neuropeptides stimulate human osteoblast activity and promote gap junctional intercellular...

Neuropeptides 47 (2013) 179–186

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Neuropeptides

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Neuropeptides stimulate human osteoblast activity and promote gap junctionalintercellular communication

Wenhui Ma a,1, Xuemin Zhang b,1, Shushan Shi c, Yingze Zhang a,⇑a Department of Orthopedic Surgery, The Third Hospital of Hebei Medical University, Shijiazhuang, 050051 Hebei Province, Chinab Department of Orthopedic Surgery, Hebei National Defence Construction Hospital, Shijiazhuang, 050081 Hebei Province, Chinac Department of Orthopedic Surgery, General Hospital of Beijing Military Region, Beijing 100072, China

a r t i c l e i n f o a b s t r a c t

Article history:Available online 4 February 2013

Keywords:NeuropeptideOsteoblastGap junction intercellular communication

0143-4179/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.npep.2012.12.002

⇑ Corresponding author. Tel.: +86 311 88603682; faE-mail addresses: hmq318@hotmail.com (W. M

(X. Zhang), shushan98@126.com (S. Shi), zyz9820@161 These two authors contributed equally to this wor

Neuropeptides released from the skeletal nerve fibers have neurotransmitter and immunoregulatoryroles; they exert paracrine biological effects on bone cells present close to the nerve endings expressingthese signaling molecules. The aims of this study were a systematic investigation of the effects of the neu-ropeptides substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide(VIP), Neuropeptide Y (NPY) and tyrosine hydroxylase (TH) on the cell viability and function of the humanosteoblasts, and comparing their difference in the role of regulating bone formation. Cultures of normalhuman osteoblasts were treated with SP, CGRP, VIP, NPY or TH at three concentrations. We found thateach of the five neuropeptides induced increases in cell viability of human osteoblasts. The stimulatoryaction of NPY was the highest, followed by VIP, SP and TH, while CGRP had the lowest stimulatory effect.The viability index of osteoblasts was inversely associated with the concentration of neuropeptides, andpositively with the time of exposure. Moreover, the five neuropeptides increased the ALP activity andosteocalcin to different extents in a dose-dependent manner. The GJIC of osteoblasts was significantlypromoted by neuropeptides. The results demonstrated that neuropeptides released from skeletal nerveendings after a stimulus appeared to be able to induce the proliferation and activity of osteoblasts viaenhancing GJIC between cells, and further influence the bone formation. These findings may contributetoward a better understanding of the neural influence on bone remodeling and improving treatmentsrelated to bone diseases.

1. Introduction

Bone alters its metabolic and anabolic activities in response tothe variety of systemic and local factors such as hormones andgrowth factors. Classical observations about the abundance of thenerves fibers in bone also predict a paradigm that the nervous sys-tem influences bone metabolism. Many of the nerve-derived sig-naling molecules that may act as efferent agents on the bonecells were fallen into the category of neuropeptides. Five neuro-peptides have been known to be involved in the control of bonehomeostasis, including calcitonin gene-related peptide (CGRP),Neuropeptide Y (NPY), substance P (SP), vasoactive intestinal pep-tide (VIP) and tyrosine hydroxylase (TH) (Shi and Bladock, 2012;Togari et al., 1997; Strange-Vognsen et al., 1997). Of them, themost intensive studies have so far been performed on CGRP. It isabundantly distributed in bone via sensory nerves, especially in

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x: +86 311 87023626.a), sunnyz98@yahoo.com.cn3.com (Y. Zhang).

the epiphyseal trabecular bones, which strongly supports thatCGRP influences bone metabolism (Imai and Matsusue, 2002).However, in the current study our focus is to compare the effectof all five neuropeptides on human osteoblast so as to acquire morecomprehensive knowledge about neuropeptides regulating boneremodeling.

NPY, a classic neuronal regulator of energy homeostasis, is nowalso known to be involved in the control of bone homeostasis (Leeand Herzog, 2009). But its role in osteoblast activity and the biolog-ical functions involving NPY receptors in bone homeostasis remainto be clarified. Functional analysis made by Teixeira (Teixeira et al.,2009) revealed the osteogenic potential of osteoprogenitor cellssignificantly stimulated by NPY, probably due to the down-regula-tion of Y1 receptor. Furthermore, Lundberg (Lundberg et al., 2007)suggested that the greater number of mesenchymal progenitorsand the altered Y1 receptor expression within bone cells in the ab-sence of Y2 receptors are a likely mechanism for the greater bonemineralization in vivo and in vitro.

In addition, some observations showed that VIP, a neuropeptidepresent in peptidergic skeletal nerve fibers, could regulate theactivities of osteoblasts and osteoclasts, suggesting the existence

180 W. Ma et al. / Neuropeptides 47 (2013) 179–186

of a neuro-osteogenic interplay in bone metabolism (Persson andLerner, 2011; Lundberg et al., 2000). Meanwhile, many articles alsodemonstrated the distribution of nerve fibers containing SP, an-other sensory nerve-specific neuropeptide, and TH, the rate-limit-ing enzyme of catecholamine (Imai and Matsusue, 2002). Thedistinct effects of SP and catecholamines on the bone cells togetherwith their in vivo influences manifested by experimental denerva-tion studies suggested that the sensory and sympathetic nervesseemed to play important roles in bone metabolism through con-trolling vascularization and matrix differentiation during skeletalgrowth (Opolka et al., 2012; Adamus and Dabrowski, 2001; Shihand Bernard, 1997; Goto et al., 2007).

These data support the hypothesis that neuropeptide signalingstimulates bone formation and inhibits bone resorption. But, noinformation about comparative studies the effect of neuropeptideson bone remodeling is available to date. Hence, to further test thishypothesis we have first fully investigated the role of each of thefive neuropeptides. In addition, the ALP activity and osteocalcinwere included to exemplify some features of the osteoblast. So,the study examined the effects of neuropeptides on osteoblast interms of cell viability levels, ALP activity, and osteocalcin determi-nations in culture supernatants.

Gap junctions formed by connexins (Cx) play an important rolein transmitting signals between bone cells such as osteoblasts andosteoclasts, cells responsible for bone formation and bone remod-eling, respectively. GJIC has been demonstrated to mediate the pro-cess of osteoblast differentiation and bone formation. Furthermore,GJIC propagates Ca2+ signaling, conveys anabolic effects of hor-mones and growth factors, and regulates gene transcription ofosteoblast differentiation markers (Stains and Civitelli, 2005; Wró-bel et al., 2011). To date, no available information on the effects ofneuropeptides on GJIC is available. Therefore, we also first investi-gated the effects of neuropeptides on GJIC in osteoblasts. It seemedto allow some initial extrapolations to the possible mechanism ofneuropeptides regulating bone remodeling.

Table 1The ALP activity and osteocalcin contained in osteoblasts and fibroblasts.

2. Materials and methods

2.1. Cell culture

The normal human osteoblast frozen in liquid nitrogen in ourlab was thawed. After the cells were subcultured, the thawed cellsshowed more than 85% of vitality and the same characteristics asbefore in terms of growth, morphology, and genetic characteristics(Ma et al., 2010). The cells were cultured in Dulbecco Modified Ea-gle Medium (DMEM) (Gibco, USA) supplemented with 10% fetalbovine serum (Gibco, USA), 100 U/ml penicillin and 100 lg/mlstreptomycin. Incubation was conducted at 5% CO2 at 37 �C. Themedium was changed three times during one week. Cells of thesecond or third passage were used for the experiment. Cells grow-ing in medium without any of neuropeptides were used as con-trols. The neuropeptides used in the study were humanneuropeptide Y (NPY) (Sigma, USA), substance P (SP) (Sigma,USA), calcitonin gene-related peptide (CGRP) (Sigma, USA), andvasoactive intestinal peptide (VIP) (Sigma, USA), tyrosine hydroxy-lase (TH) (Sigma, USA). All neuropeptides were diluted with ser-um-free DMEM to corresponding conceration.

Group ALP (U/L) Osteocalcin (ng/ml)

Osteoblast 34.61 ± 2.94 0.189 ± 0.006Fibroblast 20.85 ± 0.69 0.049 ± 0.004Unpaired t-test <0.0001 <0.0001P values (t = 10.6) (t = 44.5)

Values of the ALP activity and osteocalcin are expressed as mean ± SD. The results ofunpaired t-test demonstrate that the levels of alkaline phosphatase and osteocalcinare significantly higher in osteoblasts than fibroblasts. Statistical significance isnoted by bold text.

2.2. Cell viability

Cell viability was measured by the MTT (3–4, 5-dimethylthia-zol2, 5-diphenyltetrazolium bromide) assay. Cells were seeded into96-well plate (clear bottom), at a density of 10,000 cells per well in100 ll medium, incubated for 24 h, and treated with five neuro-peptides at final concentrations of 1 lg/ml, 0.1 lg/ml and

0.01 lg/ml, using 6 replicates per concentration per treatment.After 24, 48 and 72 h, respectively, 20 ll of 5 mg/ml MTT solutionwere added to each well and incubated in a humidified atmosphereof 5% CO2 at 37 �C for 5 h. After incubation, the cells were washedwith PBS solution. Subsequently, 100 ll of isopropanol acid 4% andhydrochloric acid were added to each well, and the cells were incu-bated at room temperature for 10 min. The absorbance was mea-sured by Elisa Reader (Organon Teknika Reader 230S, Austria) at492 nm with 620 nm as reference.

2.3. ALP activity

The cells (2 � 104 cell in 100 ll) from the second passage wereseeded in four 24-well plates and incubated for 3–5 days near con-fluence and were treated with the NPY, SP, CGRP, VIP, TH at finalconcentrations of 0.1 lg/ml, respectively. After 24 h, the cells intwo plates were measured for ALP activity. For simulating the clin-ical mode of administration, the cells in the two remaining platescontinued to be cultured for 3 days, and then treated with five neu-ropeptides again. After incubation for 24 h, the cells were assayed.The determinations were performed using 6 replicates each treat-ment. To assay the ALP activity, the remaining medium was re-moved, and the cells were washed with PBS, digested with 0.25%trypsin 1 min. Then, the suspension of cells were collected inEppendorf tubes, respectively, added distilled water in each tube,freezed with liquid nitrogen and thawed repeatedly for 3–4 timesto destroy cell membrane. After centrifugation, the supernatantswere collected to measure with Automatic Biochemistry Analyzer(Hitachi 7150, Japan) at 405 nm. All treatments were comparedagainst control wells (cell culture with ordinary DMEM withoutneuropeptides and BMP).

2.4. Osteocalcin assay

This assay for the osteocalcin was performed with radioimmu-noassay (RIA). Cells were cultured and treated in the way describedabove in ALP assay. The NPY, SP, CGRP, VIP, TH were added at finalconcentrations of 0.1 lg/ml, respectively. The osteocalcin kit usedin the study was purchased from China Institute of Atomic Energy.For measuring the osteocalcin, the supernatants were collected inEppendorf tubes, respectively, to perform the determinations. A to-tal of 6 replicates were used per treatment.

2.5. Gap junctional intercellular communication (GJIC) in humanosteoblasts

The effects of the neuropeptides on GJIC were determined byfluorescence recovery after photobleaching (FRAP) technique usinga laser scanning confocal microscope (LSCM). Cells were seededinto 96-well plate, at a density of 5000 cells per well, incubatedto confluence. The cells were rinsed by D-Hank’s for two timesand treated with 10 lmol/L CFDA (5, 6-carboxy fluorescein diace-tate) (Sigma, USA), 37 �C for 90 min, rinsed by D-Hank’s again.

Fig. 1. The thawed osteablasts showed the stable biological characteristics. (A) Immunohistochemical staining suggests that the type I collagen is mainly produced by thecells. (B) The bone nodules are observed with fluorochrome-tetracycline staining. (C) Chromosome analysis indicates that there are 23 pairs of chromosomes, and abnormalchromosome is not detected, suggesting that the obtained cells are normal human cells.

W. Ma et al. / Neuropeptides 47 (2013) 179–186 181

Then, cells were treated with the NPY, SP, CGRP, VIP, TH at finalconcentrations of 0.1 lg/ml, and their GJIC were measured withMRC-1024 laser scanning confocal microscope imaging system(Bio-Rad, USA). The rate of fluorescence recovery (R) at 10 minafter photobleaching was adopted as the functional index of GJIC.

2.6. Statistical analyses

For each parameter of neuropeptides, mean and standard devi-ation for three concentrations at any time points were reported.Two-way ANOVA analysis was applied to address the followingthree questions: (i) does exposure time have the interaction withconcentration, (ii) does exposure time affect the result, and (iii)does concentration affect the result. For analyzing the differencesamong five neuropeptides, additional comparisons were madeemploying one-way ANOVA analysis. Additionally, for each neuro-peptide, the difference between the group treated once and the onetreated twice was explored using unpaired t-test. SAS softwarepackage (version 9.1, SAS Institute Inc., Cary, NC, USA) was usedfor the statistical analysis. Statistical significance was assigned toP < 0.05.

3. Results

3.1. Osteoblast culturing and identifying

The cellular morphology of human osteoblast was observedusing a phase-contrast microscope. In general, Osteoblasts werefusiform or polygon, with plenty cytoplasm and big oval nucleus.The thawed cells showed the stable characteristics as before (Maet al., 2010). As the specific osteoblast markers, alkaline phospha-tase and osteocalcin were assayed. The result demonstrated thattheir levels were significantly higher than fibroblast (Table 1).Immunohistochemical staining suggested that the type I collagenwas mainly produced by the cells (Fig. 1). Chromosome analysisindicated that there were 23 pairs of chromosomes, and abnormalchromosome was not detected, suggesting that the obtained sam-ples were normal human cells which were still normal followingfrozen reservation. The bone nodules were observed with fluoro-chrome-tetracycline staining, suggesting that the cultured cells inthis study had the osteogenesis ability.

3.2. Cell viability

The index (OD value) of viable cells exposed to five neuropep-tides at the concentrations of 1 lg/ml, 0.1 lg/ml and 0.01 lg/mlfor 24, 48 and 72 h were all significantly higher than those of theblank control group. Additionally, significant differences in OD va-lue were also observed among five neuropeptides employing one-way ANOVA in any concentrations at any time points (Table 2).Furthermore, two-way ANOVA analysis of OD value of each neuro-peptide revealed the interaction between the exposure time andthe concentration of neuropeptides. Meanwhile, one-way ANOVAanalysis showed that OD value was influenced by both the concen-

tration of each neuropeptide and the exposure time (Tables 3 and4) (Fig. 2). The data indicated that OD value was inversely associ-ated with the concentration of the neuropeptides, and positivelywith the time of exposure.

3.3. ALP activity

The ALP activity of osteoblasts exposed to the five neuropep-tides at the concentration of 0.1 lg/ml was evaluated using the col-orimetric assay, respectively (Table 5). All of the results of analysisbased on one-way ANOVA showed the significant differenceamong the five neuropeptides. The stimulation action of 0.1 lg/ml NPY was the highest. Further unpaired t-test comparisons dem-onstrated that the level of ALP activity for osteoblasts treated twicewith neuropeptides were significantly higher than that of osteo-blasts treated only once, suggesting that all stimuli increased ALPactivity in a dose-dependent manner (Fig. 3).

3.4. Osteocalcin level

The osteocalcin level of osteoblasts exposed to neuropeptides of0.1 lg/ml was evaluated using radioimmunoassay (Table 6). All ofthe results of analysis based on one-way ANOVA showed the sig-nificant difference among the five neuropeptides. The cells treatedwith NPY had the highest osteocalcin level. Further unpaired t-testcomparisons showed that neuropeptides also increased osteocalcinlevel in a dose-dependent manner (Fig. 4).

3.5. Gap junctional intercellular communication (GJIC) in humanosteoblasts

The FRAP method was used to determine the GJIC. The ratio offluorescence recovery (R) of cells exposed to the five neuropeptidesat the concentration of 0.1 lg/ml were between 15.7 ± 0.4 to27.0 ± 1.9, whereas the cells in the control group had an R- valueof 6.8 ± 0.4, demonstrating that the GJIC of osteoblasts was signif-icantly promoted by the neuropeptides (P < 0.05). The R- value ofcells exposed to 0.1 lg/ml NPY (27.0 ± 1.9) was the highest, fol-lowed by VIP (23.9 ± 0.5), SP (20.0 ± 0.4) and TH (18.0 ± 0.2),whereas the R- value in CGRP-treated cells was the lowest (Table7).

4. Discussion

Bone is abundantly innervated by small diameter sensorynerves in the periosteum, bone marrow, and vascular canals (Imaiand Matsusue, 2002; Hill and Elde, 1991; Mach et al., 2002). Thehomeostatic nature of bone remodeling has become a notion fur-ther supported lately by the demonstration that neuropeptidesand their receptors regulate the function of osteoblast and osteo-clast in vivo. Because of the complexity of the mechanisms regulat-ing bone remodeling, the experimental and mechanistic studiesbased on in vivo murine genetic and pharmacologic models hadtheir limitation to some extent. While in vitro studies have been

lues⁄

Table 3Results of the effects of the concentration on the OD values.

Time 24 h 48 h 72 h

Treatments F value P values F value P values F value P values

NPY 248.9 <0.0001 1042.8 <0.0001 222.2 <0.0001SP 285.3 <0.0001 450.6 <0.0001 426.2 <0.0001CGRP 322.2 <0.0001 286.8 <0.0001 220.1 <0.0001VIP 879.2 <0.0001 719.6 <0.0001 576.8 <0.0001TH 559.6 <0.0001 705.3 <0.0001 459.0 <0.0001

For each neuropeptide, further one-way ANOVA is performed to analyze the effectsof the concentration on the OD values. The results are showed in above table, alongwith the appropriate P-values. Statistical significance is noted by bold text.

Table 4Results of the effect of the exposure time on the OD values.

Concentration 1 lg/ml 0.1 lg/ml 0.01 lg/ml

Treatments F value P values F value P values F value P values

NPY 33.7 <0.0001 14.9 <0.0001 28.4 <0.0001SP 54.5 <0.0001 76.9 <0.0001 81.2 <0.0001CGRP 141.0 <0.0001 170.8 <0.0001 217.6 <0.0001VIP 127.7 <0.0001 111.0 <0.0001 186.6 <0.0001TH 211.4 <0.0001 302.6 <0.0001 213.9 <0.0001

For each neuropeptide, further one-way ANOVA is performed to analyze the effectof the exposure time on the OD values. The results are showed in above table, alongwith the appropriate P-values. Statistical significance is noted by bold text.

a valuable method for evaluating cell interactions with the stimu-lating signals. Also, the proliferation and differentiation of theosteoblast cells are essential prerequisites for bone formation.Many studies have shown favorable results following the use ofdifferent osteoblast cells (Teixeira et al., 2009; Lundberg et al.,2007; Persson and Lerner, 2011; Lundberg et al., 2000). However,previous researches based on osteoblast cell mainly derived fromanimal or osteosarcoma have prevented us from better under-standing the ‘‘osteo-neuromediators’’ which was implicated inthe control of osteoblast activity and thus bone formation. So, weused the normal human osteoblast cell lines established by ourlab (Ma et al., 2010) in this study, which should markedly improvethe reliability of the research. The cells reserved with liquid nitro-gen frozen method were thawed. They were fusiformed-shapedand had plentiful processes. Furthermore, the cells had the osteo-genic characteristics, with high expression of alkaline phosphataseand osteocalcin. According to the author, the cells cultured had themorphology and characteristics for osteoblast cells, and could beused in the subsequent experiments.

This study is unique in which the dose-dependent effects ofNPY, SP, CGRP, VIP or TH on human osteoblast were comprehen-sively investigated, looking specifically at cellular proliferationand function. The effects of five neuropeptides on osteoblasts werealso compared.

Our results indicated that all of the neuropeptides led to in-crease in the cell viability of human osteoblasts. NPY caused thehighest cell viability, followed by VIP, SP, TH, CGRP, and controlgroup. Many studies have been conducted to evaluate the osteo-genic action of different neuropeptides. In line with the presentstudy, Teixeira et al. Teixeira et al. (2009), Persson and Lerner(2011) found that the osteogenic potential of NPY with osteoblastphenotype markers being significantly enhanced in osteoprogeni-tor cells stimulated by NPY, probably due to the down-regulationof Y1 receptor. Lee (Lee et al., 2010) also demonstrated that theNPY system, via the Y1 receptor, directly inhibits the differentia-tion of mesenchymal progenitor cells as well as the activity of ma-ture osteoblasts. For VIP, Persson (Persson and Lerner, 2011)observed that VIP affected the expression of RANKL, OPG, and M-CSF in osteoblasts and stromal cells suggesting an important role

Fig. 2. Cell viability levels after treatments with neuropeptides in three exposure times. Results are the average of percentage of change in relation to controls. Cells weretreated for 24, 48 or 72 h with different neuropeptides at indicated concentrations. Straight lines solid diamond markers designate 24 h treatment; short dashed lines solidsquare markers indicate 48 h treatment; long dashed lines solid triangle markers represent 72 h treatment. In this assay, cell viability levels gradually dropped with theincreasing concentration of neuropeptides.

Table 5The ALP activity of the human osteoblast treated with neuropeptides (U/L).

Treatment NPY VIP SP CGRP TH Control One-way ANOVA (treatments) P values⁄

Treated once 3.96 ± 0.30 3.16 ± 0.27 3.00 ± 0.20 1.94 ± 0.23 2.52 ± 0.29 1.00 ± 0.20 <0.0001 (F = 85.0)Treated twice 5.56 ± 0.50 5.02 ± 0.32 4.72 ± 0.36 3.14 ± 0.29 3.90 ± 0.27 1.54 ± 0.18 <0.0001 (F = 96.9)Unpaired t-test 0.0003 <0.0001 <0.0001 <0.0001 <0.0001 0.0021P values# (t = 6.2) (t = 9.9) (t = 9.4) (t = 7.3) (t = 7.8) (t = 4.5)

The table demonstrates the ALP activity of the osteoblasts treated once or twice with 0.1 lg/ml NPY, VIP, SP, CGRP and TH. Values of the ALP activity are expressed asmean ± SD. The P values⁄ represent the results of one-way ANOVA analysis to the data among different treatments. And unpaired t-test is done to analyze the differencebetween the group treated once and one treated twice, of which the result is represented by the P values#. Statistical significance is noted by bold text.

Fig. 3. ALP activity after treatments with neuropeptides in osteoblastic cells.Results are the average of ALP activity. Cells were treated once or twice withdifferent neuropeptides at the concentration of 0.1 lg/ml. The stimulation action ofNPY is the highest. The level of ALP activity for osteoblasts treated twice withneuropeptides were significantly higher than that of osteoblasts treated only once(⁄P < 0.05).

for VIP in bone remodeling. Moreover, Lundberg (Lundberg et al.,1999) reported that VIP could stimulate osteoblastic ALP biosyn-thesis and bone noduli formation by a mechanism mediated bycyclic AMP. Numerous studies have demonstrated that CGRP wasexpressed endogenously by the osteoblasts. Furthermore, trans-genic mice with osteoblasts overexpressing CGRP were character-ized by increased bone formation rate and enhanced bonevolume, suggesting that CGRP indeed acted on bone metabolismnot only via nervous route but also via autocrine loop (Villaet al., 2003; Kawase et al., 2003; Vignery and McCarthy, 1996).Additionally, TH is the rate-limiting enzyme of catecholamine. InBjurholm’s study of osteoblastic cells, receptors to CGRP, VIP, nor-adrenaline (NA) and NPY were demonstrated, and the stimulatoryeffects of different neuropeptides and norepinephrine (NE) on cyc-lic AMP formation in four different osteoblastic cell lines and inisolated neonatal mouse calvarial bone cells had been reported(Bjurholm et al., 1992).

Our study showed that these neuropeptides increased not onlycell viability but also cell differentiation. In the present study, weused ALP activity and osteocalcin as markers for osteoblastic differ-entiation. All tested neuropeptides had higher ALP activities andosteocalcin compared with the control group. The ALP activityand osteocalcin were also up-regulated by all stimuli in a dose-dependent manner, consistent with Goto’s, Lundberg’s, and Bjur-holm’s findings in rat osteoblastic cells (Goto et al., 2007; Lundberget al., 1999). Moreover, the ALP activity and osteocalcin levels werethe highest after NPY treatment and the lowest after CGRP, whichsuggested that NPY action was the most powerful, CGRP action wasthe weakest and VIP, SP or TH intermediate between the other two.

The stimulatory action of the neuropeptides on human osteo-blast indicated that they were involved in modulating bone metab-olism in some way. We believed that these neuropeptides couldinfluence cellular biological characteristics via binding with corre-sponding receptors and activating specific signals. But, for neuro-peptides, the further mechanism of action after binding withreceptors and the specificity of the various responses need to befurther solved.

Several groups have studied neuropeptide actions on osteoblas-tic cell proliferation, differentiation, or function, but few haveexamined the effects of various concentrations of neuropeptides.We observed in the current study that osteoblast viability was in-versely associated with the concentration of neuropeptides (0.01–1 lg/ml), and positively with the time of exposure. Higher concen-tration (1 lg/ml, corresponding to 10�7 M) neuropeptides had thelow effect on osteoblast viability. The experimental studies per-formed by Adamus et al. were in agreement with our findings. InAdamus’s study, the alkaline phosphatase activity decreased withhigher concentration of SP (10�8 M) treatment Adamus andDabrowski, 2001. Another recent study found that higher concen-tration of SP (10�7–10�5 M) dose-dependently inhibited alkalinephosphatase activity and bone nodule formation, as well as geneexpression for osteocalcin in fetal rat calvarial osteoblasts (Azumaet al., 2004). In contrast to our findings, however, Shih’s studyshowed that similar concentration of SP (ranging from 10�8 M to

Table 6The osteocalcin of the human osteoblast treated with neuropeptides (ng/ml).

Treatment NPY VIP SP CGRP TH Control One-way ANOVA (Treatments) P values⁄

Treated once 0.27 ± 0.01 0.24 ± 0.01 0.23 ± 0.01 0.17 ± 0.01 0.20 ± 0.02 0.08 ± 0.02 <0.0001 (F = 87.0)Treated twice 0.54 ± 0.03 0.49 ± 0.01 0.46 ± 0.03 0.31 ± 0.02 0.38 ± 0.01 0.19 ± 0.03 <0.0001 (F = 127.1)Unpaired t-test 0.0002 <0.0001 <0.0001 0.0002 0.0002 0.0007P values# (t = 15.5) (t = 62.1) (t = 13.0) (t = 16.9) (t = 16.8) (t = 6.3)

The table displays the osteocalcin of the osteoblasts treated once or twice with 0.1 lg/ml NPY, VIP, SP, CGRP and TH. Values of the osteocalcin are expressed as mean ± SD. TheP values⁄ represent the results of one-way ANOVA analysis of the data among different treatments. Meanwhile, the P values# represent the results of unpaired t-testperformed between the group treated once and one treated twice. Statistical significance is noted by bold text.

Fig. 4. Osteocalcin level after treatments with neuropeptides in osteoblastic cells.Results are the average of osteocalcin level. Cells were treated once or twice withdifferent neuropeptides at the concentration of 0.1 lg/ml. The cells treated withNPY have the highest osteocalcin level. Moreover, the cells treated twice withneuropeptides have significantly higher osteocalcin level (⁄P < 0.05).

10�6 M) increased, rather than decreased, the number and size ofbone colonies in a concentration-dependent manner (Shih and Ber-nard, 1997). Similarly, Goto (Goto et al., 2007) found that higherconcentration of SP (ranging from 10�8 M to 10�6 M) increasedthe size of bone colonies in rat calvarial osteoblastic cells. Likewise,Wang (Wang et al., 2010) found that CGRP (10�10–10�8 M) stimu-lated mouse bone marrow stromal cells (BMSC) proliferation, up-regulated the expression of osteoblastic genes, and increased ALPactivity and mineralization in the BMSCs. The different results inour study could be due to different protocols, such as the variouscell lines used in studies, the duration of culturing, the diluentfor neuropeptides, and methods used to evaluate cell viability.But we just initially investigated three orders of magnitude forthe concentration of the neuropeptides in the study. The relativelymost effective dose of action is still not clear. In addition, we sup-posed that higher concentration neuropeptides may stimulate thefeedback inhibition of neuropeptides in some way.

Collectively, these results suggested that neuropeptides couldstimulate osteogenesis in human osteoblast. Previously it was ob-served that neuropeptide depletion in rats caused trabecular boneloss and inhibited bone formation (Offley et al., 2005), and hered-itary small fiber sensory neuropathy in man was associated withneuropeptide loss, reduced bone mineral density (BMD) and in-creased bone fragility (Pearson et al., 1975; Maayan et al., 2002;Maayan et al., 2001). These diverse data supported the hypothesisthat neuropeptides signaling contributes to the maintenance of

Table 7The ratio of fluorescence recovery (R) of cells exposed to the neuropeptides.

Treatment NPY SP VIP

R value (%) 27.0 ± 1.9 20.0 ± 0.4 23.9 ± 0.5

The table shows the ratio of fluorescence recovery of the osteoblasts treated with 0.1 lg/The P values represent the results of one-way ANOVA analysis of the data among differ

bone mass by regulating osteogenic activity in osteoblast. How-ever, few studies have evaluated the mechanism for neuropeptidesregulating osteogenic behavior of osteoblasts. Signal factors,depending on their own properties and their impact on messengersubstance, can affect cell differentiation and bone formation. Fur-thermore, signal transmission has an essential role in cell activa-tion. Gap junctional intercellular communication (GJIC) mediatedby connexins, in particular connexin 43 (C�43), plays importantroles in regulating signal transmission among different cells.Thereby, the investigation of GJIC may have important implicationsfor understanding the mechanism for neuropeptides modulatingbone formation. Many studies have shown that GJIC regulatesosteoblast formation, differentiation, survival and apoptosis. More-over, osteocytes utilize GJIC to coordinate bone remodeling in re-sponse to anabolic factors and mechanical loading. Thesetransmembrane channels allow continuity of cytoplasms andmediate the transfer of molecules between communicating cells(Civitelli, 2008; Unger et al., 1999; Cherian et al., 2005; Civitelli,2008). The biological importance of the communication mediatedby connexin-forming channels in bone development is revealedby the low bone mass and osteoblast dysfunction in the C�43-nullmice and the skeletal malformations observed in occulodentodigi-tal dysplasia (ODDD) caused by mutations in the C�43 gene(McLachlan et al., 2008; Watkins et al., 2011; Lecanda et al.,2000; Chung et al., 2006; Laird, 2008). But there are no availabledata on the effects of neuropeptides on GJIC between osteoblastsso far.

The novelty of this current work is that we confirmed the stim-ulatory effect of the neuropeptides on GJIC in osteoblasts with theFRAP method. The R- value of cells exposed to NPY was the highest,followed by VIP, SP, TH and CGRP, suggesting that NPY had themost powerful action of regulating osteoblast activity via promot-ing the GJIC between cells. This result was consistent with previousobservations in the present study which show the strongest role ofNPY in the modulation of osteoblast viability, ALP, and osteocalcinproduction. According to the author’s opinion, gap junctional com-munication may serve as a means by which osteoblasts could workin synchrony and propagate locally generated signals throughoutthe skeletal tissue. Thus, we supposed that the five neuropeptidesmay enhance the GJIC between osteoblasts, and then promote thetransfer of molecules between communicating cells. In this way,they would improve the response of osteoblast to various stimulat-ing signals. These data provided further support for the hypothesisthat stimulatory effects of the neuropeptides on osteoblast and

CGRP TH Control P values

15.7 ± 0.4 18.0 ± 0.2 6.8 ± 0.4 <0.0001 (F = 404.6)

ml NPY, VIP, SP, CGRP and TH. Values of the osteocalcin are expressed as mean ± SD.ent treatments. Statistical significance is noted by bold text.

osteogenesis activity were not only due to a directly binding andactivating their receptors, but also via a direct action on the GJICthat was involved in coordinating bone remodeling. This study alsopresented a novel interest point in neuropeptides controlling bonehomeostasis.

Taken together, neuropeptides stimulated osteoblast cell viabil-ity and function in a dose-dependent manner. In addition, thestimulatory effects of the neuropeptides on osteoblast cell were in-versely associated with the concentration of the neuropeptides,and positively with the time of exposure. Moreover, neuropeptidescould enhance the GJIC between osteoblasts. Given all the observa-tions, it was tempting to assume that neuropeptides could be phys-iologic activators of bone formation in vitro, membrane-boundreceptors were essential in maintaining skeletal homeostasisthrough regulating proliferation and activation of osteoblasts bypromoting signal transmission among bone cells. The regulationof the GJIC between osteoblasts could be another mechanism forneuropeptides in modulating bone metabolism. Furthering ourunderstanding of the mechanisms underlying the involvement ofthe neuropeptide system in the control of bone homeostasis couldlead to the development of therapies to improve bone mass in pa-tients with diseases such as osteoporosis. In addition, consideringthe important role of the GJIC in regulating various signal transmis-sion between communicating cells, we have another hypothesisthat the pain signal transmission may be prevented to relieve thebone pain via inhibiting the gap junctions. However, we emphasizethat the results reported here are preliminary and restricted by aninadequate knowledge of the biology of cell and by the superficialunderstanding of the mechanism of bone remodeling. Many fac-tors, such as the specificity of the neuropeptide effects and effec-tive concentration that are closer to the physiological state,should be taken into account in later work. Future studies examin-ing the role of neuropeptides signaling in animal models are alsorequired to further investigate these hypotheses.

In summary, we have shown that human osteoblasts were reg-ulated by the five neuropeptides (i.e. SP, CGRP, VIP, NPY and TH),and they exhibited enhancing reactions when the cell–cell commu-nication was stimulated by the neuropeptides signals in the cellcultures.

Acknowledgements

This study receives no financial support. We are grateful to MrsHuang jinxiang for assistance with tissue culture. We also expressour gratitude to Wu Shuaishuai, Xu Bin, and Liu Song for their ef-forts in data preparation.

References

Adamus, M.A., Dabrowski, Z.J., 2001. Effect of the neuropeptide substance P on therat bone marrow-derived osteogenic cells in vitro. J. Cell. Biochem. 81, 499–506.

Azuma, H., Kido, J., Ikedo, D., Kataoka, M., Nagata, T., 2004. Substance P enhances theinhibition of osteoblastic cell differentiation induced by lipopolysaccharidefrom porphyromonas gingivalis. J. Periodontol. 75, 974–981.

Bjurholm, A., Kreicbergs, A., Schultzberg, M., Lerner, U.H., 1992. Neuroendocrineregulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS17/2.8, MC3T3-E1, and Saos-2) and primary bone cells. J. Bone Miner. Res. 7,1011–1019.

Cherian, P.P., Siller-Jackson, A.J., Gu, S., Wang, X., Bonewald, L.F., Sprague, E., Jiang,J.X., 2005. Mechanical strain opens connexin43 hemichannels in osteocytes: anovel mechanism for the release of prostaglandin. Mol. Biol. Cell 16, 3100–3106.

Chung, D.J., Castro, C.H., Watkins, M., Stains, J.P., Chung, M.Y., Szejnfeld, V.L.,Willecke, K., Theis, M., Civitelli, R., 2006. Low peak bone mass and attenuatedanabolic response to parathyroid hormone in mice with an osteoblast-specificdeletion of connexin43. J. Cell Sci. 119, 4187–4198.

Civitelli, R., 2008. Cell–cell communication in the osteoblast/osteocyte lineage.Arch. Biochem. Biophys. 473, 188–192.

Civitelli, R., 2008. Connexin43 modulation of osteoblast/osteocyte apoptosis: apotential therapeutic target? J. Bone Miner. Res. 23, 1709–1711.

Goto, T., Nakao, K., Gunjigake, K.K., Kido, M.A., Kobayashi, S., Tanaka, T., 2007.Substance P stimulates late-stage rat osteoblastic bone formation throughneurokinin-1 receptors. Neuropeptides 41, 25–31.

Hill, E.L., Elde, R., 1991. Distribution of CGRP-, VIP-, D beta H-, SP-, and NPY-immunoreactive nerves in the periosteum of the rat. Cell Tissue Res. 264, 469–480.

Imai, S., Matsusue, Y., 2002. Neuronal regulation of bone metabolism andanabolism: calcitonin gene-related peptide-, substance P-, and tyrosinehydroxylase-containing nerves and the bone. Microsc. Res. Tech. 58, 61–69.

Kawase, T., Okuda, K., Burns, D.M., 2003. Immature human osteoblastic MG63 cellspredominantly express a subtype 1-like CGRP receptor that inactivatesextracellular signal response kinase by a cAMP-dependent mechanism. Eur. J.Pharmacol. 470, 125–137.

Laird, D.W., 2008. Closing the gap on autosomal dominant connexin-26 andconnexin-43 mutants linked to human disease. J. Biol. Chem. 283, 2997–3001.

Lecanda, F., Warlow, P.M., Sheikh, S., Furlan, F., Steinberg, T.H., Civitelli, R., 2000.Connexin43 deficiency causes delayed ossification, craniofacial abnormalities,and osteoblast dysfunction. J. Cell Biol. 151, 931–944.

Lee, N.J., Herzog, H., 2009. NPY regulation of bone remodelling. Neuropeptides 43(6), 457–463.

Lee, N.J., Doyle, K.L., Sainsbury, A., Enriquez, R.F., Hort, Y.J., Riepler, S.J., Baldock, P.A.,Herzog, H., 2010. Critical role for Y1 receptors in mesenchymal progenitor celldifferentiation and osteoblast activity. J. Bone Miner. Res. 25, 1736–1747.

Lundberg, P., Boström, I., Mukohyama, H., Bjurholm, A., Smans, K., Lerner, U.H.,1999. Neuro-hormonal control of bone metabolism: vasoactive intestinalpeptide stimulates alkaline phosphatase activity and mRNA expression inmouse calvarial osteoblasts as well as calcium accumulation mineralized bonenodules. Regul. Pept. 85, 47–58.

Lundberg, P., Lie, A., Bjurholm, A., Lehenkari, P.P., Horton, M.A., Lerner, U.H., Ransjö,M., 2000. Vasoactive intestinal peptide regulates osteoclast activity via specificbinding sites on both osteoclasts and osteoblasts. Bone 27, 803–810.

Lundberg, P., Allison, S.J., Lee, N.J., Baldock, P.A., Brouard, N., Rost, S., Enriquez, R.F.,Sainsbury, A., Lamghari, M., Simmons, P., Eisman, J.A., Gardiner, E.M., Herzog, H.,2007. Greater bone formation of Y2 knockout mice is associated with increasedosteoprogenitor numbers and altered Y1 receptor expression. J. Biol. Chem. 282,19082–19091.

Ma, W.H., Zhang, X.M., Shi, S.S., Li, Y.F., 2010. Establishment and identification of anormal human osteoblast system. J. Clin. Rehabil. Tissue Eng. Res. 14, 2661–2666.

Maayan, C., Becker, Y., Gesundheit, B., Girgis, S.I., 2001. Calcitonin gene relatedpeptide in familial dysautonomia. Neuropeptides 35, 189–195.

Maayan, C., Bar-On, E., Foldes, A.J., Gesundheit, B., Pollak, R.D., 2002. Bone mineraldensity and metabolism in familial dysautonomia. Osteoporos. Int. 13, 429–433.

Mach, D.B., Rogers, S.D., Sabino, M.C., Luger, N.M., Schwei, M.J., Pomonis, J.D., Keyser,C.P., Clohisy, D.R., Adams, D.J., O’Leary, P., Mantyh, P.W., 2002. Origins of skeletalpain: sensory and sympathetic innervation of the mouse femur. Neuroscience113, 155–166.

McLachlan, E., Plante, I., Shao, Q., Tong, D., Kidder, G.M., Bernier, S.M., Laird, D.W.,2008. ODDD-linked Cx43 mutants reduce endogenous Cx43 expression andfunction in osteoblasts and inhibit late stage differentiation. J. Bone Miner. Res.23, 928–938.

Offley, S.C., Guo, T.Z., Wei, T., Clark, J.D., Vogel, H., Lindsey, D.P., Jacobs, C.R., Yao, W.,Lane, N.E., Kingery, W.S., 2005. Capsaicin-sensitive sensory neurons contributeto the maintenance of trabecular bone integrity. J. Bone Miner. Res. 20, 257–267.

Opolka, A., Straub, R.H., Pasoldt, A., Grifka, J., Grässel, S., 2012. Substance P andnorepinephrine modulate murine chondrocyte proliferation and apoptosis.Arthritis Rheum. 64, 729–739.

Pearson, J., Dancis, J., Axelrod, F., Grover, N., 1975. The sural nerve in familialdysautonomia. J. Neuropathol. Exp. Neurol. 34, 413–424.

Persson, E., Lerner, U.H., 2011. The neuropeptide VIP regulates the expression ofosteoclastogenic factors in osteoblasts. J. Cell. Biochem. 112, 3732–3741.

Shi, Y.C., Bladock, P.A., tul=0?>Shi and Bladock, 2012. Central and peripheralmechanisms of the NPY system in the regulation of bone and adipose tissue.Bone 50, 430–436.

Shih, C., Bernard, G.W., 1997. Neurogenic substance P stimulates osteogenesisin vitro. Peptides 18, 323–326.

Stains, J.P., Civitelli, R., 2005. Cell-to-cell interactions in bone. Biochem. Biophys.Res. Commun. 328, 721–727.

Strange-Vognsen, H.H., Arnbjerg, J., Hannibal, J., 1997. Immunocytochemicaldemonstration of pituitary adenylate cyclase activating polypeptide (PACAP)in the porcine epiphyseal cartilage canals. Neuropeptides 31 (2), 137–141.

Teixeira, L., Sousa, D.M., Nunes, A.F., Sousa, M.M., Herzog, H., Lamghari, M., 2009.NPY revealed as a critical modulator of osteoblast function in vitro: newinsights into the role of Y1 and Y2 receptors. J. Cell. Biochem. 107, 908–916.

Togari, A., Arai, M., Mizutani, S., Mizutani, S., Koshihara, Y., Nagatsu, T., 1997.Expression of mRNAs for neuropeptide receptors and beta-adrenergic receptorsin human osteoblasts and human osteogenic sarcoma cells. Neurosci. Lett. 233,125–128.

Unger, V.M., Kumar, N.M., Gilula, N.B., Yeager, M., 1999. Three-dimensionalstructure of a recombinant gap junction membrane channel. Science 283,1176–1180.

Vignery, A., McCarthy, T.L., 1996. The neuropeptide calcitonin gene-related peptidestimulates insulin-like growth factor I production by primary fetal ratosteoblasts. Bone 18, 331–335.

Villa, I., Dal Fiume, C., Maestroni, A., Rubinacci, A., Ravasi, F., Guidobono, F., 2003.Human osteoblast-like cell proliferation induced by calcitonin-related peptidesinvolves PKC activity. Am. J. Physiol. Endocrinol. Metab. 284, E627–E633.

Wang, L., Shi, X., Zhao, R., Halloran, B.P., Clark, D.J., Jacobs, C.R., Kingery, W.S., 2010.Calcitonin-gene-related peptide stimulates stromal cell osteogenicdifferentiation and inhibits RANKL induced NF-kappaB activation,osteoclastogenesis and bone resorption. Bone 46, 1369–1379.

Watkins, M., Grimston, S.K., Norris, J.Y., Guillotin, B., Shaw, A., Beniash, E., Civitelli,R., 2011. Osteoblast connexin43 modulates skeletal architecture by regulatingboth arms of bone remodeling. Mol. Biol. Cell 22, 1240–1251.

Wróbel, E., Leszczynska, J., Przybylski, J., 2011. Role of gap junctions in bone tissue.Postepy Biochem. 57, 411–417.

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