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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 cytotoxicity and apoptosis of HepG2 cellsReverse transfection of suspended HepG2 cells were performed with scrambled siRNA (Control) or siRNA of PKR for 24 hours and the transfected cells were cultured in regular media for another 24 hours (A, B). Cells were then harvested, and RT-PCR and western blot analysis were performed to detect the gene, protein and phosphorylation levels of PKR to confirm that the PKR gene was silenced and the activity of PKR was suppressed (A). LDH release, Caspase-3 activity, and DNA fragmentation were assayed (B). In (C), reverse transfection of scramble siRNA (■, control) or siRNA of PKR (▨, siPKR) was performed followed by forward transfection of empty vector pCMV6-XL5 (pCMV) or the plasmid containing PKR cDNA sequence (hPKR). Cells were then harvested and caspase-3 activity was assayed (C). Data expressed as average of three (A) or nine (B, C) samples ± SD from three independent experiments. One-way ANOVA with Tukey’s post hoc method was used for analyzing the differences between treatment groups. ★, significantly higher (B, C) or lower (A, C), than control, p<0.01. #, significantly lower than siPKR-CMV in Fig. C, p<0.01.
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Figure 3: Role of PKR in cytotoxicity and apoptosis of HepG2 cellsReverse transfection of suspended HepG2 cells were performed with scrambled siRNA (Control) or siRNA of PKR for 24 hours and the transfected cells were cultured in regular media for another 24 hours (A, B). Cells were then harvested, and RT-PCR and western blot analysis were performed to detect the gene, protein and phosphorylation levels of PKR to confirm that the PKR gene was silenced and the activity of PKR was suppressed (A). LDH release, Caspase-3 activity, and DNA fragmentation were assayed (B). In (C), reverse transfection of scramble siRNA (■, control) or siRNA of PKR (▨, siPKR) was performed followed by forward transfection of empty vector pCMV6-XL5 (pCMV) or the plasmid containing PKR cDNA sequence (hPKR). Cells were then harvested and caspase-3 activity was assayed (C). Data expressed as average of three (A) or nine (B, C) samples ± SD from three independent experiments. One-way ANOVA with Tukey’s post hoc method was used for analyzing the differences between treatment groups. ★, significantly higher (B, C) or lower (A, C), than control, p<0.01. #, significantly lower than siPKR-CMV in Fig. C, p<0.01.

Mentions: The siRNA targeting PKR employed in the present study markedly inhibited the gene and protein expression of PKR, and thereby reduced the level of phosphorylated PKR (Fig. 3A). Silencing PKR with this siRNA increased the activity of caspase-3 significantly (Fig. 3B) but not the release of LDH (Fig. 3B), and induced fragmentation of chromatin DNA (Fig. 3B), suggesting that PKR has an anti-apoptotic role in HepG2 cells. To confirm the role of PKR in apoptosis, we over-expressed and rescued the PKR expression level in PKR-silenced cells and found that the caspases-3 activity was reduced to levels close to control (Fig. 3C). Taken together, these results suggest that PKR plays an anti-apoptotic role in HepG2 cells. To further confirm a catalytic role of PKR in regulating apoptosis, we inhibited the activity of PKR with a pharmaceutical inhibitor of PKR (59-61) and found that, similar to the siRNA of PKR, the PKR inhibitor also induced apoptosis in HepG2 cells, as evidenced by the caspase-3 activity and DNA fragmentation (supplementary Fig. 1). Considering the negative effect of palmitate on the activity of PKR, we therefore proposed that palmitate induces apoptosis, in part, by repressing PKR.


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 cytotoxicity and apoptosis of HepG2 cellsReverse transfection of suspended HepG2 cells were performed with scrambled siRNA (Control) or siRNA of PKR for 24 hours and the transfected cells were cultured in regular media for another 24 hours (A, B). Cells were then harvested, and RT-PCR and western blot analysis were performed to detect the gene, protein and phosphorylation levels of PKR to confirm that the PKR gene was silenced and the activity of PKR was suppressed (A). LDH release, Caspase-3 activity, and DNA fragmentation were assayed (B). In (C), reverse transfection of scramble siRNA (■, control) or siRNA of PKR (▨, siPKR) was performed followed by forward transfection of empty vector pCMV6-XL5 (pCMV) or the plasmid containing PKR cDNA sequence (hPKR). Cells were then harvested and caspase-3 activity was assayed (C). Data expressed as average of three (A) or nine (B, C) samples ± SD from three independent experiments. One-way ANOVA with Tukey’s post hoc method was used for analyzing the differences between treatment groups. ★, significantly higher (B, C) or lower (A, C), than control, p<0.01. #, significantly lower than siPKR-CMV in Fig. C, p<0.01.
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Figure 3: Role of PKR in cytotoxicity and apoptosis of HepG2 cellsReverse transfection of suspended HepG2 cells were performed with scrambled siRNA (Control) or siRNA of PKR for 24 hours and the transfected cells were cultured in regular media for another 24 hours (A, B). Cells were then harvested, and RT-PCR and western blot analysis were performed to detect the gene, protein and phosphorylation levels of PKR to confirm that the PKR gene was silenced and the activity of PKR was suppressed (A). LDH release, Caspase-3 activity, and DNA fragmentation were assayed (B). In (C), reverse transfection of scramble siRNA (■, control) or siRNA of PKR (▨, siPKR) was performed followed by forward transfection of empty vector pCMV6-XL5 (pCMV) or the plasmid containing PKR cDNA sequence (hPKR). Cells were then harvested and caspase-3 activity was assayed (C). Data expressed as average of three (A) or nine (B, C) samples ± SD from three independent experiments. One-way ANOVA with Tukey’s post hoc method was used for analyzing the differences between treatment groups. ★, significantly higher (B, C) or lower (A, C), than control, p<0.01. #, significantly lower than siPKR-CMV in Fig. C, p<0.01.
Mentions: The siRNA targeting PKR employed in the present study markedly inhibited the gene and protein expression of PKR, and thereby reduced the level of phosphorylated PKR (Fig. 3A). Silencing PKR with this siRNA increased the activity of caspase-3 significantly (Fig. 3B) but not the release of LDH (Fig. 3B), and induced fragmentation of chromatin DNA (Fig. 3B), suggesting that PKR has an anti-apoptotic role in HepG2 cells. To confirm the role of PKR in apoptosis, we over-expressed and rescued the PKR expression level in PKR-silenced cells and found that the caspases-3 activity was reduced to levels close to control (Fig. 3C). Taken together, these results suggest that PKR plays an anti-apoptotic role in HepG2 cells. To further confirm a catalytic role of PKR in regulating apoptosis, we inhibited the activity of PKR with a pharmaceutical inhibitor of PKR (59-61) and found that, similar to the siRNA of PKR, the PKR inhibitor also induced apoptosis in HepG2 cells, as evidenced by the caspase-3 activity and DNA fragmentation (supplementary Fig. 1). Considering the negative effect of palmitate on the activity of PKR, we therefore proposed that palmitate induces apoptosis, in part, by repressing PKR.

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