Ben-Zion Levi

Interleukin 6 Induces the Expression of Vascular Endothelial Growth Factor

Angiogenesis, the formation of new blood vessels, is induced by various growth factors and cytokines that act either directly or indirectly. Vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells and therefore has a central role in physiological events of angiogenesis. Interleukin-6 (IL-6) expression on the other hand is elevated in tissues that undergo active angiogenesis but does not induce proliferation of endothelial cells. We demonstrate using Northern analysis that treatment of various cell lines with IL-6 for 6-48 h results in a significant induction of VEGF mRNA. The level of induction is comparable to the documented induction of VEGF mRNA by hypoxia or cobalt chloride, an activator of hypoxia-induced genes. In addition, it is demonstrated by transient transfection assays that the effect of IL-6 is mediated not only by DNA elements at the promoter region but also through specific motif(s) located in the 5′-untranslated region (5′-UTR) of VEGF mRNA. Our results imply that IL-6 may induce angiogenesis indirectly by inducing VEGF expression. It is also shown that the 5′-UTR is important for the expression of VEGF. The 5′-UTR of VEGF is exceptionally long (1038 base pairs) and very rich in G + C. This suggests that secondary structures in the 5′-UTR might be essential for VEGF expression through transcriptional and post-transcriptional control mechanisms.

Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription.

Vascular Endothelial Growth Factor (VEGF) is a very potent angiogenic agent that has a central role in normal physiological angiogenesis as well as in tumor angiogenesis. VEGF expression is induced by hypoxia and hypoglycemia, and thus was suggested to promote neovascularization during tumor outgrowth. Yet, the molecular mechanism that governs VEGF expression is not fully characterized. VEGF induction is attributed in part to increased levels of transcription and RNA stability. Previously, we demonstrated that the 5′ Untranslated Region (5′ UTR) of VEGF has an important regulatory role in its expression. VEGF has an exceptionally long 5′ UTR (1038 bp) which is highly rich in G+C nucleotides. This suggests that secondary structures in the 5′ UTR might be essential for VEGF expression through transcriptional and post-transcriptional control mechanisms, as demonstrated for other growth factors. In this communication, we provide evidence that a computer predicted Internal Ribosome Entry Site (IRES) structure is biologically active and is located at the 3′ end of the UTR. In addition, the results demonstrate that an alternative transcriptional initiation site for VEGF exists in the 5′ UTR of VEGF. This alternative initiation site is 633 bp downstream of the main transcription start site and the resulting 5′ UTR includes mainly the IRES structure. Therefore, our results suggest that VEGF is subjected to regulation at either translational level through a mechanism of ribosome internal initiation and/or transcriptional level through alternative initiation.

Vascular endothelial growth factor and its receptors.

Vascular endothelial growth factor (VEGF) is a highly specific mitogen for vascular endothelial cells and an angiogenic factor that is structurally related to platelet derived growth factor (PDGF). It is also known as the vascular permeability factor (VPF) because it efficiently potentiates the permeabilization of blood vessels. Five types of VEGF mRNA encoding VEGF species which differ in their molecular mass and in their biological properties are transcribed from a single gene as a result of alternative splicing. VEGFs are produced and secreted by several normal cell types including smooth muscle, luteal and adrenal cortex cells. VEGFs are also produced by different tumorigenic cells, and appear to play a major role in tumour angiogenesis. Antibodies directed against VEGF can inhibit the growth of a variety of VEGF producing tumours. Of the various VEGF species, the best characterized is the 165 amino acid long form (VEGF165). VEGF165 is a heparin binding growth factor, and its interaction with VEGF receptors on the cell surface of vascular endothelial cells depends on the presence of heparin-like molecules. Several cell types which do not proliferate in response to VEGF such as bovine corneal endothelial cells, HeLa cells and human melanoma cells also express cell surface VEGF receptors, but the function of the VEGF receptors in these cells is unclear. Recently, the tyrosine-kinase receptors encoded by the flt and KDRflk-1 genes were found to function as VEGF165 receptors.

Cloning of a negative transcription factor that binds to the upstream conserved region of moloney murine leukemia virus

The long terminal repeat of Moloney murine leukemia virus (MuLV) contains the upstream conserved region (UCR). The UCR core sequence, CGCCATTTT, binds a ubiquitous nuclear factor and mediates negative regulation of MuLV promoter activity. We have isolated murine cDNA clones encoding a protein, referred to as UCRBP, that binds specifically to the UCR core sequence. Gel mobility shift assays demonstrate that the UCRBP fusion protein expressed in bacteria binds the UCR core with specificity identical to that of the UCR-binding factor in the nucleus of murine and human cells. Analysis of full-length UCRBP cDNA reveals that it has a putative zinc finger domain composed of four C2H2 zinc fingers of the GLI subgroup and an N-terminal region containing alternating charges, including a stretch of 12 histidine residues. The 2.4-kb UCRBP message is expressed in all cell lines examined (teratocarcinoma, B- and T-cell, macrophage, fibroblast, and myocyte), consistent with the ubiquitous expression of the UCR-binding factor. Transient transfection of an expressible UCRBP cDNA into fibroblasts results in down-regulation of MuLV promoter activity, in agreement with previous functional analysis of the UCR. Recently three groups have independently isolated human and mouse UCRBP. These studies show that UCRBP binds to various target motifs that are distinct from the UCR motif: the adeno-associated virus P5 promoter and elements in the immunoglobulin light- and heavy-chain genes, as well as elements in ribosomal protein genes. These results indicate that UCRBP has unusually diverse DNA-binding specificity and as such is likely to regulate expression of many different genes.

Selective Binding of VEGF to One of the Three Vascular Endothelial Growth Factor Receptors of Vascular Endothelial Cells

VEGF and VEGF are vascular endothelial growth factor splice variants that promote the proliferation of endothelial cells and angiogenesis. VEGF contains the 44 additional amino acids encoded by exon 7 of the VEGF gene. These amino acids confer upon VEGF a heparin binding capability which VEGF lacks. I-VEGF bound to three vascular endothelial growth factor (VEGF) receptors on endothelial cells, while I-VEGF bound selectively only to the flk-1 VEGF receptor which corresponds to the larger of the three VEGF receptors. The binding of I-VEGF to flk-1 was not affected by the removal of cell surface heparan sulfates or by heparin. Both VEGF and VEGF inhibited the binding of I-VEGF to a soluble extracellular domain of the flk-1 VEGF receptor in the absence of heparin. However, heparin potentiated the inhibitory effect of VEGF by 2-3-fold. These results contrast with previous observations which have indicated that the binding of I-VEGF to the flk-1 receptor is strongly dependent on heparin-like molecules. Further experiments showed that the receptor binding ability of VEGF is susceptible to oxidative damage caused by oxidants such as HO or chloramine-T. VEGF was also damaged by oxidants but to a lesser extent. Heparin or cell surface heparan sulfates restored the flk-1 binding ability of damaged VEGF but not the receptor binding ability of damaged VEGF. These observations suggest that alternative splicing can generate a diversity in growth factor signaling by determining receptor recognition patterns. They also indicate that the heparin binding ability of VEGF may enable the restoration of damaged VEGF function in processes such as inflammation or wound healing.

Cell cycle-specific action of nerve growth factor in PC12 cells: differentiation without proliferation.

PC12 cells were manipulated in such a way as to permit the study of differentiation‐specific responses independently from proliferative responses. Cells were starved for serum then exposed to nerve growth factor (NGF) or serum. Following addition of serum, cells incorporated thymidine in a synchronous manner. Subsequent to the wave of DNA synthesis, the cell number increased approximately two‐fold. Addition of NGF to serum‐starved cultures had no measurable effect on either parameter. Neurite outgrowth was more rapid and extensive and appearance of Na+ channels, measured as saxitoxin binding sites, more rapid than when NGF was added to exponentially‐growing cells. Epidermal growth factor receptors were heterologously down‐regulated by NGF with similar kinetics under both conditions. Induction of the proto‐oncogene c‐fos by NGF was also greater in the serum‐starved cells than in exponentially‐growing cultures. These results indicated that serum starvation resulted in synchronisation of the cultures and that NGF action may be cell cycle‐specific. Analysis of the cellular response to NGF at different times during the cell cycle showed that c‐fos was induced in the G1 phase but not in S or G2. Fluorescence‐activated cell sorter analysis demonstrated that addition of NGF to exponentially‐growing cells, resulted in their accumulation in a G1‐like state. With regard to the study of the mechanism of NGF action, these results illustrate that measurements of NGF effects on specific components in the signal transduction pathway may be confounded by the use of exponentially‐growing cultures.

IFN-Stimulated Gene 15 Is Synergistically Activated Through Interactions Between the Myelocyte/Lymphocyte-Specific Transcription Factors, PU.1, IFN Regulatory Factor-8/IFN Consensus Sequence Binding Protein, and IFN Regulatory Factor-4: Characterization of a New Subtype of IFN-Stimulated Response Element

Type I IFNs cause the induction of a subset of genes termed IFN-stimulated genes (ISGs), which harbor a specific DNA element, IFN-stimulated response element (ISRE). This ISRE confers the responsiveness to the IFN signal through the binding of a family of transcription factors designated IFN regulatory factors (IRFs). Some IRFs can bind to the DNA alone, such as IRF-1, which elicits transcriptional activation, or IRF-2, which leads to transcriptional repression. In addition, these factors associate with IRF-8/IFN consensus sequence binding protein (ICSBP), an immune cell-restricted IRF, and the assembled heterocomplexes lead to synergistic repression of ISRE elements. ISG15 is a prototype ISG that contains a well-characterized ISRE. Here we show that PU.1, an ETS member essential for myeloid/lymphoid cell differentiation, forms heterocomplexes with the immune-restricted IRFs, IRF-8\/ICSBP and IRF-4, which lead to transcriptional activation of ISG15. These data allowed the characterization of a subset of ISREs designated ETS/IRF response element (EIRE), which are differentially regulated in immune cells. EIREs are unique in their ability to recruit different factors to an assembled enhanceosomes. In nonimmune cells the factors will mainly include IRF members, while cell type-restricted factors, such as PU.1, IRF-8\/ICSBP, and IRF-4, will be recruited in immune cells. IRF heterocomplex formation leads to transcriptional repression, and conversely, PU.1/IRFs heterocomplex formation leads to transcriptional activation. The fact that IRF-8\/ICSBP is an IFN-γ-induced factor explains why some of the EIREs are also induced by type II IFN. Our results lay the molecular basis for the unique regulation of ISGs, harboring EIRE, in immune cells.

Structural and functional features of a specific nucleosome containing a recognition element for the thyroid hormone receptor

The Xenopus thyroid hormone receptor βA (TRβA) gene contains an important thyroid hormone response element (TRE) that is assembled into a positioned nucleosome. We determine the translational position of the nucleosome containing the TRE and the rotational positioning of the double helix with respect to the histone surface. Histone H1 is incorporated into the nucleosome leading to an asymmetric protection to micrococcal nuclease cleavage of linker DNA relative to the nucleosome core. Histone H1 association is without significant consequence for the binding of the heterodimer of thyroid hormone receptor and 9‐cis retinoic acid receptor (TR/RXR) to nucleosomal DNA in vitro, or for the regulation of TRβA gene transcription following microinjection into the oocyte nucleus. Small alterations of 3 and 6 bp in the translational positioning of the TRE in chromatin are also without effect on the transcriptional activity of the TRβA gene, whereas a small change in the rotational position of the TRE (3 bp) relative to the histone surface significantly reduces the binding of TR/RXR to the nucleosome and decreases transcriptional activation directed by TR/RXR. Our results indicate that the specific architecture of the nucleosome containing the TRE may have regulatory significance for expression of the TRβA gene.

Review: ICSBP/IRF-8 Transactivation: A Tale of Protein-Protein Interaction

Interferon (IFN) consensus sequence binding protein (ICSBP) is a member of a family of transcription factors termed IFN regulatory factors (IRF) and is also called IRF-8. Its expression is restricted mainly to cells of the immune system, and it plays a key role in the maturation of macrophages. ICSBP exerts its activity through the formation of different DNA-binding heterocomplexes. The interacting partner dictates a specific DNA recognition sequence, thus rendering ICSBP dual transcriptional activity, that is, repression or activation. Accordingly, such DNA elements were identified at the promoter regions of target genes that manifest macrophage action. A specific module (IRF association domain [IAD]) within ICSBP and a PEST domain located on the interacting partners mediate this association. Thus, ICSBP serves as an excellent prototype, demonstrating how a small subset of transcription factors can regulate gene expression in a spatial, temporal, and delicate tuning through combinatorial protein-protein interactions on different enhanceasomes.

Insect Cell-derived VP2 of Infectious Bursal Disease Virus Confers Protection Against the Disease in Chickens

Infectious bursal disease virus (IBDV) has become a major problem in recent years. Conventional vaccines make use of attenuated or inactivated viral strains, but these are gradually losing their effectiveness. We investigated the possibility of using purified VP2, a subunit of IBDV structural protein expressed in insect cells, as a vaccine. The VP2 gene was cloned into pAcYM1. The cloned gene was expressed in a baculovirus system, giving rise to a high quantity of recombinant VP2 (rVP2) protein. The length of the VP2 is 453 amino acids, and it contains two additional amino acids of the baculovirus at the carboxyl terminus. The molecular mass of the protein is about 48 kD. The rVP2 protein reacted with antibodies raised against viral VP2 and had a similar molecular weight. This protein was tested in a controlled vaccination experiment and compared with an inactivated commercial vaccine. High levels of antibodies were raised by the vaccinated birds. The vaccinated birds were challenged with a pathogenic viral strain. rVP2-vaccinated chickens exhibited high resistance to the virus. No mortality or weight changes in the bursa of Fabricius were observed in the vaccinated birds, whereas in the negative control birds, vaccinated with phosphate buffer, up to 50% mortality was found. Higher levels of antibodies were found by enzyme-linked immunosorbent assay in birds vaccinated with rVP2 compared with those vaccinated with the commercial vaccine. This study suggests the potential use of the isolated rVP2 as a subunit vaccine.