Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents

Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1–4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix–binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases

Role of ADAMTS2, 3 and 14 in physiological and pathological processes

Enzymes of the ADAMTS family are closely related to MMPs and ADAMs. They further contain specific domains, such as a variable number of a domain known as « ThromboSPondin type I repeats » (TSP I). Amongs this family, ADAMTS2, 3 and 14 share a strong homology of sequence and structure. The primary function of ADAMTS2, 3 and 14, also known as aminoprocollagen peptidases, is to process fibrillar procollagen into mature molecules by excising the amino-propeptide. The lack of activity of ADAMTS2 is responsible for a inherited disease in human and in animal (Ehlers Danlos syndrom type VII C or dermatosparaxis) characterized by an extreme skin fragility. Analysis of ADAMTS-2 knock-out mice (Adamts2-/-) has shown also that male are sterile, suggesting that ADAMTS-2 have functions independent of its aminoprocollagen peptidase activity. In the first part of the results, we demonstrate that ADAMTS-2 inhibits angiogenesis, independently of its catalytic activity. ADAMTS-2 is able to reduce proliferation of endothelial cells, and to induce their retraction and detachment from the substrate resulting in apoptosis. Dephosphorylation of Erk1/2 and MLC largely precedes the ADAMTS-2 induced morphological alterations. In 3-D culture models, ADAMTS-2 strongly reduced branching of capillary-like structures formed by endothelial cells and their long-term maintenance and inhibited vessels formation in embryoid bodies (EB). Growth and vascularization of tumors formed in nude mice by HEK 293-EBNA cells expressing ADAMTS-2 were drastically reduced. A similar anti-tumoral activity was observed when using cells expressing recombinant deleted forms of ADAMTS-2, including catalytically inactive enzyme. Nucleolin, a nuclear protein also found to be associated with the cell membrane, was identified as a potential receptor mediating the antiangiogenic properties of ADAMTS-2.In following parts of results, we studied the functions of ADAMTS2, 3 and 14 in two different models: zebrafish models and mice models without expression of one of the aminoprocollagen peptidases.Zebrafish models: Four orthologues for ADAMTS2, 3 and 14 were identified in zebrafish (two orthologues for ADAMTS-2, one for ADAMTS3 and one for ADAMTS14). These enzymes present each a specific expression profil in RT-PCR and in situ hybridization studies. ADAMTS3 is the most expressed during the embryonic period. In situ hybridization demonstrate a strong expression of ADAMTS3 in the neural tube and in specific parts of the brains, suggesting a function during brain development. Repression of ADAMTS-3 expression by specific morpholinos induces lethality at an early stage of development. Mice models: Constitutive knock-out of Adamts3 lead to embryonic lethality around 15 dpc. Histological studies of 12.5 dpc, 13.5 dpc and 14.5 dpc Adamts3-/- embryos demonstrated an implication of Adamts3 in development of the brain, the liver and most probably of the heart.Constitutive knock-out of Adamts14 lead to a reduction of body size and of ear size. Histological studies of Adamts14-/- bones and ears suggested an implication of Adamts14 in growth regulation of bones and/or cartilage

Inherited Proteoglycan Biosynthesis Defects—Current Laboratory Tools and Bikunin as a Promising Blood Biomarker

Proteoglycans consist of proteins linked to sulfated glycosaminoglycan chains. They constitute a family of macromolecules mainly involved in the architecture of organs and tissues as major components of extracellular matrices. Some proteoglycans also act as signaling molecules involved in inflammatory response as well as cell proliferation, adhesion, and differentiation. Inborn errors of proteoglycan metabolism are a group of orphan diseases with severe and irreversible skeletal abnormalities associated with multiorgan impairments. Identifying the gene variants that cause these pathologies proves to be difficult because of unspecific clinical symptoms, hardly accessible functional laboratory tests, and a lack of convenient blood biomarkers. In this review, we summarize the molecular pathways of proteoglycan biosynthesis, the associated inherited syndromes, and the related biochemical screening techniques, and we focus especially on a circulating proteoglycan called bikunin and on its potential as a new biomarker of these diseases

Effect of ADAMTS-2, a metalloproteinase containing a disintegrin domain and thrombospondin type I repeats, during angiogenesis in vitro and in vivo

Formation of new blood vessels (angiogenesis) is a key step during the development of various pathologies, including cancer. Enzymes of the ADAMTS family are closely related to MMPs and ADAMs. They further contain specific domains, such as the ‘‘Thrombospondin type I’’ (TSP1) repeats, that are able to strongly repress angiogenesis, as described for thrombospondin-1 and -2, and for ADAMTS-1 and -8. The primary function of ADAMTS-2 is to process collagen type I, II and III precursors into mature molecules by excising the aminopropeptide. We further hypothesized that it could modulate angiogenesis through its TSP1 repeats. This hypothesis was investigated using different in vitro experimental models of angiogenesis. Recombinant ADAMTS-2 induced morphological changes in human umbilical vein endothelial cells (HUVEC) and human microvessel endothelial cells (HMEC), and significantly reduced their proliferation, attachment and spreading. Similar effects were observed when using inactive ADAMTS-2 mutated at the Zn2+-binding catalytic site. ADAMTS-2 did not alter the initial steps of formation of capillary-like structures by HUVEC in vitro. However, these structures appeared much less stable and were more rapidly disrupted in presence of ADAMTS-2 than in control conditions. ADAMTS-2 was also tested in an ex vivo angiogenesis model using aortic rings from rats and mice, wild type or KO for ADAMTS-2. Outgrowth of capillaries was slightly increased from aortas of ADAMTS-2 KO mice (TS2-/-) as compared to aortas from control animals (TS2+/+), while addition of full size recombinant ADAMTS-2 reduced the formation of capillary structures from rat aortas, suggesting its anti-angiogenic activity. Choroidal neovascularization induced in TS2+/+ or TS2-/- mice by LASER burns was used as in vivo model to confirm the in vitro and ex vivo results. Several genes involved in the healing and angiogenesis processes (fibrillar collagens, VEGF, TGF-beta and CTGF) were not differently regulated in TS2+/+ and TS2-/- mice at 5 days

Etude d'un nouveau variant d'épissage alternatif de la Neuropilin-1: rôle antagoniste dans la régulation de la progression tumorale?

Neuropilin-1 (NRP1) is a transmembrane glycoprotein and a co-receptor for several growth factors, for example some variants of the Vascular Endothelial Growth Factor A (VEGF-A). It largely contributes to the regulation of angiogenesis but also to cancer formation. NRP1 can be considered as a proteoglycan as glycosaminoglycans side chains can be added on serine 612. Currently, six splice variants of NRP1 have been described. An additional form was recently identified in our laboratory. Depending upon the cell types, it represents 20-30% of the total amount of NRP1. As compared to the full size NRP1 (NRP1-FS), 7 amino acids are deleted. As the missing sequence is located 2 amino acids downstream of the Ser612 required for glycosaminoglycans addition, this process could be somehow affected and the function of the protein could be modified. The glycosylation of NRP1-FS and -Δ7 was analyzed in different cells overexpressing each isoform. Western blotting analyses suggested that NRP1-Δ7 was less glycosylated than NRP1-FS. Prostate cancer cells (PC3) were engineered to express NRP1-FS or –Δ7 only in the presence of doxycycline. The migration of these cells was analyzed by scratch assay, with or without doxycycline in the medium. As compared to controls and to NRP1-FS-expressing cells, production of NRP1-Δ7 was linked to a reduction of cell migration. A DNA dosage showed that NRP1-FS enhanced cell proliferation, while NRP1-Δ7 reduced it. Tumor growth was assessed in vitro by a culture in soft agar. As compared to control conditions, expression of NRP1-FS by doxycycline increased colonies formation. By contrast, NRP1-Δ7 inhibited colonies number, suggesting an inhibition of tumorigenesis by this variant. As PC3 cells express basal level of endogenous NRP1, this suggests some competitive inhibition of NRP1 functions by NRP1-Δ7. Finally, the function of each variant was investigated in vivo in a model of injection in the flanks of nude mice of PC3 cells conditionally expressing NRP1-FS or -Δ7. As compared to the control, NRP1-FS increased tumor size and weight. By sharp contrast, the expression of NRP1-Δ7 was associated with a reduction of tumorigenicity. Cells with forced expression of NRP1-Δ7 also developed fewer blood vessels as compared to the control. These results suggest that NRP1-Δ7 have an antagonistic action on cancer formation and angiogenesis.Study of the Neuropilin isoforms produced by alternative splicing: functional implications for the regulation of angiogenesis and tumor progressio

A Novel Physiological Glycosaminoglycan-Deficient Splice Variant of Neuropilin-1 Is Anti-Tumorigenic In Vitro and In Vivo.

Neuropilin-1 (NRP1) is a transmembrane protein acting as a co-receptor for several growth factors and interacting with other proteins such as integrins and plexins/semaphorins. It is involved in axonal development, angiogenesis and cancer progression. Its primary mRNA is subjected to alternative splicing mechanisms generating different isoforms, some of which lack the transmembrane domain and display antagonist properties to NRP1 full size (FS). NRP1 is further post-translationally modified by the addition of glycosaminoglycans (GAG) side chains through an O-glycosylation site at serine612. Here, we characterized a novel splice variant which has never been investigated, NRP1-Δ7, differing from the NRP1-FS by a deletion of 7 amino acids occurring two residues downstream of the O-glycosylation site. This short sequence contains two aspartic residues critical for efficient glycosylation. As expected, the high molecular weight products appearing as a smear in SDS-PAGE and reflecting the presence of GAG in NRP1-FS were undetectable in the NRP1-Δ7 protein. NRP1-Δ7 mRNA was found expressed at an appreciable level, between 10 and 30% of the total NRP1, by various cells lines and tissues from human and murine origin. To investigate the biological properties of this isoform, we generated prostatic (PC3) and breast (MDA-MB-231) cancer cells able to express recombinant NRP1-FS or NRP1-Δ7 in a doxycycline-inducible manner. Cells with increased expression of NRP1-Δ7 were characterized in vitro by a significant reduction of proliferation, migration and anchorage-independent growth, while NRP1-FS had the expected opposite "pro-tumoral" effects. Upon VEGF-A165 treatment, a lower internalization rate was observed for NRP1-Δ7 than for NRP1-FS. Finally, we showed that NRP1-Δ7 inhibited growth of prostatic tumors and their vascularization in vivo. This report identifies NRP1-Δ7 as a splice variant displaying anti-tumorigenic properties in vitro and in vivo, emphasizing the need to consider this isoform in future studies

SLC10A7, an orphan member of the SLC10 family involved in congenital disorders of glycosylation

International audienceSLC10A7, encoded by the so-called SLC10A7 gene, is the seventh member of a human sodium/bile acid cotransporter family, known as the SLC10 family. Despite similarities with the other members of the SLC10 family, SLC10A7 does not exhibit any transport activity for the typical SLC10 substrates and is then considered yet as an orphan carrier. Recently, SLC10A7 mutations have been identified as responsible for a new Congenital Disorder of Glycosylation (CDG). CDG are a family of rare and inherited metabolic disorders, where glycosylation abnormalities lead to multisystemic defects. SLC10A7-CDG patients presented skeletal dysplasia with multiple large joint dislocations, short stature and amelogenesis imperfecta likely mediated by glycosaminoglycan (GAG) defects. Although it has been demonstrated that the transporter and substrate specificities of SLC10A7, if any, differ from those of the main members of the protein family, SLC10A7 seems to play a role in Ca2+ regulation and is involved in proper glycosaminoglycan biosynthesis, especially heparan-sulfate, and N-glycosylation. This paper will review our current knowledge on the known and predicted structural and functional properties of this fascinating protein, and its link with the glycosylation process