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Protein kinase Cdelta stimulates proteasome-dependent degradation of C/EBPalpha during apoptosis induction of leukemic cells.

Zhao M, Duan XF, Zhao XY, Zhang B, Lu Y, Liu W, Cheng JK, Chen GQ - PLoS ONE (2009)

Bottom Line: More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment.Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT

Background: The precise regulation and maintenance of balance between cell proliferation, differentiation and death in metazoan are critical for tissue homeostasis. CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been implicated as a key regulator of differentiation and proliferation in various cell types. Here we investigated the potential dynamic change and role of C/EBPalpha protein during apoptosis induction.

Methodology/principal findings: Upon onset of apoptosis induced by various kinds of inducers such as NSC606985, etoposide and others, C/EBPalpha expression presented a profound down-regulation in leukemic cell lines and primary cells via induction of protein degradation and inhibition of transcription, as assessed respectively by cycloheximide inhibition test, real-time quantitative RT-PCR and luciferase reporter assay. Applying chemical inhibition, forced expression of dominant negative mutant and catalytic fragment (CF) of protein kinase Cdelta (PKCdelta), which was proteolytically activated during apoptosis induction tested, we showed that the active PKCdelta protein contributed to the increased degradation of C/EBPalpha protein. Three specific proteasome inhibitors antagonized C/EBPalpha degradation during apoptosis induction. More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment. Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.

Conclusions/significance: These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

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Related in: MedlinePlus

The decrease of C/EBPα protein during apoptosis involves induction of protein degradation and inhibition of transcription.(A) After pretreatment with 25 nM of NSC606985 or 0.5 µM of etoposide for 12 hours, NB4 cells were incubated with 10 µg/ml CHX for indicated hours. Then, C/EBPα protein was detected with β-actin as a loading control. Folds of decrease of C/EBPα protein/β-actin ratios against untreated cells are shown as means±SD of three independent experiments. (B) NB4 (top) and U937 cells(bottom) were treated respectively with 25 nM and 50 nM of NSC606985 (left) or 0.5 µM and 1 µM etoposide (right) for hours as indicated, and CEBPA mRNA level was detected by real-time quantitative RT-PCR. (C) NB4 (top) and U937 cells (bottom) were transfected with CEBPA promotor-luciferase plasmid together with pRL-SV40 vector. After 24 hours of transfection, these cells were treated respectively with 25 nM and 50 nM of NSC606985 (left) or 0.5 µM and 1 µM etoposide (right) for hours as indicated. Then, the cells were harvested and the relative luciferase activities were measured. The columns represent means of fold changes against untreated cells, with the bar as S.D. of three independent experiments each with triplicates The symbol * indicates P value compared with untreated cells.
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pone-0006552-g002: The decrease of C/EBPα protein during apoptosis involves induction of protein degradation and inhibition of transcription.(A) After pretreatment with 25 nM of NSC606985 or 0.5 µM of etoposide for 12 hours, NB4 cells were incubated with 10 µg/ml CHX for indicated hours. Then, C/EBPα protein was detected with β-actin as a loading control. Folds of decrease of C/EBPα protein/β-actin ratios against untreated cells are shown as means±SD of three independent experiments. (B) NB4 (top) and U937 cells(bottom) were treated respectively with 25 nM and 50 nM of NSC606985 (left) or 0.5 µM and 1 µM etoposide (right) for hours as indicated, and CEBPA mRNA level was detected by real-time quantitative RT-PCR. (C) NB4 (top) and U937 cells (bottom) were transfected with CEBPA promotor-luciferase plasmid together with pRL-SV40 vector. After 24 hours of transfection, these cells were treated respectively with 25 nM and 50 nM of NSC606985 (left) or 0.5 µM and 1 µM etoposide (right) for hours as indicated. Then, the cells were harvested and the relative luciferase activities were measured. The columns represent means of fold changes against untreated cells, with the bar as S.D. of three independent experiments each with triplicates The symbol * indicates P value compared with untreated cells.

Mentions: To test whether C/EBPα expression is regulated at the post-transcriptional level during apoptosis, NB4 cells were treated with 10 µg/ml cycloheximide (CHX) and/or NSC606985 or etoposide for the different times. As shown in Figure 2A, half-life of C/EBPα protein in NB4 cells was more than 8 hours. In NSC606985 or etoposide-treated cells, however, C/EBPα protein disappeared or decreased to less than 50% at 4 hours after CHX blockage of protein synthesis, indicating that the stabilization of the C/EBPα protein was also lowered upon apoptosis induction. Evan so, we continued to dynamically measure the mRNA level of CEBPA in NSC606985- or etoposide-treated NB4 and U937 cells by real-time quantitative RT-PCR. The results revealed that NSC606985/etoposide-treated NB4 cells also presented reduced CEBPA mRNA, which initially appeared at 6 hours and 24 hours after treatment of NSC606985 and etoposide respectively, and became more significant later (Figure 2B). The reduced CEBPA mRNA could also be seen in NSC606985/etoposide-treated U937 cells, but CEBPA mRNA also experienced a rapid but temporary increase before the down-regulation in U937 cells (Figure 2B). This was also true for C/EBPα protein in U937 but not in NB4 cells treated by both NSC606985 and etoposide, although statistic significance was absent (Figure 1A/B). Furthermore, a CEBPA promoter-driven luciferase reporter assay also supported NSC606985 and etoposide significantly inhibited CEBPA gene promoter-driven luciferase transcription in both NB4 and U937 cells (Figure 2C). It should be point out that although the transitory elevation of CEBPA gene promoter-driven transcription could also be tested in U937 cells under the treatment of NSC606985 or etoposide, the early elevation of the reporter gene is very weak which indicated that there may be other mechanisms involved in the transient elevation of CEBPA mRNA. All these results proposed that both reduced transcription and increased degradation contributed to the down-regulation of C/EBPα expression during apoptosis induction.


Protein kinase Cdelta stimulates proteasome-dependent degradation of C/EBPalpha during apoptosis induction of leukemic cells.

Zhao M, Duan XF, Zhao XY, Zhang B, Lu Y, Liu W, Cheng JK, Chen GQ - PLoS ONE (2009)

The decrease of C/EBPα protein during apoptosis involves induction of protein degradation and inhibition of transcription.(A) After pretreatment with 25 nM of NSC606985 or 0.5 µM of etoposide for 12 hours, NB4 cells were incubated with 10 µg/ml CHX for indicated hours. Then, C/EBPα protein was detected with β-actin as a loading control. Folds of decrease of C/EBPα protein/β-actin ratios against untreated cells are shown as means±SD of three independent experiments. (B) NB4 (top) and U937 cells(bottom) were treated respectively with 25 nM and 50 nM of NSC606985 (left) or 0.5 µM and 1 µM etoposide (right) for hours as indicated, and CEBPA mRNA level was detected by real-time quantitative RT-PCR. (C) NB4 (top) and U937 cells (bottom) were transfected with CEBPA promotor-luciferase plasmid together with pRL-SV40 vector. After 24 hours of transfection, these cells were treated respectively with 25 nM and 50 nM of NSC606985 (left) or 0.5 µM and 1 µM etoposide (right) for hours as indicated. Then, the cells were harvested and the relative luciferase activities were measured. The columns represent means of fold changes against untreated cells, with the bar as S.D. of three independent experiments each with triplicates The symbol * indicates P value compared with untreated cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2719015&req=5

pone-0006552-g002: The decrease of C/EBPα protein during apoptosis involves induction of protein degradation and inhibition of transcription.(A) After pretreatment with 25 nM of NSC606985 or 0.5 µM of etoposide for 12 hours, NB4 cells were incubated with 10 µg/ml CHX for indicated hours. Then, C/EBPα protein was detected with β-actin as a loading control. Folds of decrease of C/EBPα protein/β-actin ratios against untreated cells are shown as means±SD of three independent experiments. (B) NB4 (top) and U937 cells(bottom) were treated respectively with 25 nM and 50 nM of NSC606985 (left) or 0.5 µM and 1 µM etoposide (right) for hours as indicated, and CEBPA mRNA level was detected by real-time quantitative RT-PCR. (C) NB4 (top) and U937 cells (bottom) were transfected with CEBPA promotor-luciferase plasmid together with pRL-SV40 vector. After 24 hours of transfection, these cells were treated respectively with 25 nM and 50 nM of NSC606985 (left) or 0.5 µM and 1 µM etoposide (right) for hours as indicated. Then, the cells were harvested and the relative luciferase activities were measured. The columns represent means of fold changes against untreated cells, with the bar as S.D. of three independent experiments each with triplicates The symbol * indicates P value compared with untreated cells.
Mentions: To test whether C/EBPα expression is regulated at the post-transcriptional level during apoptosis, NB4 cells were treated with 10 µg/ml cycloheximide (CHX) and/or NSC606985 or etoposide for the different times. As shown in Figure 2A, half-life of C/EBPα protein in NB4 cells was more than 8 hours. In NSC606985 or etoposide-treated cells, however, C/EBPα protein disappeared or decreased to less than 50% at 4 hours after CHX blockage of protein synthesis, indicating that the stabilization of the C/EBPα protein was also lowered upon apoptosis induction. Evan so, we continued to dynamically measure the mRNA level of CEBPA in NSC606985- or etoposide-treated NB4 and U937 cells by real-time quantitative RT-PCR. The results revealed that NSC606985/etoposide-treated NB4 cells also presented reduced CEBPA mRNA, which initially appeared at 6 hours and 24 hours after treatment of NSC606985 and etoposide respectively, and became more significant later (Figure 2B). The reduced CEBPA mRNA could also be seen in NSC606985/etoposide-treated U937 cells, but CEBPA mRNA also experienced a rapid but temporary increase before the down-regulation in U937 cells (Figure 2B). This was also true for C/EBPα protein in U937 but not in NB4 cells treated by both NSC606985 and etoposide, although statistic significance was absent (Figure 1A/B). Furthermore, a CEBPA promoter-driven luciferase reporter assay also supported NSC606985 and etoposide significantly inhibited CEBPA gene promoter-driven luciferase transcription in both NB4 and U937 cells (Figure 2C). It should be point out that although the transitory elevation of CEBPA gene promoter-driven transcription could also be tested in U937 cells under the treatment of NSC606985 or etoposide, the early elevation of the reporter gene is very weak which indicated that there may be other mechanisms involved in the transient elevation of CEBPA mRNA. All these results proposed that both reduced transcription and increased degradation contributed to the down-regulation of C/EBPα expression during apoptosis induction.

Bottom Line: More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment.Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT

Background: The precise regulation and maintenance of balance between cell proliferation, differentiation and death in metazoan are critical for tissue homeostasis. CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been implicated as a key regulator of differentiation and proliferation in various cell types. Here we investigated the potential dynamic change and role of C/EBPalpha protein during apoptosis induction.

Methodology/principal findings: Upon onset of apoptosis induced by various kinds of inducers such as NSC606985, etoposide and others, C/EBPalpha expression presented a profound down-regulation in leukemic cell lines and primary cells via induction of protein degradation and inhibition of transcription, as assessed respectively by cycloheximide inhibition test, real-time quantitative RT-PCR and luciferase reporter assay. Applying chemical inhibition, forced expression of dominant negative mutant and catalytic fragment (CF) of protein kinase Cdelta (PKCdelta), which was proteolytically activated during apoptosis induction tested, we showed that the active PKCdelta protein contributed to the increased degradation of C/EBPalpha protein. Three specific proteasome inhibitors antagonized C/EBPalpha degradation during apoptosis induction. More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment. Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.

Conclusions/significance: These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

Show MeSH
Related in: MedlinePlus