Cooperativity between two NF-kappa B complexes, mediated by high-mobility-group protein I(Y), is essential for cytokine-induced expression of the E-selectin promoter.

Cytokine-induced expression of the E-selectin gene requires the promoter binding and interaction of the transcription factors NF-kappa B and ATF. Here we have further analyzed the E-selectin promoter and revealed an additional region (nucleotides -140 to -105 [-140/-105]) which is essential in controlling promoter activation by cytokines. We identified high-mobility-group protein I(Y) [HMG-I(Y)] interacting specifically at two sites within this region. We noted that one of the HMG-I(Y)-binding sites overlaps a sequence element (-127/-118) diverging at only one position from the NF-kappa B consensus binding sequence. This led us to ask whether the -127/-118 element represents a second functional NF-kappa B-binding site within the E-selectin promoter. Using specific antisera, we show that p50, p65, and, interestingly, RelB are components of the complex interacting at this site. Mutational analysis of the -127/-118 NF-kappa B site indicates that both NF-kappa B and HMG-I(Y) binding at this site are essential for interleukin-1 induction of the promoter. We demonstrate that the binding affinity of the p50 subunit of NF-kappa B to both NF-kappa B sites within the E-selectin promoter is significantly enhanced by HMG-I(Y). In addition, an essential role for cooperative interaction between the two NF-kappa B complexes is shown by the requirement for both NF-kappa B sites to mediate E-selectin promoter activation by interleukin-1 and p50/p65 expression. We conclude that HMG-I(Y) mediates binding of a distinct NF-kappa B complex at two sites within the E-selectin promoter. Furthermore, a unique cooperativity between these NF-kappa B complexes is essential for induced E-selectin expression. These results suggest mechanisms by which NF-kappa B complexes are involved in specific gene activation.

Rates of mutation to growth factor autonomy and tumorigenicity differ in hematopoietic stem and precursor cells expressing the multilineage colony-stimulating factor gene.

At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-P1 or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.

Cyclic AMP-independent ATF family members interact with NF-kappa B and function in the activation of the E-selectin promoter in response to cytokines.

We previously reported that NF-kappa B and a complex we referred to as NF-ELAM1 play a central role in cytokine-induced expression of the E-selectin gene. In this study we identify cyclic AMP (cAMP)-independent members of the ATF family binding specifically to the NF-ELAM1 promoter element. The NF-ELAM1 element (TGACATCA) differs by a single nucleotide substitution from the cAMP-responsive element consensus sequence. We demonstrate that this sequence operates in a cAMP-independent manner to induce transcription and thus define it as a non-cAMP-responsive element (NCRE). We show that ATFa is a component of the NF-ELAM1 complex and its overexpression activates the E-selectin promoter. In addition, ATFa, ATF2, and ATF3 interact directly with NF-kappa B in vitro, linking two unrelated families of transcription factors in a novel protein-protein interaction. Furthermore, we demonstrate that the ability of overexpressed NF-kappa B to transactivate the E-selectin promoter in vivo is dependent on the NF-ELAM1 complex. Our results suggest that a direct interaction between ATFs and NF-kappa B is, at least in part, the mechanism by which these factors specifically regulate E-selectin promoter activity.

RZRs, a new family of retinoid-related orphan receptors that function as both monomers and homodimers.

Members of the superfamily of nuclear receptors share the greatest homology in their DNA-binding domains. We have used reverse transcription-polymerase chain reaction and highly degenerate primers based on the amino acid sequence of the zinc finger motif of known nuclear receptors to identify novel members of the family. Starting with rat brain RNA, we have isolated an orphan receptor that we call RZR beta. The sequence of its nearly full-length complementary DNA shows great similarity to RZR alpha, a receptor we recently identified from human umbilical vein endothelial cells. These RZR subtypes represent members of a new family of orphan nuclear receptors that most likely regulate specific gene expression. Sequence comparison with other known nuclear receptors reveals great similarity for both RZR subtypes to retinoic acid and retinoid-X receptors. By Northern blot analyses, we found RZR beta messenger RNA only in brain, whereas RZR alpha is expressed in many tissues. We show here that the RZRs bind as monomers to natural retinoid response elements formed by (A/G)GGTCA half-sites. However, a T-residue in the -1 position of this motif greatly enhances the DNA binding affinity of RZRs, whereas the -2 position has no influence. We show that RZRs can bind as homodimers on response elements formed by palindromes, inverted palindromes, or direct repeats of two TAGGTCA half-sites. Interestingly, these response elements display dramatically reduced affinity for retinoic acid receptor-retinoid-X receptor heterodimers. Thus, the 5'-flanking sequence of hexameric half-sites appears to be crucial to direct the activity of several nuclear receptors. On monomeric as well as dimeric binding sites, RZRs show constitutive transactivational activity that can be enhanced by unidentified components of fetal calf serum.

Commonly used cat reporter vectors contain a cAMP-inducible, cryptic enhancer that co-operates with NF-kappa B-sites.

Commercially available and widely used cat expression vectors were found to contain a forskolin (Fs)-inducible element capable of co-operation with NF-kappa B-sites in test promoters. An alternative NF-kappa B-dependent reporter system is presented that allows investigation of the effects of Fs and other agents that augment intracellular cyclic AMP.

Vasoactive intestinal peptide: expression of the prohormone in bacterial cells.

A complementary DNA (cDNA) to the messenger RNA for the preprohormone of human vasoactive intestinal peptide (VIP) has been isolated and characterized. This cDNA extends from 65 bases 5' of the AUG translation start codon through the entire 3' untranslated region. Using this cDNA we have constructed expression plasmids which allow the synthesis of 120 out of the 150 amino acids of the prohormone in E. coli. This portion of the prohormone gene was either fused to a segment of a bacteriophage structural gene or expressed alone. When expression was induced the fusion protein constituted 15% of the total bacterial cell protein while the prohormone alone was 5%. Both proteins are recognized by antiserum raised against porcine VIP. They provide protein to study the precursor-product relationship of the hormone plus the possibility of identifying cryptic regulatory peptides contained within the prohormone.

Evidence for three classes of avian sarcoma viruses: comparison of the transformation-specific proteins of PRCII, Y73, and Fujinami viruses.

The gag-linked transformation-specific proteins (polyproteins( of PRCII, Fujinami, and Y73 avian sarcoma viruses have been compared by tryptic peptide mapping. In addition to shared gag peptides, PRCII polyprotein p105 and FSV polyprotein p140 were found to have seven methionine-containing and five cysteine-containing tryptic peptides in common. These represent the majority of the non-gag peptides for each virus. In contrast, no overlap was detected with the non-gag peptides of the Y73 polyprotein p90. Examination of the tryptic phosphopeptides of p105, p140, and p90 labeled by their associated protein kinases gave similar results. Although the major phosphopeptides of p105 and p140 comigrated, they were distinct from the phosphopeptide of p90. Three classes of transformation-specific proteins can now be identified among known avian sarcoma viruses. After the pp60src of Rous sarcoma virus and B77 virus, the proteins of PRCII and Fujinami virus form a second class and Y73--currently the only representative--characterizes the third. Despite their structural differences, these viruses may share a common mechanism of transformation, effected by their associated protein kinases.

Transcriptional control of endogenous virus genes in murine lymphocytes.

The expression of endogenous retrovirus in murine lymphocytes is under genetic control and also depends on the differentiation state of the lymphocytes. We have used a cDNA probe complementary to induced virus RNA to quantify transcription of virus sequences in lymphocytes from mitogen-stimulated lymphocytes of the AKR, 129/J and Balb/c mice. Balb/c lymphocytes show the clearest case for induction of new virus sequences in response to stimulation. All strains including 129/J show expression of virus sequences in unstimulated control lymphocytes. The data indicate that mitogen induction of endogenous retrovirus is regulated at the transcriptional level.

Identification of nuclear receptor mRNAs by RT-PCR amplification of conserved zinc-finger motif sequences.

Highly degenerate PCR primers were designed based on the amino acid sequence of the zinc finger motifs of known nuclear receptor members. Reverse transcription (RT)-PCR was performed using these degenerate primers starting with total RNA isolated from human umbilical vein endothelial cells (HUVEC). We identified 13 nuclear receptors of which three were novel. Of the novel orphans two-RZR alpha and PHR-1-were further characterised. Cloning of their cDNAs and sequence comparisons revealed that RZR alpha is related to Drosophila DHR3, RAR and RXR. PHR-1 turned out to be the human homologue of the recently published murine LRH-1. By Northern blot analyses we found RZR alpha mRNA expressed in a variety of organs, whereas a high amount of PHR-1 mRNA was found in pancreas and less in liver. This technique not only aides in the identification of additional members of the nuclear receptor superfamily but also measures relative changes in the expression of nuclear receptors between two different biological states, for example, normal and disease states.

Two distinct NF-kappa B complexes differing in their larger subunit bind the E-selectin promoter kappa B element.

We have investigated the proteins binding the E-selectin promoter NF-kappa B element in its natural DNA context, using probes extending beyond the NF-kappa B recognition decamer. In band shift assays, we detected two distinct NF-kappa B complexes using nuclear extracts from several cytokine-induced cells. Subunit-specific antisera as blockers of complex formation plus DNA-protein cross-linking experiments revealed the faster migrating form to contain the NF-kappa B p50 plus p65 subunits. In contrast, the slower migrating form is composed of p50 plus the p65-related p75 protein. We show as the crucial determinant in generation of the larger complex the presence of more than five basepairs extra DNA sequence downstream of the NF-kappa B-site. Although no specific sequence is required in this 3' extended DNA to bind the larger complex, an intact kappa B binding site is. This may be explained by a requirement for activated p50 as part of this complex. The potential for a regulatory role for the p75 containing complex on the E-selectin promoter is discussed.

Involvement of HIV nef protein in abnormal hematopoiesis in AIDS: in vitro study on bone marrow progenitor cells.

In previous studies (1-4), we described disrupted hematopoiesis in long-term human bone marrow cultures infected with HIV. Conditioned medium (CM) from HIV-1 non-productively infected liquid cultures inhibits the proliferation of granulo-monocytic progenitor cells (CFU-GM) in clonogenic assays. A growth-inhibitory soluble factor was therefore suspected and we report here that this factor has been identified as the nef gene product. These nef-containing supernatants are specifically neutralized by anti-nef antibodies and recombinant nef has the same growth inhibitory properties. We used a nef-deficient virus to confirm that nef protein is responsible for the in vitro growth inhibition of CFU-GM.

Transcription factor-induced, phased bending of the E-selectin promoter.

E-selectin is an endothelial adhesion molecule that is critically involved in neutrophil adhesion and recruitment. All DNA elements required for interleukin-1 inducibility have been located in the proximal promoter: an NF-ELAM1/ATF site, two NF-kappa B sites (I and II), the NF-ELAM2 element and a TATA box. We show here that interleukin-1 induced promoter activity is exquisitely sensitive to the spatial arrangements of these elements. Phasing of the ATF and NF-kappa B II elements indicates that their relative helix orientation is more important than distance per se. This sensitivity is partly due to a requirement for correctly oriented, transcription factor-induced DNA-bending. (i) Band shift analyses with permuted ATF- and NF-kappa B elements show that their associated factors all bend DNA. (ii) One can functionally replace the NF-ELAM1/ATF element by a subset of a panel of DNA fragments that contain defined bends in various planes. We conclude that the main role of the factors binding at the NF-ELAM1/ATF element is to alter the conformation of the E-selectin promoter, presumably looping distant enhancer elements into each other's proximity.

ATF-a0, a novel variant of the ATF/CREB transcription factor family, forms a dominant transcription inhibitor in ATF-a heterodimers.

We have isolated a cDNA encoding a variant of the transcription factor ATF-a (called ATF-a0) by screening a HeLa cDNA expression library with a regulatory element of the E-selectin promoter, NF-ELAM1/delta A. Relative to full-length ATF-a, the ATF-a0 cDNA contains a large in-frame deletion of 525 base pairs that removes the P/S/T-rich putative transactivation domain. Using reverse-transcription-polymerase chain reaction and Northern blot hybridization to characterize ATF-a0 expression, we found that putative mRNAs for ATF-a0 and ATF-a are present at varying ratios in different tissues. Full-length ATF-a is a transcriptional activator for the NF-ELAM1/delta A site of the E-selectin promoter. In contrast, we show ATF-a0 has no measurable transactivating function on this element. Moreover, we demonstrate that co-expressed ATP-a0 and ATF-a preferentially heterodimerize. In the heterodimer ATF-a0 is a dominant inhibitor that completely blocks the transactivating activity of ATF-a. Both forms of ATF-a bind the p50 subunit of NF-kappa B as shown by affinity chromatography. ATF-a0 appears to be a splice variant similar to the one found for ATF-2, its closest homologue in structure and function. Taken together, our results suggest that ATF-a0 is an important member of the ATF family with a negative regulatory role in transactivation.

Murine endothelial leukocyte-adhesion molecule 1 is a close structural and functional homologue of the human protein.

Human endothelial leukocyte-adhesion molecule 1 (ELAM-1), a cell-surface glycoprotein expressed solely on cytokine-activated endothelial cells, mediates the adhesion of blood neutrophils, memory T-cells and some monocytes. ELAM-1, also known as E-selectin or leukocyte endothelial-cell-adhesion molecule 2, is a member of the lectin/epidermal-growth-factor/complement-regulatory-protein-like cell-adhesion molecule family, which includes structurally related molecules referred to as selectins. They are all involved in cell/cell adhesion, playing roles in leukocyte trafficking which are currently only partially defined. We report here the isolation and characterization of the murine equivalent of human ELAM-1. Murine ELAM-1 is encoded by a single-copy gene, spanning about 13 kb, which is structurally organized into 14 exons and 13 introns; very similar to that of its human counterpart. The exon/intron architecture exactly parallels the domain structure of the encoded protein. A murine ELAM-1-specific cDNA was cloned from heart tissue of an interleukin-1-(IL-1)-treated mouse. Its nucleotide sequence shows an overall similarity of 70% to human ELAM-1 cDNA. Transiently expressed in Cos cells, the encoded protein promotes the adhesion between recombinant cells and both human polymorphic nuclear cells, as well as HL60 cells expressing S-Lewis-x sugar moiety. Northern blot studies revealed by far the highest expression of the murine ELAM-1 gene in heart tissue and only low expression in lung tissue of IL-1-treated mice. Within the promoter, most of the recently identified regulatory elements are conserved. An exception is the nuclear factor (NF) kappa B box sequence, which, in the murine ELAM-1 promoter, does not correspond to the consensus NF kappa B sequence (Lenardo and Baltimore, 1989). Band-shift analyses show no binding to NF kappa B-like proteins. However, fusion of the murine ELAM-1 promoter to a chloramphenicol acetyltransferase reporter confers cytokine-inducible transcription, although at a lower level, when compared to the human ELAM-1 promoter. Our results demonstrate the existence of a murine homologue of the human gene and demonstrate for adhesion functional equivalence between the homologous proteins from the two species. In addition, we provide the first evidence of the utility of the murine model in addressing biological questions about the role which ELAM-1 plays in inflammation.

Distamycin prolongs E-selectin expression by interacting with a specific NF-kappaB-HMG-I(Y) binding site in the promoter.

The E-selectin cell adhesion protein plays a critical role in mediating adherence of leukocytes to endothelium at sites of inflammation. Cytokine-induced E-selectin expression on the surface of endothelial cells is transient; mRNA expression peaks at 3-4 h after induction and returns to basal levels within 24 h. The mechanism for this transcriptional down-modulation is not known. Promoter binding factors responsible for induced gene expression include NF-kappaB, which binds at three sites within the E-selectin promoter, and HMG-I(Y), which binds to the A/T-rich core found at the centre of these binding sites. Distamycin is an antibiotic that also binds A/T-rich DNA and inhibits HMG-I(Y) DNA binding. To study the role of HMG-I(Y) in E-selectin expression, we have examined the effect of distamycin on the cytokine-induced E-selectin expression cycle. We found that distamycin prolonged E-selectin expression, both by sustaining mRNA transcription and by extending the transcript's half-life. The distamycin effect on transcription was mediated through one of the three NF-kappaB-HMG-I(Y) binding sites (NF-kappaBII) within the promoter. This suggests that the NF-kappaB-HMG-I(Y) complex interacting at the NF-kappaBII site plays a role not only in cytokine induction of E-selectin expression, but also in its down-modulation.

Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells.

Human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied. Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size. Mammalian hGM-CSF was purified 200,000-fold using immunoaffinity and lectin chromatography. Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation. Following enzymatic deglycosylation, immunoreactivity was measured by radioimmunoassay and biological activity was measured in vitro on responsive human primary cells. Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4- to 8-fold. Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E. coli. The E. coli produced-protein--lacking any carbohydrate--had by far the highest specific activity observed for the recombinant hGM-CSFs.

Labile proteins play a dual role in the control of endothelial leukocyte adhesion molecule-1 (ELAM-1) gene regulation.

Endothelial leukocyte adhesion molecule-1 (ELAM-1) is a membrane protein exclusively expressed on endothelial cells, where it plays a key role in the inflammatory response by adhering to a subset of leukocytes. The expression of the ELAM-1 gene is very tightly regulated. ELAM-1 is undetectable in uninduced cells, and it is transiently expressed following cytokine induction. Treatment of resting endothelial cells with three different protein synthesis inhibitors, cycloheximide (CHX), anisomycin, and emetine, caused an increase in the steady-state level of ELAM-1 mRNA above that observed with IL (interleukin)-1 alone. Furthermore, ELAM-1 mRNA was found in the presence of all three protein synthesis inhibitors without IL-1 treatment. Analysis of the mRNA half-life indicated that the protein synthesis inhibitors act, in part, by stabilizing ELAM-1 mRNA. In addition, protein synthesis inhibitors potentiate the effect of IL-1 beta at the level of transcription initiation as shown by nuclear run-on experiments. The NF kappa B-like binding activity to the ELAM-1 promoter sequence induced by IL-1 beta is augmented by inhibitors of protein synthesis. The NF kappa B binding sequence was found to be necessary and sufficient for superinduction of the ELAM-1 gene by CHX. These results show that regulation at the level of protein synthesis is implicated in the overall regulation of ELAM-1 gene expression. Mechanisms which could explain these effects are discussed.

Inducible inhibition of eukaryotic gene expression.

A regulatable binary expression system for eukaryotes was recently developed based on the tetracycline repressor and its operator. Here we show that this system can be successfully applied to express antisense RNA and completely inhibit gene expression in a tetracycline-repressible fashion.