’-TGCATCTCCAGCCTTCTTGG-5’ pan), and reverse transcri- Runx2 NM_001278483.1 F: 5’-CAGACACAATCCTCCCCACC-3’ 190 bed to cDNA using the Pri- R: 3’-GCCAGAGGCAGAAGTCAGAG-5’ meScript RT Reagent Kit with gDNA Erase (Takara Bio). The resulting cDNA was amplified by PCR with the Platinum PCR SuperMix (Invitrogen). And the PCR product identified by agarose gel electrophoresis ap- paratus (BIO-RAD PAc300, America).

Quantitative real-time polymerase chain reaction

BMSCs were induced to osteoblast with osteogenic medium. The cells divided into the

following four groups: CGRP group, CGRP8-37

group, CGRP+CGRP8-37 group and control group. All of these groups cultured 1 or 5 days. Fol- lowing up, we detected the mRNA transcription levels of osteogenesis-related factors by quantitative real time PCR analysis. The process of extraction of mRNA and reverse transcription as above stated. Quantitative Real- time polymerase chain reaction amplifications labeled with SYBR Premix Ex Taq (Takara Bio) were performed in a Roche 480 Light Cycler (Roche, Mannheim, Germany). The sequences of primers used in these experiments were listed in Table 1. GAPDH was used as an inter- Figure 1. PCR and Western blot demonstrated the nal control. presence of the calcitonin gene-related protein (CGRP) receptors in rat bone mesenchymal stem Western blot analysis cells (rBMSCs). PCR showed CGRP receptors (RAMP1 and CRLR) mRNA were expressed in the rBMSCs. (A) Western blot analysis of this experiment was M: DL 1000DNA Maker (100-1000 bp); 1: GAPDH; 2: carried out using the whole-cell lysate. The CRLR; 3: RAMP1. The proteins of RAMP1 and CRLR were discovered by western blot (B). cells were washed with ice-cold phosphate- buffered thrice, and directly lysed in a 0.5 ml/ well lysis buffer (150 mmol/l NaCl, 20 mmol/l The animals used in this study were maintained Tris, pH 7.5, 0.1% Triton X-100, 1 mmol/l phe- and used in accordance with guidelines estab- nylmethylsulfonyl fluoride [PMSF] and 10 μg/ lished by the Institutional Animal Care and Use ml aprotonin). The lysate was sonicated, boiled Committee of Shandong University in Jinan, for 5 min and centrifuged at 7500 rpm for 10 Shandong Province, PR China. min at 4°C. The concentration of protein was

1076 Int J Clin Exp Pathol 2017;10(2):1074-1083 Effect of CGRP on proliferation and osteogenesis in rBMSCs

linked goat-anti rabbit IgG (Santa Cruz, CA, USA), the blots were visualized with an ECL kit. GAPDH protein expression was used as an in- ternal control.

Cell proliferation assay

The status of rBMSCs cell proliferation was evaluated using Cell Counting Kit-8 (CCK- 8, Dojindo, Japan) according to the manufacturer’s instruction. BMSCs were seeded on 96-well plates (5×103/ well) and treated with CGRP at concentration of 0, 10-6 mol/L, 10-7 mol/L, 10-8 mol/ L and 10-9 mol/L for 1, 3, 5, 7 days in order to identified optimal concentration of CG- RP. Then BMSCs were seeded on 96-well plates (5×103/ well) and treated with differ-

ent concentration of CGRP8-37 into each well. After half an hour, we added CGRP (10-8 Figure 2. Effect of CGRP on proliferation in rBMSCs (A). The effect of CGRP mol/L) and made the final with different concentrations on proliferation in rBMSCs was detected in 1, 3, 5 and 7 days. The proliferation of cells was measured by Cell Counting Kit-8 concentration of CGRP8-37 of -6 (CCK-8) assay. The proliferation curve showed the concentration (10-9 mol/ each wells at 10 mol/l, 5× l~10-6 mol/l) could increased the OD value of CCK-8, comparing with control 10-7 mol/l, 10-7 mol/l, 5×10 -8 group. But 10-8~10-6 mol/l CGRP could promote cell proliferation more obvi- mol/l, and 10-8 mol/l. After ously (P<0.05), and there was no significant difference between the latter 3 and 5 days, the cells were three groups. Effect of CGRP on proliferation in rBMSCs (B). The effect of 8-37 washed with PBS twice, and CGRP8-37 on proliferation in rBMSCs, which was the antagonism of CGRP, was tested by Cell Counting Kit-8 (CCK-8) assay. We added different concentra- 10 μl of CCK-8 solution was -8 tion of CGRP8-37 and 10 mol/l CGRP each well in 1 and 5 days. The result added to each well. After in- of CCK-8 demontrated 5 to 100 times of CGRP8-37 could antagonised the -8 cubated for 2 hours in a st- effect of CGRP, but the antagonism at 5×10 mol/l is not obvious. **P<0.05 andard incubator, the absor- vs control group. bance was measured at 450 nm under spectrophotometer, measured by BCA protein assay kit (Pierce, and a proliferation curve was generated based USA). Western blot analyses were then per- on the absorbance and culture time. formed using SDS-PAGE 4-12% Bis-Tris gradi- ent gels and 0.45 μm Invitrolon polyvinylinde- Alkaline phosphatase activity ne fluoride membranes (Invitrogen, USA). After transfer, the membrane was blocked with a The BMSCs were seeded on 6-well plates incu- solution of 1% Tween 20 (TBS-T) containing bated with the osteogenic medium, which were

5% non-fat milk for 2 h at room temperature divided into control group, CGRP group, CGRP8-

(25°C). The blots were incubated at 4°C over- 37 group and CGRP+CGRP8-37 group. At 1 and 5 night with primary antibodies against CRLR days time point, each well was assayed for (1:1000), Ramp1 (1:1000), Runx2 (1:1000) alkaline phosphatase activity. ALP activity was and BSP (1:1000) (CST, USA) diluted in TBST. determined using a commercially available kit After washing and incubation with secondary (Jiancheng, China) according to the manufac- antibodies of horseradish peroxidase (HRP)- turer’s instruction. The absorbance was mea-

1077 Int J Clin Exp Pathol 2017;10(2):1074-1083 Effect of CGRP on proliferation and osteogenesis in rBMSCs

fixed in 95% alcohol solution and then stained with 2% Ali- zarin red (Sigma) for 10 min- utes. After staining, each well was eluted with 10% cetylpy- ridinium chloride (Sigma) for 1 hour to determine the level of the calcium salts in the cell matrix. The total absorbance of the concentration of soluble calcium ion was measured with a spectrophotometer at 562 nm wave long.

Statistical analysis

The result of experiment shown as means ± SEM. Statis- tically significant differences (P<0.05) between the various groups was measured using One-way ANOVA by a SPSS 12.0 statistical software pa- ckage (SAS, Cary, NC, USA).

Results

Expression of CGRP receptors in rats bone mesenchymal stem cells

PCR and Western-Blot was used to measure the mRNA and protein expression of CGRP1 receptors of RAMP1 and CRLR in rBMSCs. The result of PCR demonstrated that CGRP1 receptors (RAM- P1 and CRLR) mRNA were ex- Figure 3. Effect of CGRP and CGRP8-37 on the mRNA expression of Runx2 (A), BSP (B), ALP (C) in BMSCs. The expression of Runx2, BSP, ALP mRNA pressed in the rBMSCs (Fig- were siginificantly increased in the CGRP-treat group. At the same time, the ure 1A). In addition, RAMP1 increase was attenuated in CGRP group and CGRP+CGRP group. There 8-37 8-37 and CRLR protein were dete- was no significant difference between the two groups. *P<0.05 vs CGRP8-37 and CGRP+CGRP group; **P<0.05 vs control group. cted in rBMSCs by Western 8-37 blot (Figure 1B). sured with a spectrophotometer at 405 nm Effect of CGRP on proliferation in rBMSCs wave long. ALP activity from each well was normalized to total protein, which was quantified The experimental groups in different concen- using a BCA protein assay kit. trations of CGRP were detected at 1, 3, 5, 7 days by Cell Counting Kit-8 (CCK-8) assay. The Mineralization proliferation curve showed that the period of cell growth could be divided into 3 stages, Four groups of cells were incubated continu- including detention phase, logarithmic phase, ously in osteogenic medium for the mineraliza- stationary phase. Compared with control group, tion assay in the third week. The cells were 10-9~10 -6 mol/l CGRP could increase the OD

1078 Int J Clin Exp Pathol 2017;10(2):1074-1083 Effect of CGRP on proliferation and osteogenesis in rBMSCs

Runx2. The addition of CGRP to the cell media significantly increased BMSC gene expression of BSP, ALP, Runx2 in rBMSCs at 1 and 5 days of cell culture (P<0.05), and the control group of the expression of bone related factors are lower than the other three groups (P<0.05) (Figure 3).

Effect of CGRP on alkaline phosphatase activity in rBMSCs

Osteoblastic differentiation of rat bone mesenchymal stem cells caused by CGRP was evaluated by testing alkaline phosphatase for 1 and 5 days. Comparing with control groups, Figure 4. Alkaline phosphatase protein expression the activity of alkaline phosphatase on three groups expect control group were increased of rBMSC after stimulation with CGRP and CGRP8-37. Cell lysates were used for analyzing alkaline phos- (P<0.05). At the same time, the activity of al- phatase activity was normalized to the amount of cell kaline phosphatase on CGRP group was high- protein. The CGRP siginificantly promoted alkaline er than CGRP8-37 and CGRP+CGRP8-37 group phosphatase activity in rBMSCs at each point. The (P<0.05). No siginificant difference was de- effect of CGRP8-37 was slighter than CGRP. *P<0.05 tected between CGRP8-37 and CGRP+CGRP8-37 vs CGRP8-37 and CGRP+CGRP8-37 group; **P<0.05 vs control group. group (P>0.05). In addition, CGRP increased alkaline phosphatase activity in rBMSCs in a time-dependent manner (Figure 4). value of CCK-8. The data suggested that all the experimental concentration could promo- Effect of CGRP on osteogenic protein expres- te cell proliferation (P<0.05), while 10-8~10 -6 sion in rBMSCs mol/L CGRP could promote cell proliferation more obviously (P<0.05), and there was no sig- Similarly, we evaluated that CGRP’s effect on nificant difference between the latter three osteoblastic differentiation after osteogenic groups (Figure 2A). Therefore, we choose the induction of rat bone mesenchymal stem cells lowest effective concentration (10-8 mol/l) as by western blot. The total proteins of rBMSCS optimal CGRP experimental concentration. were collected at 1 and 5 days to examine the protein level of Runx2, BSP. As shown in Figure

CGRP8-37 optimum concentration selection 5A and 5B, the protein level of Runx2 and BSP on CGRP group were significantly higher than In addition, we choose the minimum effective that in the other three groups (P<0.05). At the -8 concentration of 10 mol/l alpha-CGRP as ex- same time, the protein level of Runx2 and BSP perimental concentration to explore the effec- on CGRP8-37 group and CGRP+CGRP8-37 were tive inhibition concentration of CGRP recep- also higher than in control group (P<0.05). tor antagonist. After 3, 5 days, we tested absorbance of each group, the result showed The formation of mineral nodules that 5 to 100 times of CGRP8-37 could antago- nized the effect of CGRP (P>0.01) , but the The minerlization of rBMSCs was measured antagonism at 5×10-8 mol/l was not obvious by Alizarin red staining after continuous osteo- -8 (P>0.05) (Figure 2B), so we chose 10-7 mol/l genic induction of CGRP treatment (10 mol/l) as CGRP experimental concentration. for 21 days. After Alizarin Red staining (Figure 8-37 6), the mineralized nodules of CGRP group was Effect of CGRP on osteogenic genes expres- most of all. On the contrary, the control group sion in rBMSCs was the least.

Osteogenic genes expression of rat bone mar- In addition, we tested the calcium ion concen- row cells after osteogenic induction was evalu- tration of each well after Alizarin Red staining ated by real time quantitative PCR technique (Figure 6). The calcium ion concentration of and total mRNA were isolated at 1 and 5 days. control group was significantly lower than the Gene of ostogenic gene included BSP, ALP and other three groups (P<0.05). Meanwhile, the

1079 Int J Clin Exp Pathol 2017;10(2):1074-1083 Effect of CGRP on proliferation and osteogenesis in rBMSCs

Discussion

Numbers of studies prove that the nervous system influence bone metabolism and take part in bone reconstruction and fracture or other post-traumatic bone repair response. During fracture healing, newly formed CGRP positive fibers can be found in areas with high bone formation rates [15]. Familial dysautonoma is a genetic disorder that disturbs cells in the auto- nomic nervous system. Patients with familial dysautonoma suffer from a loss of unmyelinat- ed axons with a reduction in neuropeptipe signaling, a poor bone quality and increased risk of bone fractures [15-17]. All of these findings suggest that the nervous system plays an important role in development and regeneration of bone tissue.

Genetically, in the process of new bone formation, neuropeptide promote osteogenesis by influencing the bone cells proliferation and differentiation. Numbers of experimental results show that CGRP promote cells proliferation, including human endothelial cells [18, 19], osteoblasts, epithelial cell [20, 21], Schwann cell [22] and so on. In this experiment, we determined the alpha-CGRP effects on osteopro- genitor cellular proliferation by using Cell Co- unting Kit-8. After treatment 7 days, we found the concentrations of alpha-CGRP between 10-6 and 10-8 mol/l could promote rat bone marrow mesenchymal cells proliferation. The resulted proved that alpha-CGRP could promote the proliferation of marrow mesenchymal cells and further increase the speed of bone formation to improve the efficiency of osteogenesis.

The current study also demonstrated rBMSCs expressed mRNA and proteins for CRLR and Figure 5. Effect of CGRP and CGRP on the protein 8-37 RAMP1 receptors. As well as, the mRNAs of expression in rat BMSCs. A: Runx2, BSP protein levels of four groups in 1 day. Runx2 and BSP were up- CRL and RAMP1 have been detected in the regulated in CGRP-treat group, CGRP8-37-treat group human MG63 osteoblastic cell line, the mouse and CGRP+CGRP8-37-treat group. But the expression MC3T3-E1 osteoblastic cell line, human prima- of Runx2, BSP in CGRP-treat group were higher than ry osteoblast cell cultures, and in rat primary others. B: Runx2, BSP protein levels of four groups in calvarial steoblasts [23]. These results further 5 day. Runx2, BSP expression significant increased suggest that CGRP could affect the process in CGRP-treat group. *P<0.05 vs CGRP8-37 and CGRP+CGRP8-37 group; **P<0.05 vs control group. of osteogenesis differentiation of bone mesenchymal stem cells through its receptor. calcium ion concentration of CGRP group was On the other hand, CGRP involved in bone for- more than CGRP+CGRP8-37 group and CGRP8-37 mation, bone metabolism and bone repair pro- group. There were no differences between the cess, and play a role in the early stages of the last two groups (P>0.05). mineralized induction. The CGRP positive nerve

1080 Int J Clin Exp Pathol 2017;10(2):1074-1083 Effect of CGRP on proliferation and osteogenesis in rBMSCs

bone maker, including BSP, ALP and Runx2. It is an activa- tor early stage of bone formation. The stimulatory effects of CGRP on BMSC osteoblastic differentiation were only observed prior to the onset of mineralization [27]. Our result is meeting with the findings that αCGRP knockout mice have decreased bone formation, and normal osteoblast numbers [28]. Collectively, these valuable data demon- strate CGRP promote osteoblastic differentiation in bone marrow osteoprogenitors. The stimulatory effects of CGRP were restricted to the earlier stages of osteoblast development, which suggested that it’s osteogenic effects are primarily due to its ability to direct stromal progenitors in- Figure 6. The formation of mineral nodules in rBMSCs with CGRP and CGRP8- 37 treatment (A). The mineral nodules of rBMSCs were measured by Alizarin to the osteoblastic lineage. red staining after continuous osteogenic induction for 3 weeks. The mineral nodules in CGRP-treat group were most in quantity than the others. At Interestingly, we also found the same, the quantity of mineralized nodules in CGRP8-37-treat group and that CGRP treatment in- CGRP+ CGRP -treat group were more than those in the control group. (A) 8-37 8-37 creased the expression of Control group; (B) CGRP group; (C) CGRP8-37 group; (D) CGRP+CGRP8-37 group. Calciumion concentration measurement were used to test the differentiation osteogenesis related cytoki- and osteogenesis ability of rBMSCs in the later period (B). The concentra- nes, but the function of CG- tion of calcium ion in control group was the lowest. And the concentration of RP8-37 was slight comparing calcium ion was incresaed remarkerable in CGRP-treat group. No significant with CGRP. However, the in- morphological changes were observed CGRP -treat group and CGRP+ 8-37 creased in bone formation CGRP -treat group. *p < 0.05 vs CGRP and CGRP+CGRP group; **p 8-37 8-37 8-37 in rats treated with CGRP < 0.05 vs control group. 8-37 was an unexpected result.

CGRP8-37 antagonizes signal- fibers could be observed in femoral epiphyseal ing via the CGRP1 receptor and is a part of ossification center of rats at 10 day after birth. CGRP [29, 30]. This receptor is composed by a It was suggested synchronizing regeneration of heterodimer of the calcitonin receptor-like CGRP nerve fibers during the development of receptor (CRLR) and receptor activity modifying bone [24]. CGRP positive nerve could be found protein 1 (RAMP1). In addition, CGRP is also during bone defect repair and fracture repair thought to signal through a putative CGRP2 process [25]. A variety of animal of osteoblast receptor. Specifically, AMY(1(a)) (calcitonin cultures have been proved that CGRP increased receptor/RAMP1) and AM(2) (CL/RAMP3) cAMP [26]. All of these suggested CGRP partici- receptors can be activated by CGRP but are pated in bone metabolism, and increased bone relatively insensitive to CGRP(8-37). Some formation. In order to study the influence of pharmacological characterization of receptors CGRP during osteogenetic differentiation of related to CGRP has revealed that some of bone marrow stromal stem cells, we added these receptors may explain CGRP2 receptor exogenous alpha-CGRP to detected osteogen- pharmacology [29]. Thus we speculated that esis related cytokines. In our study, we found the CGRP2 receptor could also contribute to that the osteogenic induction with CGRP for 1 bone formation. The result is similar to and 5 days moderately enhanced expression Susannah J’s result that systemic administra- levels of osteogenic transcription factors and tion of CGRP8-37 over a 10 day period after uni-

1081 Int J Clin Exp Pathol 2017;10(2):1074-1083 Effect of CGRP on proliferation and osteogenesis in rBMSCs lateral fatigue loading increased bone forma- odontal tissue repair. Periodontol 2000 2012; tion in the periosteal, endosteal and intracorti- 59: 185-202. cal envelopes [25]. [2] Jones KB, Mollano AV, Morcuende JA, Cooper RR, Saltzman CL. Bone and brain: a review of In addition, we found after 3 weeks CGRP and neural, hormonal, and musculoskeletal con- nections. Iowa Orthop J 2004; 24: 123-32. CGRP8-37 enhanced the nodule formation and increased the concentration of calcium ion. [3] Masi L. Crosstalk between the brain and bone. However a large number of studies have shown Clin Cases Miner Bone Metab 2012; 9: 13-16. [4] Lv L, Wang Y, Zhang J, Zhang T, Li S. Healing of that activate the osteogenic differentiation in periodontal defects and calcitonin gene relat- the early stage. As we mentioned above, CGRP ed peptide expression following inferior alveo- enhanced expression levels of osteogenic tran- lar nerve transection in rats. J Mol Histol 2014; scription factors and bone maker at day 1 and 45: 311-320. 5 after osteogenic induction. The generation of [5] Eftekhari S, Edvinsson L. Calcitonin gene-relat- mineralized nodules is a long process and influ- ed peptide (cgrp) and its receptor components ence by a variety of factor. We inferred that in human and rat spinal trigeminal nucleus CGRP cause related pathways downstream of and spinal cord at c1-level. BMC Neurosci osteogenesis related factors in early phase 2011; 12: 1-18. which lead to the enhanced formation of miner- [6] Caviedes-Bucheli J, Moreno JO, Carreño CP, alized nodules. Delgado R, Garcia DJ, Solano J, Diaz E, Munoz HR. The effect of single-file reciprocating sys- In conclusion, our data support the concept tems on substance p and calcitonin gene-related peptide expression in human periodontal that there are CGRP receptors in rats bone ligament. Int Endod J 2012; 46: 419-426. mesenchymal stem cells. The present study [7] Dakhama A, Larsen GL, Gelfand EW. Calcitonin also suggests that alpha-CGRP improve the gene-related peptide: role in airway homeosta- proliferation of rats bone mesenchymal stem sis. Curr Opin Pharmacol 2004; 4: 215-220. cells. Furthermore, these results clearly indi- [8] Sample SJ, Heaton CM, Mary B, Bleedorn JA, cated that alpha-CGRP conducive to the differ- Racette MA, Hao Z, Muir P. Role of calcitonin entiation of rBMSCs into osteoblasts. However, gene-related peptide in functional adaptation the molecular mechanism of CGRP on osteo- of the skeleton. PLoS One 2014; 9: e113959- genic differentiation is still not clear. Indeed, e113959. the effects of the mechanism between osteo- [9] Irie K, Hara-Irie F, Ozawa H, Yajima T. Calcitonin genic differentiation and CGRP still need fur- gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in ther investigation to promote bone formation bone remodeling. Microsc Res Tech 2002; 58: during periodontal regeneration. 85-90. [10] Wang L, Wang N, Li M, Wang K. To investigate Acknowledgements the role of the nervous system of bone in ste- roid-induced osteonecrosis in rabbits. Osteo- This research was supported by the National poros Int 2010; 21: 2057-2066. Natural Science Foundation of China (8127- [11] Uzan B, de Vernejoul MC, Cressent M. RAMPs 1138). and CRLR expressions in osteoblastic cells after dexamethasone treatment. Biochem Bio- Disclosure of conflict of interest phys Res Commun 2004; 321: 802-808. [12] Schinke T, Liese S, Priemel M, Haberland M, None. Schilling AF, Catala-Lehnen P, Blicharski D, Rueger JM, Gagel RF, Emeson RB, Amling M. Address correspondence to: Shu Li, Department of Decreased bone formation and osteopenia Periodontology, School of Stomatology Shandong in mice lacking alpha-calcitonin gene-related University; Shandong Provincial Key Laboratory of peptide. J Bone Miner Res 2004; 19: 2049- Oral Biomedicine, 44-1 West Wenhua Road, Jinan 2056. 250012, Shandong, PR China. Tel: +86 531 8838 [13] Haegerstrand A, Dalsgaard CJ, Jonzon B, 2769; Fax: +86 531-8838 2923; E-mail: lishu@sdu. Larsson O, Nilsson J. Calcitonin gene-related edu.cn peptide stimulates proliferation of human endothelial cells. Proc Natl Acad Sci U S A 1990; References 87: 3299-3303. [14] Li S, Tu Q, Zhang J, Stein G, Lian J, Yang PS, [1] Christoph AR, Giulio R, Salvatore B, William VG. Chen J. Systemi-cally transplanted bone mar- Advanced regenerative technologies for peri- row stromal cells contributing to bone tissue

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regeneration. J Cell Physiol 2008; 215: 204- [24] Hara-Irie F, Amizuka N, Ozawa H. Immunohis- 209. tochemical and ultrastructural localization of [15] Li J, Kreicbergs A, Bergström J, Stark A, Ahmed cgrp-positive nerve fibers at the epiphyseal M. Site-specific CGRP innervation coincides trabecules facing the growth plate of rat fe- with bone formation during fracture healing murs. Bone 1996; 18: 29-39. and modeling: a study in rat angulated tibia. [25] Sample SJ, Zhengling H, Wilson AP, Wilson J Orthop Res 2007; 25: 1204-1212. PM. Role of calcitonin gene-related peptide in [16] Pearson J, Dancis J, Axelrod F, Grover N. The bone repair after cyclic fatigue loading. PLoS sural nerve in familial dysautonomia. J Neuro- One 2011; 6: e20386. pathol Exp Neurol 1975; 34: 413-24. [26] Bjurholm A, Kreicbergs A, Schultzberg M, [17] Maayan C, Bar-On E, Foldes AJ, Gesundheit Lerner UH. Neuroendocrine regulation of cyclic B, Pollak RD. Bone mineral density and me- AMP formation in osteoblastic cell lines (UMR- tabolism in familial dysautonomia. Osteoporos 106-01, ROS 17/ 2.8, MC3T3-E1, and Saos-2) Int 2002; 13: 429-433. and primary bone cells. J Bone Miner Res [18] Tuo Y, Guo X, Zhang X, Wang Z, Zhou J, Xia 1992; 7: 1011-1019. L, Jin D. The biological effects and mecha- [27] Wang L, Shi XR, Halloran BP, Clark DJ, Jacobs nisms of calcitonin gene-related peptide on CR, Kingery WS. Calcitonin-gene-related pep- human endothelial cell. J Recept Signal Trans- tide stimulates stromal cell osteogenic differ- duct Res 2013; 33: 114-123. entiation and inhibits RANKL induced NF- [19] Mapp PI, McWilliams DF, Turley MJ, Hargin E, kappaB activation, osteoclastogenesis and Walsh DA. A role for the sensory neuropeptide bone resorption. Bone 2010; 46: 1369-1379. calcitonin gene-related peptide in endothelial [28] Huebner AK, Schinke T, Priemel M, Schilling cell proliferation in vivo. Br J Pharmacol 2012; S, Schilling AF, Emeson RB, Rueger JM, Amling 166: 1261-1271. M. Calcitonin deficiency in mice progressive- [20] Hoffmann P, Hoeck K, Deters S, Werner-Martini ly results in high bone turnover. J Bone Miner I, Schmidt WE. Substance p and calcitonin Res 2006; 21: 1924-1934. gene related peptide induce tgf-alpha expres- [29] Hay DL. What makes a CGRP2 receptor? Clin sion in epithelial cells via mast cells and fibro- Exp Pharmacol Physiol 2007; 34: 963-971. blasts. Regul Pept 2010; 161: 33-37. [30] Lerner UH, Persson E. Osteotropic effects by [21] Kawanami Y, Morimoto Y, Kim H, Nakamura the neuropeptides calcitonin gene-related pep- T, Machida K, Kido T, Asonuma E, Yatera K, tide, substance P and vasoactive intestinal Yoshii C, Kido M. Calcitonin gene-related pep- peptide. J Musculoskelet Neuronal Interact tide stimulates proliferation of alveolar epithe- 2008; 8: 154-165. lial cells. Respir Res 2009; 10: 8. [22] Toth CC, Willis D, Twiss JL, Walsh S, Martinez JA, Liu WQ, Midha R, Zochodne DW. Locally synthesized calcitonin gene-related peptide has a critical role in peripheral nerve regeneration. J Neuropathol Exp Neurol 2009; 68: 326-337. [23] Wang L, Shi X, Rong Z, Halloran BP, Clark DJ, Jacobs CR, Kingery WS. Calcitonin-gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits rankl induced nf-κb activation, osteoclastogenesis and bone resorption. Bone 2010; 46: 1369-79.

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