Charles A. Kunsch

NF-kappa B subunit-specific regulation of the interleukin-8 promoter.

Interleukin-8 (IL-8), a chemotactic cytokine for T lymphocytes and neutrophils, is induced in several cell types by a variety of stimuli including the inflammatory cytokines IL-1 and tumor necrosis factor alpha TNF-alpha. Several cis elements, including a binding site for the inducible transcription factor NF-kappa B, have been identified in the regulatory region of the IL-8 gene. We have examined the ability of various NF-kappa B subunits to bind to, and activate transcription from, the IL-8 promoter. A nuclear complex was induced in phorbol myristate acetate-treated Jurkat T cells which bound specifically to the kappa B site of the IL-8 promoter and was inhibited by addition of purified I kappa B alpha to the reaction mixture. Only antibody to RelA (p65), but not to NFKB1 (p50), NFKB2 (p50B), c-Rel, or RelB was able to abolish binding, suggesting that RelA is a major component in these kappa B binding complexes. Gel mobility shift analysis with in vitro-translated and purified proteins indicated that whereas the kappa B element in the human immunodeficiency virus type 1 long terminal repeat bound to all members of the kappa B/Rel family examined, the IL-8 kappa B site bound only to RelA and to c-Rel and NFKB2 homodimers, but not to NFKB1 homodimers or heterodimers of NFKB1-RelA. Transient transfection analysis demonstrated a kappa B-dependent expression of the IL-8 promoter in a human fibrosarcoma cell line (8387) and in Jurkat T lymphocytes. Cotransfection with various NF-kappa B subunits indicated that RelA and c-Rel, but neither NFKB1 nor heterodimeric NFKB1-RelA, was able to activate transcription from the IL-8 promoter. Furthermore, cotransfection of NFKB1 and RelA, although able to support activation from the human immunodeficiency virus type 1 long terminal repeat, failed to activate expression from the IL-8 promoter. Antisense oligonucleotides to RelA, but not NFKB1, inhibited phorbol myristate acetate-induced IL-8 production in Jurkat T lymphocytes. These data demonstrate the differential ability of members of the kappa B/Rel family to bind to, and activate transcription from, the IL-8 promoter. Furthermore, while providing a novel example of a kappa B-regulated promoter in which the classical NF-kappa B complex is unable to activate transcription from the kappa B element, these data provide direct evidence for the role of RelA in regulation of IL-8 gene expression.

Use of a Whole Genome Approach To Identify Vaccine Molecules Affording Protection against Streptococcus pneumoniae Infection

ABSTRACT Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniaeinfection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.

PDEF, a Novel Prostate Epithelium-specific Ets Transcription Factor, Interacts with the Androgen Receptor and Activates Prostate-specific Antigen Gene Expression

Prostate cancer, the most frequent solid cancer in older men, is a leading cause of cancer deaths. Although proliferation and differentiation of normal prostate epithelia and the initial growth of prostate cancer cells are androgen-dependent, prostate cancers ultimately become androgen-independent and refractory to hormone therapy. The prostate-specific antigen (PSA) gene has been widely used as a diagnostic indicator for androgen-dependent and -independent prostate cancer. Androgen-induced and prostate epithelium-specific PSA expression is regulated by a proximal promoter and an upstream enhancer via several androgen receptor binding sites. However, little progress has been made in identifying androgen-independent regulatory elements involved in PSA gene regulation. We report the isolation of a novel, prostate epithelium-specific Ets transcription factor, PDEF (prostate-derived Etsfactor), that among the Ets family uniquely prefers binding to a GGAT rather than a GGAA core. PDEF acts as an androgen-independent transcriptional activator of the PSA promoter. PDEF also directly interacts with the DNA binding domain of androgen receptor and enhances androgen-mediated activation of the PSA promoter. Our results, as well as the critical roles of other Ets factors in cellular differentiation and tumorigenesis, strongly suggest that PDEF is an important regulator of prostate gland and/or prostate cancer development.

Local Control of Granule Cell Generation by Cerebellar Purkinje Cells

Cerebellar Purkinje cells were ablated by the specific expression of diphtheria toxin in these cells in transgenic mice. Purkinje cell degeneration during early postnatal development shows a zonally restricted pattern which has been exploited in order to look for local secondary effects. The most obvious early effect is the alignment of gaps in the Purkinje cell layer with dramatically thinned zones in the overlying EGL, the germinal layer from which granule cells are generated. Within these EGL zones in the transgenic mutant, markers that distinguish matrix from mantle cells demonstrate a preferential loss of the proliferative cells. Comparison of BrdU incorporation in the mutant vs wild-type confirms the reduction in proliferation. In the mutant, in situ labeling of DNA fragmentation associated with apoptotic cell death shows abundant labeling of granule cells that have exited the EGL, but not of progenitor cells in the EGL. Thus, although a trophic role for Purkinje cells has been well documented, these observations further suggest a mitogenic role which can be exerted locally.

Isolation of cDNA Clones Encoding Eight Different Human G Protein γ Subunits, Including Three Novel Forms Designated the γ4, γ10, and γ11 Subunits

With the growing awareness that the G protein β and γ subunits directly regulate the activities of various enzymes and ion channels, the importance of identifying and characterizing these subunits is underscored. In this paper, we report the isolation of cDNA clones encoding eight different human γ subunits, including three novel forms designated γ4, γ10, and γ11. The predicted protein sequence of γ4 shares the most identity (60-77%) with γ2, γ3, and γ7 and the least identity (38%) with γ1. The γ4 is modified by a geranylgeranyl group and is capable of interacting with both β1 and β2 but not with β3. The predicted protein sequence of γ10 shows only modest to low identity (35-53%) with the other known γ subunits, with most of the differences concentrated in the N-terminal region, suggesting γ10 may interact with a unique subclass of α. The γ10 is modified by a geranylgeranyl group and is capable of interacting with β1 and β2 but not with β3. Finally, the predicted protein sequence of γ11 shows the most identity to γ1 (76% identity) and the least identity to the other known γ (33-44%). Unlike most of the other known γ subunits, γ11 is modified by a farnesyl group and is not capable of interacting with β2. The close resemblance of γ11 to γ1 raises intriguing questions regarding its function since the mRNA for γ11 is abundantly expressed in all tissues tested except for brain, whereas the mRNA for γ1 is expressed only in the retina where the protein functions in phototransduction.

Characterization of NERF, a novel transcription factor related to the Ets factor Elf-1.

We have cloned the gene for a novel Ets-related transcription factor, new Ets-related factor (NERF), from human spleen, fetal liver, and brain. Comparison of the deduced amino acid sequence of NERF with those of other members of the Ets family reveals that the level of homology to ELF-1, which is involved in the regulation of several T- and B-cell-specific genes, is highest. Homologies are clustered in the putative DNA binding domain in the middle of the protein, a basic domain just upstream of this domain, and several shorter stretches of homology towards the amino terminus. The presence of two predominant NERF transcripts in various fetal and adult human tissues is due to at least three alternative splice products, NERF-1a, NERF-1b, and NERF-2, which differ in their amino termini and their expression in different tissues. Only NERF-2 and ELF-1, and not NERF-1a and NERF-1b, function as transcriptional activators of the lyn and blk gene promoters, although all isoforms of NERF bind with affinities similar to those of ELF-1 to a variety of Ets binding sites in, among others, the blk, lck, lyn, mb-1, and immunoglobulin H genes and are expressed at similar levels. Since NERF and ELF-1 are coexpressed in B and T cells, both might be involved in the regulation of the same genes.