Limits...
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.

Show MeSH
Gene silencing of cathepsin H was carried out by electroporatic gene transfer of small interfering RNA. siCTSH targeting cathepsin H or siControl (300 pmol/cuvette each) was transfected with pCAGGS-BMP4 (30 μg/cuvette). (a) Quantitative RT-PCR at day 3. Cathepsin H mRNA expression was inhibited significantly. (b) Western blot analysis for BMP4 in the culture medium at day 3 showed marked increase of BMP4. (c) Alcian blue staining of micromass showed significant increase by gene silencing of cathepsin H. *P < 0.05, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3842059&req=5

fig4: Gene silencing of cathepsin H was carried out by electroporatic gene transfer of small interfering RNA. siCTSH targeting cathepsin H or siControl (300 pmol/cuvette each) was transfected with pCAGGS-BMP4 (30 μg/cuvette). (a) Quantitative RT-PCR at day 3. Cathepsin H mRNA expression was inhibited significantly. (b) Western blot analysis for BMP4 in the culture medium at day 3 showed marked increase of BMP4. (c) Alcian blue staining of micromass showed significant increase by gene silencing of cathepsin H. *P < 0.05, ***P < 0.001.

Mentions: RNA interference for cathepsin H was carried out by electroporatic transfer of small interfering RNA molecule. After cotransfection of siCTSH or siControl (300 pmol/cuvette) with pCAGGS-BMP4 (30 μg/cuvette) by electroporation, cells were cultured in micromass. qRT-PCR at day 3 showed suppression of cathepsin H mRNA expression. Cathepsin H gene silencing slightly increased the expression level of BMP4 by 1.5 ± 0.4-folds (Figure 4(a)). Western blotting for BMP4 showed that cathepsin H gene silencing remarkably increased BMP4 protein expression by 2.9 ± 1.4-folds (Figure 4(b)). Alcian blue staining of micromass at day 6 showed significant increase in siCTSH transfected cells (Figure 3(c)).


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

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

Gene silencing of cathepsin H was carried out by electroporatic gene transfer of small interfering RNA. siCTSH targeting cathepsin H or siControl (300 pmol/cuvette each) was transfected with pCAGGS-BMP4 (30 μg/cuvette). (a) Quantitative RT-PCR at day 3. Cathepsin H mRNA expression was inhibited significantly. (b) Western blot analysis for BMP4 in the culture medium at day 3 showed marked increase of BMP4. (c) Alcian blue staining of micromass showed significant increase by gene silencing of cathepsin H. *P < 0.05, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Gene silencing of cathepsin H was carried out by electroporatic gene transfer of small interfering RNA. siCTSH targeting cathepsin H or siControl (300 pmol/cuvette each) was transfected with pCAGGS-BMP4 (30 μg/cuvette). (a) Quantitative RT-PCR at day 3. Cathepsin H mRNA expression was inhibited significantly. (b) Western blot analysis for BMP4 in the culture medium at day 3 showed marked increase of BMP4. (c) Alcian blue staining of micromass showed significant increase by gene silencing of cathepsin H. *P < 0.05, ***P < 0.001.
Mentions: RNA interference for cathepsin H was carried out by electroporatic transfer of small interfering RNA molecule. After cotransfection of siCTSH or siControl (300 pmol/cuvette) with pCAGGS-BMP4 (30 μg/cuvette) by electroporation, cells were cultured in micromass. qRT-PCR at day 3 showed suppression of cathepsin H mRNA expression. Cathepsin H gene silencing slightly increased the expression level of BMP4 by 1.5 ± 0.4-folds (Figure 4(a)). Western blotting for BMP4 showed that cathepsin H gene silencing remarkably increased BMP4 protein expression by 2.9 ± 1.4-folds (Figure 4(b)). Alcian blue staining of micromass at day 6 showed significant increase in siCTSH transfected cells (Figure 3(c)).

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