Fas ligand (CD95L) is synthesized both on the cell surface membrane and in a soluble form. Although CD95L contributes to immune privilege in the cornea and testis, the functions of these alternatively processed proteins are not well understood. Some reports suggest that the cytotoxicity of soluble CD95L is insignificant, whereas others show potent responses in vivo, including hepatocyte apoptosis that causes liver failure. We show here that extracellular matrix proteins interact with soluble CD95L and potentiate its pro-apoptotic activity. The cytotoxicity of supernatants from CD95L-expressing cells was increased by incubation on tissue culture plates coated with these matrix proteins; this effect was mediated by trimeric soluble CD95L. With the use of immunoprecipitation, it was found that CD95L binds directly to fibronectin. In addition, immunohistochemical analysis of the cornea revealed that soluble CD95L binds primarily to extracellular matrix. The retention of soluble CD95L on extracellular matrices is likely to play an important role in the development of peripheral tolerance in immune-privileged sites.
"It has also been reported that naturally cleaved FasL may dampen apoptotic activity by competing with mFasL or blocking Fas receptors (Gregory et al., 2011; Knox et al., 2003; O' Reilly et al., 2009; Suda et al., 1996; Tanaka et al., 1998). When binding to the matrix proteins or cross-linking by antibodies, the cytotoxicity of naturally cleaved soluble FasL was potentiated in vitro and in vivo (Aoki et al., 2001; Schneider et al., 1998). Recombinant soluble FasL corresponding to the entire extracellular domain of FasL shows considerable cytotoxicity compared with naturally cleaved sFasL, although lower than membrane-bound FasL (Hohlbaum et al., 2000; Suda et al., 1996). "
[Show abstract][Hide abstract] ABSTRACT: FasL is the most extensively studied apoptosis ligand. In 2000, tilapia FasL was identified using anti-human FasL monoclonal antibody by Evans's research group. Recently, a tilapia FasL-like protein of smaller molecule weight was predicted in Genbank (XM_003445156.2). Based on several clues drawn from previous studies, we cast doubt on the authenticity of the formerly identified tilapia FasL. Conversely, using reverse transcription polymerase chain reaction (RT-PCR), the existence of the predicted FasL-like was verified at the mRNA level. Through multiple alignments, this FasL-like protein was found to be highly similar to the FasL of the Japanese flounder. Moreover, we artificially expressed the functional region of the predicted protein and later confirmed its apoptosis-inducing activity using a methyl thiazolyl tetrazolium (MTT) assay, Annexin-V / Propidium iodide (PI) double staining, and DNA fragment detection. Supported by these evidences, we suggest that the predicted protein is the authentic tilapia FasL. To advance this research further, tilapia FasL mRNA and its protein across different tissues were quantified. High expression levels were identified in the tilapia immune system and sites where active cell turnover conservatively occurs. In this regard, FasL may assume an active role in the immune system and cell homeostasis maintenance in tilapia, similar to that shown in other species. In addition, because the distribution pattern of FasL mRNA did not synchronize with that of the protein, post-transcriptional expression regulation is suggested. Such regulation may be dominated by potential adenylate- and uridylate-rich elements (AREs) featuring AUUUA repeats found in the 3' untranslated region (UTR) of tilapia FasL mRNA.
"This is in contrast to an experimental form of FasL that corresponds to the entire extracellular domain , . On the other hand, sFasL bound to extracellular matrix proteins is cytotoxic and FasL has been localized to the extracellular matrix in the anterior chamber of the eye . Thus, whether FasL accumulates in the ocular environment as full-length mFasL or truncated sFasL, matrix-associated or not, could influence its functional consequences. "
[Show abstract][Hide abstract] ABSTRACT: Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.
PLoS ONE 03/2011; 6(3):e17659. DOI:10.1371/journal.pone.0017659 · 3.23 Impact Factor
"To test whether cell-bound human mCD95L could trigger an increase in motility and/or invasiveness of CD95 apoptosis-resistant tumors, we incubated MCF7(FB) cells with a cell line expressing authentic unmodified human mCD95L (CT26L; Aoki et al, 2001). When mixed at a ratio of 3.3:1, CT26L cells induced maximal motility of MCF7(FB) cells (Figure 1E) although significant increase of motility was observed when mixed at a ratio of 1:1 or 6.7:1. "
[Show abstract][Hide abstract] ABSTRACT: The apoptosis-inducing death receptor CD95 (APO-1/Fas) controls the homeostasis of many tissues. Despite its apoptotic potential, most human tumors are refractory to the cytotoxic effects of CD95 ligand. We now show that CD95 stimulation of multiple apoptosis-resistant tumor cells by CD95 ligand induces increased motility and invasiveness, a response much less efficiently triggered by TNFalpha or TRAIL. Three signaling pathways resulting in activation of NF-kappaB, Erk1/2 and caspase-8 were found to be important to this novel activity of CD95. Gene chip analyses of a CD95-stimulated tumor cell line identified a number of potential survival genes and genes that are known to regulate increased motility and invasiveness of tumor cells to be induced. Among these genes, urokinase plasminogen activator was found to be required for the CD95 ligand-induced motility and invasiveness. Our data suggest that CD95L, which is found elevated in many human cancer patients, has tumorigenic activities on human cancer cells. This could become highly relevant during chemotherapy, which can cause upregulation of CD95 ligand by both tumor and nontumor cells.
The EMBO Journal 09/2004; 23(15):3175-85. DOI:10.1038/sj.emboj.7600325 · 10.43 Impact Factor
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