Homeostasis is a tightly regulated phenomenon, which requires the continuous interplay of both cell death and cell survival pathways. NF-κB and PI3K/Akt are important regulators of cellular homeostasis. In the context of FasL-mediated apoptosis, NF-κB primarily exerts a pro-survival signal, probably as a defense mechanism in order to protect cells from superfluous death ligand-induced apoptosis (48
). We found that FasL-induced NF-κB activity was ROS dependent in 293T cells, with H2
being the predominant species responsible for NF-κB activation (). This is consistent with previous studies showing that transcriptional inhibition of FasL using antioxidants also leads to the inhibition of NF-κB and a net inhibitory effect on FasL (49
Both PI3K/Akt and NF-κB play a role in protecting cells from undergoing apoptosis (53
). However, this is the first study that demonstrates an inhibitory effect of PI3K/Akt on FasL-induced NF-κB activity. This result is certainly surprising, given that FasL positively stimulates both PI3K/Akt and NF-κB activity ( and ). However, given that PI3K/Akt has also been shown to play a pro-apoptotic role in response to FasL (42
), inhibition of NF-κB fits well with the model of PI3K/Akt-induced apoptosis, since NF-κB is a strong anti-apoptotic signaling molecule and its inhibition may be required for potent induction of cell death (57
Furthermore, this is the first study that demonstrates that c-FLIP can inhibit FasL-induced NF-κB activity (). c-FLIP is a known antioxidant that is regulated by the NF-κB signaling pathway (59
), and c-FLIP in turn can lead to a direct and potent increase in NF-κB activity (10
). The role of c-FLIP as an oncogenic factor in cancer progression and chemo-resistance has been well documented (60
). However, studies assessing its role in tumors predominantly involve high levels of c-FLIP expression (more than 100ng/ml). The ‘physiological’ expression levels of c-FLIP vary depending on cell type, and are typically extremely low, which has hindered the delineation of c-FLIP function under normal cellular conditions (16
). In fact, basal c-FLIP levels were undetectable even in 293T cells in our study (), which has also been corroborated by others (61
). However, there have been a few important studies assessing the role of c-FLIP under physiological conditions, the overall results of which are reaffirmed in our study. For example, Kreuz et al. suggested that physiological levels of c-FLIP exerted an inhibitory effect on FasL-induced NF-κB-driven interleukin-8 activity in a caspase-dependent manner (16
). Also, Chang et al. demonstrated that c-FLIP may play a dual role depending upon its expression levels – at low levels that mimic physiological conditions, c-FLIP is pro-apoptotic, whereas at higher protein levels of c-FLIP such as that observed in tumors, c-FLIP promotes survival (23
). Two recent studies investigating the role of c-FLIP and its processed forms in FasL signaling by Neumann et al. and Fricker et al. from the German Cancer Research Center in Germany have suggested a similar pro-apoptotic effect for c-FLIP at physiological concentrations using a systems biology approach (64
). Overall, our results effectively complement the data presented by the aforementioned studies, while demonstrating a novel role for PI3K/Akt in regulating c-FLIP-mediated inhibition of NF-κB upon FasL treatment.
We demonstrate that at low levels, c-FLIP alone activates NF-κB as expected. However, in the presence of FasL, NF-κB activity was inhibited by c-FLIP in a dose-dependent manner (). Furthermore, low levels of c-FLIP also inhibited H2
levels in the presence of FasL (), which suggested that the inhibition of NF-κB by c-FLIP might be due at least partially to its antioxidant properties, which has been shown to be important in FasL signaling (67
). However, c-FLIP can further inhibit NF-κB even in the presence of NAC, suggesting that a ROS-independent mechanism of NF-κB regulation by c-FLIP may also exist. We now know from corroborating evidence provided by the studies performed in Germany that c-FLIP-driven inhibition of NF-κB may be due to direct interaction of the p43 form of c-FLIP with the IKKγ subunit. The increased processing of c-FLIP upon FasL stimulation observed in our study supports this idea (). Secondly, c-FLIP is capable of inhibiting NF-κB activity even when IKK is upregulated using constitutively active IKKγ construct (). Finally, much higher inhibition of NF-κB is observed when transfected with the p43-FLIP construct as compared to c-FLIPL
. However, the important aspect of this result is the fact that c-FLIP seems to play a regulatory role depending upon its expression levels, with higher levels leading to an exactly opposite effect as proposed by the recent studies (64
). Such concentration-dependent duality in function for c-FLIP is very significant in that it suggests an extremely critical function for c-FLIP in regulation of the cell death. Some of the important findings in 293T cells have also been confirmed using Jurkat T-lymphocytes, which offers further credibility to this study, and makes these findings translatable to physiological conditions (Supplemental Fig. 2
We also observe that both PI3K/Akt and c-FLIP positively regulate each other in the presence of FasL (), and can both inhibit NF-κB, which has not been reported previously. A feedback loop is observed between c-FLIP and Akt, leading to an overall decrease in NF-κB levels, which is corroborated by data observed with co-expression of c-FLIP and Akt-WT (). The modulation of c-FLIP by PI3K/Akt was observed only in c-FLIP-transfected cells (), which suggests that either any increase in endogenous c-FLIP expression with Akt stimulation is undetectable, or that there might be a high turnover of c-FLIP protein, which has also been observed by others (16
). Our results also show that c-FLIP promotes PI3K/Akt-mediated down-regulation of NF-κB, which may be due to the antioxidant properties of c-FLIP, as suggested by recapitulation of this effect upon co-treatment with NAC. Also, we modulated levels of the regulatory IKKγ subunit () and not other subunits of the IKK complex because both c-FLIP and v-FLIP have been previously shown to directly act on IKKγ, thereby exerting effects on overall NF-κB levels (68
As mentioned earlier, processing of c-FLIP was important for inhibition of NF-κB. This is supported by the fact that c-FLIP is found to be processed in the presence of FasL and not in its absence at least in 293T cells (compare lanes 2 and 4 in ), and opposite effects are seen with c-FLIP modulation of NF-κB depending upon either the presence or absence of FasL (). Assessment of NF-κB activity using deletion mutants of c-FLIP that lack one or more important domains indicate that p22-FLIP was particularly effective in inhibiting NF-κB, leading to a much higher decrease in NF-κB activity as compared to full-length c-FLIP (). Although previous studies show that p22-FLIP promotes NF-κB activity, the seeming inconsistency can be explained by the fact that only low levels of c-FLIP (up to 4ng/ml) were used in our study as compared to the much higher levels in previous work. This provides further validation to the dichotomy observed with NF-κB regulation in the context of c-FLIP. Interestingly, the non-S-nitrosylable mutant of c-FLIP caused further inhibition of NF-κB activity, with levels even lower than that observed with p22-FLIP. This suggests that not only is the processing of c-FLIP important for its inhibitory effect on NF-κB, but post-translational modifications such as S-nitrosylation may impede this down-regulation under physiological conditions, and may have an important role to play in c-FLIP associated pathophysiological effects.
In addition to the effects of Akt on c-FLIP, previous studies have also shown that the stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) pathway may also regulate c-FLIP, and can potentially play a regulatory role in the inhibition of NF-κB. However, our data indicates that modulation of JNK activity (using the JNK inhibitor SP600125) did not lead to any significant changes in Akt levels, nor affected downstream proteins at the time-points included in this study (see Supplemental Fig. 1
). However, treatment with SP600125 was able to counter the increases in NF-κB activity when Akt was repressed (see Supplemental Fig. 1D
), suggesting that Akt and JNK may have some form of cross-talk, which needs to be investigated further. In addition, no significant changes in FasR levels were observed for the time-points assayed, suggesting that inhibitory effects of c-FLIP on NF-κB may be independent of FasR levels (Supplemental Fig. 1
FasL is an important stimulator of apoptosis, and we observe a decrease in NF-κB levels with an increase in c-FLIP levels in our system. Since NF-κB is an important pro-survival factor, cells transfected with low levels of c-FLIP show a higher level of FasL-dependent apoptosis as compared to non-transfected cells (). Thus, our study purports the importance of c-FLIP mediated down-regulation of NF-κB as a novel and significant mechanism in induction of FasL-induced apoptosis. Also, in our system, we observe an increase in apoptosis but a decrease in c-FLIP-mediated H2
production. Although this may be counter-intuitive, given that a positive co-relation exists between ROS levels and degree of apoptosis, several studies also show that ROS may also have anti-apoptotic effects (70
). On the other hand, this may suggest that inhibition of NF-κB by physiological levels of c-FLIP may have a more significant impact on overall apoptosis as compared to the putative protective mechanism as a result of the anti-oxidant effects of c-FLIP. Therefore, our model may serve as an alternate and updated representation of the role of c-FLIP signaling in the context of FasL-induced inflammatory response under physiological conditions (). The data presented in this article recapitulates some of the important conclusions drawn by recent studies investigating the physiological role of c-FLIP, and highlights the duality of c-FLIP signaling based upon levels of both c-FLIP itself and of FasL, and the resulting effect on overall apoptosis (). In addition, our data also hints at the importance of other factors such as NO (which contributes to S
-nitrosylation of c-FLIP), which may have an influence on the function of c-FLIP, and will be pursued in the future.
Model for c-FLIP-induced apoptosis at low levels of c-FLIP expression
We believe that this study would help pave the way for further understanding of FasL-mediated inflammatory response, and shed new light on the role of PI3K/Akt and c-FLIP in apoptosis, which are traditionally understood to be pro-survival factors. Particularly, such studies are important to extend the understanding of c-FLIP under normal biological conditions including growth and immune response, and may lay the foundation for detailed mechanistic studies on c-FLIP and its role in apoptosis.