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Lithium chloride enhances cathepsin H expression and BMP-4 degradation in C3H10T1/2 cells.

Kishimoto KN, Itoi E - Biomed Res Int (2013)

Bottom Line: The effect of canonical Wnt/β-catenin signaling on chondrogenic differentiation induced by transfection of BMP4 expressing plasmid was analyzed.Although BMP4 mRNA expression was not affected by LiCl, LiCl decreased BMP4 protein accumulation.These results suggested that cathepsin H is regulated by Wnt/β-catenin signaling and plays an important role in the regulation of BMP4 biological activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

ABSTRACT
The effect of canonical Wnt/β-catenin signaling on chondrogenic differentiation induced by transfection of BMP4 expressing plasmid was analyzed. Lithium chloride (LiCl) which mimics canonical Wnt/β-catenin signaling was added to cells transfected with BMP4 expressing plasmid. Although BMP4 mRNA expression was not affected by LiCl, LiCl decreased BMP4 protein accumulation. Gene expression analysis exhibited upregulation of cathepsin H by LiCl treatment. Gene silencing of cathepsin H enhanced BMP4 protein accumulation from BMP4 expressing cells. These results suggested that cathepsin H is regulated by Wnt/β-catenin signaling and plays an important role in the regulation of BMP4 biological activity.

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Related in: MedlinePlus

(a) Alcian blue staining of cultured micromass and quantification. C3H10T1/2 cells were transfected with BMP4 expressing plasmid, pCAGGS-BMP4. Cultured micromass was stained at day 6. The indicated amount of plasmid was supplemented in a 4 mm cuvette (600 μL) at the electroporation. (b) Quantitative RT-PCR for BMP4, Sox9, Aggrecan, and Col2a1 mRNA at day 3. BMP4 mRNA expression levels and chondrogenic properties were increased in the dose dependent manner of pCAGGS-BMP4. *P < 0.05, ***P < 0.001.
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fig1: (a) Alcian blue staining of cultured micromass and quantification. C3H10T1/2 cells were transfected with BMP4 expressing plasmid, pCAGGS-BMP4. Cultured micromass was stained at day 6. The indicated amount of plasmid was supplemented in a 4 mm cuvette (600 μL) at the electroporation. (b) Quantitative RT-PCR for BMP4, Sox9, Aggrecan, and Col2a1 mRNA at day 3. BMP4 mRNA expression levels and chondrogenic properties were increased in the dose dependent manner of pCAGGS-BMP4. *P < 0.05, ***P < 0.001.

Mentions: To confirm the chondrogenesis, pCAGGS-BMP4 transfected C3H10T1/2 cells were cultured in micromass. From day 3, the cells metamorphosed into round shape. Alcian blue staining at day 6 exhibited positive stain. The intensity of Alcian blue staining showed dose-dependent increase depending on the amount of plasmid used at electroporation (Figure 1(a)). Gene expression profiles were analyzed by qRT-PCR at day 3. BMP4 mRNA expression increased in the dose-dependent manner of the plasmid amount. Sox9 mRNA expression was not affected by the dose of plasmid. Aggrecan and Col2a1 mRNA showed marked dose-dependent increase (Figure 1(b)).


Lithium chloride enhances cathepsin H expression and BMP-4 degradation in C3H10T1/2 cells.

Kishimoto KN, Itoi E - Biomed Res Int (2013)

(a) Alcian blue staining of cultured micromass and quantification. C3H10T1/2 cells were transfected with BMP4 expressing plasmid, pCAGGS-BMP4. Cultured micromass was stained at day 6. The indicated amount of plasmid was supplemented in a 4 mm cuvette (600 μL) at the electroporation. (b) Quantitative RT-PCR for BMP4, Sox9, Aggrecan, and Col2a1 mRNA at day 3. BMP4 mRNA expression levels and chondrogenic properties were increased in the dose dependent manner of pCAGGS-BMP4. *P < 0.05, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3842059&req=5

fig1: (a) Alcian blue staining of cultured micromass and quantification. C3H10T1/2 cells were transfected with BMP4 expressing plasmid, pCAGGS-BMP4. Cultured micromass was stained at day 6. The indicated amount of plasmid was supplemented in a 4 mm cuvette (600 μL) at the electroporation. (b) Quantitative RT-PCR for BMP4, Sox9, Aggrecan, and Col2a1 mRNA at day 3. BMP4 mRNA expression levels and chondrogenic properties were increased in the dose dependent manner of pCAGGS-BMP4. *P < 0.05, ***P < 0.001.
Mentions: To confirm the chondrogenesis, pCAGGS-BMP4 transfected C3H10T1/2 cells were cultured in micromass. From day 3, the cells metamorphosed into round shape. Alcian blue staining at day 6 exhibited positive stain. The intensity of Alcian blue staining showed dose-dependent increase depending on the amount of plasmid used at electroporation (Figure 1(a)). Gene expression profiles were analyzed by qRT-PCR at day 3. BMP4 mRNA expression increased in the dose-dependent manner of the plasmid amount. Sox9 mRNA expression was not affected by the dose of plasmid. Aggrecan and Col2a1 mRNA showed marked dose-dependent increase (Figure 1(b)).

Bottom Line: The effect of canonical Wnt/β-catenin signaling on chondrogenic differentiation induced by transfection of BMP4 expressing plasmid was analyzed.Although BMP4 mRNA expression was not affected by LiCl, LiCl decreased BMP4 protein accumulation.These results suggested that cathepsin H is regulated by Wnt/β-catenin signaling and plays an important role in the regulation of BMP4 biological activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

ABSTRACT
The effect of canonical Wnt/β-catenin signaling on chondrogenic differentiation induced by transfection of BMP4 expressing plasmid was analyzed. Lithium chloride (LiCl) which mimics canonical Wnt/β-catenin signaling was added to cells transfected with BMP4 expressing plasmid. Although BMP4 mRNA expression was not affected by LiCl, LiCl decreased BMP4 protein accumulation. Gene expression analysis exhibited upregulation of cathepsin H by LiCl treatment. Gene silencing of cathepsin H enhanced BMP4 protein accumulation from BMP4 expressing cells. These results suggested that cathepsin H is regulated by Wnt/β-catenin signaling and plays an important role in the regulation of BMP4 biological activity.

Show MeSH
Related in: MedlinePlus