Korean J Physiol Pharmacol.  2013 Oct;17(5):447-453. 10.4196/kjpp.2013.17.5.447.

Nectandrin A Enhances the BMP-Induced Osteoblastic Differentiation and Mineralization by Activation of p38 MAPK-Smad Signaling Pathway

• Kim DY
• Kim GW
• Chung SH

• KMID: 1500241
• DOI: http://doi.org/10.4196/kjpp.2013.17.5.447

Abstract

Osteoblastic activity of nectandrin A was examined in C2C12 cells. Nectandrin A enhances the BMP-induced osteoblastic differentiation and mineralization, manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and increased calcium contents. In C2C12 cells co-transfected with expression vector encoding Smad4 and Id1-Luc reporter, nectandrin A increased Id1 luciferase activity in a concentration-dependent manner, when compared to that in BMP-2 treated cells, indicating that Smad signaling pathway is associated with nectandrin A-enhanced osteoblastic differentiation in C2C12 cells. In addition, nectandrin A activated p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and phosphorylated form of pSmad1/5/8 and alkaline phosphatase activity were both decreased when the cells were pretreated with SB203580, a p38 MAPK inhibitor, suggesting that p38 MAPK might be an upstream kinase for Smad signaling pathway. Taken together, nectandrin A enhances the BMP-induced osteoblastic differentiation and mineralization of C2C12 cells via activation of p38 MAPK-Smad signaling pathway, and it has a therapeutic potential for osteoporosis by promoting bone formation.

Keyword

C2C12; Nectandrin A; p38 MAPK; Smad

MeSH Terms

Alkaline Phosphatase
Antigens, Differentiation
Calcium
Imidazoles
Luciferases
Osteoblasts*
Osteogenesis
Osteoporosis
p38 Mitogen-Activated Protein Kinases
Phosphotransferases
Protein Kinases
Pyridines
Up-Regulation
Alkaline Phosphatase
Antigens, Differentiation
Calcium
Imidazoles
Luciferases
Phosphotransferases
Protein Kinases
Pyridines
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 Effects of nectandrin A on BMP-2-inudced osteoblastic differentiation and mineralization in C2C12 cells. (A) Cells were cultured in a 96-well plate for 24 h and then the medium was replaced with DMEM containing 5% FBS and rhBMP-2 (100 ng/ml) in the presence or absence of nectandrin A (differentiation day 0). Cell viability assay was then performed on the differentiation day 3. Medium was changed every third day. (B) ALP enzyme activity and Alizarin Red staining were performed on the differentiation day 7 and 14, respectively. (C, D) ALP activity and calcium contents were measured at different time points after the induction of C2C12 differentiation in the presence or absence of nectandrin A. *p<0.05 and **p<0.01 compared to rhBMP-2 treated group.

• 

  Fig. 2 Effects of nectandrin A on BMP-2-induced osteoblastic differentiation markers and transcription factors. C2C12 cells were cultured in a 6-well plate for 1 day and then the medium was replaced with DMEM containing 5% FBS and rhBMP-2 (100 ng/ml) in the presence or absence of nectandrin A (differentiation day 0). Medium was changed every third day. Total RNA was prepared and mRNA expression levels of Id1, Runx2, ALP, OCN, Osx and Col-I were analyzed by real-time PCR. The mRNA expression levels of Id1 (A) and Runx2 (B) were analyzed on day 3 and mRNA expression levels of ALP (C), OCN (D) and Osx (E) were analyzed on day 7. mRNA expression level of Col-I was analyzed on day 14 (F). Each value represents the mean±SEM of three independent experiments. *p<0.05 and **p<0.01 compared to rhBMP-2 treated group.

• 

  Fig. 3 Effects of nectandrin A on Id1 and Smad activation. (A) Id1 activation was measured by luciferase reporter assay using Id1-promoter-driven luciferase plasmid (Id1-Luc)-transfected C2C12 cells. Cells was cultured in a 6-well plate for 1 day and then incubated with DMEM containing 5% FBS in the presence or absence of rhBMP-2 with/without nectandrin A for 1 day. (B) The effect of nectandrin A on the activation of Smad was evaluated by Western blot analysis. Cells were cultured in a 6-well plate for 1 day and then incubated with DMEM containing 5% FBS in the presence or absence of rhBMP-2 (100 ng/ml) with/without nectandrin A for indicated time periods. (C) Cells were transfected with pCMV-Smad4-Flag with or without Id1-Luc reporter and then cells were incubated with DMEM containing 5% FBS and 100 ng/ml rhBMP-2 in the presence or absence of nectandrin A for 4 h. *p<0.05 compared to rhBMP-2 treated group.

• 

  Fig. 4 Involvement of p38 MAPK in the nectandrin A-enhanced osteoblastic differentiation. (A) Cells were cultured in a 6-well plate for 1 day and then incubated with DMEM containing 5% FBS in the presence or absence of BMP-2 and/or nectandrin A for up to 120 min. (B) Phosphorylated levels of p38 and Smad1/5/8 were determined after C2C12 cells were treated with nectantrin A as indicated in the presence of BMP-2. (C) Cells in a 6-well plate were treated with indicated concentrations of SB203580 for 1 h, followed by 15 min treatment with 100 ng/ml rhBMP-2 with/without 20 µM nectandrin A. (D) On differentiation day 6, ALP enzyme activity was determined as described in 'Methods'. *p<0.05 compared to rhBMP-2 plus nectandrin A treated group.

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