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Identification of C-terminal Hsp70-interacting protein as a mediator of tumour necrosis factor action in osteoblast differentiation by targeting osterix for degradation.

Xie J, Gu J - J. Cell. Mol. Med. (2015)

Bottom Line: But less is known regarding the molecular mechanisms that mediate the effect of TNF-α on osteoblast function.Co-immunoprecipitation and ubiquitination assays revealed ubiquitinated Osx, confirmed that CHIP indeed interacted with Osx and identified K55 and K386 residues as the ubiquitination sites in Osx, Luciferase reporter assay and Small interfering RNAs examined whether TNF-α target the bone morphogenetic protein signalling through CHIP.We found that the K55 and K386 residues are ubiquitination site(s) in Osx, and that TNF-α inhibits osteoblast differentiation by promoting Osx degradation through up-regulation of E3 ubiquitin ligase CHIP in osteoblast.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China.

No MeSH data available.


Related in: MedlinePlus

TNF-α increases CHIP expression. (A) C2C12 or MC3T3-E1 cells were cultured in medium with PBS or TNF-α (5.0–10 ng/ml) for 24 and 48 hrs. The expression of CHIP and actin mRNA was determined by real-time RT-PCR. The values are the mean ± SD of three loadings. *P < 0.05 versus the PBS-treated group. (B) C2C12 or MC3T3-E1 cells were treated with 5.0–10 ng/ml TNF-α for 48 hrs. CHIP and β-actin protein levels were measured by Western blot analysis. (C) Correlation between TNF-α-induced apoptosis and CHIP expression. Both cell types were treated with TNF-α (5.0–10 ng/ml) or PBS for 24 and 48 hrs, and the cell lysates were used to measure caspase-3 activity or were submitted to a MTT assay. The values are the mean ± SD of three loadings. *P < 0.05 versus the PBS-treated group.
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fig01: TNF-α increases CHIP expression. (A) C2C12 or MC3T3-E1 cells were cultured in medium with PBS or TNF-α (5.0–10 ng/ml) for 24 and 48 hrs. The expression of CHIP and actin mRNA was determined by real-time RT-PCR. The values are the mean ± SD of three loadings. *P < 0.05 versus the PBS-treated group. (B) C2C12 or MC3T3-E1 cells were treated with 5.0–10 ng/ml TNF-α for 48 hrs. CHIP and β-actin protein levels were measured by Western blot analysis. (C) Correlation between TNF-α-induced apoptosis and CHIP expression. Both cell types were treated with TNF-α (5.0–10 ng/ml) or PBS for 24 and 48 hrs, and the cell lysates were used to measure caspase-3 activity or were submitted to a MTT assay. The values are the mean ± SD of three loadings. *P < 0.05 versus the PBS-treated group.

Mentions: Because TNF-α has been reported to enhance the expression of the E3 ligase Smurf1, we determined whether TNF-α had a similar effect on the expression of the CHIP protein. The C2C12 and MC3T3-E1 osteoblast precursor cell lines were treated with PBS or 5–10 ng/ml TNF-α, and the expression levels of the CHIP mRNA were examined by RT-PCR. The results show that the dose of TNF-α (10 ng/ml) significantly enhanced CHIP levels in both cell lines at 48 hrs (Fig.1A). Consistent with these mRNA results, TNF-α enhanced CHIP protein expression, as determined by western blot analysis at 48 hrs (Fig.1B). To confirm that up-regulated CHIP expression is associated with TNF-α-induced apoptosis, we analysed the caspase-3 activity and cell viability of both cell lines, which were treated with 10 ng/ml TNF-α for 24 and 48 hrs, using MTT (Fig.1C). At the same time, CHIP mRNA expression in the same samples was detected by RT-PCR. The results showed that the osteoblasts were morphologically normal and had normal caspase-3 activity and that CHIP expression was increased when the cells were treated with 10 ng/ml TNF-α. However, higher concentrations (>10 ng/ml) resulted in the death of the C2C12 and MC3T3-E1 cells.


Identification of C-terminal Hsp70-interacting protein as a mediator of tumour necrosis factor action in osteoblast differentiation by targeting osterix for degradation.

Xie J, Gu J - J. Cell. Mol. Med. (2015)

TNF-α increases CHIP expression. (A) C2C12 or MC3T3-E1 cells were cultured in medium with PBS or TNF-α (5.0–10 ng/ml) for 24 and 48 hrs. The expression of CHIP and actin mRNA was determined by real-time RT-PCR. The values are the mean ± SD of three loadings. *P < 0.05 versus the PBS-treated group. (B) C2C12 or MC3T3-E1 cells were treated with 5.0–10 ng/ml TNF-α for 48 hrs. CHIP and β-actin protein levels were measured by Western blot analysis. (C) Correlation between TNF-α-induced apoptosis and CHIP expression. Both cell types were treated with TNF-α (5.0–10 ng/ml) or PBS for 24 and 48 hrs, and the cell lysates were used to measure caspase-3 activity or were submitted to a MTT assay. The values are the mean ± SD of three loadings. *P < 0.05 versus the PBS-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: TNF-α increases CHIP expression. (A) C2C12 or MC3T3-E1 cells were cultured in medium with PBS or TNF-α (5.0–10 ng/ml) for 24 and 48 hrs. The expression of CHIP and actin mRNA was determined by real-time RT-PCR. The values are the mean ± SD of three loadings. *P < 0.05 versus the PBS-treated group. (B) C2C12 or MC3T3-E1 cells were treated with 5.0–10 ng/ml TNF-α for 48 hrs. CHIP and β-actin protein levels were measured by Western blot analysis. (C) Correlation between TNF-α-induced apoptosis and CHIP expression. Both cell types were treated with TNF-α (5.0–10 ng/ml) or PBS for 24 and 48 hrs, and the cell lysates were used to measure caspase-3 activity or were submitted to a MTT assay. The values are the mean ± SD of three loadings. *P < 0.05 versus the PBS-treated group.
Mentions: Because TNF-α has been reported to enhance the expression of the E3 ligase Smurf1, we determined whether TNF-α had a similar effect on the expression of the CHIP protein. The C2C12 and MC3T3-E1 osteoblast precursor cell lines were treated with PBS or 5–10 ng/ml TNF-α, and the expression levels of the CHIP mRNA were examined by RT-PCR. The results show that the dose of TNF-α (10 ng/ml) significantly enhanced CHIP levels in both cell lines at 48 hrs (Fig.1A). Consistent with these mRNA results, TNF-α enhanced CHIP protein expression, as determined by western blot analysis at 48 hrs (Fig.1B). To confirm that up-regulated CHIP expression is associated with TNF-α-induced apoptosis, we analysed the caspase-3 activity and cell viability of both cell lines, which were treated with 10 ng/ml TNF-α for 24 and 48 hrs, using MTT (Fig.1C). At the same time, CHIP mRNA expression in the same samples was detected by RT-PCR. The results showed that the osteoblasts were morphologically normal and had normal caspase-3 activity and that CHIP expression was increased when the cells were treated with 10 ng/ml TNF-α. However, higher concentrations (>10 ng/ml) resulted in the death of the C2C12 and MC3T3-E1 cells.

Bottom Line: But less is known regarding the molecular mechanisms that mediate the effect of TNF-α on osteoblast function.Co-immunoprecipitation and ubiquitination assays revealed ubiquitinated Osx, confirmed that CHIP indeed interacted with Osx and identified K55 and K386 residues as the ubiquitination sites in Osx, Luciferase reporter assay and Small interfering RNAs examined whether TNF-α target the bone morphogenetic protein signalling through CHIP.We found that the K55 and K386 residues are ubiquitination site(s) in Osx, and that TNF-α inhibits osteoblast differentiation by promoting Osx degradation through up-regulation of E3 ubiquitin ligase CHIP in osteoblast.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China.

No MeSH data available.


Related in: MedlinePlus