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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 an AICAR treatment on the differentiation of MC3T3-E1 cells. AICAR (0.5 mM), a AMP kinase stimulator, was added after the cells reached confluency. Total RNA was collected at 14, 21, 28 days. Col-I (A) and OCN (B) mRNA expression were increased by the AICAR treatment. Results were expressed as fold increase over the control values at 14 days. The result was the representative of three different experiments. (C) ALP activity of MC3T3-E1 cells by an AICAR treatment. ALP activity was evaluated biochemically. ALP activity was increased compared to the control significantly (* p < 0.05, and ** p < 0.01).
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Figure 5: Effects of an AICAR treatment on the differentiation of MC3T3-E1 cells. AICAR (0.5 mM), a AMP kinase stimulator, was added after the cells reached confluency. Total RNA was collected at 14, 21, 28 days. Col-I (A) and OCN (B) mRNA expression were increased by the AICAR treatment. Results were expressed as fold increase over the control values at 14 days. The result was the representative of three different experiments. (C) ALP activity of MC3T3-E1 cells by an AICAR treatment. ALP activity was evaluated biochemically. ALP activity was increased compared to the control significantly (* p < 0.05, and ** p < 0.01).

Mentions: Because the results above demonstrated that adiponectin and AICAR activated the AMP kinase signaling pathway in wild-type MC3T3-E1 cells, we examined whether or not the activation of this pathway would lead to the differentiation and mineralization of the cells. AICAR (0.5 mM) was added after the cells reached confluency, and the total RNA was collected on days 14, 21, and 28. Real-time PCR showed that in contrast to the results for siRNA-AdipoR1, the AICAR treatments increased the expression levels of Col-I and OCN mRNA (Figs. 5A and 5B, respectively). ALP activities were significantly greater than those of the control at 0.1 and 0.5 mM concentrations of AICAR and in a 14-day culture (p < 0.05) (Fig. 5C). Moreover, both von Kossa and Alizarin red stainings showed that the mineralization of MC3T3-E1 cells was augmented by 0.01–0.5 mM AICAR on day 28 (Fig. 6A). The quantification of the Alizarin red staining showed that these augmentations were significant (p < 0.001) (Fig. 6B). Thus, the activation of AMP kinase seemed to promote the differentiation and mineralization of MC3T3-E1 cells.


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 an AICAR treatment on the differentiation of MC3T3-E1 cells. AICAR (0.5 mM), a AMP kinase stimulator, was added after the cells reached confluency. Total RNA was collected at 14, 21, 28 days. Col-I (A) and OCN (B) mRNA expression were increased by the AICAR treatment. Results were expressed as fold increase over the control values at 14 days. The result was the representative of three different experiments. (C) ALP activity of MC3T3-E1 cells by an AICAR treatment. ALP activity was evaluated biochemically. ALP activity was increased compared to the control significantly (* p < 0.05, and ** p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2214728&req=5

Figure 5: Effects of an AICAR treatment on the differentiation of MC3T3-E1 cells. AICAR (0.5 mM), a AMP kinase stimulator, was added after the cells reached confluency. Total RNA was collected at 14, 21, 28 days. Col-I (A) and OCN (B) mRNA expression were increased by the AICAR treatment. Results were expressed as fold increase over the control values at 14 days. The result was the representative of three different experiments. (C) ALP activity of MC3T3-E1 cells by an AICAR treatment. ALP activity was evaluated biochemically. ALP activity was increased compared to the control significantly (* p < 0.05, and ** p < 0.01).
Mentions: Because the results above demonstrated that adiponectin and AICAR activated the AMP kinase signaling pathway in wild-type MC3T3-E1 cells, we examined whether or not the activation of this pathway would lead to the differentiation and mineralization of the cells. AICAR (0.5 mM) was added after the cells reached confluency, and the total RNA was collected on days 14, 21, and 28. Real-time PCR showed that in contrast to the results for siRNA-AdipoR1, the AICAR treatments increased the expression levels of Col-I and OCN mRNA (Figs. 5A and 5B, respectively). ALP activities were significantly greater than those of the control at 0.1 and 0.5 mM concentrations of AICAR and in a 14-day culture (p < 0.05) (Fig. 5C). Moreover, both von Kossa and Alizarin red stainings showed that the mineralization of MC3T3-E1 cells was augmented by 0.01–0.5 mM AICAR on day 28 (Fig. 6A). The quantification of the Alizarin red staining showed that these augmentations were significant (p < 0.001) (Fig. 6B). Thus, the activation of AMP kinase seemed to promote the differentiation and mineralization of MC3T3-E1 cells.

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