Nrp1, a Neuronal Regulator, Enhances DDR2-ERK-Runx2 Cascade in Osteoblast Differentiation via Suppression of DDR2 Degradation.
BACKGROUND
Osteoblastogenesis is under delicate control by multiple factors and hormones. Recent reports indicated the involvement of immunological and neuronal regulators. However, the role of neuropilin 1 (Nrp1) in osteoblastogenesis remains obscure.


METHODS
Real-time PCR was carried out to detect the mRNA of osteoblastic markers, Nrp1, and discoidin domain receptor 2 (DDR2). Immunoblot was performed to test the protein of Nrp1 and DDR2. Osteogenic differentiation was evaluated by mRNA analysis of osteogenic markers, and determination of ALP activity and OCN secretion. The intercellular signaling effectors were examined by immunoblot. Immunofluorescent assays were performed to detect the localization of Nrp1 and DDR2. Half-life determination assay was executed to test the DDR2 stability.


RESULTS
The expression of Nrp1 paralleled with that of DDR2 during osteoblastogensis. Nrp1 overexpression enhanced DDR2-induced stimulation of osteoblastogensis, whereas Nrp1 silencing caused attenuation. Nrp1 overexpression increased the phosphorylation of DDR2, ERK1/2 and Runx2. Nrp1 co-localized with DDR2 in the cellular membrane of differentiated MC3T3-E1. Enhanced or attenuated Nrp1 expression did not alter the mRNA transcript of DDR2. Nrp1 overexpression prolonged the half-life of DDR2 protein.


CONCLUSIONS
Our results originally demonstrated the stimulatory role of Nrp1 in DDR2-induced osteoblast differentiation, providing molecular evidence for exploiting Nrp1 and DDR2 as targets to treat bone-related disease.
Center for Translational Medicine, The First Affiliated Hospital of Xi'an Jiaotong University College of Medicine, Xi'an, People's Republic of China.
Yan
Y
Zhang
Jin
J
Su
Yue
Y
Teng
Jian
J
Zhang
Jiang
J
Wang
Kun
K
Li
Libo
L
Yao
Xu
X
Li
D016475:3T3 Cells; D000818:Animals; D002454:Cell Differentiation; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D000070818:Discoidin Domain Receptors; D006207:Half-Life; D020935:MAP Kinase Signaling System; D051379:Mice; D039942:Neuropilin-1 / Q000235:genetics* / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D011975:Receptors, Mitogen / Q000235:genetics* / Q000378:metabolism*
D050659:Core Binding Factor Alpha 1 Subunit; D011975:Receptors, Mitogen; C493716:Runx2 protein, mouse; D039942:Neuropilin-1; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases