Cross-linking of Fas (CD95) induces apoptosis, a response that has been reported to depend upon the Ras activation pathway. Since many examples of apoptosis have been reported to involve AP-1 and/or the AP-1-activating enzyme Jun kinase (JNK), downstream effectors of Ras or Ras-like small GTP-binding proteins, we evaluated the role of these molecules in Fas-mediated apoptosis. Although cross-linking of Fas on Jurkat T cells did result in JNK activation, increased activity was observed relatively late, being detectable only after 60 min of stimulation. Expression of a dominant negative form of SEK1 that blocked Fas-mediated induction of JNK activity had no effect on Fas-mediated apoptosis. Furthermore, maximally effective concentrations of anti-Fas did not cause JNK activation if apoptosis was blocked by a cysteine protease inhibitor, suggesting that under these conditions, activation of JNK may be secondary to the stress of apoptosis rather than a direct result of Fas engagement. Despite the activation of JNK, there was no induction of AP-1 activity as determined by gel shift assay or induction of an AP-1-responsive reporter. The lack of a requirement for AP-1 induction in Fas-mediated death was further substantiated with Jurkat cells that were stably transfected with a dominant negative cJun, TAM-67. While TAM-67 effectively prevented AP-1-dependent transcription of both the interleukin-2 and cJun genes, it had no effect on Fas-induced cell death, even at limiting levels of Fas signaling. Thus, induction of JNK activity in Jurkat cells by ligation of Fas at levels sufficient to cause cell death is likely a result, rather than a cause, of the apoptotic response, and AP-1 function is not required for Fas-induced apoptosis.

Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

The exquisite sensitivity of thymocytes to steroid-induced apoptosis, the steroidogenic potential of thymic epithelial cells, and the ability of steroid synthesis inhibitors to enhance antigen-specific deletion of thymocytes in fetal thymic organ cultures suggest a role for glucocorticoids in thymocyte development. To address this further, transgenic mice that express antisense transcripts to the glucocorticoid receptor (GR) specifically in immature thymocytes were generated. The consequent hyporesponsiveness of thymocytes to glucocorticoids was accompanied by a reduction in thymic size, primarily owing to a decrease in the number of CD4+CD8+ cells. While an enhanced susceptibility to T cell receptor (TCR)-mediated apoptosis appeared to be partially responsible for this reduction, thymocyte loss could also be detected before thymocytes progressed to the CD4+CD8+ TCR alpha beta-expressing stage. These results suggest that glucocorticoids are necessary for survival and maturation of thymocytes, and are consistent with a role for steroids in both the transition from CD4-CD8- to CD4+CD8+ cells and the survival of CD4+CD8+ cells stimulated via the TCR.

Intracellular Ca2+ plays a central role in controlling lymphocyte function. Nonetheless, critical gaps remain in our understanding of the mechanisms that regulate its concentration. Although Ca2+-release-activated calcium (CRAC) channels are the primary Ca2+ entry pathways in T cells, additional pathways appear to be operative in B cells. Our efforts to delineate these pathways in primary murine B cells reveal that Ca2+-permeant nonselective cation channels (NSCCs) operate in a cooperative fashion with CRAC. Interestingly, these non-CRAC channels are selectively activated by mechanical stress, although the mechanism overlaps with BCR-activated pathways, suggesting that they may operate in concert to produce functionally diverse Ca2+ signals. NSCCs also regulate the membrane potential, which activates integrin-dependent binding of B cells to extracellular matrix elements involved in their trafficking and localization within secondary lymphoid organs. Thus, CRAC and distinct Ca2+ permeant NSCCs are differentially activated by the BCR and mechanical stimuli and regulate distinct aspects of B cell physiology.