Dataset Information

Functional Roles of Netrin-1 in Osteoblast Differentiation.

ABSTRACT: Recent studies have demonstrated that netrin-1 plays a vital role in bone metabolism. Previous studies have shown that osteoblasts produce netrin-1 which affects osteoclast differentiation. However, the role of netrin-1 in osteoblast differentiation is not well understood. In this study, we explored the roles of netrin-1 in osteoblasts.Quantitative reverse-transcriptase polymerase chain reaction (qPCR), RNA interference for netrin receptors, the generation of netrin-1 plasmid, transfection of plasmids, and cell proliferation assay were performed.During osteoblast differentiation by ascorbic acid, netrin-1 expression was significantly decreased. Gene expression related with osteoblast differentiation was down-regulated by netrin-1 treatment. We also found that osteoblast differentiation by bone morphogenetic protein-4 (BMP-4) was inhibited in the presence of recombinant netrin-1. Forced expression of both BMP-4 and netrin-1 significantly decreased alkaline phosphatase expression. On the other hand, Unc5b, neogenin, and A2b which belong to netrin receptors were expressed by osteoblasts. Moreover, alkaline phosphatase expression was significantly decreased by knockdown for the combination of two receptors among these receptors.Netrin-1 is involved in the regulation of osteoblast differentiation.

SUBMITTER: Sato T

PROVIDER: S-EPMC5461440 | biostudies-literature | 2017 May-Jun

REPOSITORIES: biostudies-literature

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Functional Roles of Netrin-1 in Osteoblast Differentiation.

Sato Tsuyoshi T Kokabu Shoichiro S Enoki Yuichiro Y Hayashi Naoki N Matsumoto Masahito M Nakahira Mitsuhiko M Sugasawa Masashi M Yoda Tetsuya T

In vivo (Athens, Greece) 20170501 3

<h4>Aim</h4>Recent studies have demonstrated that netrin-1 plays a vital role in bone metabolism. Previous studies have shown that osteoblasts produce netrin-1 which affects osteoclast differentiation. However, the role of netrin-1 in osteoblast differentiation is not well understood. In this study, we explored the roles of netrin-1 in osteoblasts.<h4>Materials and methods</h4>Quantitative reverse-transcriptase polymerase chain reaction (qPCR), RNA interference for netrin receptors, the generati ...[more]

PMID: 28438858

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Project description:Insulin-like growth factor binding protein 2 (IGFBP-2) is important for acquisition of normal bone mass in mice; however, the mechanism by which IGFBP-2 functions is not defined. These studies investigated the role of IGFBP-2 in stimulating osteoblast differentiation. MC-3T3 preosteoblasts expressed IGFBP-2, and IGFBP-2 knockdown resulted in a substantial delay in osteoblast differentiation, reduced osteocalcin expression and Alizarin red staining. These findings were replicated in primary calvarial osteoblasts obtained from IGFBP-2(-/-) mice, and addition of IGFBP-2 rescued the differentiation program. In contrast, overexpression of IGFBP-2 accelerated the time course of differentiation as well as increasing the total number of differentiating cells. By day 6, IGFBP-2-overexpressing cells expressed twice as much osteocalcin as control cultures and this difference persisted. To determine the mechanism by which IGFBP-2 functions, the interaction between IGFBP-2 and receptor tyrosine phosphatase ? (RPTP?) was examined. Disruption of this interaction inhibited the ability of IGFBP-2 to stimulate AKT activation and osteoblast differentiation. Knockdown of RPTP? enhanced osteoblast differentiation, whereas overexpression of RPTP? was inhibitory. Adding back IGFBP-2 to RPTP?-overexpressing cells was able to rescue cell differentiation via enhancement of AKT activation. To determine the region of IGFBP-2 that mediated this effect, an IGFBP-2 mutant that contained substitutions of key amino acids in the heparin-binding domain-1 (HBD-1) was prepared. This mutant had a major reduction in its ability to stimulate differentiation of calvarial osteoblasts from IGFBP-2(-/-) mice. Addition of a synthetic peptide that contained the HBD-1 sequence to calvarial osteoblasts from IGFBP-2(-/-) mice rescued differentiation and osteocalcin expression. In summary, the results clearly demonstrate that IGFBP-2 stimulates osteoblast differentiation and that this effect is mediated through its heparin-binding domain-1 interacting with RPTP?. The results suggest that stimulation of differentiation is an important mechanism by which IGFBP-2 regulates the acquisition of normal bone mass in mice.

Dataset Information

Functional Roles of Netrin-1 in Osteoblast Differentiation.

Publications

Functional Roles of Netrin-1 in Osteoblast Differentiation.

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