Functional divergence between 2 chemokines is conferred by single amino acid change.

CXCL4 and CXCL4L1 are 2 closely related CXC chemokines that exhibit potent antiangiogenic activity. Because interactions with glycosaminoglycans play a crucial role in chemokines activity, we determined the binding parameters of CXCL4 and CXCL4L1 for heparin, heparan sulfate, and chondroitin sulfate B. We further demonstrated that the Leu67/His67 substitution is critical for the decrease in glycan binding of CXCL4L1 but also for the increase of its angiostatic activities. Using a set of mutants, we show that glycan affinity and angiostatic properties are not completely related. These data are reinforced using a monoclonal antibody that specifically recognizes structural modifications in CXCL4L1 due to the presence of His67 and that blocks its biologic activity. In vivo, half-life and diffusibility of CXCL4L1 compared with CXCL4 is strongly increased. As opposed to CXCL4L1, CXCL4 is preferentially retained at its site of expression. These findings establish that, despite small differences in the primary structure, CXCL4L1 is highly distinct from CXCL4. These observations are not only of great significance for the antiangiogenic activity of CXCL4L1 and for its potential use in clinical development but also for other biologic processes such as inflammation, thrombosis or tissue repair.

Translokin is an intracellular mediator of FGF-2 trafficking.

Basic fibroblast growth factor (bFGF or FGF-2) exerts its pleiotropic activities both as an exogenous and an intracellular factor. FGF-1 and FGF-2 are prototypes for this dual signalling, but the mechanisms of their intracellular actions remain unknown. Here we show that Translokin, a cytoplasmic protein of relative molecular mass 55,000 (M(r) 55K), interacts specifically with the 18K form of FGF-2. Translokin is ubiquitously expressed and colocalizes with the microtubular network. As Translokin does not interact with FGF-1, we used a strategy based on FGF-1-FGF-2 chimaeras to map the interacting regions in FGF-2 and to generate Nb1a2, a non-interacting variant of FGF-2. Although most of the FGF-2 properties are preserved in Nb1a2, this variant is defective in intracellular translocation and in stimulating proliferation. The fusion of a nuclear localization signal to Nb1a2 restores its mitogenic activity and its nuclear association. Inhibiting Translokin expression by RNA interference reduces the translocation of FGF-2 without affecting the intracellular trafficking of FGF-1. Our data show that the nuclear association of internalized FGF-2 is essential for its mitogenic activity and that Translokin is important in this translocation pathway.

Translational induction of VEGF internal ribosome entry site elements during the early response to ischemic stress.

Vascular endothelial growth factor-A (VEGF), a powerful factor involved in vasculogenesis and angiogenesis, is translationally regulated through 2 independent internal ribosome entry sites (IRESs A and B). IRESs enable an mRNA to be translated under conditions in which 5'-cap-dependent translation is inhibited, such as low oxygen stress. In the VEGF mRNA, IRES A influences translation at the canonical AUG codon, whereas the 5' IRES B element regulates initiation at an upstream, in frame CUG. In this study, we have developed transgenic mice expressing reporter genes under the control of these 2 IRESs. We reveal that although these IRESs display low activity in embryos and adult tissues, they permit efficient translation at early time points in ischemic muscle, a stress under which cap-dependent translation is inhibited. These results demonstrate the in vivo efficacy of the VEGF IRESs in response to a local environmental stress such as hypoxia.

Control of the vascular endothelial growth factor internal ribosome entry site (IRES) activity and translation initiation by alternatively spliced coding sequences.

The vascular endothelial growth factor-A (VEGF) gene locus contains eight exons that span 14 kb. Alternative splicing generates multiple, different mRNAs that in turn translate into at least five protein isoforms. While the canonical AUG start codon is located at position 1039 in exon 1, there also exists an upstream, in-frame CUG initiation codon that drives expression of L-VEGF, containing an additional 180 amino acids. Two separate internal ribosome entry sites (IRES) regulate the activity of each initiation codon. Thus the 5'-UTR of VEGF, which comprises the majority of exon 1, consists of IRES B, the CUG, IRES A, and the AUG, from 5' to 3'. Previously, it has been shown that IRES B regulates initiation at the CUG and IRES A regulates AUG usage. In this study, we have found evidence that the exon content of the VEGF mRNA, determined through alternative splicing, controls IRES A activity. While the CUG is most efficient at initiating translation, transcripts that lack both exons 6 and 7 and therefore contain an exon 5/8 junction lack AUG-initiated translation. The process of splicing is not responsible for this start codon selection since transfection of genomic and cDNA VEGF sequences give the same expression pattern. We hypothesize that long range tertiary interactions in the VEGF mRNA regulate IRES activity and thus control start codon selection. This is the first report describing the influence of alternatively spliced coding sequences on codon selection by modulating IRES activity.