2. RECEPTOR-LIGAND SYSTEMS WITH PRIMARILY

PROAPOPTOTIC FUNCTIONS

2.1. The CD95 (Fas/APO-1) system

2.1.1. CD95 and CD95L: discovery of the first direct apoptosis-inducing receptor-ligand system

In 1989, two monoclonal antibodies, anti-APO-1 and anti-Fas, were described. Although anti-APO-1 was reported to bind to a cell surface protein of approximately 48 kD, anti-Fas was thought to bind to a cell surface receptor of approximately 200 kD, suggesting that APO-1 and Fas would be two different antigens. Both anti-APO-1 and anti-Fas rapidly induced cell death in a variety of human cancer cells by a then very poorly understood cellular process known as apoptosis. The systematic and specific employment of these two antibodies in the following decade most likely uncovered more mysteries and contributed more to our understanding of apoptosis than any other approach, possibly only matched on the antiapoptotic side by the study of Bcl-2.

The first question addressed by the groups working with anti-APO-1 and anti-Fas was to which receptors the respective antibodies bind. Whereas the group led by Shigekazu Nagata in Osaka, Japan, performed an expression cloning strategy with anti-Fas, Peter Krammer’s team in Heidelberg, Germany, pursued a classical purification approach with anti-APO-1. The surprising result was that, despite the indications of the original biochemical characterization, anti-Fas and anti-APO-1 recognized the same antigen. They both bound to a cellular receptor of 48 kD now commonly referred to as CD95 or Fas (APO-1) (Itoh et al., 1991; Oehm et al., 1992). The cloning of CD95 revealed that it contained three cysteine-rich domains (CRDs) in its extracellular portion, qualifying it as a new addition to the TNFRSF, which at the time only consisted of a handful of members and was not yet referred to as a superfamily for exactly this reason. The functional dissection of the intracellular domain of CD95, on the other hand, revealed the presence of a discrete domain that was required for induction of cell death. This domain is now known as the previously mentioned death domain (DD). Employment of the DD of CD95 in a yeast two-hybrid screen by the groups of Vishva Dixit in Ann Arbor, Michigan, and David Wallach in Rehovot, Israel (Boldin et al., 1995; Chinnaiyan et al., 1995), led to the discovery of FADD (MORT1), a protein that contained a DD and a second, DD-like domain, which is now known as a death effector domain (DED).

At this point, it remained a mystery how binding of FADD to CD95 induced the drastic biochemical changes characteristic of apoptosis. However, the discovery of the death-inducing signaling complex (DISC), induced when anti-APO-1 cross-links CD95, illuminated the picture. Kischkel et al. found that receptor cross-linking resulted in recruitment of FADD and two other proteins, as revealed by two-dimensional (2D) gel electrophoresis of the protein complex that forms when CD95 is cross-linked by anti-APO-1 (Kischkel et al., 1995). The two spots turned out to be processed and unprocessed forms of the same enzyme. This enzyme was first named FLICE, for FADD-like ICE (interleukin [IL]-1–converting enzyme), or MACH, and it was codiscovered by the Wallach team and by a concerted effort of the Dixit and Krammer teams. FLICE/MACH is now known as caspase-8. Caspase-8 is present in the cytosol as a proenzyme. It is activated by a conformational change induced by FADDmediated recruitment to CD95. Caspase-8 activation at the CD95 DISC then triggers a caspase cascade, which induces the execution of the apoptotic cell death program. The identification of caspase-8 recruitment and activation at the DISC provided the missing link between extracellular cross-linking of a receptor and intracellular activation of an enzymatic event, which triggers an entire proteolytic cascade responsible for all the biochemical hallmarks of apoptosis. This transition from the outside to the inside of the cell possibly resembles one of the most striking and beautiful examples of energy translation across a membrane in cell biology.

An entirely different line of research was opened when it was found by the Nagata team that murine CD95 was mutated in mice, which had long been studied as a model system for lupus erythematosus. These mice, known as lpr mice, carried a mutation in the murine CD95 gene that interfered with its proper expression. As a result, these mice exhibit a massive accumulation of lymphocytes. This had been mistaken for lymphoproliferation (lpr) due to the fact that, before the advent of apoptosis research, scientists automatically assumed that accumulation of cells would be due to abnormally high proliferation rather than a block in cell death. However, it turned out that T cells in lpr mice have a defect in cell death and that they therefore accumulate. A similar phenotype had been observed in gld (generalized lymphoproliferative disease) mice and elegant bone marrow transplant experiments by Cohen and Eisenberg (Chapel Hill, North Carolina) had suggested that the genes mutated in lpr and gld mice encoded an interacting pair of proteins on interacting cells (Cohen et al., 1992). When the CD95 ligand (CD95L/FasL/APO1L) was then cloned by the Nagata team, this time

employing both a purification and an expression cloning approach, this prediction turned out to be true: the gene encoding CD95L is mutated in gld mice. However, this finding did not yet explain the biological phenomenon responsible for the lpr and gld phenotypes. This was solved when the Krammer team and two other groups led by Ann Marshak-Rothstein (Boston, Massachusetts) and Douglas R. Green (San Diego, California), respectively, discovered that the interaction between CD95 and CD95L is responsible for a major portion of activationinduced cell death (AICD) in T cells (Ju et al., 1995; Dhein et al., 1995; Zhang et al., 1997). Subsequently, CD95L was also found to be responsible for the long-known perforin-independent killing activity of CD8+ cytotoxic T cells. Thus the CD95 system does not only play a role in the homeostatic regulation of the immune system, but is also employed in the defense mechanisms used by the immune system to fight infection.

When research into the CD95/CD95L system began, it was hoped that agonists of CD95 would hold the promise that TNF unfortunately could not keep as a result of the detrimental effects associated with its systemic application. These hopes were, however, immediately crushed to pieces when it became clear that animals systemically treated with antibodies to CD95 or with recombinant CD95L died within hours due to fulminant hepatitis.

As disappointing as this outcome first appeared at the time of its discovery, it sparked another line of research. Apparently, the CD95L had an enormous capacity to harm normal human tissue. It was, however, not known to what extent this function was actually involved in various pathological conditions in which apoptotic damage occurred to normal tissue. Thus began the investigation of the role of the CD95/CD95L system in various diseases in which normal tissue is damaged. In the meantime, there is ample evidence for the involvement of the CD95 system in various diseases, including very strong evidence for its involvement in graft-versus- host disease and some forms of acute hepatitis, but also data that indicate a role for this system in acute myocardial infarction, stroke, and spinal cord injury, among others. An additional and rather unexpected, but potentially quite broad, application of CD95L inhibitors was suggested recently when it was shown that instead of inducing apoptosis, CD95 can also induce migration in certain cancer cells, and that inhibition of CD95L was able to halt this effect. Although we are only beginning to understand the molecular mechanisms of this type of signaling induced by CD95L-mediated stimulation of CD95, its elucidation will be of pivotal importance to identify markers for those types or individual cases of cancer that

may respond to a CD95L-inhibiting therapy by exhibiting less migration and hence a reduction in metastasis. Recently, the first biotherapeutic inhibitor of CD95L, a soluble CD95-Fc fusion protein, successfully passed phase I clinical testing. It will be interesting to see how its further clinical testing will unfold. Therefore, it seems that for the CD95 system, as in the case of TNF (which is covered later in this chapter), the most prominent medical applications will most likely derive from antagonizing rather than stimulating it. It should, however, be mentioned that there are attempts to also use certain recombinant forms of CD95L as cancer therapeutics. Obviously such trials must be carried out with extreme care not to harm any tissues, especially the liver.

2.1.2. Biochemistry of CD95 apoptosis signaling

CD95 is ubiquitously expressed, though predominantly in the thymus, liver, heart, and kidney. In contrast, CD95L expression is very restricted: it is primarily expressed by activated T cells. Even if CD95 and CD95L are mostly expressed as membrane-bound proteins, soluble forms of both receptor and ligand also have been reported. The exact roles of soluble CD95 and CD95L are still unclear. However, soluble CD95 is thought to counteract CD95-induced apoptosis, whereas soluble CD95L, generated by metalloprotease-mediated cleavage, has been shown to either possess killing activity or to act as an inhibitor of membrane-bound CD95L, the outcome depending on how the signaling events triggered by receptor cross-linking are integrated in the particular target cell. Even though to date the CD95 system is the best characterized direct apoptosis-inducing receptor-ligand system, it can also trigger other signaling outcomes, ranging from proliferation to proinflammatory signaling and even to increased motility. However, the induction of apoptosis remains its most prominent function.

CD95-induced apoptosis is triggered when CD95Lmediated cross-linking of CD95 receptors, preassembled on the surface of the cell, leads to the recruitment of the adaptor protein FADD, the proteases caspase-8 and caspase-10, and the cellular FLICE-like inhibitory protein (cFLIP). Together, these proteins constitute the DISC. The exact molecular mechanism of CD95 activation by its ligand-induced cross-linking has only recently been uncovered. In a first step, stimulation of CD95 by its ligand results in the stabilization of an open conformation of the intracellular domain (ICD) of CD95. This open conformation contains two newly formed helices: the stem helix, created by the fusion between two helices of CD95, and a C-terminal helix. As a result of