Cyclosporin A-sensitive transcription factor Egr-3 regulates Fas ligand expression.

Activation-induced transcriptional upregulation of the ligand for Fas (FasL) and the resulting apoptosis of Fas-bearing cells constitute essential steps in a host of normal and pathological processes. Here we describe an activation-inducible cis-acting regulatory element in the fasL promoter that is required for gene expression. Oligonucleotide competition and antibody supershift analyses identified two activation-induced DNA-binding species: Egr-1 (NGFI-A, krox-24, zif268, TIS-8), a transcription factor that has been implicated in growth, differentiation, and apoptosis; and Egr-3 (PILOT), a transcription factor of no previously known function. Activation-induced expression of Egr-3, like that of FasL, was inhibited by cyclosporin A, whereas expression of Egr-1 was unaffected. Transient expression of Egr-3 alone increased fasL promoter activity in a cyclosporin A-insensitive manner, whereas expression of Egr-1 had little effect. Moreover, endogenous fasL mRNA was induced in nonlymphoid cells by forced expression of Egr-3 in the absence of any other stimulus. These studies identify a critical Egr family-binding site in the fasL promoter and demonstrate that activation-induced Egr-3, but not Egr-1, directly upregulates fasL transcription in response to activating stimuli.

Mechanism of B cell antigen receptor function: transmembrane signaling and triggering of apoptosis.

The antigen receptor of B lymphocytes (BCR) plays important roles in virtually every stage in the development, inactivation, or activation of B cells. The BCR is a complex of membrane immunoglobulin (mIg) and a heterodimer of two transmembrane polypeptides called Ig-alpha and Ig-beta. Site directed mutation of the mu immunoglobulin heavy chain has demonstrated that the mu transmembrane domain plays a key role in the assembly of mIgM with Ig-alpha/Ig-beta. In addition, there is a strong correlation between the ability of various mutant mIgM molecules to associate with Ig-alpha/Ig-beta and their ability to induce signal transduction reactions such as protein tyrosine phosphorylation and phosphoinositide breakdown. The cytoplasmic domains of Ig-alpha and Ig-beta share a region of limited homology with each other and with components of the T cell antigen receptor and of the Fc receptor. The presence of regions of the cytoplasmic domains of Ig-alpha or Ig-beta including this conserved amino acid sequence motif is sufficient to confer signaling function on chimeric transmembrane proteins. Both Ig-alpha and Ig-beta chimeras are capable of inducing all of the BCR signaling events tested. Based on these and related observations, we propose that the motifs act to initiate the BCR signaling reactions by binding and activating tyrosine kinases. Among the important events mediated by BCR signaling is induced expression of a series of genes referred to as early response genes. In B cells these include transcription factors and at least one component that regulates signaling events. One of these genes, c-myc, appears to play an important role in mediating apoptosis in B cells stimulated via the BCR complex.

Induction of early response genes by cross-linking membrane Ig on B lymphocytes.

Cross-linking of membrane Ig (mIg) on B lymphocytes induces protein tyrosine phosphorylation and phosphoinositide hydrolysis, events that are thought to mediate the diverse biologic responses of B cells to Ag binding. mIg stimulation also induces the expression of the putative transcriptional regulators c-myc, c-fos, egr-1, and jun-B. In this report, normal murine B cells and two murine B lymphoma cell lines were examined for the induction of mRNA expression of seven early response genes first identified in fibroblasts. Expression of four of the seven genes (nur77, nup475, pip92, and 3CH134), encoding two putative transcriptional regulators, a protein of unknown function, and a putative protein phosphatase, was induced after cross-linking of mIg in resting B cells isolated from mouse spleen. In the 2PK-3 and WEHI-231 B lymphoma cell lines three and two, respectively, of these four genes were induced. Expression of these genes could be induced in 2PK-3 cells by activating the phosphoinositide-signaling pathway independently of the tyrosine phosphorylation pathway by signaling through an M1 muscarinic acetylcholine receptor introduced by transfection. Additionally, in all but one case, these early response genes could be induced by directly activating protein kinase C with phorbol esters. In the cell line 2PK-3, the gene 3CH134 was not induced by phorbol ester treatment, but was induced by elevation of intracellular calcium. Thus, a subset of the early response genes identified in serum-stimulated fibroblasts is also induced by Ag-receptor stimulation in B lymphocytes, and this induction appears to be mediated by the phosphoinositide signaling pathway and, for the most part, protein kinase C.

Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ.

The immunosuppressive effects of glucocorticoids arise largely by inhibition of cytokine gene expression, which has been ascribed to interference between the glucocorticoid receptor and transcription factors such as AP-1 and NF-kappa B as well as by competition for common coactivators. Here we show that glucocorticoid-induced inhibition of interleukin-2 mRNA expression in activated normal T cells required new protein synthesis, suggesting that this phenomenon is secondary to expression of glucocorticoid-regulated genes. One of the most prominent glucocorticoid-induced genes is glucocorticoid-induced leucine zipper (GILZ), which has been reported to inhibit activation-induced up-regulation of Fas ligand (FasL) mRNA. Indeed, transient expression of GILZ in Jurkat T cells blocked induction of a reporter construct driven by the FasL promoter. This could be accounted for by GILZ-mediated inhibition of Egr-2 and Egr-3, NFAT/AP-1-inducible transcription factors that bind a regulatory element in the FasL promoter and up-regulate FasL expression. GILZ also potently inhibited AP-1-driven and IL-2 promoter-driven reporter constructs, and recombinant GILZ specifically interacted with c-Fos and c-Jun in vitro and inhibited the binding of active AP-1 to its target DNA. Whereas homodimerization of GILZ required the presence of its leucine zipper, the interaction with c-Fos and c-Jun occurred through the N-terminal 60-amino acid region of GILZ. Thus, GILZ represents a glucocorticoid-induced gene product that can inhibit a variety of activation-induced events, at least in part by direct interference with AP-1, and is therefore a candidate for a mediator of glucocorticoid-induced immunosuppression.

Role of Egr-2 in up-regulation of Fas ligand in normal T cells and aberrant double-negative lpr and gld T cells.

We previously identified a Fas ligand regulatory element (FLRE) in the Fas ligand (fasL) promoter that binds Egr family proteins and demonstrated that Egr-3 (PILOT) but not Egr-1 (NGFI-A, Krox-24, Tis-8, and Zif-268) induces transcription of fasL. The aberrant CD4(-)CD8(-) T cells from lpr/lpr and gld/gld mice, which have mutations in the genes encoding Fas and FasL, respectively, have an activated phenotype and constitutively express high levels of fasL mRNA, prompting us to ask what role if any the FLRE and Egr family proteins have in this aberrant expression of fasL. Unstimulated MRL-lpr/lpr and C3H-gld/gld CD4(-)CD8(-) T cells constitutively contained high levels of two proteins that bound to the FLRE. Supershift analysis revealed these proteins to be Egr-1 and Egr-2 (Krox-20); Egr-3 was not detected. Activation of normal lymph node cells resulted in increased expression of Egr-1, -2, and -3. As with egr-3, expression of egr-2 was blocked by cyclosporin A. Although overexpressed Egr-1 was ineffective, overexpressed Egr-2 was as potent as Egr-3 in inducing fasL promoter-dependent reporter constructs in T cell hybridomas and HeLa cells, and both up-regulated endogenous fasL mRNA in HeLa cells. FasL-dependent reporter constructs in MRL-lpr/lpr and C3H-gld/gld CD4(-)CD8(-) T cells were constitutively active, and this activity was largely prevented by mutation of the critical Egr family binding element. Thus, Egr-2, in addition to Egr-3, regulates FasL expression in activated normal T cells, and Egr-2 is likely to play a direct role in aberrant fasL up-regulation in lpr/lpr and gld/gld CD4(-)CD8(-) T cells.

Sequential involvement of NFAT and Egr transcription factors in FasL regulation.

The critical function of NFAT proteins in maintaining lymphoid homeostasis was revealed in mice lacking both NFATp and NFAT4 (DKO). DKO mice exhibit increased lymphoproliferation, decreased activation-induced cell death, and impaired induction of FasL. The transcription factors Egr2 and Egr3 are potent activators of FasL expression. Here we find that Egr2 and Egr3 are NFAT target genes. Activation of FasL occurs via the NFAT-dependent induction of Egr3, as demonstrated by the ability of exogenously provided NFATp to restore Egr-dependent FasL promoter activity in DKO lymph node cells. Further, Egr3 expression is enriched in Th1 cells, suggesting a molecular basis for the known preferential expression of FasL in the Th1 versus Th2 subset.