The aim of our laboratory is to define molecular mechanisms governing programmed cell death and to reveal alterations of such mechanisms which can lead to disease.
- Intracellular transduction of apoptotic signals generated from the surface receptor Fas (APO-1/CD95). We have provided evidence suggesting that surface crosslinking of Fas generates ceramide accumulation through the activation of an acidic sphingomyelinase (ASM), a type-C phospholipase responsible for membrane sphingomyelin hydrolysis. Although multiple signaling pathways originate from Fas, ceramide produced by ASM appears relevant for the progression of the apoptotic signal, as cellular mutants defective in Fas "death domain" expression, fail to activate ASM and are resistant to Fas-induced death signals. In "death domain" defective mutants, Fas-derived signals induce changes in cyclin-dependent kinases (CDKs) activity, suggesting that changes in CDKs activity are not sufficient for apoptosis induction. We are currently investigating Fas-induced upstream activators of ASM, as well as downstream targets of ceramide. We recently identified GD3 ganglioside as a key mediator for the progression of ceramide and Fas-induced apoptotic signals in hematopoietic cells. By adoptive transfer of ASM into ASM-deficient cells we could show that ASM-deirived ceramide is required for GD3 accumulation and efficent apoptosis of lymphoid cells. GD3 can directly relocate to mitochondria, causing mitochondrial damage, in a bcl2-controlled manner.
- Relevance of the Fas system in tissue homeostasis and disease. We have investigated the role of the Fas system in the lymphoid tissue associated with the human intestinal mucosa, a major peripheral lymphoid compartment heavily and persistently challenged by antigen. Lamina propria T lymphocytes (T-LPL), unlike peripheral blood T lymphocytes (T-PBL), are extremely sensitive to Fas-mediated signals, express detectable amounts of Fas Ligand (FasL) in vivo and display significant "spontaneous apoptosis" in vitro. Exploiting the "natural" sensitivity of freshly isolated T-LPL to Fas-generated death signaling, we investigated soluble or cellular factors affecting the susceptibility to Fas-induced apoptosis in primary lymphoid cells. We also recently studied the expression and function of Fas and FasL in the thyroids from patients with Hashimoto's thyroiditis. The constitutive expression of FasL and the inducible expression of Fas by IL-1, revealed a novel apoptogenic mechanism based on omotypic cellular interactions leading to tissue destruction and desease. On the other hand, a minor role seems to be played by thyroid infiltrating cytotoxic T lymphocytes. A different mechanism is likely to be at work in autoimmune diabetes, where IL-1-induced upregulation of Fas via nitric oxide primes beta cells for destruction by FasL+ infiltrating T lymphocytes. A critical role for islet alpha cells in preventing the T cell infiltration is suggested by data gathered in the NOD mouse model. Fas/FasL interactions therefore contribute significantly to the pathogenesis of organ-specific autoimmunity.
- Ceramide-mediated pathways and cell differentiation. We have suggested that different cytokines can modulate dendritic cells differentiation and function through a common ceramide-mediated pathway. The relevance of ceramides as intracellular messengers involved in a variety of cellular adaptive responses is currently being investigated. In Salmonella-infected macrophages, an ASM-dependent ceramide-mediated pathway is responsible for SEK1 activation.
- Caspases in cell differentation and death. We found a number of potential cellular substrates for caspases by "in silico" screening of public protein databases. One of them is the PML/RARalpha fusion protein responsible for the arrest of differentiation in acute promyeloblastic leukemia. The role of other potential substrates, and of natural inhibitors of caspases, in the differentiation and death of selected cell types, is currently being investigated.