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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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Related in: MedlinePlus

Effects of palmitate and oleate on the protein level of Bcl-2HepG2 cells were cultured in regular media until reaching 90% confluency, and then exposed to different levels of palmitate (A), 0.7 mM palmitate or 0.7 mM oleate (B) for 24 hours. The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA), in which the concentration of FFAs was 0 (A, B). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of Bcl-2. Quantified Bcl-2 protein level by normalizing to beta actin levels and expressed as average of three 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 lower than control, i.e., regular media with BSA, p<0.01.
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Figure 4: Effects of palmitate and oleate on the protein level of Bcl-2HepG2 cells were cultured in regular media until reaching 90% confluency, and then exposed to different levels of palmitate (A), 0.7 mM palmitate or 0.7 mM oleate (B) for 24 hours. The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA), in which the concentration of FFAs was 0 (A, B). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of Bcl-2. Quantified Bcl-2 protein level by normalizing to beta actin levels and expressed as average of three 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 lower than control, i.e., regular media with BSA, p<0.01.

Mentions: Palmitate decreased the protein level of Bcl-2 (Fig. 4A). Oleate did not have a significant effect on the protein level of Bcl-2 (Fig. 4B). It is unclear from the literature how palmitate would regulate the Bcl-2 protein. However, as illustrated in Figs. 2 and 4, palmitate, concomitantly, decreased the phosphorylation of PKR and the protein level of Bcl-2, suggesting a potential association between PKR and Bcl-2. To confirm this association and test the involvement of PKR in mediating the effect of palmitate on Bcl-2, gene silencing and over-expression of PKR were performed.


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

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

Effects of palmitate and oleate on the protein level of Bcl-2HepG2 cells were cultured in regular media until reaching 90% confluency, and then exposed to different levels of palmitate (A), 0.7 mM palmitate or 0.7 mM oleate (B) for 24 hours. The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA), in which the concentration of FFAs was 0 (A, B). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of Bcl-2. Quantified Bcl-2 protein level by normalizing to beta actin levels and expressed as average of three 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 lower than control, i.e., regular media with BSA, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664847&req=5

Figure 4: Effects of palmitate and oleate on the protein level of Bcl-2HepG2 cells were cultured in regular media until reaching 90% confluency, and then exposed to different levels of palmitate (A), 0.7 mM palmitate or 0.7 mM oleate (B) for 24 hours. The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA), in which the concentration of FFAs was 0 (A, B). After treatment, the cells were harvested, and western blot analysis was performed to detect the protein level of Bcl-2. Quantified Bcl-2 protein level by normalizing to beta actin levels and expressed as average of three 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 lower than control, i.e., regular media with BSA, p<0.01.
Mentions: Palmitate decreased the protein level of Bcl-2 (Fig. 4A). Oleate did not have a significant effect on the protein level of Bcl-2 (Fig. 4B). It is unclear from the literature how palmitate would regulate the Bcl-2 protein. However, as illustrated in Figs. 2 and 4, palmitate, concomitantly, decreased the phosphorylation of PKR and the protein level of Bcl-2, suggesting a potential association between PKR and Bcl-2. To confirm this association and test the involvement of PKR in mediating the effect of palmitate on Bcl-2, gene silencing and over-expression of PKR were performed.

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