Fas/Fas ligand pathway

The orphan nuclear receptor Nur77/N10 has recently been demonstrated to be involved in apoptosis of T cell hybridomas. We report here that chronic expression of Nur77/N10 in thymocytes of transgenic mice results in a dramatic reduction of CD4+CD8+ double-positive as well as CD4+CD8and CD4-CD8+ single-positive cell populations due to an early onset ofapoptosis. CD4-CD8double-negative and CD25+ precursor cells, however, are unaffected. Moreover, nur77/NJO-transgenic thymocytes show increased expression of Fas ligand (FasL), while the levels of the Fas receptor (Fas) are not increased. The mouse spontaneous mutant gld (generalized lymphoproliferative disease) carries a point mutation in the extracellular domain of the FasL gene that abolishes the ability of FasL to bind to Fas. Thymuses from nur77/N1O-transgenic mice on a gW/gld background have increased cellularity and an almost normal profile of thymocyte subpopulations. Our results demonstrate that one pathway of apoptosis triggered by Nur77/N10 in doublepositive thymocytes occurs through the upregulation of FasL expression resulting in increased signaling through Fas. The analysis of the molecular mechanisms leading to apoptosis is crucial to understand differentiation and developmental processes, in particular in the immune system. During T cell development, most self-reacting immature thymocytes are eliminated by clonal deletion (negative selection) to establish immunological self-tolerance (1). This death occurs by apoptosis after an immature CD4+CD8+ double-positive T cell encounters antigen presented by self-major histocompatibility complex (1-3). The vast majority of apoptotic cells in the thymus, however, are probably a reflection of failure to undergo positive selection (4). Apoptosis in thymocytes induced by engagement of the T cell antigen receptor requires new gene transcription (5), but relatively little is known about the molecular mechanisms that mediate this response. Using subtractive hybridization, a differentially expressed cDNA, nur77/N10, has been recently isolated from libraries prepared from dying T cell hybridomas (6) or thymocytes (7). Moreover, blocking Nur77/N10 with a dominant negative (6) or antisense construct (7) and inhibition of the DNA binding activity of Nur77/N10 by the immunosuppressive drug cyclosporin A (8) prevent T cell receptor (TCR)-mediated apoptosis in hybridomas. Originally, nur77/N10 (also called NGFI-B in rat) was identified as an immediate-early gene that can be induced by a variety of stimuli in PC12 pheochromocytoma cells (9) and in fibroblasts (10, 11), suggesting that it has an important function in mediating responses to various cell stimulatory signals. Nur77/N10, a zinc finger transcription factor, shares structural features of the steroid/thyroid receptor superfamily, but because no ligand has been shown to regulate its transcriptional activity, it is referred to as an orphan nuclear The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. receptor. N10/Nur77 is expressed in many tissues including thymus (11) and binds to the regulatory element 5'AAAGGTCA-3' as a monomer (12, 13). Recently, it has been demonstrated that it can also form heterodimers with the receptors for 9-cis-retinoic acid (14, 15). So far, genes that potentially are regulated by Nur77/N10 encode steroidogenic enzymes (16). However, mice with a targeted disruption of Nur77/N10 maintain normal steroidogenesis (17). To investigate the role of this orphan nuclear receptor during T cell development further, we generated transgenic mice overexpressing a full-length nur77/N10 cDNA in the thymus. Our data show that chronic expression of Nur77/N10 in transgenic thymocytes is sufficient to induce apoptotic death of CD4+CD8+ double-positive cells. We also show that while expression of the Fas receptor is not increased, mRNA and protein levels of the Fas ligand (FasL) are upregulated. Indeed, crossing the nur77/N10-transgene into a FasL-mutant mouse strain efficiently reduces the apoptotic death of thymocytes and results in increased cellularity of double-positive cells. MATERIALS AND METHODS Mice. The coding region of the mouse nur77/N10 cDNA (11) was placed under the control of the mouse proximal lck promoter (3.2 kb; ref. 18). To obtain high translation efficiency, the initiation codon of nur77/NJO is proceeded by the f-globin initiation signal. Human growth hormone gene sequences (2.1 kb) were added to confer stability to the mRNA transcripts, and a 2.0-kb fragment encompassing the human CD2 gene locus control region (LCR; ref. 19) was inserted at the 3' end (CD2 3'-LCR). Generation of transgenic mice and PCR genotyping of tail DNA was performed as described (20). Mice carrying the autosomal recessive mutation gld (B6Smn.C3H-Fasl19d) were obtained from The Jackson Laboratory. To discriminate mice carrying the wild-type Fas ligand gene or the mutated gld allele, a PCR reaction using tail DNA was performed (20 cycles: 1 min at 95°C, 1 min at 44°C, 1 min at 72°C; primer sequences: 5'-CAACATATCTCAACTCTC-3' and 5'-AAGACTCTCATTCAAGAC-3') followed by a ligation chain reaction (21) with specific primers for the two genotypes (15 cycles: 1 min at 95°C, 4 min at 57°C; wild type-specific primer, 5'-AATTTTGAGGAATCTAAGACCT-3'; gld-specific primer, 5'-AATTTTGAGGAATCTAAGACCC-3'; ligation partner for both reactions, 5'-TTTTCGGCTTGTATAAGGTTTA-3'). Wild typeand gld-specific primers were end-labeled at their 5' end with ['y-32P]ATP, and separate reactions together with the 5'-phosphorylated ligation partner were performed. Reaction products were separated in 15% denaturing polyacrylamid gels (ratio 1:20 N,N'methylene-bisacrylamide/acrylamide, 8 M urea, 90 mM Tris, 90 mM boric acid, 1 mM EDTA) and visualized by autoradiography. Abbreviation: TCR, T cell receptor. *F.W. and R.-P.R. contributed equally to this report. tPresent address: Stanley S. Scott Cancer Center, Louisiana State University Medical Center, New Orleans, LA 70112. tTo whom reprint requests should be addressed.