The interleukin 2 receptor alpha chain/CD25 promoter is a target for nuclear factor of activated T cells.

The expression of the murine interleukin (IL)-2 receptor alpha chain/CD25 is strongly induced at the transcriptional level after T cell activation. We show here that nuclear factor of activated T cell (NF-AT) factors are involved in the control of CD25 promoter induction in T cells. NF-ATp and NF-ATc bind to two sites around positions -585 and -650 located upstream of the proximal CD25 promoter. Immediately 3' from these NF-AT motifs, nonconsensus sites are located for the binding of AP-1-like factors. Mutations of sites that suppress NF-AT binding impair the induction and strong NF-ATp-mediated transactivation of the CD25 promoter in T cells. In T lymphocytes from NF-ATp-deficient mice, the expression of CD25 is severely impaired, leading to a delayed IL-2 receptor expression after T cell receptor (TCR)/CD3 stimulation. Our data indicate an important role for NF-AT in the faithful expression of high affinity IL-2 receptors and a close link between the TCR-mediated induction of IL-2 and IL-2 receptor alpha chain promoters, both of which are regulated by NF-AT factors.

GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction.

Triggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E. Flory, A. Hoffmeyer, U. Smola, U. R. Rapp, and J. T. Bruder, J. Virol. 70:2260-2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation.

Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site.

Transforming growth factor beta (TGF-beta) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-beta on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (proto-enhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-beta-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-beta and cyclosporin A in El4 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.

Signal-specific and phosphorylation-dependent RelB degradation: a potential mechanism of NF-kappaB control.

RelB is an unusual member of the Rel/NF-kappaB family of transcription factors which are involved in oncogenic processes. Due to a relaxed control by the IkappaBs, the cytosolic NF-kappaB inhibitors, RelB is constitutively expressed in the nuclei of lymphoid cells. We show here that RelB is inducibly degraded upon activation of T cells in a fashion similar to the IkappaBs. However, RelB degradation differs from that of IkappaBs since it is not induced by TNFalpha but only by T cell receptor or TPA/ionomycin stimulation. Moreover, RelB degradation occurs in three steps: (i) after stimulation RelB is rapidly phosphorylated at amino acids Thr84 and Ser552 followed by (ii) an N-terminal cut and, finally, (iii) the complete degradation in the proteasomes. Since mutation of the two phosphoacceptor sites to non-acceptor sites abolished RelB phosphorylation in vivo and led to the stabilization of the mutated RelB(DM), site-specific phosphorylation appears to be a necessary prerequisite for RelB degradation. RelB is a crucial regulator of NF-kappaB-dependent gene expression. Thus, the signal-induced degradation of RelB should be an important control mechanism of NF-kappaB activity.

The role of NF-AT transcription factors in T cell activation and differentiation.

The family of genuine NF-AT transcription factors consists of four members (NF-AT1 [or NF-ATp], NF-AT2 [or NF-ATc], NF-AT3 and NF-AT4 [or NF-ATx]) which are characterized by a highly conserved DNA binding domain (is designated as Rel similarity domain) and a calcineurin binding domain. The binding of the Ca(2+)-dependent phosphatase calcineurin to this region controls the nuclear import and exit of NF-ATs. This review deals (1) with the structure of NF-AT proteins, (2) the DNA binding of NF-AT factors and their interaction with AP-1, (3) NF-AT target genes, (4) signalling pathways leading to NF-AT activation: the role of protein kinases and calcineurin, (5) the nuclear entry and exit of NF-AT factors, (6) transcriptional transactivation by NF-AT factors, (7) the structure and expression of the chromosomal NF-AT2 gene, and (8) NF-AT factors in Th cell differentiation. The experimental data presented and discussed in the review show that NF-AT factors are major players in the control of T cell activation and differentiation and, in all likelihood, also of the cell cycle and apoptosis of T lymphocytes.

STAT6 and the regulation of CD23 expression in B-chronic lymphocytic leukemia.

High CD23 expression is a hallmark of B-CLL cells. It is lost during in vitro culture and can be reinduced by IL-4, albeit to a lower extent than in normal B cells. To elucidate the events controlling CD23 expression in B-CLL cells, the IL-4 mediated induction of STAT6 was investigated. Western-blot analysis demonstrated that B-CLL cells contain comparable amounts of STAT6. Electrophoretic mobility shift assays (EMSA) showed no constitutive nuclear translocation of STAT6. IL-4 induced the translocation of STAT6 in B-CLL cells from all 22 patients investigated. The increase was transient, dose and time dependent without a distinct difference between B-CLL cells and non-malignant B cells. However, in contrast to normal B lymphocytes no strict correlation between CD23 expression and STAT6 activation was detected in B-CLL. Therefore further signalling pathways and transcription factors in addition to STAT6 have to be activated to explain the high expression of CD23 in B-CLL cells. For example, STAT1 which is induced by IFN-gamma and binds to the classical STAT6 site. It might be involved in the strong induction of CD23 on B-CLL cells after cotreatment with IL-4 and IFN-gamma, while in non-malignant B lymphocytes IFN-gamma leads to a reduction of IL-4 mediated CD23 expression.

Rel/NF-kappa B transcription factors and cancer.

The transcription factors of the Rel/NF-kappa B family play a central role in immune regulation. In addition, they may be involved in the pathogenesis of certain tumors, especially those of hematopoietic origin. In this review evidence is summarized indicating an association between dysregulated NF-kappa B-dependent gene expression and tumorigenesis. Furthermore, possible molecular mechanisms through which NF-kappa B might exert these effects, such as disturbance of cell cycle control or apoptosis, are outlined. Potential therapeutic approaches based on these findings are also described. Rel/NF-kappa B factors are presented here as an important example of the growing number of transcription factors possibly involved in oncogenesis.

Inefficient termination of antigen responses in NF-ATp-deficient mice.

In order to elucidate the role of NF-ATp, one of the most prominent members of family of NF-AT transcription factors in peripheral T lymphocytes, in T cell activation and differentiation we created NF-ATp-deficient mice by gene targeting. Such NF-ATp-/- mice are born and appear to develop a normal immune system. Apart from clear-cut defects in the synthesis of mRNAs for Th2-type lymphokines, such as IL-4, IL-5, IL-10 and IL-13, in primary and secondary stimulations of spleen cells in vitro, of a distinct impaired deletion of V beta 11+/CD4+ T lymphocytes from these mice was detected after superantigen injection. Moreover, NF-ATp-/- mice older than 6 weeks show an 2-5 fold increase in number of lymphocytes. This is correlated with an increased expression of activation markers CD44 and CD69 and decreased expression of CD62.

Nuclear NF-ATp is a hallmark of unstimulated B cells from B-CLL patients.

B lymphocytes from the peripheral blood of patients with chronic lymphocytic leukaemia (CLL) were analysed for the nuclear presence and DNA binding of a panel of transcription factors which are involved in the gene control of lymphoid cells. The following transcription factors were studied: the Octamer factors Oct-1 and Oct-2, members of the AP-1 factor family, NF-AT factors, in particular NF-ATp, and members of the Rel/NF-kB family. We show that the constitutive nuclear translocation of NF-ATp, a member of the growing family of NF-AT factors, is a hallmark of nonstimulated B cells from CLL patients that distinguishes B-CLL cells from 'normal' B lymphocytes. Constitutive nuclear appearance was also observed for NF-kB2/p52. Constitutive binding of further factor proteins to DNA, such as JunD, c-Fos and FosB, was detected in several patients whereas the localisation and DNA binding of other factors such as c-Jun, RelA/p65 and c-Rel was unaltered. It is remarkable that in B-CLL cells the nuclear appearance and DNA binding of specific transcription factors is dramatically affected whereas other members of the same factor family remained unaltered in these leukemic cells. It remains to be shown which molecular events lead to the specific 'pre-activation', i.e. constitutive nuclear translocation and DNA binding, of these members of NF-AT, NF-kB and AP-1 factor families.

Yersinia enterocolitica leads to transient induction of TNF-alpha and activates NF-kappaB in synovial fibroblasts.

OBJECTIVE: The importance of the presence of bacterial antigen or even living bacteria for the pathogenesis of reactive arthritis has been discussed increasingly ever since bacterial antigen was found in inflamed joints. Bacteria may persist in the body and drive the local immune response, maintaining arthritis. Cytokines, in particular tumor necrosis factor-alpha (TNF-alpha) are essential for bacterial elimination. In reactive arthritis, the course of the disease is influenced by several cytokines, including TNF-alpha. TNF-alpha expression can be mediated by transcription factor nuclear factor-kappa B (NF-kappaB). Moreover, TNF-alpha is also one of the strongest activators of NF-kappaB. METHODS: In vitro expression of TNF-alpha and activation of NF-kappaB in synovial fibroblasts after infection with Yersinia enterocolitica or Salmonella enteritidis was analysed by electrophoretic mobility shift assay, Western blot assay and real-time PCR. RESULTS: We found that infection of synovial fibroblasts with yersinia and salmonellae lead to the transient expression of TNF-alpha mRNA and induction of NF-kappaB. CONCLUSION: Induction of TNF-alpha in synovial fibroblasts after infection with yersiniae or salmonellae might be insufficient to eliminate bacteria, and this could allow the intracellular persistence of these bacteria. Our results therefore support the hypothesis that a permissive cytokine pattern might contribute to the pathogenesis of reactive arthritis.

BCR/ABL modulates the cytokine and retinoic acid response of c-Rel in human myeloid cells.

A human myeloid cell line, Mo7, and a daughter cell line expressing the bcr/abl oncogene, Mo7-P210, were used in a comparative study analyzing the effects of p210BCR/ABL expression on tyrosine phosphorylation, specific DNA binding and expression of the proto-oncoprotein c-Rel. The steady state expression of c-Rel was indistinguishable in both cell lines. Tyrosine phosphorylation and DNA binding of c-Rel were slightly elevated in Mo7-P210 cells. Further, Mo7 and Mo7-P210 cells showed different responses concerning c-Rel after stimulation with cytokines and retinoic acid. The results presented here demonstrate that c-Rel can be modulated by hematopoietic cytokines and suggest that bcr/abl expression has an impact on these responses and that c-Rel may be a downstream effector for p210BCR/ABL.

The transcripts of Balbiani rings from Chironomus thummi. Giant RNA molecules with messenger characteristics.

Experimental results on the transcription of Balbiani rings BR 1 and BR 2 of Chironomus thummi salivary gland chromosomes are presented. The DNA of Balbiani ring 2, which is the most active puff in larvae, is transcribed into large RNA molecules of about 22 X 10(6) D which resist degradation by heating, formamide or urea treatment. The asymmetrical distribution of electrophoretic profiles of BR 2 RNA and the appearence of a symmetrical BR RNA peak in the nucleoplasm suggest the synthesis of (mainly) one RNA fraction in BR 2. The gel electrophoretic patterns of BR 1 RNA are, on the other hand, characterized by the appearance of two main fractions of high molecular weight RNA, one of which corresponds in molecular weight (about 22 X 10(6) D) to BR 2 RNA. The second RNA fraction is significantly smaller in molecular size (molecular weight: about 10 X 10(6) D) and, like the 22 X 10(6) D RNA fractions of the two Balbiani rings, resistant against heating in 8 M urea. Binding to poly (U) sepharose of a significant part of Balbiani ring RNA suggests the existence of poly (A) and/or oligo (A) sequences in the Balbiani ring RNA. -- In situ hybridization of BR RNA to the salivary gland chromosomes reveals accumulation of silver grains over the Balbiani ring regions only and demonstrates the restriction of BR DNA sequences to the corresponding Balbiani ring.

The repetition frequency of DNA in Balbiani ring 2 of Chironomus thummi.

The RNA of Balbiani ring BR2 of polytene chromosomes from Chironomus thummi salivary glands was microisolated and reassociated in the presence of an excess of total larval DNA. BR2 RNA reacts as a single component with a C0t 1/2 of 8.6. Ribosomal precursor RNA from microisolated nucleoli reassociates under identical conditions with a C0t 1/2 of 12.3. These C0t 1/2-values suggest repetition frequencies in the range of 35 and 50 for ribosomal DNA and Balbiani ring 2 DNA, respectively. The data presented here favour the view that the gene for BR2 RNA of C. thummi is internally repeated and contains only one type of DNA sequence.

A transfer RNAArg gene of Pelargonium chloroplasts, but not a 5S RNA gene, is efficiently transcribed after injection into Xenopus oocyte nuclei.

We present the primary structure of a chloroplast tRNAArgACG gene of the plant, Pelargonium zonale, and its faithful expression in Xenopus oocyte nuclei. This tRNAArg gene is located 250 bp downstream of a 5S RNA gene within a cloned 5kb long ribosomal DNA segment (Fig. 1). The Pelargonium tRNAArg gene shares 97% and 86% sequence homology with tRNAArgACG genes of Spirodela oligorhiza and Euglena gracilis chloroplasts, respectively, and also extensive homology (70%) with the corresponding gene of E. coli. It lacks an intervening sequence and, like eukaryotic tRNA genes, does not code for the 3' terminal CCA nucleotides. Moreover, the chloroplast tRNAArg gene carries all the sequence elements essential for transcription by vertebrate RNA polymerase III since it is efficiently expressed in Xenopus oocyte nuclei, even in the presence of 1 microgram/ml alpha-amanitin. In Xenopus oocyte nuclei, no transcripts of the chloroplast 5S RNA gene were detected.

The immunosuppressives FK 506 and cyclosporin A inhibit the generation of protein factors binding to the two purine boxes of the interleukin 2 enhancer.

Like Cyclosporin A (CsA), the macrolide FK 506 is a potent immunosuppressive that inhibits early steps of T cell activation, including the synthesis of Interleukin 2 (II-2) and numerous other lymphokines. The block of II-2 synthesis occurs at the transcriptional level. At concentrations that block T cell activation, FK 506 and CsA inhibit the proto-enhancer activity of Purine boxes of the II-2 promoter and the generation of lymphocyte-specific factors binding to the Purine boxes. Under the same conditions, the DNA binding of other II-2 enhancer factors remains unaffected by both compounds. These results support the view that FK 506 and CsA, which both inhibit the activity of peptidylprolyl cis/trans isomerases, suppress T cell activation by a similar, if not identical mechanism.