Neuropeptide W regulates proliferation and differentiation of ATDC5: Possible involvement of GPR7 activation, PKA and PKC‐dependent signalling cascades

Abstract Various neuropeptides related to the energy equilibrium affect bone growth in humans and animals. Neuropeptides W (NPW) are identical in the internal ligands of the two G‐protein receptors (GPRs) included in subtypes 7 and 8. Neuropeptides W inhibits proliferation in the cultivated rat calvarial osteoblast‐like (ROB) cells. This study examines the expression of NPW and GPR7 in murine chondrocyte and their function. An immunohistochemical analysis showed that NPW and GPR7 were expressed in the proliferative chondrocytes of the growth plates in the hind limbs of mice. The NPW mRNA quickly elevated in the early differentiation (7‐14 days) of ATDC5 cells, while NPW and GPR7 mRNA were reduced during the late stage (14‐21 days) of differentiation. Neuropeptide W‐23 (NPW‐23) promoted the proliferation of ATDC5 cells, which was attenuated by inhibiting the GPR7, protein kinase A (PKA), protein kinase C (PKC) and ERK1/2 pathways. Neuropeptide W‐23 enhanced the early cell differentiation, as evaluated by collagen type II and the aggrecan gene expression, which was unaffected by inhibiting the ERK1/2 pathway, but significantly decreased by inhibiting the PKA, PKC and p38 MAPK pathways. In contrast, NPW‐23 was not involved in the terminal differentiation of the chondrocytes, as evaluated by the mineralization of the chondrocytes and the activity of the alkaline phosphatase. Neuropeptides W stimulated the PKA, PKC, p38 MAPK and ERK1/2 activities in a dose‐ and time‐dependent manner in the ATDC5 cells. These results show that NPW promotes the proliferation and early differentiation of murine chondrocyte via GPR7 activation, as well as PKA and PKC‐dependent signalling cascades, which may be involved in endochondral bone formation.


| INTRODUCTION
Neuropeptides W (NPW) and Neuropeptides B (NPB) have been identified as endogenetic ligands of G-protein receptors (GPR) 7 and 8. 1,2 Both GPR7 and GPR8 are expressed in humans, but GPR8 is absent in rodents. 3 Neuropeptides W, NPB and their receptors are mainly expressed in the central and periphery issues, which are involved in many physiological processes, including inflammatory pain, energy homeostasis, cardiovascular functions, immune system, stress and the neuroendocrine and respiratory systems. [4][5][6] Previous studies have detected NPW/NPB mRNA, including bone marrow, femur and costal cartilagein, in humans, rats, pigs and chickens. 1,7,8 The effect of G protein activation was mediated by protein kinase A (PKA), protein kinase C (PKC) and the mitogen-activated protein kinases (MAPKs) cascades reaction. 9,10 The down-regulation or inhibition of PKA and PKC blocks chondrogenesis. 11,12 The proliferation and differentiation of chondrocytes are regulated by PKC-mediated p38 MAPK and the ERK1/2 signalling pathway. 13 The PKA and PKC cascades are relevant to the secret agogue effect of NPW and NPB in human adrenocortical cells. 9 Neuropeptides W stimulates the proliferation of NCI-H295 cells, which are derived from human adrenocortical carcinoma by exerting the ERK1/2 pathway, 14 which is considered a crucial growth factor in rat adrenocortical cells. 15 Neuropeptides and their receptors are expressed in bone tissue and are involved in bone development in humans and animals. [16][17][18] Neuropeptides W, NPB and their receptors are expressed and inhibited proliferative activity in cultured rat calvarial osteoblast-like (ROB) cells. 19 However, little is known about whether NPW/B can regulate endochondral bone formation. The role of NPW/B in the regulation of the chondrocyte function has not been characterized so far. Therefore, we used immunohistochemical analyses to assess the expression of NPW and it's receptor in the growth plates of mice. We also identify the role of NPW and GPR7 in chondrocyte using an excellent in vitro model cell line called ATDC5 for chondrocyte proliferation and differentiation. The ATDC5 cell line is derived from AT805 teratocarcinoma cells and is characterized as a chondrogenic cell line that is capable of differentiating into chondrocytes. 20,21 The molecular analysis of early-and late-phase differentiation markers of chondrocytes in vivo can also be mimicked by ATDC5 cells in vitro. Finally, the primers for the NPW, GPR7, p21, aggrecan, Sox-9, Coll II, Coll X, Runx2 and ribosomal protein L19 (RPL-19) were designed and synthesized by Invitrogen Co. (Guangzhou, China).

| Immunohistochemistry analysis
Slides were processed as described above for the immunohistochemical analysis. 22 The tibiofemoral joints were fixed briefly in 10% paraformaldehyde for 24 hours after the knee samples were harvested. The fixed femoral condyles were decalcified in 10% EDTA for 7 days at 37°C. After graded ethanol dehydration and dimethylbenzene vitrification, the femoral condyles were embedded in paraffin and sectioned at 5 μm thickness. The sections were then incubated with primary antibodies against anti-NPW and anti-GPR7 antibodies overnight at 4°C. The biotinylated secondary antibody and streptavidin peroxidase solution were then used to visualize the sections.

| Cell culture
ATDC5 cells were cultured in 1:1 mixed DMEM and Ham'sF-12 media containing 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO). The ATDC5 cells were cultured at 37°C in a humidified atmosphere of 5% CO 2 in air. Insulin, transferin and selenite (ITS, Sigma-Aldrich) were added to the medium to induce ATDC5 cell differentiation in the culture.

| Small interfering RNA transfections
The ATDC5 cells were transfected with small interfering RNA

| RT-PCR analysis
The total RNA was extracted using TRNzol Universal Reagent (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. The first strand of cDNA was produced using the First-Strand cDNA Synthesis Kit (Thermo Scientific, USA). The reaction conditions for the PCR and gene-specific primers (Table 1) were the same as previously above, 23 and the cDNA was initially pre-denaturated at 94°C for 10 minutes, followed by 34 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 60 seconds. The PCR products were detected using electrophores is on 1.5% agarose gel stained with ethidium bromide, and the size was compared with the DNA ladder (O'RangeRuler 100-bp DNA Ladder [MBI Fermentas, Lithuania]). The reverse transcriptase (RT-PCR) was used as a negative control, and the results were normalized by RPL-19.

| Quantitative real-time PCR
Relative quantification real-time PCR was performed with SYBR Green PCR Master Mix (Thermo Fisher Scientific, Woolston Warrington, UK) according to the manufacturer's protocols, 2μL of cDNA template, and the gene specific primer sets (Table 1). PCR was carried out in the ABI 7500 system (Applied Biosystems). Relative gene expression was calculated using the 2 -ΔΔCT method and RPL-19 was used as a housekeeping gene for normalization.

| Western blotting analysis
The Western blotting analysis was described previously. 24 Briefly, cells from different experimental conditions were lysed with ice-cold RIPA lysis buffer. The protein concentration was measured with a BCA protein assay kit according to the manufacturer's instructions.
SDS-PAGE electrophoresis was carried out with the same amounts of lysate protein (20 μg/lane) and 10% polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. After transfer nitrocellulose blots were first blocked with 3% bovine serum albumin (BSA) in PBST buffer (PBS with 0.01% Tween 20, PH 7.4), and then incubated overnight at 4°C with primary antibodies in PBST containing 1% BSA. Horseradish peroxidase-coupled secondary antibody was incubated continuously to determine the immunore activity, and enhanced chemiluminescence technique was used to detect the immunore activity.

| CCK-8 assay
ATDC5 cells were seeded in 96-well plates at the density of 1000 cells per well with 100 μL of culture medium in the 5% (v/v) Fetal Bovine Serum (FBS) culture medium. After adhesion for 24 hours, the medium was changed to serum-free and NPW-23 was added to medium with final concentrations ranging from 25 to 400 ng/mL, then the cells were re-cultured for 24 hours. The cells which were not exposed to NPW-23 were used as controls. The wells with only culture medium was served as blanks. Afterward, CCK-8 was added to each well and the plates were incubated at 37°C for additional 3 hours. Then the absorbance of each solution was measured at 450 nm using BIO-RAD 680 plate reader (Thermo, Walsam, MA, USA). All the experiments were repeated at least three times using triplicate cultures (n = 9).

| BrdU cell proliferation assay
Cell proliferation was assessed using BrdU cell proliferation assay kits according to the manufacturer's instructions. Briefly, the cells suspended in the growth medium were seeded in a 96-well plates with a density of 1 × 10 4 cells/well and grown overnight at 37°C in a humidified incubator with 5% CO 2 . The second day, the cells were incubated with NPW-23 (25-400 ng/mL) for 24 hours. Afterward, the cells were incubated with 20 μL of BrdU for another 12 hours. Fixing solution was then added and incubated for 30 minutes at room temperature.
After washing, cells were incubated with a pre-diluted detector antibody for 1 hour at room temperature. Acid stop solution was added after incubation with HRP-conjugated secondary antibody for 1 hour at room temperature. The optical density (OD) was then measured at 450 nm using an automated microplate reader.

| Alcian blue and Alizarin red staining
The cells were cultured in 12-well plates at a density of 50 000 cell/ well for 4, 7, 14 and 21 days, and were treated with or without indicated concentrations of NPW-23. Cells were then fixed in 4% (v/v) formalin for 15 minutes, and then stained with Alcian blue 8GX for 30 minutes or with 1% (w/v) Alizarin red for 10 minutes. The stained cells were washed for three times with 1× PBS, then photographed with a scanning camera.

| Alkaline phosphatase assay
ATDC5 cells were seeded in 6-well plates at a density of 100 000 cell/well and then treated with the indicated amounts of T A B L E 1 Primers used for PCR (all primers suitable for mouse cDNA, S, sense; A, antisense)

Gene Sequence
Accession number

| Statistical analysis
All the results were reported as means ± SEM for 3-5 times. ANOVA was used to analyse the differences between the groups, followed by the TukeyeKramer or Dunnett's multi-comparison test with PASW Software (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.

| Detection of NPWand GPR7 expression in the growth plate
An immunohistochemical study of the tibiofemoral joints, which used specific antibodies, detected NPW and GPR7 proteins in the round chondrocytes in the growth plate. As shown in Figure Figure 2D).

| Effect of NPW-23 on ATDC5 proliferation
As NPW and NPB were thought to act as regulators of cell proliferation, we studied the effect of NPW-23 on ATDC5 proliferation. The cell proliferation assays showed that NPW-23 (200 ng/mL) stimulated ATDC5 proliferation by more than 30% compared with the control cultures (Figures 3-6A

| Effect of NPW-23 on markers of chondrogenic differentiation
To investigate the effects of NPW-23 on the chondrogenic differ-

| The effect of NPW-23 on major regulatory signalling pathways in chondrocytes
We used RT-PCR to study the major regulators of chondrogenesis that mediate the effects of NPW-23ʹ on ATDC5 cells. As shown in Figure 5A, the NPW-23 increased early differentiation when evaluated by Coll II mRNA expression, which was sensitive to the specific inhibitors of the PKA, PKC and p38 MAPK pathways, while the ERK1/2 and JNK inhibitors had no effect. The aggrecan gene expression, which acts as a marker of early chondrocyte differentiation, showed similar results ( Figure 5B). Furthermore, p21 expression, which is essential for the chondrogenesis of ATDC5 cells, was inhib- Cell proliferation was determined by CCK-8 assay (A) and BrdU cell proliferation assay (B), respectively. C, Different concentrations of CYM 50769 for 30 min, ATDC5 cells were exposed with or without 200 ng/mL NPW-23 for 24 h, and the cell proliferation was measured by CCK-8. D, ATDC5 cells were transfected GPR7 siRNA for 36 h, and the knockdown efficiency of the GPR7 was detected by RT-PCR. E, ATDC5 cells with or without GPR7 knockdown were exposed to NPW-23 for 24 h, and the cell viability was measured by CCK-8 assay. F, Effects of protein kinase A, protein kinase C and mitogen-activated protein kinases pathway inhibitors on NPW-23-induced proliferation. ATDC5 cells were treated with various pathway inhibitors (H89; Chele; PD98059; SB203580; JNK inhibitors) for 30 min followed by treatment with NPW-23 for 24 h. CCK-8 assay was used to determine cell viability. Data is expressed as a percentage of the corresponding control. Results are shown as the mean ± SEM, and represent assays from three independent experiments. *P < 0.05, **P < 0.01 vs control group, # P < 0.05, ## P < 0.05 vs NPW-23 group.

| DISCUSSION
The G protein-coupled receptor (GPR) plays a key role in regulating bone physiology and pathology. As one subtype of the GPR family, GPR7 is expressed in many brain and periphery tissues. 25 Neuropeptides W is a natural agonist of GPR7 and has universal distribution in the brain tissue. 3,7 It is also expressed in periphery tissues and is involved in different cellular processes via GPR7, including hormonal and energy homeostasis. [26][27][28][29] Our results showed the first time that  32 We also found that NPW induced differentiation of ATDC5, which coincided with augmented Coll II  pathways. It may be true that PKC mediates the p38 MAPK signal pathway in ATDC5 cells. 13 However, further investigation is required to determine the contribution of PKA/PKC signalling in mediating GPR7 activation in murine chondrocyte.
In conclusion, this study is the first to suggest that NPW binds GPR7 in order to promote proliferation and early differentiation of murine chondrocyte. This effect was because of the activation of the adenylate cyclase/PKA and the phospholipase C/PKC cascades, as well as its promoted p38 MAPK phosphorylation. Earlier studies also suggest that NPW/NPB exists in the blood and systemic veins of rats, 41 which indicates that NPW/GPR7 also affects cartilage F I G U R E 5 Neuropeptide W-23 (NPW-23)-enhanced differentiation of ATDC5 cells through the protein kinase A, protein kinase C, ERK1/2, and p38 signalling pathways. Cells were cultured in differentiation medium (1% ITS) and incubated with or without NPW-23 (200 ng/mL). On day 7 of the culture process, the cells were in the presence or absence of specific inhibitors of the different signalling pathways (CYM50769; PD98059; SB203580; Chele; H-89). After 12 h, the total RNA was extracted and (A) Coll II, (B) aggrecan, and (C) p21 mRNA were detected by quantitative RT-PCR. Each experiment was repeated at least three times. The data represent triplicate samples in two independent experiments. *P < 0.05 vs control group, # P < 0.05 vs NPW-23 group.
functions in vivo. However, we failed to identify the possible mechanism of NPW action on chondrocyte in vivo. We hope to study this in the future.