Apoptosis, programmed cell death, is a major cellular activity and is highly involved in inflammatory responses, tissue development, control of tumor growth, and many other processes. In addition, recent studies revealed strong relationships exist between apoptosis and metabolism. For example, certain regulators of the apoptotic pathway alter the mitochondria membrane which affects cellular metabolism and ATP generation. On the other hand, metabolites such as free fatty acids (FFAs) have distinct roles in regulating cellular apoptosis. Saturated FFAs (e.g. palmitate) has been found to induce apoptosis in cardiac cells, pancreatic beta cells, breast cancer cells, and many other cell types. On the other hand, unsaturated FFAs (e.g. oleate) has been shown to protect cells from apoptosis induced by saturated FFAs, by restoring the cardiolipin levels and directing the esterification of saturated FFAs to triacylglycerol (TG) accumulation. Research has shown that palmitate-induced apoptosis in various cell types is dependent upon different mechanisms, such as the accumulation of ceramide and TG, production of reactive oxygen species (ROS) and nitric oxide (NO), reduced mitochondrial potential and the protein level of Bcl-2. However, previous studies on palmitate-induced apoptosis focused predominantly on the intracellular metabolic pathways, such as glucose metabolism, and the production of ceramide and ROS, and not on the signal transduction pathways activated by palmitate. As a result, it remains unclear how palmitate affects the Bcl-2 protein and what the intermediates are that mediate their connections. It is known that the transcription of the Bcl-2 gene is up-regulated by NF-κB, and the phosphorylation status of the Bcl-2 protein is regulated by certain protein kinases (e.g. MAPKs) or Ser/Thr protein phosphatases (e.g. PP2A). Interestingly, these regulators of Bcl-2, namely, NF-κB, MAPKs, and PP2A, are possibly associated with the double-stranded RNA-dependent protein kinase (PKR). In the present study we identified that PKR is involved, as well as how it is involved, in mediating palmitate-induced apoptosis through Bcl-2.

PKR, a protein kinase involved in a number of signal transduction pathways, is known to trigger cell defense mechanisms by regulating cellular apoptosis during virus infection. Upon auto-phosphorylation and dimerization, PKR phosphorylates the eukaryotic translation initiation factor, eIF-2α, which directly leads to cell death. PKR is known to affect other apoptosis-regulating factors, such as, NF-κB through I-κB kinase complex (IKK), protein phosphatase 2A (PP2A), and MAPKs. In the human hepatocellular carcinoma cells (HepG2 cell line), we have identified for the first time that palmitate and TNF-α down-regulate the activity of PKR, with a concomitant decrease in the Bcl-2 protein level. We identified that PKR is required to maintain the level of Bcl-2 in HepG2 cells by using pharmaceutical inhibitors of PKR activity and siRNA of the PKR gene. It is well-established that the transcription factor, NF-κB, can be activated by PKR, and is involved in up-regulating the transcription of Bcl-2. Therefore, we proposed that palmitate and TNF-α induce apoptosis, in part, by repressing the PKR- and NF-κB-mediated up-regulation of the Bcl-2 gene transcription. In addition, the phosphorylation of Bcl-2 at the different amino acid residues is important in defining the pro- or anti-apoptotic roles of Bcl-2. It has been shown that the phosphorylation of Bcl-2 at the anti-apoptotic site, Ser70, sustains the anti-apoptotic role of Bcl-2, while the phosphorylation of Bcl-2 at serine 87 results in the reduction of the anti-apoptotic function of Bcl-2, possibly by inhibiting the phosphorylation of Bcl-2 at Ser70 or destabilizing the Bcl-2 protein. We showed that palmitate and TNF-α both elicit opposing effects on these two sites: the phosphorylation at Ser70 was decreased while the phosphorylation at Ser87 was increased. Inhibitor studies and gene silencing of PKR both support the hypothesis that PKR is involved in mediating the effects of palmitate and TNF-α on the phosphorylation of Bcl-2. We further verified that PKR activates PP2A and thus decreases the phosphorylation of Bcl-2 at the pro-apoptotic site, Ser87, whereas the effect on the anti-apoptotic site, Ser 70, is less straightforward. Through co-immunoprecipitation analysis, we identified for the first time that one of the MAPKs interacts directly with both PKR and Bcl-2. This MAPK antagonizes the effects of PP2A and increases the phosphorylation of Bcl-2 at the Ser 70 residue. In summary, we have re-constructed two signaling pathways, starting from palmitate to PKR, which leads to the regulation of the protein level and the phosphorylation status of the Bcl-2 protein. Our findings provided a new mechanism that reveals how exogenous palmitate and TNF-α regulate apoptosis in HepG2 cells.

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