Limits...
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.

Show MeSH

Related in: MedlinePlus

Adiponectin R1 and R2 expression in MC3T3-E1 cells and effects of siRNA-AdipoR1 transfection. (A) Adiponectin receptor expression in MC3T3-E1 cells. Total RNA from the cells was subjected to RT-PCR. HPRT, house keeping gene, and AdipoR1 were visualized in a 2% agarose gel stained with ethidium bromide. AdipoR1 mRNA but not AdipoR2 was expressed in MC3T3-E1 cells, while both of them were expressed in 3T3-L1 cells, which were examined as a positive control. (B) Confirmation of the effect of siRNA-AdipoR1. The siRNA restrained only siAdipoR1, showing that its knock down effect was specific (p < 0.001). N; 0.2% BSA, TR; transfection reagent, CypB; transfection of siRNA-Cyclophilin B (CypB), control; transfection of non-targeting siRNA. (C) Confirmation of the effect of siRNA-CypB. (D) The durability of siRNA-AdipoR1. Total RNA was collected at 4, 7, 10 days after siRNA transfection. The knock down effect of siRNA-AdipoR1 was sustained as long as 10 days after transfection at the expression level of 14.9% of the control. Results are expressed as the mean ± SEM fold increase (n = 5) over control values. * p < 0.05, ** p < 0.01, *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2214728&req=5

Figure 1: Adiponectin R1 and R2 expression in MC3T3-E1 cells and effects of siRNA-AdipoR1 transfection. (A) Adiponectin receptor expression in MC3T3-E1 cells. Total RNA from the cells was subjected to RT-PCR. HPRT, house keeping gene, and AdipoR1 were visualized in a 2% agarose gel stained with ethidium bromide. AdipoR1 mRNA but not AdipoR2 was expressed in MC3T3-E1 cells, while both of them were expressed in 3T3-L1 cells, which were examined as a positive control. (B) Confirmation of the effect of siRNA-AdipoR1. The siRNA restrained only siAdipoR1, showing that its knock down effect was specific (p < 0.001). N; 0.2% BSA, TR; transfection reagent, CypB; transfection of siRNA-Cyclophilin B (CypB), control; transfection of non-targeting siRNA. (C) Confirmation of the effect of siRNA-CypB. (D) The durability of siRNA-AdipoR1. Total RNA was collected at 4, 7, 10 days after siRNA transfection. The knock down effect of siRNA-AdipoR1 was sustained as long as 10 days after transfection at the expression level of 14.9% of the control. Results are expressed as the mean ± SEM fold increase (n = 5) over control values. * p < 0.05, ** p < 0.01, *** p < 0.001.

Mentions: We first investigated AdipoR1 and AdipoR2 expressions in MC3T3-E1 cells. By using RT-PCR, we confirmed that AdipoR1 mRNA, but not AdipoR2, was expressed in MC3T3-E1 cells (Fig. 1A). To elucidate the action of adiponectin through AdipoR1, we performed experiments using an RNA interference to block the receptor expression. Real-time PCR revealed that a treatment with 50-nM siRNA-AdipoR1 blocked the expression of AdipoR1 mRNA (Fig. 1B), while a treatment with cyclophilin B (CypB) siRNA blocked the expression of CypB mRNA (Fig. 1C), showing that the knockdown effect of siRNA-AdipoR1 was specific. The effect of knockdown by siRNA-AdipoR1 persisted for as long as 10 days after the transfection, when the expression level was still suppressed by 85.1% in comparison to that in the control (Fig. 1D).


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)

Adiponectin R1 and R2 expression in MC3T3-E1 cells and effects of siRNA-AdipoR1 transfection. (A) Adiponectin receptor expression in MC3T3-E1 cells. Total RNA from the cells was subjected to RT-PCR. HPRT, house keeping gene, and AdipoR1 were visualized in a 2% agarose gel stained with ethidium bromide. AdipoR1 mRNA but not AdipoR2 was expressed in MC3T3-E1 cells, while both of them were expressed in 3T3-L1 cells, which were examined as a positive control. (B) Confirmation of the effect of siRNA-AdipoR1. The siRNA restrained only siAdipoR1, showing that its knock down effect was specific (p < 0.001). N; 0.2% BSA, TR; transfection reagent, CypB; transfection of siRNA-Cyclophilin B (CypB), control; transfection of non-targeting siRNA. (C) Confirmation of the effect of siRNA-CypB. (D) The durability of siRNA-AdipoR1. Total RNA was collected at 4, 7, 10 days after siRNA transfection. The knock down effect of siRNA-AdipoR1 was sustained as long as 10 days after transfection at the expression level of 14.9% of the control. Results are expressed as the mean ± SEM fold increase (n = 5) over control values. * p < 0.05, ** p < 0.01, *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Adiponectin R1 and R2 expression in MC3T3-E1 cells and effects of siRNA-AdipoR1 transfection. (A) Adiponectin receptor expression in MC3T3-E1 cells. Total RNA from the cells was subjected to RT-PCR. HPRT, house keeping gene, and AdipoR1 were visualized in a 2% agarose gel stained with ethidium bromide. AdipoR1 mRNA but not AdipoR2 was expressed in MC3T3-E1 cells, while both of them were expressed in 3T3-L1 cells, which were examined as a positive control. (B) Confirmation of the effect of siRNA-AdipoR1. The siRNA restrained only siAdipoR1, showing that its knock down effect was specific (p < 0.001). N; 0.2% BSA, TR; transfection reagent, CypB; transfection of siRNA-Cyclophilin B (CypB), control; transfection of non-targeting siRNA. (C) Confirmation of the effect of siRNA-CypB. (D) The durability of siRNA-AdipoR1. Total RNA was collected at 4, 7, 10 days after siRNA transfection. The knock down effect of siRNA-AdipoR1 was sustained as long as 10 days after transfection at the expression level of 14.9% of the control. Results are expressed as the mean ± SEM fold increase (n = 5) over control values. * p < 0.05, ** p < 0.01, *** p < 0.001.
Mentions: We first investigated AdipoR1 and AdipoR2 expressions in MC3T3-E1 cells. By using RT-PCR, we confirmed that AdipoR1 mRNA, but not AdipoR2, was expressed in MC3T3-E1 cells (Fig. 1A). To elucidate the action of adiponectin through AdipoR1, we performed experiments using an RNA interference to block the receptor expression. Real-time PCR revealed that a treatment with 50-nM siRNA-AdipoR1 blocked the expression of AdipoR1 mRNA (Fig. 1B), while a treatment with cyclophilin B (CypB) siRNA blocked the expression of CypB mRNA (Fig. 1C), showing that the knockdown effect of siRNA-AdipoR1 was specific. The effect of knockdown by siRNA-AdipoR1 persisted for as long as 10 days after the transfection, when the expression level was still suppressed by 85.1% in comparison to that in the control (Fig. 1D).

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