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Repression of PKR mediates palmitate-induced apoptosis in HepG2 cells through regulation of Bcl-2.

Yang X, Chan C - Cell Res. (2009)

Bottom Line: Palmitate down-regulates the activity of PKR and thereby decreases the level of Bcl-2 protein, mediated in part by reduced activation of the NF-kappaB transcription factor.The decrease in the phosphorylation of Bcl-2 at Ser70 upon exposure to palmitate is mediated by inhibition of PKR and possibly by c-Jun N-terminal kinase (JNK), whereas the phosphorylation of Bcl-2 at Ser87 is unaffected by palmitate or PKR.In summary, PKR mediates the regulation of the protein level and the phosphorylation status of Bcl-2, providing a novel mechanism of palmitate-induced apoptosis in HepG2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824, USA.

ABSTRACT
The present study shows that double-stranded RNA-dependent protein kinase (PKR) regulates the protein expression level and phosphorylation of Bcl-2 and plays an anti-apoptotic role in human hepatocellular carcinoma cells (HepG2). In various types of cells, saturated free fatty acids (FFAs), such as palmitate, have been shown to induce cellular apoptosis by several mechanisms. Palmitate down-regulates the activity of PKR and thereby decreases the level of Bcl-2 protein, mediated in part by reduced activation of the NF-kappaB transcription factor. In addition to the level of Bcl-2 protein, the phosphorylation of Bcl-2 at different amino acid residues, such as Ser70 and Ser87, is also important in regulating cellular apoptosis. The decrease in the phosphorylation of Bcl-2 at Ser70 upon exposure to palmitate is mediated by inhibition of PKR and possibly by c-Jun N-terminal kinase (JNK), whereas the phosphorylation of Bcl-2 at Ser87 is unaffected by palmitate or PKR. In summary, PKR mediates the regulation of the protein level and the phosphorylation status of Bcl-2, providing a novel mechanism of palmitate-induced apoptosis in HepG2 cells.

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Role of PKR in regulating the Nuclear NF-κB p65 levelReverse transfection of scramble siRNA (control, the first two lanes) or siRNA of PKR (siPKR, the third and fourth lane) was performed followed by forward transfection of empty vector pCMV6-XL5 (pCMV) or the plasmid containing PKR cDNA sequence (hPKR) (A). Cells were then harvested and the nuclear extract was separated from the cytoplasmic fraction, and western blot analysis was performed to detect the level of Bcl-2 in the cytoplasmic fraction and the levels of NF-kB p65 in both the nuclear extract and the cytoplasmic fraction. TBP and beta actin were also measured as loading controls for the nuclear extracts and the cytoplasmic fraction, respectively (A). 90%-confluent HepG2 cells were exposed to a cell-permeable inhibitor of NF-kB, SN50 (18 μM), or its negative control, SN50M (18 μM), in regular media for 24 hours (B). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of Bcl-2 in the whole cell lysates (B). 90%-confluent HepG2 cells were exposed to 0.7 mM palmitate or oleate for 24 hours (C). The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of NF-kB p65 in the nuclear extracts (C).
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Figure 6: Role of PKR in regulating the Nuclear NF-κB p65 levelReverse transfection of scramble siRNA (control, the first two lanes) or siRNA of PKR (siPKR, the third and fourth lane) was performed followed by forward transfection of empty vector pCMV6-XL5 (pCMV) or the plasmid containing PKR cDNA sequence (hPKR) (A). Cells were then harvested and the nuclear extract was separated from the cytoplasmic fraction, and western blot analysis was performed to detect the level of Bcl-2 in the cytoplasmic fraction and the levels of NF-kB p65 in both the nuclear extract and the cytoplasmic fraction. TBP and beta actin were also measured as loading controls for the nuclear extracts and the cytoplasmic fraction, respectively (A). 90%-confluent HepG2 cells were exposed to a cell-permeable inhibitor of NF-kB, SN50 (18 μM), or its negative control, SN50M (18 μM), in regular media for 24 hours (B). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of Bcl-2 in the whole cell lysates (B). 90%-confluent HepG2 cells were exposed to 0.7 mM palmitate or oleate for 24 hours (C). The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of NF-kB p65 in the nuclear extracts (C).

Mentions: Thus far, we showed that palmitate down-regulated the activity of PKR, which played an anti-apoptotic role in HepG2 cells. We identified that by suppressing the PKR activity, palmitate down-regulated the expression level of Bcl-2. Although the mechanism is not fully understood, the positive effect of PKR on the anti-apoptotic protein Bcl-2 could serve as one of the potential pathways by which PKR protects HepG2 cells from apoptosis. It is known that PKR phosphorylates I- κB, which then releases and activates NF-κB (11, 12), the key transcription factor that up-regulates the transcription of Bcl-2 in HepG2 cells (41, 42). Indeed, silencing the gene expression of PKR decreased the level of NF-κB in the nucleus (Fig. 6A, comparing lanes 1 and 3), while over-expressing PKR increased it (Fig. 6A, comparing lanes 1 and 2). Moreover, the decreased level of NF-κB in the nucleus was restored by rescuing the PKR expression in PKR-silenced cells (Fig. 6A, comparing lanes 3 and 4). Furthermore, the PKR inhibitor also decreased the level of NF-κB p65 in the nuclear extract of HepG2 cells (Supplementary Fig. 2B). These results suggest that PKR regulates the activity of NF-κB in HepG2 cells. It has been shown that NF-κB plays a key role in facilitating the transcription of the Bcl-2 gene in liver tumor cell lines, such as U937 and HepG2 cells, and the inhibition of the NF-κB results in down-regulation of the Bcl-2 gene expression (41, 42). Therefore, it is expected that the protein level of Bcl-2 modifies in correspondence with the nuclear level of NF-κB, as shown in Fig. 6A, in which the gene expression level of PKR is modulated. We further confirmed the role of NF-κB in regulating the expression level of Bcl-2 with an inhibitor of NF-κB, NF-κB SN50 (Fig. 6B). Thus, from our results and the literature data, we propose that the transcription factor, NF-κB, mediates PKR regulation of Bcl-2 expression in HepG2 cells. Indeed, we also observed that palmitate decreased the level of NF-κB in the nucleus (Fig. 6C).


Repression of PKR mediates palmitate-induced apoptosis in HepG2 cells through regulation of Bcl-2.

Yang X, Chan C - Cell Res. (2009)

Role of PKR in regulating the Nuclear NF-κB p65 levelReverse transfection of scramble siRNA (control, the first two lanes) or siRNA of PKR (siPKR, the third and fourth lane) was performed followed by forward transfection of empty vector pCMV6-XL5 (pCMV) or the plasmid containing PKR cDNA sequence (hPKR) (A). Cells were then harvested and the nuclear extract was separated from the cytoplasmic fraction, and western blot analysis was performed to detect the level of Bcl-2 in the cytoplasmic fraction and the levels of NF-kB p65 in both the nuclear extract and the cytoplasmic fraction. TBP and beta actin were also measured as loading controls for the nuclear extracts and the cytoplasmic fraction, respectively (A). 90%-confluent HepG2 cells were exposed to a cell-permeable inhibitor of NF-kB, SN50 (18 μM), or its negative control, SN50M (18 μM), in regular media for 24 hours (B). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of Bcl-2 in the whole cell lysates (B). 90%-confluent HepG2 cells were exposed to 0.7 mM palmitate or oleate for 24 hours (C). The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of NF-kB p65 in the nuclear extracts (C).
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Figure 6: Role of PKR in regulating the Nuclear NF-κB p65 levelReverse transfection of scramble siRNA (control, the first two lanes) or siRNA of PKR (siPKR, the third and fourth lane) was performed followed by forward transfection of empty vector pCMV6-XL5 (pCMV) or the plasmid containing PKR cDNA sequence (hPKR) (A). Cells were then harvested and the nuclear extract was separated from the cytoplasmic fraction, and western blot analysis was performed to detect the level of Bcl-2 in the cytoplasmic fraction and the levels of NF-kB p65 in both the nuclear extract and the cytoplasmic fraction. TBP and beta actin were also measured as loading controls for the nuclear extracts and the cytoplasmic fraction, respectively (A). 90%-confluent HepG2 cells were exposed to a cell-permeable inhibitor of NF-kB, SN50 (18 μM), or its negative control, SN50M (18 μM), in regular media for 24 hours (B). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of Bcl-2 in the whole cell lysates (B). 90%-confluent HepG2 cells were exposed to 0.7 mM palmitate or oleate for 24 hours (C). The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of NF-kB p65 in the nuclear extracts (C).
Mentions: Thus far, we showed that palmitate down-regulated the activity of PKR, which played an anti-apoptotic role in HepG2 cells. We identified that by suppressing the PKR activity, palmitate down-regulated the expression level of Bcl-2. Although the mechanism is not fully understood, the positive effect of PKR on the anti-apoptotic protein Bcl-2 could serve as one of the potential pathways by which PKR protects HepG2 cells from apoptosis. It is known that PKR phosphorylates I- κB, which then releases and activates NF-κB (11, 12), the key transcription factor that up-regulates the transcription of Bcl-2 in HepG2 cells (41, 42). Indeed, silencing the gene expression of PKR decreased the level of NF-κB in the nucleus (Fig. 6A, comparing lanes 1 and 3), while over-expressing PKR increased it (Fig. 6A, comparing lanes 1 and 2). Moreover, the decreased level of NF-κB in the nucleus was restored by rescuing the PKR expression in PKR-silenced cells (Fig. 6A, comparing lanes 3 and 4). Furthermore, the PKR inhibitor also decreased the level of NF-κB p65 in the nuclear extract of HepG2 cells (Supplementary Fig. 2B). These results suggest that PKR regulates the activity of NF-κB in HepG2 cells. It has been shown that NF-κB plays a key role in facilitating the transcription of the Bcl-2 gene in liver tumor cell lines, such as U937 and HepG2 cells, and the inhibition of the NF-κB results in down-regulation of the Bcl-2 gene expression (41, 42). Therefore, it is expected that the protein level of Bcl-2 modifies in correspondence with the nuclear level of NF-κB, as shown in Fig. 6A, in which the gene expression level of PKR is modulated. We further confirmed the role of NF-κB in regulating the expression level of Bcl-2 with an inhibitor of NF-κB, NF-κB SN50 (Fig. 6B). Thus, from our results and the literature data, we propose that the transcription factor, NF-κB, mediates PKR regulation of Bcl-2 expression in HepG2 cells. Indeed, we also observed that palmitate decreased the level of NF-κB in the nucleus (Fig. 6C).

Bottom Line: Palmitate down-regulates the activity of PKR and thereby decreases the level of Bcl-2 protein, mediated in part by reduced activation of the NF-kappaB transcription factor.The decrease in the phosphorylation of Bcl-2 at Ser70 upon exposure to palmitate is mediated by inhibition of PKR and possibly by c-Jun N-terminal kinase (JNK), whereas the phosphorylation of Bcl-2 at Ser87 is unaffected by palmitate or PKR.In summary, PKR mediates the regulation of the protein level and the phosphorylation status of Bcl-2, providing a novel mechanism of palmitate-induced apoptosis in HepG2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824, USA.

ABSTRACT
The present study shows that double-stranded RNA-dependent protein kinase (PKR) regulates the protein expression level and phosphorylation of Bcl-2 and plays an anti-apoptotic role in human hepatocellular carcinoma cells (HepG2). In various types of cells, saturated free fatty acids (FFAs), such as palmitate, have been shown to induce cellular apoptosis by several mechanisms. Palmitate down-regulates the activity of PKR and thereby decreases the level of Bcl-2 protein, mediated in part by reduced activation of the NF-kappaB transcription factor. In addition to the level of Bcl-2 protein, the phosphorylation of Bcl-2 at different amino acid residues, such as Ser70 and Ser87, is also important in regulating cellular apoptosis. The decrease in the phosphorylation of Bcl-2 at Ser70 upon exposure to palmitate is mediated by inhibition of PKR and possibly by c-Jun N-terminal kinase (JNK), whereas the phosphorylation of Bcl-2 at Ser87 is unaffected by palmitate or PKR. In summary, PKR mediates the regulation of the protein level and the phosphorylation status of Bcl-2, providing a novel mechanism of palmitate-induced apoptosis in HepG2 cells.

Show MeSH
Related in: MedlinePlus