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Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells.

Kanazawa I, Yamaguchi T, Yano S, Yamauchi M, Yamamoto M, Sugimoto T - BMC Cell Biol. (2007)

Bottom Line: AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection.The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings.Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Internal Medicine 1, Shimane University School of Medicine, Japan. ippei.k@med.shimane-u.ac.jp

ABSTRACT

Background: Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells.

Results: Adiponectin receptor type 1 (AdipoR1) mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase) was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR), in the cells. AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01-0.5 mM) in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01-1.0 mug/ml) also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively.

Conclusion: Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

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Effects of siRNA-AdipoR1 transfection on the mineralization of MC3T3-E1 cells. Mineralization of MC3T3-E1 was evaluated by von Kossa and Alizarin red stainings. (A) Plate view of von Kossa and Alizarin red stainings in cultured MC3T3-E1. (B) Quantification of Alizarin red staining via extraction with etylpyridinium chloride. The amount of released dye was quantified by microplate reader at 550 nm. Mineralizarion was significantly decreased in siRNA-AdipoR1 treatment compared to the control (*** p < 0.001). The result was the representative of three different experiments.
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Figure 3: Effects of siRNA-AdipoR1 transfection on the mineralization of MC3T3-E1 cells. Mineralization of MC3T3-E1 was evaluated by von Kossa and Alizarin red stainings. (A) Plate view of von Kossa and Alizarin red stainings in cultured MC3T3-E1. (B) Quantification of Alizarin red staining via extraction with etylpyridinium chloride. The amount of released dye was quantified by microplate reader at 550 nm. Mineralizarion was significantly decreased in siRNA-AdipoR1 treatment compared to the control (*** p < 0.001). The result was the representative of three different experiments.

Mentions: To examine the effect of AdipoR1 knockdown on MC3T3-E1 cells, we analyzed the differentiation and mineralization of the siRNA-transfected cells. The total RNA was collected 4, 7, and 10 days after the siRNA treatment. Real-time PCR showed reduced collagen-I (Col-I) and osteocalcin (OCN) mRNA levels by the siRNA-AdipoR1 treatments (Figs. 2A and 2B, respectively). After the siRNA-AdipoR1 transfection, the ALP activity was significantly lower than that of the control on day 14 (p < 0.001) (Fig. 2C), and both von Kossa and Alizarin red stainings showed that the mineralization was apparently inhibited by siRNA-AdipoR1 on day 28 (Fig. 3A). The quantification of the Alizarin red staining showed that this inhibition was significant (p < 0.001) (Fig. 3B). These results suggested that adiponectin promotes the differentiation and mineralization of MC3T3-E1 cells via AdipoR1.


Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells.

Kanazawa I, Yamaguchi T, Yano S, Yamauchi M, Yamamoto M, Sugimoto T - BMC Cell Biol. (2007)

Effects of siRNA-AdipoR1 transfection on the mineralization of MC3T3-E1 cells. Mineralization of MC3T3-E1 was evaluated by von Kossa and Alizarin red stainings. (A) Plate view of von Kossa and Alizarin red stainings in cultured MC3T3-E1. (B) Quantification of Alizarin red staining via extraction with etylpyridinium chloride. The amount of released dye was quantified by microplate reader at 550 nm. Mineralizarion was significantly decreased in siRNA-AdipoR1 treatment compared to the control (*** p < 0.001). The result was the representative of three different experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2214728&req=5

Figure 3: Effects of siRNA-AdipoR1 transfection on the mineralization of MC3T3-E1 cells. Mineralization of MC3T3-E1 was evaluated by von Kossa and Alizarin red stainings. (A) Plate view of von Kossa and Alizarin red stainings in cultured MC3T3-E1. (B) Quantification of Alizarin red staining via extraction with etylpyridinium chloride. The amount of released dye was quantified by microplate reader at 550 nm. Mineralizarion was significantly decreased in siRNA-AdipoR1 treatment compared to the control (*** p < 0.001). The result was the representative of three different experiments.
Mentions: To examine the effect of AdipoR1 knockdown on MC3T3-E1 cells, we analyzed the differentiation and mineralization of the siRNA-transfected cells. The total RNA was collected 4, 7, and 10 days after the siRNA treatment. Real-time PCR showed reduced collagen-I (Col-I) and osteocalcin (OCN) mRNA levels by the siRNA-AdipoR1 treatments (Figs. 2A and 2B, respectively). After the siRNA-AdipoR1 transfection, the ALP activity was significantly lower than that of the control on day 14 (p < 0.001) (Fig. 2C), and both von Kossa and Alizarin red stainings showed that the mineralization was apparently inhibited by siRNA-AdipoR1 on day 28 (Fig. 3A). The quantification of the Alizarin red staining showed that this inhibition was significant (p < 0.001) (Fig. 3B). These results suggested that adiponectin promotes the differentiation and mineralization of MC3T3-E1 cells via AdipoR1.

Bottom Line: AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection.The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings.Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Internal Medicine 1, Shimane University School of Medicine, Japan. ippei.k@med.shimane-u.ac.jp

ABSTRACT

Background: Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells.

Results: Adiponectin receptor type 1 (AdipoR1) mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase) was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR), in the cells. AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01-0.5 mM) in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01-1.0 mug/ml) also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively.

Conclusion: Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

Show MeSH
Related in: MedlinePlus