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The death receptor pathway is coupled to the mitochondria apoptosis pathway. However, mitochondrial participation, which is stimulated by Bid but suppressed by Bcl-2/Bcl-xL, is required in certain cells (Type II), but not in others (Type I). While these differences were originally characterized in the lymphoid cell lines, the typical Type II cells are represented by hepatocytes in vivo. The molecular mechanisms that distinguish Type II from Type I cells and the regulation are not fully understood. Fas can be sequestered by the HGF receptor c-Met and high doses of HGF can promote cell death by freeing Fas from c-Met complex. We thus reasoned that treatment of the Type II cells with high doses of HGF could enhance Fas-mediated apoptosis and spare the mitochondria amplification. Indeed, such treatment led to increased apoptosis in Type II lymphoid cells, which could not be blocked by Bcl-xL. Moreover, significant hepatocyte apoptosis was induced by this scheme in the absence of Bid with increased dissociation of Fas from c-Met. These findings indicate that high doses of HGF could be used to promote apoptosis in Type II cells bypassing the requirement for mitochondria activation. J. Cell. Physiol. 213: 556–563, 2007. © 2007 Wiley-Liss, Inc.
Two major apoptosis pathways are present in the mammalian cells. The death receptor pathway is initiated at the cell surface via the death receptor, Fas, TNF-R1 and TRAIL-receptors (Ashkenazi and Dixit, 1998). The mitochondria pathway is initiated intracellularly at the level of mitochondria and regulated by the Bcl-2 family proteins (Green and Kroemer, 2004). The death receptor Fas can be activated by its natural ligand, FasL, or by agonistic antibodies. Upon ligation Fas receptors recruit the adapter molecule, FADD, which recruits the initiator caspase, caspase-8. The complex of Fas, FADD and caspase-8 is called the death inducing signaling complex (DISC), which is responsible for the activation of caspase-8 and the downstream effector caspases, committing the cell to apoptosis.
The death receptor pathway can be coupled to the mitochondria pathway mainly via the activation of the pro-death Bcl-2 family protein, Bid, which is cleaved by caspase-8 (Gross et al., 1999; Yin et al., 1999; Zhao et al., 2001). Early studies in the lymphoid cell lines indicated that cells with similar sensitivity to anti-Fas-induced apoptosis could differ in the dependence on the mitochondria pathway. Thus effective killing does not require mitochondrial involvement in Type I cells, such as SKW6.4 and H9, but does in Type II cells, such as Jurkat and CEM (Scaffidi et al., 1998). Consequently over-expression of the anti-death Bcl-2 or Bcl-xL could block Fas-mediated apoptosis in Type II cells, but not in Type I cells (Scaffidi et al., 1998; Sun et al., 2002).
Such a characterization is not an artifact of in vitro cell lines. It has been demonstrated that Fas-mediated apoptosis in primary hepatocytes requires the mitochondrial participation. Thus over-expression of Bcl-2 or Bcl-xL in hepatocytes suppresses apoptosis (Lacronique et al., 1996; de la Coste et al., 1999) and deletion of Bcl-xL in the liver leads to spontaneous apoptosis (Takehara et al., 2004). The deletion of Bid also renders hepatocytes resistant to Fas-mediated apoptosis (Yin et al., 1999; Li et al., 2002). These data clearly indicate that hepatocytes are in vivo representatives of the Type II cells.
Mitochondria activation, as characterized by the release of cytochrome c and membrane depolarization, occurs in both Type I and Type II cells, which can be equally blocked by Bcl-2 (Scaffidi et al., 1998). This indicates that the two types of cells do not differ in the mitochondria activation, which is intact in both. But other fundamental differences make the mitochondria pathway more important in Type II cells, but not in Type I cells. One such difference could be that the DISC assembly is rapid and potent in Type I cells, but weak and delayed in Type II cells (Scaffidi et al., 1998). This leads to a strong caspase-8 activation in the former, but weak caspase-8 activation in the latter (Scaffidi et al., 1998). As such, the downstream caspase-3 activation is also ineffective in Type II cells, such that it could not overcome the inhibitory effects of XIAP, which binds to it (Li et al., 2002; Sun et al., 2002). The mitochondria pathway is required to reverse XIAP inhibition via the release of Smac, which binds to XIAP and liberates the suppressed caspase-3 (Du et al., 2000; Li et al., 2002; Sun et al., 2002).
It is not fully clear as to why DISC assembly upon Fas engagement is different in the two types of cells. One possibility is the cellular location where the DISC is assembled could affect the quality of the DISC (Lee et al., 2006). Another hypothesis is that the level or status of Fas available for the ligation by either the agonistic antibody or the natural ligand, FasL, could be different. The available Fas may be less in the Type II cells, either at the absolute number per cell, or at the relative number due to functional defect. Recently it has been shown that c-Met, the receptor for hepatocyte growth factor (HGF), can bind to and sequester Fas in several types of epithelial cells, including hepatocytes as well as endothelial cells, which prevents the bound Fas from being activated by its ligand or the agonistic antibodies (Wang et al., 2002; Smyth and Brady, 2005). Disruption of c-Met/Fas interactions, such as by high doses of HGF, could increase the sensitivity of these cells to Fas-mediated apoptosis (Wang et al., 2002; Smyth and Brady, 2005). Based on these findings, we postulated that disruption of such an interaction in the Type II cells may increase the amount of Fas available for ligation and thus enhance the activation of caspase-8, leading to stronger downstream caspase-3 activation and bypassing the requirement for the mitochondria pathway.
The current study examined this hypothesis and found that co-treatment of the Type II lymphoid cells with the anti-Fas antibody and a high dose of HGF did enhance apoptosis, which could not be blocked by the over-expression of Bcl-xL. Furthermore, high doses of HGF could sensitize bid-deficient hepatocytes to anti-Fas treatment in both in vivo and in vitro models with increased dissociation of Fas from c-Met. As the result, the combined HGF and anti-Fas caused significant liver injury in bid-deficient mice, which would be otherwise resistant to anti-Fas alone. These findings thus suggest that disruption of c-Met/Fas interaction could facilitate the conversion of the Type II response to the Type I response by enhancing Fas signaling and eliminating the requirement for the mitochondrial participation.
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Fas is physiologically activated by its natural ligand, FasL, in vivo. Experimentally, Fas can also be potently activated by the agonistic antibodies, such as the CH11 clone of anti-human Fas antibody and the Jo2 clone of anti-mouse Fas antibody. The availability of Fas receptor, either physically or functionally, and its ability to recruit FADD and caspase-8 seems to be the main determining factor in the regulation of the quantity and quality of the DISC and therefore the activation of caspase-8. It is assumed that the level of caspase-8 activity in turn affects whether the mitochondrial pathway is required for the effective transmission of death signal to the downstream effector caspases, such as caspase-3 (Scaffidi et al., 1998). Low levels of caspase-8 activity will not generate sufficient amounts of activated caspase-3 to overcome the inhibitory effects of XIAP, which, however, could be reversed by the mitochondria-released Smac (Du et al., 2000; Li et al., 2002; Sun et al., 2002). This seems to be the case for Type II cells, which require the mitochondrial participation for an effective Fas-mediated killing.
We hypothesized that by increasing the Fas availability on the cell surface of the Type II cells we could enhance Fas-mediated caspase-8 activation and promote cell death without a significant contribution of the mitochondria pathway. There are plenty of examples that death signals could promote apoptosis by increasing the availability of Fas molecules on the cell surface. This could be achieved by the upregulation of Fas expression at the transcription level (Ashkenazi and Dixit, 1998), the promotion of the transportation of existing Fas molecules from the intracellular location to the cell surface (Bennett et al., 1998; Sodeman et al., 2000), and the release of Fas from the complex with c-Met on the cell surface (Wang et al., 2002; Smyth and Brady, 2005).
Previous studies have shown that the natural ligand of c-Met, HGF, although non-toxic by itself, could disrupt c-Met-Fas interaction, thereby increasing the amount of free Fas and enhancing anti-Fas-mediated cell death (Wang et al., 2002). Indeed, when treating the Type II cells with an anti-Fas antibody in the presence of HGF, cell death was significantly increased and the need for mitochondria activation was dramatically reduced. The finding is made not only in the lymphoid cell lines, but also in primary hepatocytes and in an animal model of liver injury. The significance of HGF treatment is more noticeable in bid-deficient hepatocytes, which expressed a high level of c-Met in association with Fas (Fig. 5). The level of c-Met in Jurkat cells is much lower than in hepatocytes and endothelial cells (Smyth and Brady, 2005 and data not shown), which could contribute to their generally higher sensitivity to Fas-mediated killing. However, it is interesting to note that HGF treatment could still enhance the killing in Jurkat cells so that the mitochondria amplification could be spared. Thus the balance of the various components in controlling Fas-mediated killing could be quite delicate and a small shift could change the outcome.
The ability of HGF to enhance the killing independently from mitochondria activation is determined in Bcl-xL or Bcl-2 over-expressed cells, or in bid-deficient cells, in which mitochondria activation is suppressed or lacking, respectively. However, the enhancing effect of HGF seems to require the presence of caspase-8 and is not observed for apoptosis induced by chemicals that directly activate the mitochondria pathway. Moreover, the amount of c-Met that could be immunoprecipitated with Fas was reduced in the presence of HGF. All the evidence supports the notion that HGF works at the Fas receptor level, that Fas signaling strength can be an important contributing factor in determining Type II versus Type I response and that Fas-c-Met interaction can serve as a regulatory mechanism. On the other hand, we certainly could not exclude any additional mechanisms by which the conversion of Type II cells to Type I cells by HGF could occur. These mechanisms might include a direct impact on the intracellular location where the DISC is assembled, which could also affect the quality of the DISC (Lee et al., 2006).
Genetic evidence supports the regulation of c-Met on Fas-mediated apoptosis in vivo. Transgenic mice over-expressing the extracellular domain of c-Met (with no receptor tyrosine kinase activity associated with the intracellular domain) were resistant to anti-Fas-induced liver injury (Wang et al., 2002), most likely due to the increased sequestration of Fas in the complex. On the other hand, conditional knockout of c-Met gene in the liver rendered the mice hypersensitive to anti-Fas antibody-induced hepatocyte apoptosis and liver injury (Huh et al., 2004). HGF, when used at a low dose or when applied several days before the death stimulation, can be associated with a protective effect by initiating c-Met-mediated delayed transcriptional response, which could lead to the upregulation of certain protective molecules, such as Bcl-xL or Mcl-1, via the PI3-kinase/Akt pathway (Kosai et al., 1998; Suzuki et al., 2000; Schulze-Bergkamen et al., 2004; Khai et al., 2006). However, when applied at a higher dose and at the same time of Fas stimulation, the disruption of c-Met/Fas complex could constitute an independent pro-death effect that could overcome the slow-acting transcription-dependent protective effect. The toxic effect of HGF had been well noted in several earlier reports (Conner et al., 1999; Matteucci et al., 2003; Rasola et al., 2004). Furthermore, transgenic mice over-expressing dHGF chronically in the liver (under the albumin promoter) were paradoxically more sensitive to Fas-mediated apoptosis (R. Zarnegar, unpublished observations). This study thus took the advantage of this unique action of HGF and examined the issue of whether enhanced Fas signaling in the Type II cells could allow the bypass of the mitochondria requirement by the use of high doses of HGF to disrupt the c-Met-Fas interaction.
In conclusion, our study demonstrates a plausible mechanism by which Type I and Type II response could be regulated. Furthermore, it also points out a potential use of high doses of HGF in promoting apoptosis in cells otherwise resistant to Fas-mediated killing due to mutations in the mitochondria pathway. The latter may be explored for cancer therapy, such as for the leukemia/lymphoma of the Type II category.