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

Rikang Wang,Guangqian Zhou,Chaojun Zheng,Xinshu Xie,Rifang Liao,Wenyu Jiang

doi:10.1111/jcmm.14118

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.

Highlights

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]
These data show that the protein kinase A (PKA), protein kinase C (PKC), p38 mitogen‐activated protein kinases (MAPKs) and ERK1/ 2 signalling pathways mediate Neuropeptide W‐23 (NPW‐23), which increases the early differentiation of ATDC5 cells, but the JNK1/2 pathway is not related to this process
Our results showed the first time that NPW and GPR7 are expressed in the ATDC5 cells and growth plates of the mice

Summary

| 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]. 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 be mimicked by ATDC5 cells in vitro

| MATERIALS AND METHODS

| RESULTS

Findings

| DISCUSSION