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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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Silencing of C/EBPα expression by siRNAs enhances NSC606985/etoposide-induced apoptosis in leukemic cells.(A) U937 cells were stably transfected with siRNA C1–3 against C/EBPα or negative control vector (NC), and C/EBPα and Bcl-2 proteins were blotted with β-actin as a loading control. (B) U937 cells with stable transfections of C1-C3 or NC were treated with 2 µM etoposide or 200 nM NSC606985 for 24 hours, and apoptotic sub-G1 cells% were determined on flow cytometry. (C) U937 cells with stable transfections of C1-C3 or NC were treated with 2 µM etoposide or 200 nM NSC606985 for 36 hours, and annexin-V+ cells% were determined on flow cytometry. The values represent mean±S.D. of triplicates in an independent experiment, which was repeated more than three times with the same results. The symbols * and # represent P<0.01 and <0.05 compared with NC cells with the corresponding treatment.
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pone-0006552-g007: Silencing of C/EBPα expression by siRNAs enhances NSC606985/etoposide-induced apoptosis in leukemic cells.(A) U937 cells were stably transfected with siRNA C1–3 against C/EBPα or negative control vector (NC), and C/EBPα and Bcl-2 proteins were blotted with β-actin as a loading control. (B) U937 cells with stable transfections of C1-C3 or NC were treated with 2 µM etoposide or 200 nM NSC606985 for 24 hours, and apoptotic sub-G1 cells% were determined on flow cytometry. (C) U937 cells with stable transfections of C1-C3 or NC were treated with 2 µM etoposide or 200 nM NSC606985 for 36 hours, and annexin-V+ cells% were determined on flow cytometry. The values represent mean±S.D. of triplicates in an independent experiment, which was repeated more than three times with the same results. The symbols * and # represent P<0.01 and <0.05 compared with NC cells with the corresponding treatment.

Mentions: Finally, three pairs of siRNAs (C1–3) against C/EBPα were designed and transfected into U937 cells. With selection by G418, C2 and C3 siRNAs but not C1 siRNA significantly inhibited C/EBPα protein expression, compared with negative control (Figure 7A). The suppression of C/EBPα expression by C2 and C3 siRNAs statistically significantly enhanced NSC606985/etoposide-induced activation of caspase-3 (Figure S6) and apoptosis, the latter being determined by the percentages of sub-G1 cells (Figure 7B), annexin-V+ cells (Figure 7C) as well as cell morphology (Figure S7). On the other hand, an inducible C/EBPα-expressing cell line (U937C/EBPα) was generated using myeloid leukemic U937T cells as described above. U937empty cell line with transfection of empty vector was used as a control. As can be visualized in Figure 8A, C/EBPα protein was significantly induced at 6 days after tetracycline withdrawal in U937C/EBPα cells. Hence, we treated U937empty and U937C/EBPα cells with NSC606985 or etoposide when tetracycline was removed for 8 days. The results showed that the inducible expression of C/EBPα partially inhibited NSC606985/etoposide-induced caspase-3 action (Figure S8) and apoptosis to a statistic degree (Figure 8B/C and Figure S8). These results supported the anti-apoptotic role of C/EBPα. Additionally, conditional expression of C/EBPα protein failed to alter expressions of apoptosis-related Bcl-2, Bak, Bax and Mcl-1 genes in U937C/EBPα cells (Figure 8D), and the suppression of C/EBPα expression by siRNAs also failed to alter Bcl-2 protein level (Figure 7A).


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)

Silencing of C/EBPα expression by siRNAs enhances NSC606985/etoposide-induced apoptosis in leukemic cells.(A) U937 cells were stably transfected with siRNA C1–3 against C/EBPα or negative control vector (NC), and C/EBPα and Bcl-2 proteins were blotted with β-actin as a loading control. (B) U937 cells with stable transfections of C1-C3 or NC were treated with 2 µM etoposide or 200 nM NSC606985 for 24 hours, and apoptotic sub-G1 cells% were determined on flow cytometry. (C) U937 cells with stable transfections of C1-C3 or NC were treated with 2 µM etoposide or 200 nM NSC606985 for 36 hours, and annexin-V+ cells% were determined on flow cytometry. The values represent mean±S.D. of triplicates in an independent experiment, which was repeated more than three times with the same results. The symbols * and # represent P<0.01 and <0.05 compared with NC cells with the corresponding treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2719015&req=5

pone-0006552-g007: Silencing of C/EBPα expression by siRNAs enhances NSC606985/etoposide-induced apoptosis in leukemic cells.(A) U937 cells were stably transfected with siRNA C1–3 against C/EBPα or negative control vector (NC), and C/EBPα and Bcl-2 proteins were blotted with β-actin as a loading control. (B) U937 cells with stable transfections of C1-C3 or NC were treated with 2 µM etoposide or 200 nM NSC606985 for 24 hours, and apoptotic sub-G1 cells% were determined on flow cytometry. (C) U937 cells with stable transfections of C1-C3 or NC were treated with 2 µM etoposide or 200 nM NSC606985 for 36 hours, and annexin-V+ cells% were determined on flow cytometry. The values represent mean±S.D. of triplicates in an independent experiment, which was repeated more than three times with the same results. The symbols * and # represent P<0.01 and <0.05 compared with NC cells with the corresponding treatment.
Mentions: Finally, three pairs of siRNAs (C1–3) against C/EBPα were designed and transfected into U937 cells. With selection by G418, C2 and C3 siRNAs but not C1 siRNA significantly inhibited C/EBPα protein expression, compared with negative control (Figure 7A). The suppression of C/EBPα expression by C2 and C3 siRNAs statistically significantly enhanced NSC606985/etoposide-induced activation of caspase-3 (Figure S6) and apoptosis, the latter being determined by the percentages of sub-G1 cells (Figure 7B), annexin-V+ cells (Figure 7C) as well as cell morphology (Figure S7). On the other hand, an inducible C/EBPα-expressing cell line (U937C/EBPα) was generated using myeloid leukemic U937T cells as described above. U937empty cell line with transfection of empty vector was used as a control. As can be visualized in Figure 8A, C/EBPα protein was significantly induced at 6 days after tetracycline withdrawal in U937C/EBPα cells. Hence, we treated U937empty and U937C/EBPα cells with NSC606985 or etoposide when tetracycline was removed for 8 days. The results showed that the inducible expression of C/EBPα partially inhibited NSC606985/etoposide-induced caspase-3 action (Figure S8) and apoptosis to a statistic degree (Figure 8B/C and Figure S8). These results supported the anti-apoptotic role of C/EBPα. Additionally, conditional expression of C/EBPα protein failed to alter expressions of apoptosis-related Bcl-2, Bak, Bax and Mcl-1 genes in U937C/EBPα cells (Figure 8D), and the suppression of C/EBPα expression by siRNAs also failed to alter Bcl-2 protein level (Figure 7A).

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