Notch1 endocytosis is induced by ligand and is required for signal transduction.

The Notch signalling pathway is widely utilised during embryogenesis in situations where cell-cell interactions are important for cell fate specification and differentiation. DSL ligand endocytosis into the ligand-expressing cell is an important aspect of Notch signalling because it is thought to supply the force needed to separate the Notch heterodimer to initiate signal transduction. A functional role for receptor endocytosis during Notch signal transduction is more controversial. Here we have used live-cell imaging to examine trafficking of the Notch1 receptor in response to ligand binding. Contact with cells expressing ligands induced internalisation and intracellular trafficking of Notch1. Notch1 endocytosis was accompanied by transendocytosis of ligand into the Notch1-expressing signal-receiving cell. Ligand caused Notch1 endocytosis into SARA-positive endosomes in a manner dependent on clathrin and dynamin function. Moreover, inhibition of endocytosis in the receptor-expressing cell impaired ligand-induced Notch1 signalling. Our findings resolve conflicting observations from mammalian and Drosophila studies by demonstrating that ligand-dependent activation of Notch1 signalling requires receptor endocytosis. Endocytosis of Notch1 may provide a force on the ligand:receptor complex that is important for potent signal transduction.

Notch4 reveals a novel mechanism regulating Notch signal transduction.

Notch4 is a divergent member of the Notch family of receptors that is primarily expressed in the vasculature. Its expression implies an important role for Notch4 in the vasculature; however, mice homozygous for the Notch4(d1) knockout allele are viable. Since little is known about the role of Notch4 in the vasculature and how it functions, we further investigated Notch4 in mice and in cultured cells. We found that the Notch4(d1) allele is not null as it expresses a truncated transcript encoding most of the NOTCH4 extracellular domain. In cultured cells, NOTCH4 did not signal in response to ligand. Moreover, NOTCH4 inhibited signalling from the NOTCH1 receptor. This is the first report of cis-inhibition of signalling by another Notch receptor. The NOTCH4 extracellular domain also inhibits NOTCH1 signalling when expressed in cis, raising the possibility that reported Notch4 phenotypes may not be due to loss of NOTCH4 function. To better address the role of NOTCH4 in vivo, we generated a Notch4 null mouse in which the entire coding region was deleted. Notch4 null mice exhibited slightly delayed vessel growth in the retina, consistent with our novel finding that NOTCH4 protein is expressed in the newly formed vasculature. These findings indicate a role of NOTCH4 in fine-tuning the forming vascular plexus.

Interleukin-1-mediated stabilization of mouse KC mRNA depends on sequences in both 5'- and 3'-untranslated regions.

mRNA transcribed from the mouse KC chemokine gene accumulated to significantly higher levels in multiple cell types after treatment with interleukin 1alpha (IL-1alpha) as compared with tumor necrosis factor-alpha (TNFalpha). Although TNFalpha and IL-1alpha both signaled the activation of nuclear factor kappaB and enhanced transcription of the KC gene with equal potency, only IL-1alpha treatment resulted in stabilization of KC mRNA. Nucleotide sequences that confer sensitivity for IL-1alpha-mediated mRNA stabilization were identified within the 5'- and 3'-untranslated regions (UTRs) of KC mRNA using transient transfection of chimeric plasmids containing specific portions of KC mRNA linked to the chloramphenicol acetyltransferase (CAT) gene. When plasmids containing either the 3'- or 5'-UTR of KC mRNA were used, the half-life of CAT mRNA was unaltered either in untreated or IL-1alpha-stimulated cells. In contrast, CAT mRNA transcribed from plasmids that contained both the 5'- and 3'-UTRs of the KC mRNA decayed more rapidly than control CAT mRNA, and this enhanced decay was prevented in cells treated with IL-1alpha. A cluster of four overlapping AUUUA motifs within the 3'-UTR was required, whereas the 5'-UTR region exhibited orientation dependence. These findings indicate that cooperative function of the two nucleotide sequences involves a distinct signaling pathway used by IL-1alpha but not TNFalpha.

Oxidized LDL modulates activation of NFkappaB in mononuclear phagocytes by altering the degradation if IkappaBs.

Oxidized low density lipoprotein (oxLDL) is known to alter the expression of inflammatory gene products in mononuclear phagocytes. The mechanisms involved in this effect were studied by examining the activation of nuclear factor kappaB (NFkappaB), a transcription factor known to be important in controlling the expression of such genes. Pretreatment of peritoneal macrophages with oxLDL modulated the activation of NFkappaB in response to either lipopolysaccharide (LPS) or the combination of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). In macrophages pretreated with oxLDL the nuclear translocation of Rel family members (RelA and c-Rel) is delayed (LPS) or markedly diminished (IFN-gamma/IL-2) and results in delayed or reduced appearance of kappaB binding activity within the nucleus. These changes in NFkappaB activation result from alterations in the stimulus-dependent degradation of IkappaB alpha and IkappaB beta. The effects of oxLDL on NFkappaB activation depend both on the degree of LDL oxidation (most potent with extensive oxidation) and on the time of exposure of the cells to the lipoprotein preparation (a minimal exposure of 6 h is required before inhibitory effects are observed). The modulation of IkappaB/NFkappaB function in cells exposed to oxLDL appears to be responsible for previously reported effects of oxLDL on chemoattractant cytokine gene expression where both inhibition and delay of such stimulus-dependent events has been observed.

Oxidized LDL potentiates LPS-induced transcription of the chemokine KC gene.

Oxidized low-density lipoprotein (LDL) has been shown to modulate the expression of multiple gene products associated with inflammation in several different cell types including mononuclear phagocytes. The reported effects vary dramatically, however, depending upon cell type, stimulus, and degree of LDL oxidation. In the present report, oxidized LDL has been found to markedly potentiate expression of the KC chemokine gene in lipopolysaccharide (LPS)-stimulated macrophages. Pretreatment of elicited mouse peritoneal macrophages with oxidized LDL but not native LDL produced a significant enhancement of LPS-induced KC mRNA expression, whereas levels of IP-10 mRNA, another CXC chemokine, were altered in opposite fashion. The alteration in KC mRNA expression was dependent upon the dose, exposure time, and extent of LDL oxidation. Oxidized LDL selectively prolonged the expression of KC mRNA. Surprisingly this was not a consequence of altered mRNA stability, but rather of prolonged transcription. These effects on KC gene transcription were in marked contrast to previous reports demonstrating inhibitory effects of oxidized LDL on LPS-induced macrophage chemokine expression. Thus extensively oxidized LDL acts on the transcriptional control process in macrophages in both positive and negative fashion on separate members of the same gene family.

Regulation of chemokine gene expression by contact hypersensitivity and by oral tolerance.

In mice with hapten-induced CH, T cells of the CD4+ and CD8+ phenotypes activated the gene for JE, whereas CD8+ T cells alone caused activation of the gene for IP-10. In animals tolerized by feeding either TNCB or OX, hapten-induced expression of IP-10 but not JE mRNA was lost. The down-regulation of IP-10 gene activation was adoptively transferred from tolerized mice to naive mice by CD4+ splenic T cells. These findings reflect the differential roles of individual T-cell subsets in both enhancing and diminishing chemokine gene expression in contact hypersensitivity reactions.

The effects of oxidized low density lipoproteins on inducible mouse macrophage gene expression are gene and stimulus dependent.

Oxidized LDL has been previously reported to suppress the expression of genes induced in mononuclear phagocytes by inflammatory stimuli. In this study we extend these findings to demonstrate that the suppressive effects of oxidized LDL vary depending upon the gene being monitored and the stimulus being used to induce or enhance its expression. The expression of a selection of LPS-inducible genes exhibited differential sensitivity to pretreatment with oxidized LDL. Furthermore, the ability of oxidized LDL to suppress gene expression varied markedly with the inducing stimulus used. TNF alpha and IP-10 mRNA expression induced by IFN gamma and IL-2 was markedly more sensitive to suppression by oxidized LDL than that induced by LPS. The cooperative effects of IFN gamma and LPS on the expression of the inducible nitric oxide synthase gene were suppressed by oxidized LDL while the antagonistic effect of IFN gamma on LPS-induced expression of the TNF receptor type II mRNA was not altered. The suppressive activity of LDL was acquired only after extensive oxidation and was localized in the extractable lipid component. These results suggest a potent and direct connection between the oxidative modification of LDL and the chronic inflammation seen in atherogenic lesions. Furthermore, the appreciable selectivity of oxidized LDL in mediating secondary control of cytokine gene expression demonstrates that the active material(s) is targeted to disrupt specific intracellular signaling pathways.

Chemokine expression in trinitrochlorobenzene-mediated contact hypersensitivity.

The expression of the murine IP-10 and MCP-1 genes has been examined in the skin of mice during contact hypersensitivity reactions to the hapten trinitrochlorobenezene (TNCB). In both naive and passively sensitized animals, challenge with TNCB resulted in elevated expression of both genes as early as 4 h as detected by Northern hybridization analysis. Twenty-four hours after challenge, expression was markedly reduced in naive animals but remained elevated in sensitized animals. This prolonged expression of chemokine gene products correlates with the tissue swelling response generally used as a measure of delayed-type hypersensitivity (DTH) in this model and suggests that the continued expression of these genes results from the stimulation of hapten-specific T helper cells. Examination of cell type expression patterns by in situ hybridization using 3H-radiolabeled riboprobes confirmed the results of Northern hybridization experiments. Both genes were expressed predominantly in cells exhibiting the morphology of connective tissue fibroblasts, although the distribution of cells positive for IP-10 mRNA expression differed from that of cells expressing MCP-1 mRNA. IP-10 expression was localized almost exclusively to a population of connective tissue cells surrounding the fur follicle. MCP-1 expression was rarely found associated with fur follicles but instead was distributed throughout the dermis in cells embedded in the collagenous extracellular matrix. Surprisingly, neither endothelial cells lining the small vessels located deep within the dermis nor epidermal keratinocytes were positive under any of the conditions utilized in the present study. Expression of both IP-10 and MCP-1 has been previously reported in a variety of distinct cell types in vitro. The present results indicate that only a subset of the cell types with such potential are stimulated to express these chemokine genes in vivo during hapten-mediated DTH responses, implying the presence of subtle cell type- and tissue-specific control mechanisms.

IFN-gamma and lipopolysaccharide differentially modulate expression of tumor necrosis factor receptor mRNA in murine peritoneal macrophages.

Expression of TNF receptor (TNFR) mRNA has been examined in murine peritoneal macrophages stimulated with LPS and/or IFN-gamma. LPS markedly enhanced expression of a heterogenous population of mRNA, which hybridized with a cDNA encoding the type II TNFR. mRNA expression was optimally induced by 4 to 8 h and returned to baseline by 24 h after stimulation. Interestingly, though IFN-gamma can synergize with LPS for the expression of TNF-alpha, it abrogated the LPS-mediated enhancement of type II TNFR in a dose-dependent fashion. IFN-alpha, though less effective, had a qualitatively comparable effect. These effects were selective for the type II TNFR because levels of mRNA encoding the type I TNFR did not vary appreciably with any of the treatments described. The effects of IFN-gamma on LPS-mediated TNFR expression were dependent on the sequence of exposure; pretreatment with IFN-gamma was most effective at blocking response to LPS, whereas IFN-gamma added 1 h after initiation of LPS treatment had little or no effect. The effects of both LPS and IFN-gamma on type II TNFR expression were mediated at least in part by modulation of transcription. The effects of both LPS and IFN-gamma were also independent of protein synthesis because inclusion of cycloheximide in the treatment protocol did not abrogate either the inductive or the suppressive effects. These findings suggest that IFN-gamma and LPS modulate the physiologic action of TNF through complex mechanisms involving effects on the transcription of TNF-alpha itself and on receptors through which it may act in autocrine or paracrine fashion.

Astrocyte expression of mRNA encoding cytokines IP-10 and JE/MCP-1 in experimental autoimmune encephalomyelitis.

Mononuclear leukocytes preferentially accumulate in the central nervous system (CNS) during the course of experimental autoimmune encephalomyelitis (EAE). To address factors that govern leukocyte trafficking in EAE, we monitored expression of mRNAs encoding IP-10 and JE/MCP-1, which are members of a family of chemoattractant cytokines. A transient burst of IP-10 and JE/MCP-1 mRNA accumulation in the CNS occurred, in close relation to the onset of histologic and clinical disease. In situ hybridizations showed, unexpectedly, that astrocytes were the major source of mRNAs encoding IP-10 and JE/MCP-1. These observations implicate astrocyte-derived cytokines as potential chemoattractants for inflammatory cells during EAE.

Lipopolysaccharide induces competence genes JE and KC in Balb/C 3T3 cells.

The expression of the early genes JE and KC has been examined in Balb/C 3T3 cells treated with bacterial lipopolysaccharide (LPS). Previous studies showed that JE and KC mRNAs are induced in murine peritoneal macrophages treated with LPS, suggesting a role for these genes in inflammatory responses. Consistent with this possibility are recently published cDNA sequences which document that both genes are members of a superfamily of inflammation- and/or growth-related cytokines. In the present study, we provide evidence that the mRNAs for JE and KC are specifically induced by LPS treatment of Balb/c 3T3 cells. The LPS-stimulated expression of JE and KC was dose dependent, and exhibited a transient time course; message levels were maximal between 2 and 4 hr and declined by 8 hr. The LPS-augmented accumulation of JE and KC occurred even in the presence of cyclohexamide, which additionally had a superinducing effect on the expression of both genes. Cyclohexamide alone, in the absence of LPS, also induced JE and KC mRNA accumulation. LPS-stimulated JE and KC mRNA expression was dependent upon the stimulation of transcription as determined by nuclear "run-on" studies. Comparative analyses indicated that, under the conditions employed, LPS was a somewhat less effective stimulant of JE expression than PDGF or EGF, and was more effective than PDGF and equivalent to EGF in its ability to augment KC accumulation. Unlike PDGF and EGF, LPS did not stimulate DNA synthesis by Balb/c 3T3 cells at any time over the 72 hr period examined. The ability of the inflammatory, non-mitogenic stimulus LPS to selectively induce JE and KC mRNA expression by fibroblasts may reflect their participation in inflammation and wound healing as secretory cells.

Growth of greenland ice sheet: measurement.

Measurements of ice-sheet elevation change by satellite altimetry show that the Greenland surface elevation south of 72 degrees north latitude is increasing. The vertical velocity of the surface is 0.20 +/- 0.06 meters per year from measured changes in surface elevations at 5906 intersections between Geosat paths in 1985 and Seasat in 1978, and 0.28 +/- 0.02 meters per year from 256,694 intersections of Geosat paths during a 548-day period of 1985 to 1986.