Polydom: a secreted protein with pentraxin, complement control protein, epidermal growth factor and von Willebrand factor A domains.

To identify extracellular proteins with epidermal growth factor (EGF) domains that are potentially involved in the control of haemopoiesis, we performed degenerate reverse-transcriptase-mediated PCR on the murine bone-marrow stromal cell line MS-5 and isolated a new partial cDNA encoding EGF-like domains related to those in the Notch proteins. Cloning and sequencing of the full-length cDNA showed that it encoded a new extracellular multi-domain protein that we named polydom. This 387 kDa mosaic protein contained a signal peptide followed by a new association of eight different protein domains, including a pentraxin domain and a von Willebrand factor type A domain, ten EGF domains, and 34 complement control protein modules. The human polydom mRNA is strongly expressed in placenta, its expression in the other tissues being weak or undetectable. The particular multidomain structure of the encoded protein suggests an important biological role in cellular adhesion and/or in the immune system.

Chromatin immunoselection defines a TAL-1 target gene.

Despite the major functions of the basic helix-loop-helix transcription factor TAL-1 in hematopoiesis and T-cell leukemogenesis, no TAL-1 target gene has been identified. Using immunoprecipitation of genomic fragments bound to TAL-1 in the chromatin of murine erythro-leukemia (MEL) cells, we found that 10% of the immunoselected fragments contained a CAGATG or a CAGGTG E-box, followed by a GATA site. We studied one of these fragments containing two E-boxes, CAGATG and CAGGTC, followed by a GATA motif, and showed that TAL-1 binds to the CAGGTG E-box with an affinity modulated by the CAGATG or the GATA site, and that the CAGGTG-GATA motif exhibits positive transcriptional activity in MEL but not in HeLa cells. This immunoselected sequence is located within an intron of a new gene co-expressed with TAL-1 in endothelial and erythroid cells, but not expressed in fibroblasts or adult liver where no TAL-1 mRNA was detected. Finally, in vitro differentiation of embryonic stem cells towards the erythro/megakaryocytic pathways showed that the TAL-1 target gene expression followed TAL-1 and GATA-1 expression. These results establish that TAL-1 is likely to activate its target genes through a complex that binds an E-box-GATA motif and define the first gene regulated by TAL-1.

Loss of TAL-1 protein activity induces premature apoptosis of Jurkat leukemic T cells upon medium depletion.

Transcriptional activation of the tal-1 gene occurs in -30% of patients with T cell Acute Lymphoblastic Leukemia and is therefore likely to be involved in human T cell leukemogenesis. However, the TAL-1 protein functional properties involved in this process have not been assessed so far. We have derived a clonal subline of the Jurkat T cell line which produced solely a mutant truncated form of TAL-1 protein. Sequencing of genomic DNA and cDNAs showed that the only transcribed tal-1 allele of this mutant subline harbored a G nucleotide insertion at codon 270. The resulting frameshift modifies TAL-1 residues 272-278 and creates a stop at codon 279. Although the deletion of the 53 carboxy-terminal residues of the TAL-1 protein did not directly affect the TAL-1 basic helix-loop-helix domain (residues 185-243), it had drastic effects on TAL-1 functional properties, since the mutant subline exhibited a dramatic decrease of protein binding activity to the TAL-1 DNA consensus sequence. Growth curves indicated that the mutant subline exhibited premature apoptosis upon medium depletion or serum reduction when compared with the parental cells. However, no difference between Jurkat and the mutant subline was observed in etoposide- or Fas/APO-1-triggered apoptosis. Stable expression of the mutant TAL-1 protein in Jurkat cells resulted in a phenotype that was similar to that of the mutant Jurkat subline, indicating that the TAL-1 mutant protein behaved like a dominant negative mutant and that the premature apoptosis of the mutant subline upon medium depletion was the consequence of the loss of TAL-1 protein activity.

Distinct DNase-I hypersensitive sites are associated with TAL-1 transcription in erythroid and T-cell lines.

The tal-1 gene, frequently activated in human T-cell acute lymphoblastic leukemia (T-ALL), is expressed in the erythroid, megakaryocytic, and mast cell lineages during normal hematopoiesis. To gain further insight into the molecular mechanisms that control tal-1 expression, we investigated tal-1 chromatin structure in erythroid/megakaryocytic cell lines and in T-cell lines either with or without tal-1 rearrangements. Tal-1 transcription was shown to be monoallelic in Jurkat, a T-cell line that expresses tal-1 in the absence of apparent genomic alteration of the locus. Methylation studies indicated that the tal-15' GC-rich region behaves like a CpG island, hypomethylated in normal cells, and methylated de novo on transcriptionally inactive alleles in established cell lines. Five major DNase-I hypersensitive sites (HS) were mapped in the tal-1 locus. HS I, IV, and V were exclusively observed in the erythroid/megakaryocytic cell lines that express tal-1 from the promoters 1a and 1b. HS II was weak in hematopoietic cell lines, absent in Hela, and greatly enhanced in Jurkat, suggesting that this region might be implicated in the cis-activation of tal-1 promoter 1b in this cell line. HS III was weak in HEL and Jurkat, and greatly enhanced in DU528, a T-cell line that bears a t (1;14) and initiates tal-1 transcription within exon 4. These results suggest that distinct regulatory elements are associated with the use of the different tal-1 promoters.

Erythroid-specific activity of the glycophorin B promoter requires GATA-1 mediated displacement of a repressor.

We have performed a detailed analysis of the cis-acting sequences involved in the erythroid-specific expression of the human glycophorin B (GPB) promoter and found that this promoter could be divided into two regions. The proximal region, -1 to -60, contains a GATA binding sequence around -37 and an SP1 binding sequence around -50. This region is active in erythroid and non-erythroid cells. The distal region, -60 to -95, contains two overlapping protein binding sites around -75, one for hGATA-1 and one for ubiquitous proteins. This distal region completely represses the activity of the proximal promoter in non-erythroid cells and defines the -95 GPB construct as a GPB promoter that displays erythroid specificity. Using site directed mutagenesis, we show that the -37 GATA and the -50 SP1 binding sites are necessary for efficient activity of the -95 GPB construct. Mutations that impair the -75 GATA-1 binding result in extinction of the -95 GPB construct activity if the -75 ubiquitous binding site is not altered, or in loss of erythroid specificity if the -75 ubiquitous binding site is also mutated. Using a cotransfection assay, we found that hGATA-1 can efficiently activate transcription of the -95 GPB construct in non-erythroid cells. This transactivation is abolished by mutations that impair either the -37 GATA-1 or the -50 SP1 binding. Mutations that impair the -75 GATA-1 binding and still allow the -75 ubiquitous binding also abolish the transactivation of the -95 GPB construct, indicating that hGATA-1 can remove repression of the GPB promoter by displacement of the ubiquitous proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential priming for endotoxin-induced circulating cytokine production by tumor necrosis factor-alpha and interleukin 1 beta.

In unprimed mice, a single injection of a non-lethal dose of lipopolysaccharide (LPS) produced a rise in tumor necrosis factor (TNF) and interleukin 6 (IL 6) activities. Peak serum concentrations were attained, respectively, 1.5 hr and 2.5 hr after the challenge. Pretreatment with recombinant human TNF-alpha (rHuTNF) had a priming effect for enhanced production of both serum cytokines without any change in kinetics. The enhancement was more pronounced in the TNF (15-fold) than in the IL 6 (4-fold) response. Recombinant murine TNF caused a comparable increase in LPS-induced cytokine release. In contrast, comparable pretreatment with another macrophage-derived cytokine, recombinant human interleukin 1 beta (HuIL1-beta), revealed a negative effect on LPS-induced TNF release whereas IL 6 in the blood reached levels similar to those found after priming with rTNF. Moreover, when administered in combination with rHuTNF, rHuIL1-beta inhibited the priming effect on TNF autocrine production.

Priming effect of muramyl peptides for induction by lipopolysaccharide of tumor necrosis factor production in mice.

Lipopolysaccharide-induced necrosis of grafted tumors was potentiated by several hydrophilic and lipophilic muramyl dipeptide (MDP) derivatives administered a few hours prior to small amounts of lipopolysaccharide (LPS) in spite of low titers of induced circulating tumor necrosis factor (TNF). However, pretreatment with MDP derivatives did increase the level of TNF in the blood of mice challenged by a greater dose of LPS. The TNF amount in 2 h postendotoxin mouse serum reached a peak when the glycopeptide had been given 6 h before the challenge, being approximately 100-fold above that obtained in unprimed mice. The cytotoxic activity in mouse serum was inhibited by rabbit antibodies raised against recombinant mouse TNF. Although there exists a toxic synergism between BCG or MDP and endotoxin, the effect of certain MDP derivatives was not related to an increased susceptibility to the toxicity of LPS.

[Protective effect of tumor necrosis factor (TNF) obtained by genetic recombination against experimental bacterial or fungal infection]. / Action protectrice du "tumor necrosis factor" (TNF) obtenu par recombinaison génétique contre l'infection expérimentale bactérienne ou fongique.

Recombinant human tumor necrosis factor (rHuTNF) enhanced nonspecific resistance of mice to various bacterial and fungal infections, indicating that the protective effect previously reported by us with serum TNF (sTNF) prepared in mice, could be attributed to this macrophage-derived factor. Comparative assays with both TNF preparations have shown that the protection against the infections challenges was largely correlated with antitumor activity. The protective effect of the rHuTNF preparation, expressed from a cDNA clone in Escherichia coli, was not due to contaminating endotoxin products. Since recombinant TNF and sTNF have no direct bactericidal or anti-fungal activity, the enhanced resistance to infections can be explained by the action of TNF on macrophages and polymorphonuclear cells. The experimental data support the interpretation that TNF has an important role in nonspecific immunity.

Production of differentiation-stimulating factor for murine leukemic myeloblast line by monocytic cells stimulated by a nonpyrogenic muramyl dipeptide derivative.

Muramyl dipeptide (MDP) and adjuvant-active derivatives were confirmed as unable directly to induce differentiation of mouse myeloid leukemia M1 cells. They were, however, found effective in stimulating either rabbit macrophages or human blood monocytes to produce differentiation-stimulating activity (D factor). The various conditioned media (CM) thus obtained were able to induce differentiation of the myeloblastic M1 cell line as indicated by the appearance of Fc receptors and inhibition of cell proliferation. Among the synthetic glycopeptides inducing the production of D factor, murabutide (MDP[Gln]-OnBu) was as effective as MDP, although it did not stimulate monocytes to simultaneously release endogenous pyrogen. The absence of pyrogenicity in murabutide CM was attested by IV or intracerebroventricular administration to rabbits. However, in the same CM, LAF(IL1) activity estimated by potentiation of the in vitro proliferative response to phytohemagglutinin of mouse thymocytes was usually higher than that induced by MDP.