Erhard Hofer

Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-beta gene family.

Human glioblastoma cells secrete a peptide, termed glioblastoma‐derived T cell suppressor factor (G‐TsF), which has suppressive effects on interleukin‐2‐dependent T cell growth. As shown here, complementary DNA for G‐TsF reveals that G‐TsF shares 71% amino acid homology with transforming growth factor‐beta (TGF‐beta). In analogy to TGF‐beta it is apparently synthesized as the carboxy‐terminal end of a precursor polypeptide which undergoes proteolytic cleavage to yield the 112 amino‐acid‐long mature form of G‐TsF. Comparison of the amino‐terminal sequence of G‐TsF with that of porcine TGF‐beta 2 and bovine cartilage‐inducing factor B shows complete homology, which indicates that we have cloned the human analogue of these factors. It is tempting to consider a role for G‐TsF in tumor growth where it may enhance tumor cell proliferation in an autocrine way and/or reduce immunosurveillance of tumor development.

T cell suppressor factor from human glioblastoma cells is a 12.5-kd protein closely related to transforming growth factor-beta.

T cell suppressor factor produced by human glioblastoma cells inhibits T cell proliferation in vitro and more specifically interferes with interleukin‐2 (IL‐2)‐dependent T cell growth. Here we report the purification of this factor from conditioned medium of the human glioblastoma cell line 308. Amino‐terminal sequence analysis of the 12.5‐kd protein demonstrates that eight out of the first 20 amino acids are identical to human transforming growth factor‐beta. Purified glioblastoma‐derived T cell suppressor factor and transforming growth factor‐beta from porcine platelets inhibit both IL‐2‐induced proliferation of ovalbumin‐specific T helper cells and lectin‐induced thymocyte proliferation with similar specific activities. If released by glioblastoma cells in vivo, the factor may contribute to impaired immunosurveillance and to the cellular immunodeficiency state detected in the patients.

Cytokine-inducible expression in endothelial cells of an I kappa B alpha-like gene is regulated by NF kappa B.

The transient expression of many different genes is mediated by the inducible transcription factor p50‐p65 NF kappa B, which in turn is regulated by complex formation with its inhibitor I kappa B alpha. We describe here that in porcine aortic endothelial cells, either IL‐1 alpha, TNF alpha or LPS upregulates an inhibitor of NF kappa B which we refer to as ECI‐6. ECI‐6 is by structural and functional criteria an I kappa B alpha protein, the porcine homologue of MAD‐3, pp40 and RL/IF‐1. We have studied the promoter of the ECI‐6/I kappa B alpha gene and provide three lines of evidence that its expression is directly regulated by NF kappa B. First, the 5′ regulatory region of ECI‐6/I kappa B alpha contains two sites that bind NF kappa B in electrophoretic mobility shift assays. Second, expression following transfection of an ECI‐6/I kappa B alpha promoter‐luciferase reporter construct is dependent on a co‐transfected NF kappa B‐p65 subunit. Third, pretreatment of endothelial cells with antioxidants, agents that inhibit activation of NF kappa B, inhibit the expression of ECI‐6/I kappa B alpha. We conclude that the regulated expression of ECI‐6/I kappa B alpha could represent a novel feedback mechanism by which NF kappa B downregulates its own activity after transient activation of target genes has been achieved.

Signaling Molecules of the NF-κB Pathway Shuttle Constitutively between Cytoplasm and Nucleus

We aimed to investigate the dynamics of the NF-κB signaling pathway in living cells using GFP variants of p65-NF-κB, IκBα, tumor necrosis factor-receptor associated factor 2 (TRAF2), the NF-κB inducing kinase (NIK) and IκB kinases (IKK1 and IKK2). Detailed kinetic analysis of constitutive nucleocytoplasmic shuttling processes revealed that IκBα enters the nucleus faster than p65. Examination of signaling molecules upstream of NF-κB and IκBα revealed a predominant cytoplasmic localization at steady state. However, after addition of leptomycin B, NIK rapidly accumulated in the nucleus, whereas we could not detect any significant effect on TRAF2 or IKK2. Using various truncation mutants of NIK, we identified a functional nuclear export signal within the COOH-terminal region 795–805, which counteracts the inherent NLS at amino acids 143–149. Prolonged incubation in the presence of LMB also leads to nuclear accumulation of IKK1, which was dependent on a lysine residue at position 44, which is also essential for kinase activity. Investigation of endogenous protein levels by immunofluorescence staining and Western blots verified the results obtained with GFP chimeras. We conclude that NF-κB·IκB complexes and the upstream signaling kinases NIK and IKK1 shuttle between cytoplasm and nucleus of nonactivated cells and that this process leads to a basal transcriptional activity of NF-κB.

Complementary DNA for human T-cell cyclophilin.

Complementary DNA encoding human cyclophilin, a specific cyclosporin A‐binding protein, has been isolated from the leukemic T‐cell line Jurkat and sequenced. Comparison of the deduced amino acid sequence with the previously determined sequence of bovine thymus cyclophilin reveals only three differences: an additional amino acid at the carboxy terminus end and two internal changes. RNA transfer blot analysis indicates an mRNA size of approximately 1 kb for human T‐cell cyclophilin. Phytohaemagglutinin and phorbol myristate acetate induction of T cells treated or not with cyclosporin A affects only marginally the level of cyclophilin mRNA. Southern blot analysis of human genomic DNA digested with different restriction enzymes strongly suggests the existence of a multigene family for cyclophilin.

Endothelial Cell Activation and Thromboregulation during Xenograft Rejection

Xenoreactive natural antibodies (XNA) and complement (C) are thought to be the two major inciting factors that result in hyperacute rejection (HAR) of an immediately vascularized, discordant xenograft within minutes to a very few hours, with destruction and infarction of the transplanted organ. If recipients are modified by various experimental modalities, such as removal and suppression of XNAand C-mediated responses, thus avoiding HAR, the process of delayed xenograft rejection (DXR) with a significant vascular component still occurs after a delay of several days or, at the most, a few weeks (Bach et al. 1993). The end result in both instances is the invariable and unacceptable loss of xenografts, which currently limits application of xenotransplantation beyond experimental protocols. The mechanisms underlying DXR are far from clear but appear not necessarily to involve XNAand C-mediated responses as those noted in HAR. Moreover, DXR can occur without the prominent participation of T lymphocytes. One of us (FHB) has suggested that the final common pathogenic mechanisms underly-

The primary transcription unit of the mouse β-Major globin gene

Abstract The transcriptional unit boundaries of the mouse β-major globin gene have been investigated in DMSO-induced murine erythroleukemia cells using mainly labeled RNA produced by in vitro transcription in isolated nuclei. Restriction fragments of a genomic β-major globin clone (Tilghman et al. 1977) representing regions of the 5′ and 3′ portions of the mRNA sequence, the large intervening sequence, as well as regions before the cap site and beyond the poly(A) site of the mRNA were recloned for use in DNA excess hybridization experiments. Equimolar amounts of labeled nascent RNA hybridized to the fragment encoding the capped 5′ end of the mRNA and to fragments extending at least 1000 nucleotides beyond the poly(A) addition site. The existence of continuous transcription throughout this region was shown by: samples of labeled RNA each specifically complementary to one of the globin region fragments all bound to a single strand, presumably the coding strand of the separated strands of the β-globin genomic clone; specific T1 oligonucleotides diagnostic of the region immediately following the poly(A) site of the nuclear RNA were identified in hnRNA labeled in isolated nuclei; the shortest labeled nascent RNA chains bound to the fragment containing the cap site and increasingly longer chains bound in the ordered set of fragments as a function of distance from the cap site. These results suggest the necessity for an endonucleolytic cleavage of a primary transcript followed by polyadenylation in the process of creating the 3′ end of this cellular mRNA, as has previously been demonstrated for every virus mRNA so far studied (Nevins and Darnell 1978; Ford and Hsu 1978; Fraser et al. 1979; Nevins et al. 1980). An analysis of the accumulation of the different RNA sequences labeled in vivo demonstrated a confinement of the sequences transcribed past the poly(A) site exclusively to the nucleus and indicated an even higher turnover rate for these than for the intervening sequences. In addition to the equimolar amounts of labeled nascent RNA complementary to DNA fragments from the cap site through and past the poly(A) site, a smaller amount of labeled RNA hybridized reproducibly to DNA fragments upstream from the cap site. Although this RNA was complementary to the coding strand, it did not appear to be part of the majority of the globin primary transcripts based on size analysis and it was present at only 20% or less of the molarity of the RNA from the globin coding regions.

Dimethylfumarate Inhibits TNF-Induced Nuclear Entry of NF-κB/p65 in Human Endothelial Cells

Fumaric acid esters, mainly dimethylfumarate (DMF), have been successfully used to treat psoriasis. Based on previous observations that DMF inhibited expression of several TNF-induced genes in endothelial cells, we wished to explore the molecular basis of DMF function in greater detail. In first experiments we analyzed DMF effects on tissue factor expression in human endothelial cells in culture, because tissue factor is expressed by two independent sets of transcription factors, by NF-κB via TNF and by early gene response-1 transcription factor via vascular endothelial growth factor (VEGF). We show that DMF inhibits TNF-induced tissue factor mRNA and protein expression as well as TNF-induced DNA binding of NF-κB proteins, but not VEGF-induced tissue factor protein, mRNA expression, or VEGF-induced early gene response-1 transcription factor/DNA binding. To determine where DMF interferes with the TNF/NF-κB signaling cascade, we next analyzed DMF effects on IκB and on the subcellular distribution of NF-κB. DMF does not inhibit TNF-induced IκBα phosphorylation and IκB degradation; thus, NF-κB is properly released from IκB complexes even in the presence of DMF. Importantly, DMF inhibits the TNF-induced nuclear entry of NF-κB proteins, and this effect appears selective for NF-κB after the release from IκB, because the constitutive shuttling of inactive NF-κB/IκB complexes into and out from the nucleus is not blocked by DMF. Moreover, DMF does not block NF-κB/DNA binding. In conclusion, DMF appears to selectively prevent the nuclear entry of activated NF-κB, and this may be the basis of its beneficial effect in psoriasis.

Specificity, diversity, and convergence in VEGF and TNF-α signaling events leading to tissue factor up-regulation via EGR-1 in endothelial cells

Tissue factor (TF) has been shown to be up‐regulated in endothelial cells by the inflammatory cytokine tumor necrosis factor a (TNF‐α) as well as by the main angiogenic factor VEGF. Since both stimuli induce the transcription factor EGR‐1, which is critically involved in TF gene regulation, we used EGR‐1‐dependent TF induction as a model to identify potential cross‐talks between the various signal transduction cascades initiated by VEGF and TNF‐α. The data show that at the MAP kinase level, VEGF mainly activates ERK1/2 and p38 MAP kinases in human endothelial cells. TNF‐α is able to activate all three MAP kinase cascades as well as the classical inflammatory IκB/NFκB pathway. Furthermore, the MEK/ERK module of MAP kinases appears to act as the convergence point of VEGF‐ and TNF‐α‐initiated signaling cascades, which lead to the activation of EGR‐1 and subsequent TF expression, whereas the upstream signals are distinct. We found that induction of TF by VEGF via EGR‐1 is strongly PKC dependent. The TNF‐α‐initiated MEK/ERK cascade connected to EGR‐1 and TF expression is clearly less sensitive to PKC inhibition. TNF‐α‐mediated activation of MEK/ERK and EGR‐1 can be blocked by adenoviral expression of a dominant negative mutant of IKK2, whereas the VEGF signaling pathway is unaffected. Thus, our data demonstrate a new link between the classical inflammatory IKK/IκB and the MEK/ERK cascades triggered by TNF‐α. The additional finding that EGF induces ERK and EGR‐1 in a PKC independent manner and that this signal is not sufficient to up‐regulate TF emphasizes the importance of a VEGF‐specific signaling pattern for the induction of TF.— Mechtcheriakova, D., Schabbauer, G., Lucerna, M., Clauss, M., de Martin, R., Binder, B. R., Hofer, E. Specificity, diversity, and convergence in VEGF and TNF‐α signaling events leading to tissue factor up‐regulation via EGR‐1 in endothelial cells. FASEB J. 15, 230–242 (2001)

Binding of the Fap2 Protein of Fusobacterium nucleatum to Human Inhibitory Receptor TIGIT Protects Tumors from Immune Cell Attack

Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT.