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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 decreases osteoblast mineralization via CHIP. Establishment of cell lines stably expressing HA-CHIP in MC3T3-E1 cells. (A) The stable cells transduced with adenoviruses expressing CHIP siRNA were subjected to ALP staining. (B) The ALP activity of the stable cells was measured after 21 days of differentiation in the absence or presence of TNF-α. (C) The stable cells were transduced with adenoviruses expressing CHIP siRNA or the empty virus for 6 hrs, treated with 10 ng/ml TNF-α for 48 hrs and then stimulated with 40 ng/ml BMP-2 for 28 days followed by the determination of mineralization by Alizarin Red staining. Images of mineralization by Alizarin Red staining of three independent experiments. (D) Quantification of the mineralization. The results are the mean ± SD of three loadings. *P < 0.05 versus empty vector-infected TNF-α group.
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fig06: TNF-α decreases osteoblast mineralization via CHIP. Establishment of cell lines stably expressing HA-CHIP in MC3T3-E1 cells. (A) The stable cells transduced with adenoviruses expressing CHIP siRNA were subjected to ALP staining. (B) The ALP activity of the stable cells was measured after 21 days of differentiation in the absence or presence of TNF-α. (C) The stable cells were transduced with adenoviruses expressing CHIP siRNA or the empty virus for 6 hrs, treated with 10 ng/ml TNF-α for 48 hrs and then stimulated with 40 ng/ml BMP-2 for 28 days followed by the determination of mineralization by Alizarin Red staining. Images of mineralization by Alizarin Red staining of three independent experiments. (D) Quantification of the mineralization. The results are the mean ± SD of three loadings. *P < 0.05 versus empty vector-infected TNF-α group.

Mentions: To further evaluate the potential roles of CHIP on osteoblast function under conditions in which TNF-α is overexpressed, we established stable MC3T3-E1 cell lines overexpressing HA-CHIP or depleted of endogenous CHIP by siRNA. After the stable cell lines treated with TNF-α for 2 days were cultured for 21 days, ALP staining and ALP activity was performed (Fig.6A and B). At 28 days, the stable cell lines were stained with Alizarin Red to identify mineralized nodules. In the absence of TNF-α, a 40 ng/ml dose of BMP-2 enhanced the Osx protein levels and number of nodules in CHIP knockdown clones compared with the HA-CHIP-expressing clones. Similarly, the Osx protein levels and number of nodules increased in the CHIP knockdown clones compared with the controls. However, in the presence of TNF-α, there was an apparent TNF-α-induced decrease in the Osx protein levels and in mineralized nodule formation in the HA-CHIP-expressing clones compared with the CHIP knockdown clones (Fig.6C and D). These data showed that CHIP decreased the number and size of the mineralized nodules under normal and inflammatory conditions, suggesting that CHIP overexpression prevents or delays cell differentiation into mature osteoblasts and that the depletion of CHIP promotes or enhances this process.


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 decreases osteoblast mineralization via CHIP. Establishment of cell lines stably expressing HA-CHIP in MC3T3-E1 cells. (A) The stable cells transduced with adenoviruses expressing CHIP siRNA were subjected to ALP staining. (B) The ALP activity of the stable cells was measured after 21 days of differentiation in the absence or presence of TNF-α. (C) The stable cells were transduced with adenoviruses expressing CHIP siRNA or the empty virus for 6 hrs, treated with 10 ng/ml TNF-α for 48 hrs and then stimulated with 40 ng/ml BMP-2 for 28 days followed by the determination of mineralization by Alizarin Red staining. Images of mineralization by Alizarin Red staining of three independent experiments. (D) Quantification of the mineralization. The results are the mean ± SD of three loadings. *P < 0.05 versus empty vector-infected TNF-α group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549032&req=5

fig06: TNF-α decreases osteoblast mineralization via CHIP. Establishment of cell lines stably expressing HA-CHIP in MC3T3-E1 cells. (A) The stable cells transduced with adenoviruses expressing CHIP siRNA were subjected to ALP staining. (B) The ALP activity of the stable cells was measured after 21 days of differentiation in the absence or presence of TNF-α. (C) The stable cells were transduced with adenoviruses expressing CHIP siRNA or the empty virus for 6 hrs, treated with 10 ng/ml TNF-α for 48 hrs and then stimulated with 40 ng/ml BMP-2 for 28 days followed by the determination of mineralization by Alizarin Red staining. Images of mineralization by Alizarin Red staining of three independent experiments. (D) Quantification of the mineralization. The results are the mean ± SD of three loadings. *P < 0.05 versus empty vector-infected TNF-α group.
Mentions: To further evaluate the potential roles of CHIP on osteoblast function under conditions in which TNF-α is overexpressed, we established stable MC3T3-E1 cell lines overexpressing HA-CHIP or depleted of endogenous CHIP by siRNA. After the stable cell lines treated with TNF-α for 2 days were cultured for 21 days, ALP staining and ALP activity was performed (Fig.6A and B). At 28 days, the stable cell lines were stained with Alizarin Red to identify mineralized nodules. In the absence of TNF-α, a 40 ng/ml dose of BMP-2 enhanced the Osx protein levels and number of nodules in CHIP knockdown clones compared with the HA-CHIP-expressing clones. Similarly, the Osx protein levels and number of nodules increased in the CHIP knockdown clones compared with the controls. However, in the presence of TNF-α, there was an apparent TNF-α-induced decrease in the Osx protein levels and in mineralized nodule formation in the HA-CHIP-expressing clones compared with the CHIP knockdown clones (Fig.6C and D). These data showed that CHIP decreased the number and size of the mineralized nodules under normal and inflammatory conditions, suggesting that CHIP overexpression prevents or delays cell differentiation into mature osteoblasts and that the depletion of CHIP promotes or enhances this process.

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