Activation of mechanosensitive ion channel TRPV4 normalizes tumor vasculature and improves cancer therapy.

Tumor vessels are characterized by abnormal morphology and hyperpermeability that together cause inefficient delivery of chemotherapeutic agents. Although vascular endothelial growth factor has been established as a critical regulator of tumor angiogenesis, the role of mechanical signaling in the regulation of tumor vasculature or tumor endothelial cell (TEC) function is not known. Here we show that the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) regulates tumor angiogenesis and tumor vessel maturation via modulation of TEC mechanosensitivity. We found that TECs exhibit reduced TRPV4 expression and function, which is correlated with aberrant mechanosensitivity towards extracellular matrix stiffness, increased migration and abnormal angiogenesis by TEC. Further, syngeneic tumor experiments revealed that the absence of TRPV4 induced increased vascular density, vessel diameter and reduced pericyte coverage resulting in enhanced tumor growth in TRPV4 knockout mice. Importantly, overexpression or pharmacological activation of TRPV4 restored aberrant TEC mechanosensitivity, migration and normalized abnormal angiogenesis in vitro by modulating Rho activity. Finally, a small molecule activator of TRPV4, GSK1016790A, in combination with anticancer drug cisplatin, significantly reduced tumor growth in wild-type mice by inducing vessel maturation. Our findings demonstrate TRPV4 channels to be critical regulators of tumor angiogenesis and represent a novel target for anti-angiogenic and vascular normalization therapies.

Attenuated p53 activation in tumour-associated stromal cells accompanies decreased sensitivity to etoposide and vincristine.

Alterations in the tumour suppressor p53 have been reported in tumour-associated stromal cells; however, the consequence of these alterations has not been elucidated. We investigated p53 status and responses to p53-activating drugs using tumour-associated stromal cells from A375 melanoma and PC3 prostate carcinoma xenografts, and a spontaneous prostate tumour model (TRAMP). p53 accumulation after treatment with different p53-activating drugs was diminished in tumour-associated stromal cells compared to normal stromal cells. Tumour-associated stromal cells were also less sensitive to p53-activating drugs - this effect could be reproduced in normal stromal cells by p53 knockdown. Unlike normal stromal cells, tumour stromal cells failed to arrest in G(2) after etoposide treatment, failed to upregulate p53-inducible genes, and failed to undergo apoptosis after treatment with vincristine. The lower levels of p53 in tumour stromal cells accompanied abnormal karyotypes and multiple centrosomes. Impaired p53 function in tumour stroma might be related to genomic instability and could enable stromal cell survival in the destabilising tumour microenvironment.

Overexpression of neuropilin-1 promotes constitutive MAPK signalling and chemoresistance in pancreatic cancer cells.

Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). Neuropilin-1 is expressed in pancreatic cancer, but not in nonmalignant pancreatic tissue. We hypothesised that NRP-1 expression by pancreatic cancer cells contributes to the malignant phenotype. To determine the role of NRP-1 in pancreatic cancer, NRP-1 was stably transfected into the human pancreatic cancer cell line FG. Signal transduction was assessed by Western blot analysis. Susceptibility to anoikis (detachment induced apoptosis) was evaluated by colony formation after growth in suspension. Chemosensitivity to gemcitabine or 5-fluorouracil (5-FU) was assessed by MTT assay in pancreatic cancer cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. Differential expression of apoptosis-related genes was determined by gene array and further evaluated by Western blot analysis. Neuropilin-1 overexpression increased constitutive mitogen activated protein kinase (MAPK) signalling, possibly via an autocrine loop. Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by > 30% after exposure to clinically relevant levels of gemcitabine and 5-FU. In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing > 50% more cell death at clinically relevant concentrations of gemcitabine. Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1. Neuropilin-1 overexpression in pancreatic cancer cell lines is associated with (a) increased constitutive MAPK signalling, (b) inhibition of anoikis, and (c) chemoresistance. Targeting NRP-1 in pancreatic cancer cells may downregulate survival signalling pathways and increase sensitivity to chemotherapy.

Vascular endothelial growth factor-mediated autocrine stimulation of prostate tumor cells coincides with progression to a malignant phenotype.

Vascular endothelial growth factor (VEGF), which is often produced at high levels by tumor cells, is a well-known mediator of tumor angiogenesis. VEGF receptor tyrosine kinases, KDR/Flk-1 and Flt-1, have been thought to be expressed exclusively by endothelial cells. In this study, we have used a prostate tumor progression series comprised of a differentiated rat prostate epithelial cell line, NbE-1, and its highly motile clonal derivative, FB2. Injection of NbE-1 cells into the inferior vena cava of syngeneic rats indicated that these cells are nontumorigenic. Using the same model, FB2 cells generated rapidly growing and well-vascularized tumors in the lungs. NbE-1 expressed marginal levels of VEGF, whereas high levels of VEGF protein were detected in FB2-conditioned medium and in FB2 tumors in vivo. Analysis of (125)I-VEGF(165) binding to NbE-1 and FB2 cells indicated that only motile FB2 cells expressed the VEGF receptor Flt-1. Consistent with this finding, physiological concentrations of VEGF induced chemotactic migration in FB2 but not in NbE-1 cells. This is the first documentation of a functional Flt-1 receptor in prostate tumor cells. Our results suggest two roles for VEGF in tumor progression: a paracrine role as an angiogenic factor and a previously undescribed role as an autocrine mediator of tumor cell motility.

N-arginine dibasic convertase is a specific receptor for heparin-binding EGF-like growth factor that mediates cell migration.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a mitogen and chemotactic factor, binds to two receptor tyrosine kinases, erbB1 and erbB4. Now we demonstrate that HB-EGF also binds to a novel 140 kDa receptor on MDA-MB 453 cells. Purification of this receptor showed it to be identical to N-arginine dibasic convertase (NRDc), a metalloendopeptidase of the M16 family. Binding to cell surface NRDc and NRDc in solution was highly specific for HB-EGF among EGF family members. When overexpressed in cells, NRDc enhanced their migration in response to HB-EGF but not to EGF. Conversely, inhibition of endogenous NRDc expression in cells by antisense morpholino oligonucleotides inhibited HB-EGF-induced cell migration. Anti-erbB1 neutralizing antibodies completely abrogated the ability of NRDc to enhance HB-EGF-dependent migration, demonstrating that this NRDc activity was dependent on erbB1 signaling. Although NRDc is a metalloproteinase, enzymatic activity was not required for HB-EGF binding or enhancement of cell migration; neither did NRDc cleave HB-EGF. Together, these results suggest that NRDc is a novel specific receptor for HB-EGF that modulates HB-EGF-induced cell migration via erbB1.

Neuropilin-1 expression in osteogenic cells: down-regulation during differentiation of osteoblasts into osteocytes.

The expression of neuropilin-1 (NRP1), a recently described VEGF and semaphorin receptor expressed by endothelial cells (EC) but some non-EC types as well, was analyzed in osteoblasts in vitro and in vivo. Cultured MC3T3-E1 osteoblasts expressed NRP1 mRNA and bound VEGF(165) but not VEGF(121), characteristic of the VEGF isoform-specific binding of NRP1. These cells did not express VEGFR-1 or VEGFR-2 so that VEGF binding to osteoblasts was strictly NRP1-dependent. In a chick osteocyte differentiation system, NRP1 was expressed by osteoblasts but its expression was absent as the cells matured into osteocytes. Immunohistochemical localization of NRP1 within the developing bones of 36-day-old mice and embryonic Day 17 chicks demonstrated that NRP1 was expressed by osteoblasts migrating alongside invading blood vessels within the metaphysis of the growth plate, as well as by osteoblasts at the developing edge of trabeculae within the marrow cavity. On the other hand, NRP1 was not expressed by osteocytes in either species, consistent with the in vitro results. In addition to osteogenic cells, NRP1 expression by EC was observed throughout the bone. Together these results suggest that NRP1 might have a dual function in bone by mediating osteoblast function directly as well as angiogenesis.

Genomic organization of human neuropilin-1 and neuropilin-2 genes: identification and distribution of splice variants and soluble isoforms.

Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are both receptors for semaphorins, which regulate neuronal guidance, and vascular endothelial growth factor (VEGF), an angiogenic factor. The two human NRP1 and NRP2 genes were cloned, and the exon-intron boundaries were determined. The NRP1 and NRP2 genes span over 120 and 112 kb, respectively, and are composed of 17 exons. Five of the exons are identical in size in the two genes, suggesting that they arose by gene duplication. Both NRP genes are characterized by multiple alternatively spliced variants. Two NRP2 isoforms, NRP2a and NRP2b, were cloned. A striking feature of these two isoforms is that they have identical extracellular domains but have divergent transmembrane and cytoplasmic domains. In these domains, NRP2a is closer in sequence identity to NRP1 than to NRP2b. As determined by Northern blot analysis, both NRP2a and NRP2b are expressed in a variety of tissues, mostly in a nonoverlapping manner. Within NRP2a and NRP2b, there are several alternatively spliced species: NRP2a(17), NRP2a(22), NRP2b(0), and NRP2b(5). In addition to full-length NRPs, there are truncated NRPs as well, which contain only the extracellular a/CUB and b/coagulation factor domains. These genes encode proteins that are soluble (sNRP) and released by cells. In addition to s12NRP1, which was previously cloned, s11NRP1 and s9NRP2 have now been cloned. These sNRP molecules are characterized by having intron-derived sequences at their C-termini. Altogether, eight NRP isoforms are described in this report. It was concluded that there are multiple NRP1 and NRP2 isoforms including intact and soluble forms. Characterization of these isoforms should help to elucidate the function of NRPs in neuronal guidance and angiogenesis.

Neuropilin-1 expression by tumor cells promotes tumor angiogenesis and progression.

Neuropilin-1 (NRP1) is a VEGF(165) and semaphorin receptor expressed by vascular endothelial cells (EC) and tumor cells. The function of NRP1 in tumor cells is unknown. NRP1 was overexpressed in Dunning rat prostate carcinoma AT2.1 cells using a tetracycline-inducible promoter. Concomitant with increased NRP1 expression in response to a tetracycline homologue, doxycycline (Dox), basal cell motility, and VEGF(165) binding were increased three- to fourfold in vitro. However, induction of NRP1 did not affect tumor cell proliferation. When rats injected with AT2.1/NRP1 tumor cells were fed Dox, NRP1 synthesis was induced in vivo and AT2.1 cell tumor size was increased 2.5- to 7-fold in a 3-4 wk period compared to controls. The larger tumors with induced NRP1 expression were characterized by markedly increased microvessel density, increased proliferating EC, dilated blood vessels, and notably less tumor cell apoptosis compared to noninduced controls. It was concluded that NRP1 expression results in enlarged tumors associated with substantially enhanced tumor angiogenesis.

Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation.

The mechanism(s) by which localized vascular permeability and angiogenesis occur at the sites of implantation is not clearly understood. Vascular endothelial growth factor (VEGF) is a key regulator of vasculogenesis during embryogenesis and angiogenesis in adult tissues. VEGF is also a vascular permeability factor. VEGF acts via two tyrosine kinase family receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). Recent evidence suggests that neuropilin-1 (NRP1), a receptor involved in neuronal cell guidance, is expressed in endothelial cells, binds to VEGF(165) and enhances the binding of VEGF(165) to VEGFR2. We examined the spatiotemporal expression of vegf isoforms, nrp1 and vegfr2 as well as their interactions in the periimplantation mouse uterus. We observed that vegf(164) is the predominant isoform in the mouse uterus. vegf(164) mRNA accumulation primarily occurred in epithelial cells on days 1 and 2 of pregnancy. On days 3 and 4, the subepithelial stroma in addition to epithelial cells exhibited accumulation of this mRNA. After the initial attachment reaction on day 5, luminal epithelial and stromal cells immediately surrounding the blastocyst exhibited distinct accumulation of vegf(164) mRNA. On days 6-8, the accumulation of this mRNA occurred in both mesometrial and antimesometrial decidual cells. These results suggest that VEGF(164) is available in mediating vascular changes and angiogenesis in the uterus during implantation and decidualization. This is consistent with coordinate expression of vegfr2, and nrp1, a VEGF(164)-specific receptor, in uterine endothelial cells. Their expression was low during the first 2 days of pregnancy followed by increases thereafter. With the initiation and progression of implantation (days 5-8), these genes were distinctly expressed in endothelial cells of the decidualizing stroma. Expression was more intense on days 6-8 at the mesometrial pole, the presumptive site of heightened angiogenesis and placentation. However, the expression was absent in the avascular primary decidual zone immediately surrounding the implanting embryo. Crosslinking experiments showed that (125)I-VEGF(165) binds to both NRP1 and VEGFR2 present in decidual endothelial cells. These results suggest that VEGF(164), NRP1 and VEGFR2 play a role in VEGF-induced vascular permeability and angiogenesis in the uterus required for implantation. genesis 26:213-224, 2000.

A natural ErbB4 isoform that does not activate phosphoinositide 3-kinase mediates proliferation but not survival or chemotaxis.

ErbB4 is a member of the epidermal growth factor receptor (ErbB) family that mediates cellular responses activated by neuregulins (NRG) and other epidermal growth factor-like growth factors. Two naturally occurring ErbB4 isoforms, ErbB4 CYT-1 and ErbB4 CYT-2, have previously been identified. Unlike ErbB4 CYT-1, ErbB4 CYT-2 lacks a phosphoinositide 3-kinase (PI3-K)-binding site and is incapable of activating PI3-K. We have now examined the consequences of the inability of this isoform to activate PI3-K on cell proliferation, survival, and chemotaxis in response to NRG-1beta: (i) NRG-1beta stimulated proliferation of cells expressing either ErbB4 CYT-1 or ErbB4 CYT-2. Consistent with the mitogenic responsiveness, analysis of downstream signaling showed that Shc and MAPK were phosphorylated after stimulating either isoform with NRG-1beta. (ii) NRG-1beta protected cells expressing ErbB4 CYT-1 but not cells expressing ErbB4 CYT-2 from starvation-induced apoptosis as measured by effects on cell number and 4', 6-diamidino-2-phenylindole staining. Furthermore, in cells expressing ErbB4 CYT-2, Akt, a protein kinase that mediates cell survival, was not phosphorylated. (iii) NRG-1beta stimulated chemotaxis and membrane ruffling in cells expressing ErbB4 CYT-1 but not in cells expressing ErbB4 CYT-2. In summary, ErbB4 CYT-2 can mediate proliferation but not chemotaxis or survival. These results suggest a novel mechanism by which cellular responses such as chemotaxis and survival may be regulated by the expression of alternative receptor-tyrosine kinase isoforms that differ in their coupling to PI3-K signaling.

Identification of a natural soluble neuropilin-1 that binds vascular endothelial growth factor: In vivo expression and antitumor activity.

Neuropilin-1 (NRP1) is a 130-kDa transmembrane receptor for semaphorins, mediators of neuronal guidance, and for vascular endothelial growth factor 165 (VEGF(165)), an angiogenesis factor. A 2.2-kb truncated NRP1 cDNA was cloned that encodes a 644-aa soluble NRP1 (sNRP1) isoform containing just the a/CUB and b/coagulation factor homology extracellular domains of NRP1. sNRP1 is secreted by cells as a 90-kDa protein that binds VEGF(165), but not VEGF(121). It inhibits (125)I-VEGF(165) binding to endothelial and tumor cells and VEGF(165)-induced tyrosine phosphorylation of KDR in endothelial cells. The 3' end of sNRP1 cDNA contains a unique, 28-bp intron-derived sequence that is absent in full-length NRP1 cDNA. Using a probe corresponding to this unique sequence, sNRP1 mRNA could be detected by in situ hybridization differentially from full-length NRP1 mRNA, for example, in cells of liver, kidney, skin, and breast. Analysis of blood vessels in situ showed that NRP1, but not sNRP1, was expressed. sNRP1 was functional in vivo. Unlike control tumors, tumors of rat prostate carcinoma cells expressing recombinant sNRP1 were characterized by extensive hemorrhage, damaged vessels, and apoptotic tumor cells. These results demonstrate the existence of a naturally occurring, soluble NRP1 that is expressed differently from intact NRP1 and that appears to be a VEGF(165) antagonist.

Neuropilin is a mediator of angiogenesis.

Neuropilin is both a receptor for semaphorins, which are mediators of neuronal guidance, and for VEGF, an angiogenesis factor. While the function of neuropilin in the nervous system has been characterized, its role in angiogenesis is only beginning to be elucidated. This article reviews some of the structural and functional features of neuropilin and presents the experimental evidence showing that it is a mediator of angiogenesis. In particular, we show that neuropilin and its soluble isoforms regulate tumor angiogenesis and subsequent tumor growth.

Isolation of mouse stromal cells associated with a human tumor using differential diphtheria toxin sensitivity.

Tumor vascularization is accompanied by the migration of stromal cells, including endothelial cells, smooth muscle cells, and fibroblasts, into the tumor. The biological contributions of stromal cells to tumor vascularization have not been well-defined, partly due to the difficulty of culturing stromal cells in the presence of large numbers of fast-growing tumor cells. To address this problem, a strategy was devised to kill tumor cells but not stromal cells. Advantage was taken of the observation that diphtheria toxin (DT) kills human but not rodent cells. Human melanoma (MMAN) tumor cells were injected subcutaneously into nude mice. The tumors were excised, homogenized, and treated with 50 ng/ml DT for 24 hours. Elimination of melanoma cells by DT treatment was demonstrated by lack of detectable levels of microphthalmia, a transcription factor that is a marker for melanoma cells. The murine stromal cells were viable and found to be mostly smooth muscle cells. These cells constituted about 1.5% of the MMAN tumor. RNase protection assays using a specific murine vascular endothelial growth factor probe confirmed the murine origin of the stromal cells. This method allows rapid isolation of stromal cells and should facilitate biochemical and genetic analysis of tumor-stromal interactions.

The shedding of membrane-anchored heparin-binding epidermal-like growth factor is regulated by the Raf/mitogen-activated protein kinase cascade and by cell adhesion and spreading.

Heparin-binding epidermal-like growth factor (HB-EGF) is synthesized as a transmembrane precursor (HB-EGF(TM)). The addition of phorbol ester (PMA, phorbol 12-myristate 13-acetate) to cells expressing HB-EGF(TM) results in the metalloproteinase-dependent release (shedding) of soluble HB-EGF. To analyze mechanisms that regulate HB-EGF shedding, a stable cell line was established expressing HB-EGF(TM) in which the ectodomain and the cytoplasmic tail were tagged with hemagglutinin (HA) and Myc epitopes, respectively (HB-EGF(TM)HA/Myc). HB-EGF(TM)HA/Myc cleavage was followed by the appearance of soluble HB-EGFHA in conditioned medium, the loss of biotinylated cell-surface HB-EGF(TM)HA/Myc, and the appearance of a Myc-tagged cytoplasmic tail fragment in cell lysates. By using this approach, several novel metalloproteinase-dependent regulators of HB-EGF(TM) shedding were identified as follows. (i) HB-EGF(TM)HA/Myc shedding induced by PMA was blocked by the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059. PMA activated MAP kinase within 5 min, but HB-EGF(TM)HA/Myc shedding did not occur until 20 min, suggesting that MAP kinase activation was a necessary step in the pathway of PMA-induced HB-EGF(TM) cleavage. (ii) Activation of an inducible Raf-1 kinase, DeltaRaf-1:estrogen receptor, resulted in a rapid MAP kinase activation within 10 min and shedding of HB-EGF(TM)HA/Myc within 20-40 min. (iii) Serum induced MAP kinase activation and HB-EGF(TM)HA/Myc shedding that were inhibited by PD98059. (iv) Whereas PMA induced HB-EGF(TM)HA/Myc shedding in attached cells, no shedding occurred when the cells were placed in suspension. Shedding was fully restored shortly after cells were allowed to spread on fibronectin, and the extent of PMA-induced shedding increased with the extent of cell spreading. PMA induced the same level of MAP kinase activation whether the cells were attached or in suspension suggesting that although MAP kinase activation might be necessary for shedding, it was not sufficient. Taken together, these results suggest that there are two components of cell regulation that contribute to the shedding process, not previously recognized, the Raf-1/MAP kinase signal transduction pathway and cell adhesion and spreading.

AP-1 mediates stretch-induced expression of HB-EGF in bladder smooth muscle cells.

Mechanical induction of growth factor synthesis may mediate adaptive responses of smooth muscle cells (SMC) to increases in physical load. We previously demonstrated that cyclic mechanical stretch induces expression of the SMC, fibroblast, and epithelial cell mitogen heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bladder SMC, an observation that suggests that this growth factor may be involved in compensatory bladder hypertrophy. In the present study we provide evidence that the activator protein-1 (AP-1) transcription factor plays a critical role in this mechanoinduction process. Rat bladder SMC were transiently transfected with a series of 5' deletion mutants of a promoter-reporter construct containing 1. 7 kb of the mouse HB-EGF promoter that was previously shown to be stretch responsive. The stretch-mediated increase in promoter activity was completely ablated with deletion of nucleotide positions -1301 to -881. Binding of AP-1, as evaluated by electrophoretic mobility shift assay, to a synthetic oligonucleotide containing an AP-1 binding site increased in response to stretch, and binding was inhibited by excess unlabeled DNA corresponding to nucleotides -993 to -973 from the HB-EGF promoter, a region that contains a previously recognized composite AP-1/Ets site. Stretch-induced promoter activity was significantly inhibited by site-directed mutagenesis of the AP-1 or Ets components of this site. Consistent with the promoter and gel-shift studies, curcumin, an inhibitor of AP-1 activation, suppressed the HB-EGF mRNA induction after stretch. Stretch also specifically increased mRNA levels for matrix metalloproteinase (MMP)-1, the promoter of which contains a functional AP-1 element, but not for MMP-2, the promoter of which does not contain an AP-1 element. The stretch response of the MMP-1 gene was also completely inhibited by curcumin. Collectively, these findings indicate that AP-1-mediated transcription plays an important role in the regulation of gene expression in bladder muscle in response to mechanical forces.

Differential binding of vascular endothelial growth factor B splice and proteolytic isoforms to neuropilin-1.

Vascular endothelial growth factor B (VEGF-B) is expressed in various tissues, especially strongly in the heart, and binds selectively to one of the VEGF receptors, VEGFR-1. The two splice isoforms, VEGF-B(167) and VEGF-B(186), have identical NH(2)-terminal cystine knot growth factor domains but differ in their COOH-terminal domains which give these forms their distinct biochemical properties. In this study, we show that both splice isoforms of VEGF-B bind specifically to Neuropilin-1 (NRP1), a receptor for collapsins/semaphorins and for the VEGF(165) isoform. The NRP1 binding of VEGF-B could be competed by an excess of VEGF(165). The binding of VEGF-B(167) was mediated by the heparin binding domain, whereas the binding of VEGF-B(186) to NRP1 was regulated by exposure of a short COOH-terminal proline-rich peptide upon its proteolytic processing. In immunohistochemistry, NRP1 distribution was found to be overlapping or adjacent to known sites of VEGF-B expression in several tissues, in particular in the developing heart, suggesting the involvement of VEGF-B in NRP1-mediated signaling.

Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165.

Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65-75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

Molecular angiogenesis.

New insights into the mechanisms by which blood vessels develop (angiogenesis) have been gained recently, primarily by the identification of factors that inhibit and promote this process. Angiogenesis-stimulating factors are being used to promote growth of new blood vessels in ischemic disease. In contrast, anti-angiogenesis factors are being used as inhibitors of neovascularization to control tumor growth and metastasis.

Characterization of a naturally occurring ErbB4 isoform that does not bind or activate phosphatidyl inositol 3-kinase.

Receptor tyrosine kinases regulate cell behavior by activating specific signal transduction cascades. Epidermal growth factor (EGF) receptor tyrosine kinases include ErbB1, ErbB2, ErbB3 and ErbB4. ErbB4 is a tyrosine kinase receptor that binds neuregulins (NRG) and several other EGF family members. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis identified two isoforms of ErbB4 that differed in their cytoplasmic domain sequences. Specifically, RT-PCR using primers flanking the putative phosphatidyl inositol 3-kinase (PI3-K) binding site of ErbB4 generated two specific bands when human and mouse heart and kidney tissues were analysed. Cloning and sequencing of these RT-PCR products revealed that one of the ErbB4 isoforms (ErbB4 CYT-2) lacked a 16 amino acid sequence including a putative PI3-K binding site, that was present in the other isoform (ErbB4 CYT-1). RT-PCR analysis of mouse tissues suggested that the expression of ErbB4 CYT-1 and ErbB4 CYT-2 was tissue-specific. Heart, breast and abdominal aorta expressed predominantly ErbB4 CYT-1 whereas neural tissues and kidney expressed predominantly ErbB4 CYT-2. To ascertain whether the absence of the putative PI3-K binding site in ErbB4 CYT-2 also resulted in the loss of PI3-K activity, NIH3T3 cell lines overexpressing ErbB4 CYT-1 or ErbB4 CYT-2 were produced. NRG-1 bound to and stimulated equivalent tyrosine phosphorylation of both isoforms. However, unlike ErbB4 CYT-1, the ErbB4 CYT-2 isoform was unable to bind the p85 subunit of PI3-K and to stimulate PI3-K activity in these cells. Furthermore, tyrosine phosphorylation of p85 or association of PI3-K activity with phosphotyrosine was not induced in NRG-1 treated cells expressing ErbB4 CYT-2, indicating that this isoform was incapable of activating PI3-K even indirectly. It was concluded that a novel naturally occurring ErbB4 isoform exists with a deletion of the cytoplasmic domain sequence required for the activation of the PI3-K intracellular signal transduction pathway and that this is the only PI3-K binding site in ErbB4.