Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca²⁺/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells. The “death receptor” CD95 (also known as Fas) plays an essential role in ensuring immune tolerance of self antigens as well as in the elimination of the body's cells that have been infected or transformed. This receptor is engaged by the membrane-bound ligand CD95L, which can be released into blood circulation after cleavage by metalloproteases. Hitherto, most of the studies on the CD95 signal have been performed with chimeric CD95Ls that mimic the membrane-bound ligand and exhibit a level of aggregation beyond that described for the metalloprotease-cleaved ligand. Multi-aggregated CD95L elicits a caspase-driven apoptotic signal. In this study, we observe that levels of soluble and naturally processed CD95L in sera of patients suffering from lupus correlate with disease severity. Strikingly, although this soluble CD95L fails to trigger cell death unlike its chimeric version, it induces a “non-canonical” Ca²⁺/c-yes/PI3K-dependent signaling pathway that promotes the transmigration of T-lymphocytes across the endothelial barrier. These findings shed light on an entirely new role for the soluble CD95L that may contribute to local or systemic tissue damage by enhancing the infiltration of activated T-lymphocytes. Overall, these findings underline the importance of revisiting the role of this “apoptotic cytokine” in the context of chronic inflammatory disorders.

Triple-negative breast cancers (TNBC) lacking estrogen and progesterone receptors and HER2 amplification have a relatively high risk of metastatic dissemination, but the mechanistic basis for this risk is not understood. Here, we report that serum levels of CD95 ligand (CD95L) are higher in patients with TNBC than in other patients with breast cancer. Metalloprotease-mediated cleavage of CD95L expressed by endothelial cells surrounding tumors generates a gradient that promotes cell motility due to the formation of an unconventional CD95-containing receptosome called the motility-inducing signaling complex. The formation of this complex was instrumental for Nox3-driven reactive oxygen species generation. Mechanistic investigations revealed a Yes–Orai1–EGFR–PI3K pathway that triggered migration of TNBC cells exposed to CD95L. Our findings establish a prometastatic function for metalloprotease-cleaved CD95L in TNBCs, revisiting its role in carcinogenesis. Cancer Res; 73(22); 6711–21. ©2013 AACR.

We investigate the mode of action of Cateslytin, an antimicrobial peptide, on zwitterionic biomembranes by performing numerical simulations and electrophysiological measurements on membrane vesicles. Using this natural beta-sheet antimicrobial peptide secreted during stress as a model we show that a single peptide is able to form a stable membrane pore of 1 nm diameter of 0.25 nS conductance found both from calculation and electrical measurements. The resulting structure does not resemble the barrel-stave or carpet models earlier predicted, but is very close to that found in the simulation of alpha-helical peptides. Based on the simulation of a mutated peptide and the effects of small external electric fields, we conclude that electrostatic forces play a crucial role in the process of pore formation.

Glutamoptosis is the induction of apoptotic cell death as a consequence of the aberrant activation of glutaminolysis and mTORC1 signaling during nutritional imbalance in proliferating cells. The role of the bioenergetic sensor AMPK during glutamoptosis is not defined yet. Here, we show that AMPK reactivation blocks both the glutamine-dependent activation of mTORC1 and glutamoptosis in vitro and in vivo. We also show that glutamine is used for asparagine synthesis and the GABA shunt to produce ATP and to inhibit AMPK, independently of glutaminolysis. Overall, our results indicate that glutamine metabolism is connected with mTORC1 activation through two parallel pathways: an acute alpha-ketoglutarate-dependent pathway; and a secondary ATP/AMPK-dependent pathway. This dual metabolic connection between glutamine and mTORC1 must be considered for the future design of therapeutic strategies to prevent cell growth in diseases such as cancer.

CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.