We have previously shown that the angiogenic growth factor pleiotrophin (PTN) induces migration of endothelial cells through binding to its receptor protein tyrosine phosphatase beta/zeta (RPTPbeta/zeta). In this study, we show that a monoclonal antibody against alpha(nu)beta(3) but not alpha(5)beta(1) integrin abolished PTN-induced human endothelial cell migration in a concentration-dependent manner. Integrin alpha(nu)beta(3) was found to directly interact with PTN in an RGD-independent manner, whereas a synthetic peptide corresponding to the specificity loop of the beta(3) integrin extracellular domain ((177)CYDMKTTC(184)) inhibited PTN-alpha(nu)beta(3) interaction and totally abolished PTN-induced endothelial cell migration. Interestingly, alpha(nu)beta(3) was also found to directly interact with RPTPbeta/zeta, and PTN-induced Y773 phosphorylation of beta(3) integrin was dependent on both RPTPbeta/zeta and the downstream c-src kinase activation. Midkine was found to interact with RPTPbeta/zeta, but not with alpha(nu)beta(3), and caused a small but statistically significant decrease in cell migration. In the same line, PTN decreased migration of different glioma cell lines that express RPTPbeta/zeta but do not express alpha(nu)beta(3), while it stimulated migration of U87MG cells that express alpha(nu)beta(3) on their cell membrane. Overexpression or down-regulation of beta(3) stimulated or abolished, respectively, the effect of PTN on cell migration. Collectively, these data suggest that alpha(nu)beta(3) is a key molecule that determines the stimulatory or inhibitory effect of PTN on cell migration.
Heparin affin regulatory peptide (HARP) is an 18-kDa secreted growth factor that has a high affinity for heparin and a potent role on tumor growth and angiogenesis. We have previously reported that HARP is mitogenic for different types of endothelial cells and also affects cell migration and differentiation (12). In this study we examined the signaling pathways involved in the migration and tube formation on matrigel of human umbilical vein endothelial cells (HUVEC) induced by HARP. We report for the first time that receptor-type protein-tyrosine phosphatase / (RPTP/), which is a receptor for HARP in neuronal cell types, is also expressed in HU-VEC. We also document that HARP signaling through RPTP/ leads to activation of Src kinase, focal adhesion kinase, phosphatidylinositol 3-kinase, and Erk1/2. Sodium orthovanadate, chondroitin sulfate-C, PP1, wortmannin, LY294002, and U0126 inhibit HARP-mediated signaling and HUVEC migration and tube formation. In addition, RPTP/ suppression using small interfering RNA technology interrupts intracellular signals and HUVEC migration and tube formation induced by HARP. These results establish the role of RPTP/ as a receptor of HARP in HUVEC and elucidate the HARP signaling pathway in endothelial cells.
It is becoming increasingly recognized that hydrogen peroxide (HP) plays a role in cell proliferation and migration. In the present study we found that exogenous HP significantly induced human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) seems to be involved in the stimulatory effect of HP, because the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, HP significantly increased HARP mRNA and protein amounts in a concentration-and time-dependent manner. Curcumin and activator protein-1 (AP-1) decoy oligonucleotides abrogated both HPinduced HARP expression and LNCaP cell proliferation and migration. HP increased luciferase activity of the 5-flanking region of the HARP gene introduced in a reporter gene vector, an effect that was abolished when even one of the two putative AP-1 binding sites of the HARP promoter was mutated. The effect of HP seems to be due to the binding of Fra-1, JunD, and phospho-c-Jun to the HARP promoter. These results support the notion that HARP is important for human prostate cancer cell proliferation and migration, establish the role of AP-1 in the up-regulation of HARP expression by low concentrations of HP, and characterize the AP-1 dimers involved.A growing body of evidence correlates the production of reactive oxygen species with many aspects of cellular functions (1). Reactive oxygen species include the superoxide, hydrogen peroxide (HP), 2 and the hydroxyl radical. Of these, HP has the chemical stability required to establish significant steady-state concentrations in vivo (2, 3) and is small and uncharged, properties that allow it to freely diffuse across plasma membranes and make it an ideal candidate for a signaling molecule (4). Small quantities of HP are produced by all types of cells, and several signal transduction pathways in mammalian cells have been reported to be activated by HP, such as tyrosine kinases, mitogen-activated protein kinases, protein kinase C, the epidermal growth factor receptor, protein phosphatases, potassium channels, and the transcription factors activator protein-1 (AP-1), and nuclear factor B (reviewed in ref. 1).Heparin affin regulatory peptide (HARP), also called pleiotrophin or heparin-binding growth-associated molecule, is an 18-kDa secreted growth factor that displays high affinity for heparin. A growing body of evidence indicates that HARP is involved in cell proliferation, migration, and differentiation and plays a significant role in several cellular processes (5, 6). HARP seems to play a major role in physiological as well as tumor angiogenesis and is detected in various carcinomas, such as human breast and prostate cancer, neuroblastomas, gliomas, benign meningiomas, and small cell lung cancer and mammary tumors, exhibiting a proto-oncogene function (5-8).HARP is highly conserved among human, rat, bovine, and mouse species, and its gene is expressed in a highly restricted temporal and spatial pattern during development, suggesting that HARP may be an ...
1 The involvement of platelets in neovascularization was investigated in the matrigel tube formation assay, an in vitro model of angiogenesis. 2 Platelets promoted the formation of capillary-like structures (expressed as relative tube area) numberand time-dependently. Relative tube area increased from 0.98+0.02 (n=8) in the presence of 6.25610 4 , to 3.21+0.12 (n=8) in the presence of 10 6 platelets/well compared to 0.54+0.04 (n=8) in their absence. This increase was unaected by acetyl salicylic acid (ASA), apyrase, and hirudin. Photographs from representative experiments, showed that platelets adhered along the dierentiating endothelium. 3 Addition of a-thrombin (0.1 ± 1 i.u. ml 71 ), the nitric oxide (NO) donor sodium nitroprusside (SNP; 1 ± 100 mM) or the NO synthase inhibitor, L-NG-arginine-methylester (L-NAME, 30 ± 300 mM) to the assay, had no eect on tube formation compared to that seen with platelets alone. 4 Neuraminidase (0.01 i.u./10 7 platelets), which strips sialic acid residues from membrane glycoproteins, abolished the promoting eect of platelets on tube formation. The relative tube area in the presence of neuraminidase-treated platelets was 0.81+0.03 (n=8), in the presence of untreated platelets 1.69+0.09, P50.001 (n=8) and in the absence of platelets, 0.80+0.04 (n=8). The tetrapeptide Arg-Gly-Asp-Ser (RGDS; 20 ± 200 mM) which inhibits von Willebrand factor, ®brinogen and ®bronectin-mediated adhesion, had no eect on the promoting eect of platelets on tube formation. 5 These results indicate that platelets promote angiogenesis in vitro. This eect is largely independent from activation by a-thrombin, is not modi®ed by manipulating NO and prostaglandin metabolism and proceeds possibly through adhesion of the platelets to the dierentiating endothelium.
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