Tyrosine-phosphorylated Stat1 and Stat2 plus a 48-kDa protein all contact DNA in forming interferon-stimulated-gene factor 3.

Interferon alpha induction of transcription operates through interferon-stimulated-gene factor 3 (ISGF), a transcription factor two components of which are members of the newly characterized Stat family of transcription factors. Interferon alpha induces tyrosine phosphorylation of Stat1 and Stat2 proteins that associate and, together with a 48-kDa protein, form ISGF3. Evidence is presented that a heterodimer of Stat1 and Stat2 is present in ISGF3 and that Stat1 and the 48-kDa protein make precise contact, while Stat2 makes general contact, with the interferon-stimulated response element, the binding site of the ISGF3.

Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures

Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αVβ3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αVβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

Ciliary neurotrophic factor protects striatal output neurons in an animal model of Huntington disease.

Huntington disease is a dominantly inherited, untreatable neurological disorder featuring a progressive loss of striatal output neurons that results in dyskinesia, cognitive decline, and, ultimately, death. Neurotrophic factors have recently been shown to be protective in several animal models of neurodegenerative disease, raising the possibility that such substances might also sustain the survival of compromised striatal output neurons. We determined whether intracerebral administration of brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3, or ciliary neurotrophic factor could protect striatal output neurons in a rodent model of Huntington disease. Whereas treatment with brain-derived neurotrophic factor, nerve growth factor, or neurotrophin-3 provided no protection of striatal output neurons from death induced by intrastriatal injection of quinolinic acid, an N-methyl-D-aspartate glutamate receptor agonist, treatment with ciliary neurotrophic factor afforded marked protection against this neurodegenerative insult.

Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes.

A family of interferon (IFN) regulatory factors (IRFs) have been shown to play a role in transcription of IFN genes as well as IFN-stimulated genes. We report the identification of a member of the IRF family which we have named IRF-3. The IRF-3 gene is present in a single copy in human genomic DNA. It is expressed constitutively in a variety of tissues and no increase in the relative steady-state levels of IRF-3 mRNA was observed in virus-infected or IFN-treated cells. The IRF-3 gene encodes a 50-kDa protein that binds specifically to the IFN-stimulated response element (ISRE) but not to the IRF-1 binding site PRD-I. Overexpression of IRF-3 stimulates expression of the IFN-stimulated gene 15 (ISG15) promoter, an ISRE-containing promoter. The murine IFNA4 promoter, which can be induced by IRF-1 or viral infection, is not induced by IRF-3. Expression of IRF-3 as a Gal4 fusion protein does not activate expression of a chloramphenicol acetyltransferase reporter gene containing repeats of the Gal4 binding sites, indicating that this protein does not contain the transcription transactivation domain. The high amino acid homology between IRF-3 and ISG factor 3 gamma polypeptide (ISGF3 gamma) and their similar binding properties indicate that, like ISGF3 gamma, IRF-3 may activate transcription by complex formation with other transcriptional factors, possibly members of the Stat family. Identification of this ISRE-binding protein may help us to understand the specificity in the various Stat pathways.

Induction of the gene encoding mucosal vascular addressin cell adhesion molecule 1 by tumor necrosis factor alpha is mediated by NF-kappa B proteins.

Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is involved in trafficking of lymphocytes to mucosal endothelium. Expression of MAdCAM-1 is induced in the murine endothelial cell line bEnd.3 by tumor necrosis factor alpha (TNF-alpha), interleukin 1, and bacterial lipopolysaccharide. Here we show that TNF-alpha enhances expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter, confirming transcriptional regulation of MAdCAM-1. Mutational analysis of the promoter indicates that a DNA fragment extending from nt -132 to nt +6 of the gene is sufficient for TNF-alpha inducibility. Two regulatory sites critical for TNF-alpha induction were identified in this region. DNA-binding experiments demonstrate that NF-kappa B proteins from nuclear extracts of TNF-alpha-stimulated bEnd.3 cells bind to these sites, and transfection assays with promoter mutants of the MAdCAM-1 gene indicate that occupancy of both sites is essential for promoter function. The predominant NF-kappa B binding activity detected with these nuclear extracts is a p65 homodimer. These findings establish that, as with other endothelial cell adhesion molecules, transcriptional induction of MAdCAM-1 by TNF-alpha requires activated NF-kappa B proteins.

Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF.

We show that the higher plant Arabidopsis thaliana has a serine-arginine-rich (SR) protein family whose members contain a phosphoepitope shared by the animal SR family of splicing factors. In addition, we report the cloning and characterization of a cDNA encoding a higher-plant SR protein from Arabidopsis, SR1, which has striking sequence and structural homology to the human splicing factor SF2/ASF. Similar to SF2/ASF, the plant SR1 protein promotes splice site switching in mammalian nuclear extracts. A novel feature of the Arabidopsis SR protein is a C-terminal domain containing a high concentration of proline, serine, and lysine residues (PSK domain), a composition reminiscent of histones. This domain includes a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2.