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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 or an AICAR treatment on Runx-2 mRNA and protein expressions in MC3T3-E1 cells. (A and B) Total RNA was collected at 4, 7, 10 days after siRNA-AdipoR1 transfection, and cell lysate was collected at 7 days after transfection. Real-time PCR and immunoprecipitation were performed as described in MATERIALS AND METHODS. Neither Runx-2 mRNA (A) nor its protein (B) expressions were changed by blocking the receptor expression. (C and D) AICAR (0.5 mM), an AMP kinase stimulator, was added after the cells reached confluency. Total RNA was collected at 14, 21, 28 days, and cell lysate was collected at 14 days. Neither Runx-2 mRNA (C) nor its protein (D) expressions were changed by AICAR. The result was the representative of five different experiments.
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Figure 7: Effects of siRNA-AdipoR1 transfection or an AICAR treatment on Runx-2 mRNA and protein expressions in MC3T3-E1 cells. (A and B) Total RNA was collected at 4, 7, 10 days after siRNA-AdipoR1 transfection, and cell lysate was collected at 7 days after transfection. Real-time PCR and immunoprecipitation were performed as described in MATERIALS AND METHODS. Neither Runx-2 mRNA (A) nor its protein (B) expressions were changed by blocking the receptor expression. (C and D) AICAR (0.5 mM), an AMP kinase stimulator, was added after the cells reached confluency. Total RNA was collected at 14, 21, 28 days, and cell lysate was collected at 14 days. Neither Runx-2 mRNA (C) nor its protein (D) expressions were changed by AICAR. The result was the representative of five different experiments.

Mentions: Since Runx-2 (also called cbfa1, PEBP2α A) is an important transcription factor that is involved in osteoblastic differentiation and mineralization [23], we investigated whether or not adiponectin and AICAR modulate its expression. There were little or no changes in the Runx-2 mRNA levels, as determined by real-time PCR, or its protein levels (55-kDa band), as determined by immunoblotting under either siRNA-AdipoR1 transfection (Figs. 7A and 7B, respectively) or AICAR treatment (Figs. 7C and 7D, respectively).


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 or an AICAR treatment on Runx-2 mRNA and protein expressions in MC3T3-E1 cells. (A and B) Total RNA was collected at 4, 7, 10 days after siRNA-AdipoR1 transfection, and cell lysate was collected at 7 days after transfection. Real-time PCR and immunoprecipitation were performed as described in MATERIALS AND METHODS. Neither Runx-2 mRNA (A) nor its protein (B) expressions were changed by blocking the receptor expression. (C and D) AICAR (0.5 mM), an AMP kinase stimulator, was added after the cells reached confluency. Total RNA was collected at 14, 21, 28 days, and cell lysate was collected at 14 days. Neither Runx-2 mRNA (C) nor its protein (D) expressions were changed by AICAR. The result was the representative of five different experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Effects of siRNA-AdipoR1 transfection or an AICAR treatment on Runx-2 mRNA and protein expressions in MC3T3-E1 cells. (A and B) Total RNA was collected at 4, 7, 10 days after siRNA-AdipoR1 transfection, and cell lysate was collected at 7 days after transfection. Real-time PCR and immunoprecipitation were performed as described in MATERIALS AND METHODS. Neither Runx-2 mRNA (A) nor its protein (B) expressions were changed by blocking the receptor expression. (C and D) AICAR (0.5 mM), an AMP kinase stimulator, was added after the cells reached confluency. Total RNA was collected at 14, 21, 28 days, and cell lysate was collected at 14 days. Neither Runx-2 mRNA (C) nor its protein (D) expressions were changed by AICAR. The result was the representative of five different experiments.
Mentions: Since Runx-2 (also called cbfa1, PEBP2α A) is an important transcription factor that is involved in osteoblastic differentiation and mineralization [23], we investigated whether or not adiponectin and AICAR modulate its expression. There were little or no changes in the Runx-2 mRNA levels, as determined by real-time PCR, or its protein levels (55-kDa band), as determined by immunoblotting under either siRNA-AdipoR1 transfection (Figs. 7A and 7B, respectively) or AICAR treatment (Figs. 7C and 7D, respectively).

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