The idea that cross-talk exists between extrinsic and intrinsic apoptotic pathways in certain cell types is now widely accepted; after all, cross-talk is a common theme in the world of signal transduction. However, a small number of groups remain cautious in accepting this notion. Andreas Strasser and colleagues point out that many studies use agonistic antibodies to CD95 to stimulate the extrinsic pathway. In their hands, these agonistic antibodies do not behave as the physiological form of the CD95L that, they claim, is multimeric in nature (either membrane-associated CD95L or cross-linked CD95L). While they do not observe significant hepatocyte apoptosis in the presence of anti-CD95 antibodies, these cells are sensitive to the CD95L when provided in a multimeric form 12. Moreover, the apoptotic cell death observed with the multimeric ligand is not inhibited by overexpression of Bcl-2. In response to this criticism, Schmitz et al. (Krammer group) demonstrated that the differential sensitivity to Bcl-2 in type I and type II cells is maintained when the extrinsic pathway is stimulated by the CD95L rather than agonistic antibodies to CD95 13. It is important to note, however, that the CD95L used in this study was trimeric rather than multimeric and thus differs from that used in the studies by Strasser and colleagues 14.

Is the nature of the ligand solely responsible for the discrepant results obtained by the groups of Krammer/Peter and colleagues and Strasser and colleagues in hepatocytes and other type II cells? Moreover, and importantly, is the multimeric CD95L actually a more physiological form of the ligand? Would mice defective in Bid retain resistance to hepatocellular injury in response to a more physiological stimulus of the CD95 pathway?

The nature of the stimulus of the CD95 pathway does not explain the fact that Strasser and colleagues do not observe significant cell death when type II cells are treated with anti-CD95 antibodies or trimeric CD95L, whereas Krammer and colleagues and several other groups do. These discrepant results might be due to differences in the ‘tone’ of the intrinsic pathway (i.e., the sensitivity of the intrinsic pathway to apoptotic stimuli) and the tone of the intrinsic pathway might be influenced by the array of growth factors and cytokines to which the cells are exposed. Villunger et al. elegantly illustrate this concept in the September 4 issue of this journal 15. They demonstrate that the proinflammatory cytokine G-CSF stimulates the extracellular signal–regulated kinase (ERK) pathway and inhibits spontaneous apoptosis of granulocytes as well as apoptosis induced by stimuli of the intrinsic pathway (etoposide and doxorubicin) but not apoptosis induced by stimulation of the extrinsic pathway with multimerized CD95L (granulocytes are resistant to weaker stimuli of the extrinsic pathway such as agonistic antibodies to CD95 or trimerized CD95L). Similarly, G-CSF prevents the death of granulocytes stimulated by inhibitors of the mitogen-activated protein kinase (MAPK) pathway. Given that G-CSF does not directly modulate the p38 MAPK pathway, Villunger et al. suggest that both signal transduction pathways converge to regulate the expression and/or activity of molecules that interfere specifically with the intrinsic but not the extrinsic pathway. They propose that the p38 MAPK and ERK pathways inhibit the intrinsic apoptotic pathway by shifting the delicate balance between pro- and antiapoptotic Bcl-2 family members, stimulating either the overexpression of antiapoptotic Bcl-2 family members or the downregulation of proapoptotic Bcl-2 family members (although Western blot analyses failed to detect significant differences in the expression of either pro- or antiapoptotic Bcl-2 family members in response to G-CSF). This hypothesis is supported by the observation that overexpression of Bcl-2 acts like G-CSF to protect granulocytes from spontaneous apoptosis as well as apoptosis stimulated by doxorubicin, cisplatin, and p38 MAPK inhibitors but not from multimeric CD95.

Although the mechanism by which the complex interplay between the signal transduction pathways and the intrinsic apoptotic pathway remains ill defined, this interplay is no less important and might be responsible for the differences in sensitivity of cells to anti-CD95 antibodies observed by Strasser and colleagues and Krammer and colleagues, as illustrated in Fig. 2. Alterations in the tone of the intrinsic apoptotic pathway may affect the ability of cells to respond to suboptimal stimulation of the extrinsic pathway in type II cells using anti-CD95 antibodies or trimeric CD95L (type I cells, for reasons that remain obscure, appear to support productive formation of the DISC (CD95, FADD/MORT1, caspase-8), irrespective of the strength of the stimulus). Type II cells, exposed to cytokines or growth factors that activate the signal transduction pathways and increase the resistance of mitochondria to the cytochrome c–releasing effects caused by cleavage of Bid, might behave as reported by Huang et al. (Strasser group; reference 12). These cells survive a weak CD95 stimulus (anti-CD95 antibodies or trimeric CD95L) that is dependent on the intrinsic pathway but die in the presence of a strong CD95 stimulus (e.g., multimeric CD95L) that is not dependent on the intrinsic pathway. In contrast, cells maintained in an environment impoverished in cytokines/growth factors might behave as reported by Scaffidi et al. (Krammer group; reference 5). These cells exhibit a proapoptotic response to a weak CD95 stimulus that is inhibitable by Bcl-2 overexpression or Bid deficiency.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 2 How apoptotic thresholding might influence experimental outcome. Many possible explanations may account for the differential sensitivity of cells from the ‘Northern hemisphere’ labs (e.g., Krammer/Peter) versus those from the ‘Southern hemisphere’ (e.g., Strasser) to CD95 stimulation (the Krammer/Peter labs are located in Germany and the Strasser lab is in Australia, hence the Northern and Southern hemisphere references.). Herein is illustrated one hypothesis. It assumes that most cell types have an apoptotic threshold that, when breached, results in apoptotic cell death. This apoptotic threshold (represented by the red plane) requires, say, 10 arbitrary apoptotic units (which could be, among other things, a critical mass of caspase catalytic activity). The ability of a cell to cross this threshold depends on the combined contributions of the extrinsic cell death pathway (gray) and the fraction of the intrinsic pathway (blue) that can be engaged through pathway cross-talk. Type I cells (first bar), in both the Northern and Southern hemispheres, harbor a very efficient DISC mechanism that alone can contribute the necessary apoptotic units to breach the threshold. Thus, regardless of the status of the intrinsic pathway, and even if it is completely blocked by the presence of Bcl-2, the type I cell can muster the necessary currency to engage the apoptotic machinery and die. Therefore, extrinsic and intrinsic pathways in type I cells appear independent (assuming intrinsic does not talk to extrinsic). In type II cells, DISC formation is weaker and the extrinsic pathway depends more on contributions from the intrinsic pathway. These cells behave differently in the Northern hemisphere versus the Southern hemisphere. In the Northern hemisphere, the combination of the extrinsic contribution and the fraction of the intrinsic pathway that it is able to engage is sufficient for apoptosis to proceed (second bar). Attenuation of the intrinsic component, by Bcl-2 overexpression for example, reduces the total number of apoptotic units to below the apoptotic threshold and the cells survive. The extrinsic pathway, therefore, appears to be modulated by Bcl-2. In the Southern hemisphere, the number of available apoptotic units that can be contributed by the intrinsic pathway may be lower. Thus, engagement of the extrinsic pathway with ligating CD95 (Fas/Apo1) antibody or trimerized ligand is not sufficient to mediate passage across the apoptotic threshold on its own (third bar). With a more efficient stimulus, mediated by multimerized CD95L, for example (fourth bar), a critical mass of the extrinsic pathway is enlisted to breach the apoptotic threshold, regardless of the contribution from the intrinsic pathway. Cell death in this scenario is therefore insensitive to Bcl-2 overexpression. The differences observed in the Northern and Southern hemispheres might therefore be explained by a combination of (a) the number of apoptotic units within the intrinsic pathway that can be recruited by cross-talk from the extrinsic pathway (lower in the South) and (b) the strength of the apoptotic stimulus (ligating antibodies and trimerized CD95L being equivalent but weaker than multimerized CD95L).