| Apoptosis, a unique type of death with distinguished morphological changes, has emerged as a primary mediator of diverse physiologic and pathologic processes. The apoptotic death program is orchestrated by a well conserved basic death machinery consisting of pro and anti apoptotic genes. Although the machinery has been intensively studied, the question remains: how cells sense the different intrinsic as well as extrinsic signals in order to initiate this common machinery? The focus of this thesis is to address the above question, particularly on how extracellular signals trigger apoptosis, by using ligand-receptor systems and identifying some of the intermediate signaling components in the pathway. (1) NGF/p75 system. The neurotrophin receptor p75, also a member of the TNF receptor superfamily, has been observed to participate in cell death function of multiple systems, but the mechanism has remained obscure. It was observed in our lab that treatment with NGF causes terminally differentiated cultured rat oligodendrocytes to die. Using a combination of immunohistochemical and western blotting assays, a unique group of caspases has been found to be activated upon NGF treatment, including the initiator caspase-1, -2 and effector caspase-3. In addition, although caspase-8, the crucial molecule linking the receptor complex to the death machinery in the Fas/TNF system, was expressed in oligodendrocytes, it was not activated upon NGF treatment. This data would suggest that the mechanism of apoptosis by NGF through the p75 receptor is different from TNF- and Fas-mediated killing. Finally, gamma radiation of oligodendrocytes also activated a similar subset of caspases as NGF, indicating that NGF-induced oligodendrocyte apoptosis uses a similar cell death execution mechanism as injury models. (2) Isoproterenol/beta-adrenergic receptor system. Using a variety of assays (TUNEL, MTT, and caspase activation) to measure apoptosis, the previously observed cytocidal effect of isoproterenol and cAMP on S49 mouse lymphoma cells was proven to be apoptotic cell death. T cells deficient in Gsalpha or PKA were used to demonstrate that beta-adrenergic receptors initiate Gsalpha-dependent, but PKA-independent pathways leading to apoptosis. Furthermore, Src-family kinase Lck was identified as a signaling component in this pathway using a Lck-deficient cell line. The involvement of Lck was supported by direct stimulation of Lck protein kinase activity by binding to Gsalpha. The data thus reveal a new signaling pathway for Gsalpha, distinctive from the classical PKA pathway that accounts for the apoptotic actions of G protein-coupled receptors.; Finally, In Chapter II, early work identifying and characterizing a TNFR1 interacting protein will be described. Using the yeast two-hybrid system, a previously known DNA-damage responsive protein, BRE, was identified to interact with the juxtamembrane region of TNFR-1. The interaction between the p55 receptor and BRE was verified by an in vitro biochemical assay and by co-immunoprecipitation of transfected mammalian cells. Finally the functional significance of this interaction was demonstrated, proving that BRE is involved in the modulation of TNF function, particularly in NF-kB activation. |
