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Lixin Liu - One of the best experts on this subject based on the ideXlab platform.
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erratum to hclp46 increases Smad3 Protein stability via inhibiting its ubiquitin proteasomal degradation
Protein & Cell, 2015Co-Authors: Yingying Xing, Wei Wang, Qiaoyun Chu, Run Feng, Lixin LiuAbstract:Yingying Xing and Qiaoyun Chu contributed equally to this work. The online version of the original article can be found under doi:10.1007/s13238-015-0174-0.
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hclp46 increases Smad3 Protein stability via inhibiting its ubiquitin proteasomal degradation
Protein & Cell, 2015Co-Authors: Yingying Xing, Wei Wang, Qiaoyun Chu, Run Feng, Lixin LiuAbstract:Dear Editor, hCLP46 (human CAP10-like Protein 46 kDa) was initially isolated and identified from human acute myeloid leukemia transformed from myelodysplastic syndrome (MDS-AML) CD34+ cells (Teng et al., 2006) and we demonstrated previously that hCLP46 is abnormally expressed in many hematopoietic malignancies (Wang et al., 2010). Studies from its Drosophila homolog, Rumi, suggested that Notch is a potential target of hCLP46 (Acar et al., 2008). We also found that overexpression of hCLP46 enhances Notch activation and regulates cell proliferation in a cell type-dependent manner (Ma et al., 2011; Chu et al., 2013). However, hCLP46−/− embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, suggesting that hCLP46 is likely to have additional important targets during mammalian development (Fernandez-Valdivia et al., 2011). Based on the crosstalk between Notch and the transforming growth factor-β (TGF-β) signaling, we proposed that hCLP46 might be involved in TGF-β signal regulation, but the detail mechanism remains unclear. With the full length or truncated plasmids encoding hCLP46 1–120 aa (no CAP10 domain) or hCLP46 121–392 aa (with CAP10 domain), we found that overexpression of hCLP46 1–120 aa had no obvious effect on Smad3 expression, whereas both hCLP46 121–392 aa and hCLP46-full length significantly increased Smad3 Protein expression, suggesting that hCLP46 increases Smad3 expression in a CAP10 domain dependent manner (Fig. 1A and and1B).1B). To determine the mechanism through which hCLP46 regulates Smad3 expression, we generated a stable cell line inducibly overexpressing hCLP46, which is named as 293TRex-hCLP46 hereinafter. When cells were incubated with 0.5 μg/mL Tetracycline (Tet) for 24 h, the pronounced induction of hCLP46 resulted in 85% increase of Smad3 expression (Fig. 1C and and1D).1D). We then examined Smad3 Protein turnover in 293TRex-hCLP46 cells by blocking Protein synthesis. In Tet off cells, administration of CHX (50 μg/mL) caused a remarkable decrease of Smad3 in a time dependent manner (Fig. 1C and and1D).1D). The half-life is only about 0.5 h, suggesting that endogenous Smad3 undergoes fast degradation at the steady state. However, the half-life of Smad3 Protein was significantly longer in Tet on cells as compared with that observed in Tet off cells, suggesting that hCLP46 could increase Smad3 Protein stability (Fig. 1C and and1D).1D). When endogenous hCLP46 was knocked down by siRNA, significantly reduction in Smad3 expression was observed (Fig. 1E). It is notable that the mRNA levels of Smad3 were not affected by overexpression or knockdown of hCLP46 (Fig. S1A and S1B). To determine whether the degradation of Smad3 is mediated by proteasome, we treated 293TRex-hCLP46 cells with 20 μmol/L 26S proteasome inhibitor MG-132 for 3 h, which resulted in a two-fold increase in Smad3 expression in Tet off cells (Fig. 1F and and1G).1G). In the presence of MG132, hCLP46 overexpression still significantly increased Smad3 Protein level whereas hCLP46 knockdown had no obvious effect on Smad3 expression (Fig. 1F and and1G).1G). Consistently, we found that Smad3 was polyubiquitinated in cells treated with MG132 and the ubiquitination of Smad3 was attenuated by overexpression of hCLP46 (Fig. 1H), but enhanced by knockdown of endogenous hCLP46 (Fig. 1I), suggesting that hCLP46 increases Smad3 expression and Protein stability through inhibiting proteasomal degradation of Smad3. Figure 1 hCLP46 increases Smad3 expression by preventing its proteasomal degradation in 293TRex cells. (A) 293TRex cells were transfected with full length of hCLP46 or truncated plasmids encoding hCLP46 1–120 aa or hCLP46 121–392 aa for 48 h, ... We then attempted to evaluate the impact of hCLP46 on TGF-β signaling. Overexpression of hCLP46 inhibited cell viability at basal condition and further exacerbated TGF-β1 induced cell viability inhibition (2 ng/mL, 24 h) (Fig. 2A). In contrast, knockdown of hCLP46 by siRNA increased cell viability and almost totally blocked the inhibition of cell proliferation by TGF-β1 (Fig. 2B). In addition, TGF-β1 treatment significantly increased the expression of two cell cycle inhibitors (p21 and p27) as compared to control cells, which were further enhanced by overexpression of hCLP46 (Fig. 2C and and2D),2D), whereas attenuated by knockdown of hCLP46 (Fig. 2E and and2F),2F), suggesting that hCLP46 enhances TGF-β signaling by modulating Smad3 expression. Figure 2 hCLP46 enhanced TGF-β1 induced cell growth arrest and up-regulation of cell cycle inhibitors. (A) 293TRex-hCLP46 cells were cultured in absence or presence of 0.5 μg/mL Tet for 24 h and 2 ng/mL TGF-β1 was ... As Notch signaling might be involved in Smad3 regulation, we treated cells with DAPT (2 μmol/L, 12 h) or EDTA (5 mmol/L, 15 min and then replaced with fresh DMEM plus 10% FBS and cultured for additional 6 h) to inhibit or activate Notch signaling respectively. DAPT treatment resulted in dramatically decrease of the NICD expression, but has no effect on Smad3 expression (Fig. S2A–C). EDTA significantly increased NICD and Smad3 levels. However, cells with hCLP46 overexpression still had more Smad3 expression than that of control cells (Fig. S2D–F). Together, these data suggest that hCLP46 regulates Smad3 expression is not affected by Notch signaling. As the primary intracellular transducer, Smad3 is a critical mediator of the cytostatic response to TGF-β (Zhang et al., 2014). Evidence for this comes from the observation that a variety of primary cells from Smad3-null mice are partially resistant to TGF-β induced growth arrest (Datto et al., 1999; Yang et al., 1999) and exogenous overexpression of Smad3 sensitizes cells to TGF-β induced growth arrest and apoptosis (Wildey et al., 2003). Importantly, it was found that treatment with the proteasome inhibitor caused an accumulation of Smad3 Protein in absence of TGF-β1, suggesting that not only in response to TGF-β but also in a steady state, the level of Smad3 is regulated by the proteasome pathway (Inoue et al., 2004). In addition, several studies demonstrated that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-β (Guo et al., 2008) and the U-box-containing carboxyl terminus of Hsc70-interacting Protein has been identified as an E3 ubiquitin ligase to degrade Smad3 at steady state (Xin et al., 2005). In agreement with those findings, we demonstrated that hCLP46 modulates Smad3 Protein stability by inhibiting its proteasomal degradation and consequently enhances cellular sensitivity to the TGF-β signal. Our findings suggest a new function of hCLP46 in modulating critical TGF-β/Smad3-regulated processes during development and tumor progression.
Wei Wang - One of the best experts on this subject based on the ideXlab platform.
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erratum to hclp46 increases Smad3 Protein stability via inhibiting its ubiquitin proteasomal degradation
Protein & Cell, 2015Co-Authors: Yingying Xing, Wei Wang, Qiaoyun Chu, Run Feng, Lixin LiuAbstract:Yingying Xing and Qiaoyun Chu contributed equally to this work. The online version of the original article can be found under doi:10.1007/s13238-015-0174-0.
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hclp46 increases Smad3 Protein stability via inhibiting its ubiquitin proteasomal degradation
Protein & Cell, 2015Co-Authors: Yingying Xing, Wei Wang, Qiaoyun Chu, Run Feng, Lixin LiuAbstract:Dear Editor, hCLP46 (human CAP10-like Protein 46 kDa) was initially isolated and identified from human acute myeloid leukemia transformed from myelodysplastic syndrome (MDS-AML) CD34+ cells (Teng et al., 2006) and we demonstrated previously that hCLP46 is abnormally expressed in many hematopoietic malignancies (Wang et al., 2010). Studies from its Drosophila homolog, Rumi, suggested that Notch is a potential target of hCLP46 (Acar et al., 2008). We also found that overexpression of hCLP46 enhances Notch activation and regulates cell proliferation in a cell type-dependent manner (Ma et al., 2011; Chu et al., 2013). However, hCLP46−/− embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, suggesting that hCLP46 is likely to have additional important targets during mammalian development (Fernandez-Valdivia et al., 2011). Based on the crosstalk between Notch and the transforming growth factor-β (TGF-β) signaling, we proposed that hCLP46 might be involved in TGF-β signal regulation, but the detail mechanism remains unclear. With the full length or truncated plasmids encoding hCLP46 1–120 aa (no CAP10 domain) or hCLP46 121–392 aa (with CAP10 domain), we found that overexpression of hCLP46 1–120 aa had no obvious effect on Smad3 expression, whereas both hCLP46 121–392 aa and hCLP46-full length significantly increased Smad3 Protein expression, suggesting that hCLP46 increases Smad3 expression in a CAP10 domain dependent manner (Fig. 1A and and1B).1B). To determine the mechanism through which hCLP46 regulates Smad3 expression, we generated a stable cell line inducibly overexpressing hCLP46, which is named as 293TRex-hCLP46 hereinafter. When cells were incubated with 0.5 μg/mL Tetracycline (Tet) for 24 h, the pronounced induction of hCLP46 resulted in 85% increase of Smad3 expression (Fig. 1C and and1D).1D). We then examined Smad3 Protein turnover in 293TRex-hCLP46 cells by blocking Protein synthesis. In Tet off cells, administration of CHX (50 μg/mL) caused a remarkable decrease of Smad3 in a time dependent manner (Fig. 1C and and1D).1D). The half-life is only about 0.5 h, suggesting that endogenous Smad3 undergoes fast degradation at the steady state. However, the half-life of Smad3 Protein was significantly longer in Tet on cells as compared with that observed in Tet off cells, suggesting that hCLP46 could increase Smad3 Protein stability (Fig. 1C and and1D).1D). When endogenous hCLP46 was knocked down by siRNA, significantly reduction in Smad3 expression was observed (Fig. 1E). It is notable that the mRNA levels of Smad3 were not affected by overexpression or knockdown of hCLP46 (Fig. S1A and S1B). To determine whether the degradation of Smad3 is mediated by proteasome, we treated 293TRex-hCLP46 cells with 20 μmol/L 26S proteasome inhibitor MG-132 for 3 h, which resulted in a two-fold increase in Smad3 expression in Tet off cells (Fig. 1F and and1G).1G). In the presence of MG132, hCLP46 overexpression still significantly increased Smad3 Protein level whereas hCLP46 knockdown had no obvious effect on Smad3 expression (Fig. 1F and and1G).1G). Consistently, we found that Smad3 was polyubiquitinated in cells treated with MG132 and the ubiquitination of Smad3 was attenuated by overexpression of hCLP46 (Fig. 1H), but enhanced by knockdown of endogenous hCLP46 (Fig. 1I), suggesting that hCLP46 increases Smad3 expression and Protein stability through inhibiting proteasomal degradation of Smad3. Figure 1 hCLP46 increases Smad3 expression by preventing its proteasomal degradation in 293TRex cells. (A) 293TRex cells were transfected with full length of hCLP46 or truncated plasmids encoding hCLP46 1–120 aa or hCLP46 121–392 aa for 48 h, ... We then attempted to evaluate the impact of hCLP46 on TGF-β signaling. Overexpression of hCLP46 inhibited cell viability at basal condition and further exacerbated TGF-β1 induced cell viability inhibition (2 ng/mL, 24 h) (Fig. 2A). In contrast, knockdown of hCLP46 by siRNA increased cell viability and almost totally blocked the inhibition of cell proliferation by TGF-β1 (Fig. 2B). In addition, TGF-β1 treatment significantly increased the expression of two cell cycle inhibitors (p21 and p27) as compared to control cells, which were further enhanced by overexpression of hCLP46 (Fig. 2C and and2D),2D), whereas attenuated by knockdown of hCLP46 (Fig. 2E and and2F),2F), suggesting that hCLP46 enhances TGF-β signaling by modulating Smad3 expression. Figure 2 hCLP46 enhanced TGF-β1 induced cell growth arrest and up-regulation of cell cycle inhibitors. (A) 293TRex-hCLP46 cells were cultured in absence or presence of 0.5 μg/mL Tet for 24 h and 2 ng/mL TGF-β1 was ... As Notch signaling might be involved in Smad3 regulation, we treated cells with DAPT (2 μmol/L, 12 h) or EDTA (5 mmol/L, 15 min and then replaced with fresh DMEM plus 10% FBS and cultured for additional 6 h) to inhibit or activate Notch signaling respectively. DAPT treatment resulted in dramatically decrease of the NICD expression, but has no effect on Smad3 expression (Fig. S2A–C). EDTA significantly increased NICD and Smad3 levels. However, cells with hCLP46 overexpression still had more Smad3 expression than that of control cells (Fig. S2D–F). Together, these data suggest that hCLP46 regulates Smad3 expression is not affected by Notch signaling. As the primary intracellular transducer, Smad3 is a critical mediator of the cytostatic response to TGF-β (Zhang et al., 2014). Evidence for this comes from the observation that a variety of primary cells from Smad3-null mice are partially resistant to TGF-β induced growth arrest (Datto et al., 1999; Yang et al., 1999) and exogenous overexpression of Smad3 sensitizes cells to TGF-β induced growth arrest and apoptosis (Wildey et al., 2003). Importantly, it was found that treatment with the proteasome inhibitor caused an accumulation of Smad3 Protein in absence of TGF-β1, suggesting that not only in response to TGF-β but also in a steady state, the level of Smad3 is regulated by the proteasome pathway (Inoue et al., 2004). In addition, several studies demonstrated that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-β (Guo et al., 2008) and the U-box-containing carboxyl terminus of Hsc70-interacting Protein has been identified as an E3 ubiquitin ligase to degrade Smad3 at steady state (Xin et al., 2005). In agreement with those findings, we demonstrated that hCLP46 modulates Smad3 Protein stability by inhibiting its proteasomal degradation and consequently enhances cellular sensitivity to the TGF-β signal. Our findings suggest a new function of hCLP46 in modulating critical TGF-β/Smad3-regulated processes during development and tumor progression.
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Smad3 specific inhibitor naringenin decreases the expression of extracellular matrix induced by tgf β1 in cultured rat hepatic stellate cells
Pharmaceutical Research, 2006Co-Authors: Wei Wang, Han Hu, Ning Tang, Chunling Zhang, Wei Liang, Minwei WangAbstract:Purpose. During the process of liver fibrogenesis, transforming growth factor-" (TGF-") plays an essential role in modulating extracellular matrix (ECM) gene expression, and a growing body of evidence suggests that this is a Smad3-dependent process in the activated hepatic stellate cells (HSCs). Naringenin showed a significantly protective effect on experimental rat liver fibrosis, in our efforts to elucidate its antifibrosis molecular mechanisms and to find a novel target based on Smad3 signaling for challenging fibrosis diseases. Methods. In this study, reverse transcription-polymerase chain reaction and Western blot assays were used to investigate the inhibitory effect of naringenin on ECM formation induced by TGF-"1 in the HSC-T6 cells. Results. Naringenin reduced not only the accumulation of ECM, including collagen I!1 (Col I!1), fibronectin (FN), and plasminogen activator inhibitor-1 (PAI-1), but also the production of Smad3 induced by TGF-"1 in both mRNA and Protein levels in a dose-dependent manner. Moreover, naringenin selectively inhibited the transcription of Smad3, but not other Smads involved in TGF-"1 signaling pathways. Conclusion. Our data demonstrate that naringenin can exert antifibrogenic effects by directly or indirectly down-regulating Smad3 Protein expression and phosphorylation through TGF-" signaling.
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identification of novel Protein Protein interactions using a versatile mammalian tandem affinity purification expression system
Molecular & Cellular Proteomics, 2003Co-Authors: Matthew P Knuesel, Yong Wan, Zhan Xiao, Eric P Holinger, Nick Lowe, Wei Wang, Xuedong LiuAbstract:Identification of Protein-Protein interactions is essential for elucidating the biochemical mechanism of signal transduction. Purification and identification of individual Proteins in mammalian cells have been difficult, however, due to the sheer complexity of Protein mixtures obtained from cellular extracts. Recently, a tandem affinity purification (TAP) method has been developed as a tool that allows rapid purification of native Protein complexes expressed at their natural level in engineered yeast cells. To adapt this method to mammalian cells, we have created a TAP tag retroviral expression vector to allow stable expression of the TAP-tagged Protein at close to physiological levels. To demonstrate the utility of this vector, we have fused a TAP tag, consisting of a Protein A tag, a cleavage site for the tobacco etch virus (TEV) protease, and the FLAG epitope, to the N terminus of human Smad3 and SMAD4. We have stably expressed these Proteins in mammalian cells at desirable levels by retroviral gene transfer and purified native Smad3 Protein complexes from cell lysates. The combination of two different affinity tags greatly reduced the number of nonspecific Proteins in the mixture. We have identified HSP70 as a specific interacting Protein of Smad3. We demonstrated that Smad3, but not SMAD1, binds HSP70 in vivo, validating the TAP purification approach. This method is applicable to virtually any Protein and provides an efficient way to purify unknown Proteins to homogeneity from the complex mixtures found in mammalian cell lysates in preparation for identification by mass spectrometry.
Qiaoyun Chu - One of the best experts on this subject based on the ideXlab platform.
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erratum to hclp46 increases Smad3 Protein stability via inhibiting its ubiquitin proteasomal degradation
Protein & Cell, 2015Co-Authors: Yingying Xing, Wei Wang, Qiaoyun Chu, Run Feng, Lixin LiuAbstract:Yingying Xing and Qiaoyun Chu contributed equally to this work. The online version of the original article can be found under doi:10.1007/s13238-015-0174-0.
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hclp46 increases Smad3 Protein stability via inhibiting its ubiquitin proteasomal degradation
Protein & Cell, 2015Co-Authors: Yingying Xing, Wei Wang, Qiaoyun Chu, Run Feng, Lixin LiuAbstract:Dear Editor, hCLP46 (human CAP10-like Protein 46 kDa) was initially isolated and identified from human acute myeloid leukemia transformed from myelodysplastic syndrome (MDS-AML) CD34+ cells (Teng et al., 2006) and we demonstrated previously that hCLP46 is abnormally expressed in many hematopoietic malignancies (Wang et al., 2010). Studies from its Drosophila homolog, Rumi, suggested that Notch is a potential target of hCLP46 (Acar et al., 2008). We also found that overexpression of hCLP46 enhances Notch activation and regulates cell proliferation in a cell type-dependent manner (Ma et al., 2011; Chu et al., 2013). However, hCLP46−/− embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, suggesting that hCLP46 is likely to have additional important targets during mammalian development (Fernandez-Valdivia et al., 2011). Based on the crosstalk between Notch and the transforming growth factor-β (TGF-β) signaling, we proposed that hCLP46 might be involved in TGF-β signal regulation, but the detail mechanism remains unclear. With the full length or truncated plasmids encoding hCLP46 1–120 aa (no CAP10 domain) or hCLP46 121–392 aa (with CAP10 domain), we found that overexpression of hCLP46 1–120 aa had no obvious effect on Smad3 expression, whereas both hCLP46 121–392 aa and hCLP46-full length significantly increased Smad3 Protein expression, suggesting that hCLP46 increases Smad3 expression in a CAP10 domain dependent manner (Fig. 1A and and1B).1B). To determine the mechanism through which hCLP46 regulates Smad3 expression, we generated a stable cell line inducibly overexpressing hCLP46, which is named as 293TRex-hCLP46 hereinafter. When cells were incubated with 0.5 μg/mL Tetracycline (Tet) for 24 h, the pronounced induction of hCLP46 resulted in 85% increase of Smad3 expression (Fig. 1C and and1D).1D). We then examined Smad3 Protein turnover in 293TRex-hCLP46 cells by blocking Protein synthesis. In Tet off cells, administration of CHX (50 μg/mL) caused a remarkable decrease of Smad3 in a time dependent manner (Fig. 1C and and1D).1D). The half-life is only about 0.5 h, suggesting that endogenous Smad3 undergoes fast degradation at the steady state. However, the half-life of Smad3 Protein was significantly longer in Tet on cells as compared with that observed in Tet off cells, suggesting that hCLP46 could increase Smad3 Protein stability (Fig. 1C and and1D).1D). When endogenous hCLP46 was knocked down by siRNA, significantly reduction in Smad3 expression was observed (Fig. 1E). It is notable that the mRNA levels of Smad3 were not affected by overexpression or knockdown of hCLP46 (Fig. S1A and S1B). To determine whether the degradation of Smad3 is mediated by proteasome, we treated 293TRex-hCLP46 cells with 20 μmol/L 26S proteasome inhibitor MG-132 for 3 h, which resulted in a two-fold increase in Smad3 expression in Tet off cells (Fig. 1F and and1G).1G). In the presence of MG132, hCLP46 overexpression still significantly increased Smad3 Protein level whereas hCLP46 knockdown had no obvious effect on Smad3 expression (Fig. 1F and and1G).1G). Consistently, we found that Smad3 was polyubiquitinated in cells treated with MG132 and the ubiquitination of Smad3 was attenuated by overexpression of hCLP46 (Fig. 1H), but enhanced by knockdown of endogenous hCLP46 (Fig. 1I), suggesting that hCLP46 increases Smad3 expression and Protein stability through inhibiting proteasomal degradation of Smad3. Figure 1 hCLP46 increases Smad3 expression by preventing its proteasomal degradation in 293TRex cells. (A) 293TRex cells were transfected with full length of hCLP46 or truncated plasmids encoding hCLP46 1–120 aa or hCLP46 121–392 aa for 48 h, ... We then attempted to evaluate the impact of hCLP46 on TGF-β signaling. Overexpression of hCLP46 inhibited cell viability at basal condition and further exacerbated TGF-β1 induced cell viability inhibition (2 ng/mL, 24 h) (Fig. 2A). In contrast, knockdown of hCLP46 by siRNA increased cell viability and almost totally blocked the inhibition of cell proliferation by TGF-β1 (Fig. 2B). In addition, TGF-β1 treatment significantly increased the expression of two cell cycle inhibitors (p21 and p27) as compared to control cells, which were further enhanced by overexpression of hCLP46 (Fig. 2C and and2D),2D), whereas attenuated by knockdown of hCLP46 (Fig. 2E and and2F),2F), suggesting that hCLP46 enhances TGF-β signaling by modulating Smad3 expression. Figure 2 hCLP46 enhanced TGF-β1 induced cell growth arrest and up-regulation of cell cycle inhibitors. (A) 293TRex-hCLP46 cells were cultured in absence or presence of 0.5 μg/mL Tet for 24 h and 2 ng/mL TGF-β1 was ... As Notch signaling might be involved in Smad3 regulation, we treated cells with DAPT (2 μmol/L, 12 h) or EDTA (5 mmol/L, 15 min and then replaced with fresh DMEM plus 10% FBS and cultured for additional 6 h) to inhibit or activate Notch signaling respectively. DAPT treatment resulted in dramatically decrease of the NICD expression, but has no effect on Smad3 expression (Fig. S2A–C). EDTA significantly increased NICD and Smad3 levels. However, cells with hCLP46 overexpression still had more Smad3 expression than that of control cells (Fig. S2D–F). Together, these data suggest that hCLP46 regulates Smad3 expression is not affected by Notch signaling. As the primary intracellular transducer, Smad3 is a critical mediator of the cytostatic response to TGF-β (Zhang et al., 2014). Evidence for this comes from the observation that a variety of primary cells from Smad3-null mice are partially resistant to TGF-β induced growth arrest (Datto et al., 1999; Yang et al., 1999) and exogenous overexpression of Smad3 sensitizes cells to TGF-β induced growth arrest and apoptosis (Wildey et al., 2003). Importantly, it was found that treatment with the proteasome inhibitor caused an accumulation of Smad3 Protein in absence of TGF-β1, suggesting that not only in response to TGF-β but also in a steady state, the level of Smad3 is regulated by the proteasome pathway (Inoue et al., 2004). In addition, several studies demonstrated that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-β (Guo et al., 2008) and the U-box-containing carboxyl terminus of Hsc70-interacting Protein has been identified as an E3 ubiquitin ligase to degrade Smad3 at steady state (Xin et al., 2005). In agreement with those findings, we demonstrated that hCLP46 modulates Smad3 Protein stability by inhibiting its proteasomal degradation and consequently enhances cellular sensitivity to the TGF-β signal. Our findings suggest a new function of hCLP46 in modulating critical TGF-β/Smad3-regulated processes during development and tumor progression.
Thomas J Kelley - One of the best experts on this subject based on the ideXlab platform.
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Smad3 expression is regulated by mitogen activated Protein kinase kinase 1 in epithelial and smooth muscle cells
Cellular Signalling, 2007Co-Authors: Kristie R Ross, Deborah A Corey, John M Dunn, Thomas J KelleyAbstract:Abstract Smad3 is a transcription factor that mediates TGF-β1 signaling and is known to be important in many of the cellular processes that regulate fibrosis and inflammation. Although several studies have examined Smad3 activation, little is known about the control of Smad3 expression. It is well established that the mitogen-activated Protein kinase (MAPK) pathway is responsive to TGF-β1 stimulation and coordinates with SMAD signaling in many cases; therefore, the hypothesis of this study is that the MAPK pathway will be involved in the regulation of Smad3 expression. Using a Smad3 promoter construct, we demonstrate that inhibition of either c-Jun-N-terminal kinase (JNK) or p38 activity has little effect on Smad3 promoter function. Inhibition of mitogen-activated Protein kinase kinase-1 (MEK1) with either PD98059 or UO126, however, results in a substantial dose-dependent inhibition of Smad3 promoter activity. Further studies confirm that promoter activity correlates with Protein expression by demonstrating reduced Smad3 Protein expression in A549 cells and airway smooth muscle cells after treatment with MEK1 inhibitors. Positive regulation of Smad3 expression is also demonstrated by expression of a constitutively active (ca)-MEK1 construct, where the presence of ca-MEK1 resulted in increased Smad3 Protein expression. These data lead to the conclusion that MEK1 is an important regulator of Smad3 expression.
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reduced Smad3 Protein expression and altered transforming growth factor β 1 mediated signaling in cystic fibrosis epithelial cells
American Journal of Respiratory Cell and Molecular Biology, 2001Co-Authors: Thomas J Kelley, Heather L Elmer, Deborah A CoreyAbstract:Cystic fibrosis (CF) is a disease characterized by an aggressive inflammatory response in the airways. Given the antiinflammatory properties of transforming growth factor (TGF)- β 1, it was our goal to examine components of TGF- β 1–mediated signaling in both a cultured cell model and a mouse model of CF. A CF-related reduction of Protein levels of the TGF- β 1 signaling molecule Smad3 was found in both of these model systems, whereas Smad4 levels were unchanged. Functional effects of reduced Smad3 expression are manifest in our cultured cell model, as reduced basal and TGF- β 1–stimulated levels of luciferase expression using the TGF- β 1–responsive reporter construct 3TP-Lux in the CF-phenotype cells compared with control cells. However, TGF- β 1–stimulated responses using the A3-Luc reporter construct were normal in both cell lines. These results suggest that select TGF- β 1–mediated signaling pathways are impaired in CF epithelial cells. This selective loss of Smad3 Protein expression in CF epithelium...
Yingying Xing - One of the best experts on this subject based on the ideXlab platform.
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erratum to hclp46 increases Smad3 Protein stability via inhibiting its ubiquitin proteasomal degradation
Protein & Cell, 2015Co-Authors: Yingying Xing, Wei Wang, Qiaoyun Chu, Run Feng, Lixin LiuAbstract:Yingying Xing and Qiaoyun Chu contributed equally to this work. The online version of the original article can be found under doi:10.1007/s13238-015-0174-0.
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hclp46 increases Smad3 Protein stability via inhibiting its ubiquitin proteasomal degradation
Protein & Cell, 2015Co-Authors: Yingying Xing, Wei Wang, Qiaoyun Chu, Run Feng, Lixin LiuAbstract:Dear Editor, hCLP46 (human CAP10-like Protein 46 kDa) was initially isolated and identified from human acute myeloid leukemia transformed from myelodysplastic syndrome (MDS-AML) CD34+ cells (Teng et al., 2006) and we demonstrated previously that hCLP46 is abnormally expressed in many hematopoietic malignancies (Wang et al., 2010). Studies from its Drosophila homolog, Rumi, suggested that Notch is a potential target of hCLP46 (Acar et al., 2008). We also found that overexpression of hCLP46 enhances Notch activation and regulates cell proliferation in a cell type-dependent manner (Ma et al., 2011; Chu et al., 2013). However, hCLP46−/− embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, suggesting that hCLP46 is likely to have additional important targets during mammalian development (Fernandez-Valdivia et al., 2011). Based on the crosstalk between Notch and the transforming growth factor-β (TGF-β) signaling, we proposed that hCLP46 might be involved in TGF-β signal regulation, but the detail mechanism remains unclear. With the full length or truncated plasmids encoding hCLP46 1–120 aa (no CAP10 domain) or hCLP46 121–392 aa (with CAP10 domain), we found that overexpression of hCLP46 1–120 aa had no obvious effect on Smad3 expression, whereas both hCLP46 121–392 aa and hCLP46-full length significantly increased Smad3 Protein expression, suggesting that hCLP46 increases Smad3 expression in a CAP10 domain dependent manner (Fig. 1A and and1B).1B). To determine the mechanism through which hCLP46 regulates Smad3 expression, we generated a stable cell line inducibly overexpressing hCLP46, which is named as 293TRex-hCLP46 hereinafter. When cells were incubated with 0.5 μg/mL Tetracycline (Tet) for 24 h, the pronounced induction of hCLP46 resulted in 85% increase of Smad3 expression (Fig. 1C and and1D).1D). We then examined Smad3 Protein turnover in 293TRex-hCLP46 cells by blocking Protein synthesis. In Tet off cells, administration of CHX (50 μg/mL) caused a remarkable decrease of Smad3 in a time dependent manner (Fig. 1C and and1D).1D). The half-life is only about 0.5 h, suggesting that endogenous Smad3 undergoes fast degradation at the steady state. However, the half-life of Smad3 Protein was significantly longer in Tet on cells as compared with that observed in Tet off cells, suggesting that hCLP46 could increase Smad3 Protein stability (Fig. 1C and and1D).1D). When endogenous hCLP46 was knocked down by siRNA, significantly reduction in Smad3 expression was observed (Fig. 1E). It is notable that the mRNA levels of Smad3 were not affected by overexpression or knockdown of hCLP46 (Fig. S1A and S1B). To determine whether the degradation of Smad3 is mediated by proteasome, we treated 293TRex-hCLP46 cells with 20 μmol/L 26S proteasome inhibitor MG-132 for 3 h, which resulted in a two-fold increase in Smad3 expression in Tet off cells (Fig. 1F and and1G).1G). In the presence of MG132, hCLP46 overexpression still significantly increased Smad3 Protein level whereas hCLP46 knockdown had no obvious effect on Smad3 expression (Fig. 1F and and1G).1G). Consistently, we found that Smad3 was polyubiquitinated in cells treated with MG132 and the ubiquitination of Smad3 was attenuated by overexpression of hCLP46 (Fig. 1H), but enhanced by knockdown of endogenous hCLP46 (Fig. 1I), suggesting that hCLP46 increases Smad3 expression and Protein stability through inhibiting proteasomal degradation of Smad3. Figure 1 hCLP46 increases Smad3 expression by preventing its proteasomal degradation in 293TRex cells. (A) 293TRex cells were transfected with full length of hCLP46 or truncated plasmids encoding hCLP46 1–120 aa or hCLP46 121–392 aa for 48 h, ... We then attempted to evaluate the impact of hCLP46 on TGF-β signaling. Overexpression of hCLP46 inhibited cell viability at basal condition and further exacerbated TGF-β1 induced cell viability inhibition (2 ng/mL, 24 h) (Fig. 2A). In contrast, knockdown of hCLP46 by siRNA increased cell viability and almost totally blocked the inhibition of cell proliferation by TGF-β1 (Fig. 2B). In addition, TGF-β1 treatment significantly increased the expression of two cell cycle inhibitors (p21 and p27) as compared to control cells, which were further enhanced by overexpression of hCLP46 (Fig. 2C and and2D),2D), whereas attenuated by knockdown of hCLP46 (Fig. 2E and and2F),2F), suggesting that hCLP46 enhances TGF-β signaling by modulating Smad3 expression. Figure 2 hCLP46 enhanced TGF-β1 induced cell growth arrest and up-regulation of cell cycle inhibitors. (A) 293TRex-hCLP46 cells were cultured in absence or presence of 0.5 μg/mL Tet for 24 h and 2 ng/mL TGF-β1 was ... As Notch signaling might be involved in Smad3 regulation, we treated cells with DAPT (2 μmol/L, 12 h) or EDTA (5 mmol/L, 15 min and then replaced with fresh DMEM plus 10% FBS and cultured for additional 6 h) to inhibit or activate Notch signaling respectively. DAPT treatment resulted in dramatically decrease of the NICD expression, but has no effect on Smad3 expression (Fig. S2A–C). EDTA significantly increased NICD and Smad3 levels. However, cells with hCLP46 overexpression still had more Smad3 expression than that of control cells (Fig. S2D–F). Together, these data suggest that hCLP46 regulates Smad3 expression is not affected by Notch signaling. As the primary intracellular transducer, Smad3 is a critical mediator of the cytostatic response to TGF-β (Zhang et al., 2014). Evidence for this comes from the observation that a variety of primary cells from Smad3-null mice are partially resistant to TGF-β induced growth arrest (Datto et al., 1999; Yang et al., 1999) and exogenous overexpression of Smad3 sensitizes cells to TGF-β induced growth arrest and apoptosis (Wildey et al., 2003). Importantly, it was found that treatment with the proteasome inhibitor caused an accumulation of Smad3 Protein in absence of TGF-β1, suggesting that not only in response to TGF-β but also in a steady state, the level of Smad3 is regulated by the proteasome pathway (Inoue et al., 2004). In addition, several studies demonstrated that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-β (Guo et al., 2008) and the U-box-containing carboxyl terminus of Hsc70-interacting Protein has been identified as an E3 ubiquitin ligase to degrade Smad3 at steady state (Xin et al., 2005). In agreement with those findings, we demonstrated that hCLP46 modulates Smad3 Protein stability by inhibiting its proteasomal degradation and consequently enhances cellular sensitivity to the TGF-β signal. Our findings suggest a new function of hCLP46 in modulating critical TGF-β/Smad3-regulated processes during development and tumor progression.