Functional Roles of Netrin-1 in Osteoblast Differentiation

Abstract

Aim: 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.

Materials and methods: 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.

Results: 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.

Conclusion: Netrin-1 is involved in the regulation of osteoblast differentiation.

Keywords: Netrin-1; differentiation; osteoblast.

Figure 1. Ntn1 inhibits osteoblast differentiation. A. Expression of Ntn1 mRNA in MC3T3-E1 during osteoblast differentiation in the presence of ascorbic acid and β-glycerophosphate at 7 and 14 days estimated by qPCR analysis. B. Expression of Col1a mRNA in MC3T3-E1 with recombinant Ntn1 (50 and 500 ng/ml) for 3 days estimated by qPCR analysis. C. Effect of recombinant Ntn1 (500 ng/ml) on ALP activity of MC3T3-E1 at 7 days estimated by ALP activity assay. D. Effect of recombinant Ntn1 (500 ng/ml) on cell proliferation of osteoblastic cells at 3 days estimated by cell proliferation assay. Data are calculated from three repeated experiments. Recombinant Ntn1, rNtn1. # <0.05. Data are expressed as the means±S.E.M.

Figure 2. Ntn1 overexpression inhibits osteoblast differentiation. A. MC3T3-E1 cells were transfected with Ntn1 plasmid, BMP-4 plasmid, or an empty vector at 3 days estimated by qPCR analysis: (left panel) pBMP4, (right panel) pNtn4. B. Expression of Alp mRNA in BMP-4 transfected MC3T3-E1 with recombinant Ntn1 (0, 100, 500 and 1,000 ng/ml) for 3 days estimated by qPCR analysis. C. Expression of Alp and Col1a mRNA in BMP-4 and/or Ntn1 transfected MC3T3-E1: (upper panel) Alp, (lower panel) Col1a. Plasmid Ntn1, pNtn1; plasmid BMP-4, pBMP4; empty vector, MOCK; Recombinant Ntn1, rNtn1. #p<0.05. Data are expressed as the means±S.E.M

Figure 3. Netrin receptors mRNA expression in MC3T3-E1. Expression of netrin receptors in MC3T3-E1 and murine brain estimated by qPCR analysis A. DCC mRNA. B. Neo1 mRNA. C. A2b mRNA. D. DSCAM mRNA. E. Unc5a mRNA. F. Unc5b mRNA. G. Unc5c mRNA. H. Unc5d mRNA. #p<0.05, ##p<0.01. Data are expressed as the means±S.E.M.

Figure 4. The combination of netrin receptors rescues osteoblast differentiation by Ntn1. A. Effect of RNA interference to A2b, Unc5b, and Neo1 in MC3T3-E1 at 3 days estimated by qPCR analysis: (upper panel) A2b, (middle panel) Unc5b, (lower panel) Neo1. Expression of Alp expression in MC3T3-E1 transfected netrin receptors in the presence of recombinant Ntn1 estimated by qPCR analysis. B. si-CTR transfection. C, si-A2b transfection. D. si-Neo1 transfection. E. si- Unc5b transfection. F, si-A2b and si-Neo1 transfection. G, si-A2b and si-Unc5b transfection. H. si-Unc5b and si-Neo1 transfection. si-control(si-CTL), Stealth RNAi Negative Control Duplex; si-A2b, Stealth RNAi to A2b; si-Neo1, Stealth RNAi toNeo1; si-Unc5b, Stealth RNAi to Unc5b #p<0.05, ##p<0.01. Data are expressed as the means±S.E.M