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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 and CHIP prompt Osx degradation through ubiquitination. HEK293T or MC3T3-E1 cells were either transfected with M-CHIP expression vector or were cotransfected with Flag-Osx expression vector or empty vector and cultured for 48 hrs in the presence of PBS or TNF-α (10 ng/ml). (A) Endogenous ubiquitinated Osx (Ub-Osx) protein ladders were detected by the anti-ubiquitin antibody (upper panel). After stripping the antibody, the total un-ubiquitinated Osx protein levels were determined using the anti-Osx antibody (lower panel). (B) Exogenous ubiquitinated Osx (Ub-HA) protein ladders were detected by the anti-HA antibody (upper panel). After stripping the blots, the total un-ubiquitinated Osx protein levels were determined with an anti-Flag antibody (lower panel). (C) The HEK293T cells were treated with PBS or TNF-α (10 ng/ml), Flag-Osx expression was examined by Western blotting with an anti-Flag antibody. (D) The MC3T3-E1 cells were treated with MG132 for the last 12 hrs. Osx expression was examined by western blotting with an anti-Osx antibody, and β-actin protein levels were determined by western blotting with an anti-actin antibody. The results are shown for one of three independent experiments.
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fig02: TNF-α and CHIP prompt Osx degradation through ubiquitination. HEK293T or MC3T3-E1 cells were either transfected with M-CHIP expression vector or were cotransfected with Flag-Osx expression vector or empty vector and cultured for 48 hrs in the presence of PBS or TNF-α (10 ng/ml). (A) Endogenous ubiquitinated Osx (Ub-Osx) protein ladders were detected by the anti-ubiquitin antibody (upper panel). After stripping the antibody, the total un-ubiquitinated Osx protein levels were determined using the anti-Osx antibody (lower panel). (B) Exogenous ubiquitinated Osx (Ub-HA) protein ladders were detected by the anti-HA antibody (upper panel). After stripping the blots, the total un-ubiquitinated Osx protein levels were determined with an anti-Flag antibody (lower panel). (C) The HEK293T cells were treated with PBS or TNF-α (10 ng/ml), Flag-Osx expression was examined by Western blotting with an anti-Flag antibody. (D) The MC3T3-E1 cells were treated with MG132 for the last 12 hrs. Osx expression was examined by western blotting with an anti-Osx antibody, and β-actin protein levels were determined by western blotting with an anti-actin antibody. The results are shown for one of three independent experiments.

Mentions: To determine whether TNF-α promotes Osx degradation through the ubiquitin pathway, HEK293T cells transfected with the Flag-Osx and HA-UB plasmids or MC3T3-E1 cells were treated with 10 ng/ml TNF-α and the proteasome inhibitor MG132, which is an inhibitor of protein degradation through the ubiquitination pathway. Significant increases in the protein levels of both endogenous and exogenous Osx were revealed after treatment with 20 μM MG132. At the same time, we examined the Osx protein by immunoprecipitation assays using an anti-Osx antibody to precipitate the immunocomplexes and an anti-ubiquitin antibody to perform western blot analysis. This result revealed small expression of ubiquitinated Osx in vehicle-treated cells upon MG132 treatment. Interestingly, the amount of ubiquitinated Osx was significantly increased in the presence of TNF-α (Fig.2A and B). In addition, MC3T3-E1 or HEK293T cells transfected with Flag-Osx and Myc-CHIP plasmids were treated with 10 ng/ml TNF-α and 20 μM MG132, and the levels of Flag-Osx and Osx were detected by western blot analysis. As shown in Figure2C and D, 10 ng/ml of TNF-α led to a marked reduction in exogenous Osx in HEK293T cells and in endogenous Osx in MC3T3 cells. The immunoblotting results showed that the Flag-Osx protein levels decreased with increasing expressions of Myc-CHIP in the absence of TNF-α. Taken together, these results suggest that TNF-α may initiate the ubiquitin pathway to degrade Osx and that CHIP may play an important role in mediating the effects of TNF-α exposure on Osx degradation.


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 and CHIP prompt Osx degradation through ubiquitination. HEK293T or MC3T3-E1 cells were either transfected with M-CHIP expression vector or were cotransfected with Flag-Osx expression vector or empty vector and cultured for 48 hrs in the presence of PBS or TNF-α (10 ng/ml). (A) Endogenous ubiquitinated Osx (Ub-Osx) protein ladders were detected by the anti-ubiquitin antibody (upper panel). After stripping the antibody, the total un-ubiquitinated Osx protein levels were determined using the anti-Osx antibody (lower panel). (B) Exogenous ubiquitinated Osx (Ub-HA) protein ladders were detected by the anti-HA antibody (upper panel). After stripping the blots, the total un-ubiquitinated Osx protein levels were determined with an anti-Flag antibody (lower panel). (C) The HEK293T cells were treated with PBS or TNF-α (10 ng/ml), Flag-Osx expression was examined by Western blotting with an anti-Flag antibody. (D) The MC3T3-E1 cells were treated with MG132 for the last 12 hrs. Osx expression was examined by western blotting with an anti-Osx antibody, and β-actin protein levels were determined by western blotting with an anti-actin antibody. The results are shown for one of three independent experiments.
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Related In: Results  -  Collection

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fig02: TNF-α and CHIP prompt Osx degradation through ubiquitination. HEK293T or MC3T3-E1 cells were either transfected with M-CHIP expression vector or were cotransfected with Flag-Osx expression vector or empty vector and cultured for 48 hrs in the presence of PBS or TNF-α (10 ng/ml). (A) Endogenous ubiquitinated Osx (Ub-Osx) protein ladders were detected by the anti-ubiquitin antibody (upper panel). After stripping the antibody, the total un-ubiquitinated Osx protein levels were determined using the anti-Osx antibody (lower panel). (B) Exogenous ubiquitinated Osx (Ub-HA) protein ladders were detected by the anti-HA antibody (upper panel). After stripping the blots, the total un-ubiquitinated Osx protein levels were determined with an anti-Flag antibody (lower panel). (C) The HEK293T cells were treated with PBS or TNF-α (10 ng/ml), Flag-Osx expression was examined by Western blotting with an anti-Flag antibody. (D) The MC3T3-E1 cells were treated with MG132 for the last 12 hrs. Osx expression was examined by western blotting with an anti-Osx antibody, and β-actin protein levels were determined by western blotting with an anti-actin antibody. The results are shown for one of three independent experiments.
Mentions: To determine whether TNF-α promotes Osx degradation through the ubiquitin pathway, HEK293T cells transfected with the Flag-Osx and HA-UB plasmids or MC3T3-E1 cells were treated with 10 ng/ml TNF-α and the proteasome inhibitor MG132, which is an inhibitor of protein degradation through the ubiquitination pathway. Significant increases in the protein levels of both endogenous and exogenous Osx were revealed after treatment with 20 μM MG132. At the same time, we examined the Osx protein by immunoprecipitation assays using an anti-Osx antibody to precipitate the immunocomplexes and an anti-ubiquitin antibody to perform western blot analysis. This result revealed small expression of ubiquitinated Osx in vehicle-treated cells upon MG132 treatment. Interestingly, the amount of ubiquitinated Osx was significantly increased in the presence of TNF-α (Fig.2A and B). In addition, MC3T3-E1 or HEK293T cells transfected with Flag-Osx and Myc-CHIP plasmids were treated with 10 ng/ml TNF-α and 20 μM MG132, and the levels of Flag-Osx and Osx were detected by western blot analysis. As shown in Figure2C and D, 10 ng/ml of TNF-α led to a marked reduction in exogenous Osx in HEK293T cells and in endogenous Osx in MC3T3 cells. The immunoblotting results showed that the Flag-Osx protein levels decreased with increasing expressions of Myc-CHIP in the absence of TNF-α. Taken together, these results suggest that TNF-α may initiate the ubiquitin pathway to degrade Osx and that CHIP may play an important role in mediating the effects of TNF-α exposure on Osx degradation.

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