Matrix metalloproteinases (MMPs) exert both pro-and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2 ؊/؊ mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2 Matrix metalloproteinase 2 (MMP-2) (also known as gelatinase A), like many of the 23 MMPs in humans, has long been associated with angiogenesis, tumor progression, and metastasis (20, 50). More recently the critical importance of MMP-2 in breast cancer metastasis was shown by Minn et al. (52), who found that the MMP2 gene was one of the four key genes in the genomic signature associated with the most virulent breast cancer lung metastases. In this model, the development of angiogenesis was highly dependent upon MMP-2 expression (23). With other new functions of MMPs that promote cancer cell growth, invasion, metastasis, and immune cell evasion (20, 50) a more complex role for MMPs than just degradation of extracellular matrix proteins is being revealed (12, 56).Contrary to their many carcinogenic roles, several MMPs are now being recognized as cancer antitargets due to their normal physiological roles in cell signaling and tissue homeostasis that are protective against cancer (62). MMP-3 overexpression reduces 12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis (76); Mmp8 Ϫ/Ϫ mice treated with DMBA and the phorbol ester 12-myristate 13-acetate develop more skin papillomas and fibrosarcomas than wild-type mice (4), and Mmp12 Ϫ/Ϫ mice show increased lung carcinogenesis (30). The detailed study of individual MMPs is therefore necessary to understand their specific functions in different physiological and pathological processes, information that is necessary for protease drug target validation (62). Elucidation of each protease substrate repertoire or degradome is crucial to understanding the biological roles of MMPs (44). Genetic models of disease in mice are powerful, yet time-consuming (20,60,64), so high-content proteomic approaches using human cell systems offer great promise in understanding the biological functions of human proteases (60,70).

The human immunodeficiency virus (HIV) infects target cells by the capacity of its envelope glycoproteins gp120-gp41 to attach cells and induce the fusion of virus to cell membranes, a process which leads to virus entry. The receptor complex essential for HIV entry consists of the CD4 molecule and at least one of the members of the chemokine receptor family: CCR5 or CXCR4 [1,2]. Contrary to the virus entry process, the attachment of HIV particles to cells can occur even independently of CD4. We have previously demonstrated that HIV attachment is inhibited by the pseudopeptide HB-19 that binds specifically the C-terminal tail of nucleolin, a cell-surface-expressed protein identified to be implicated in HIV attachment [3][4][5]. Consequently, we have suggested that HIV attachment is achieved by the coordination of at least two events implicating on the one hand heparan sulfate proteoglycans [6,7] and on the other hand the cell surfaceexpressed nucleolin [4]. In the search for natural ligands of nucleolin that exhibit a potential inhibitory activity against HIV infection, other than midkine [8] and lactoferrin [9], here we show that pleiotrophin The growth factor pleiotrophin (PTN) has been reported to bind heparan sulfate and nucleolin, two components of the cell surface implicated in the attachment of HIV-1 particles to cells. Here we show that PTN inhibits HIV-1 infection by its capacity to inhibit HIV-1 particle attachment to the surface of permissive cells. The b-sheet domains of PTN appear to be implicated in this inhibitory effect on the HIV infection, in particular the domain containing amino acids 60-110. PTN binding to the cell surface is mediated by high and low affinity binding sites. Other inhibitors of HIV attachment known to bind specifically surface expressed nucleolin, such as the pseudopeptide HB-19 and the cytokine midkine prevent the binding of PTN to its low affinity-binding site. Confocal immunofluorescence laser microscopy revealed that the cross-linking of surface-bound PTN with a specific antibody results in the clustering of cell surface-expressed nucleolin and the colocalization of both PTN and nucleolin signals. Following its binding to surface-nucleolin, PTN is internalized by a temperature sensitive mechanism, a process which is inhibited by HB-19 and is independent of heparan and chondroitin sulfate proteoglycans. Nevertheless, proteoglycans might play a role in the concentration of PTN on the cell surface for a more efficient interaction with nucleolin. Our results demonstrate for the first time that PTN inhibits HIV infection and suggest that the cell surfaceexpressed nucleolin is a low affinity receptor for PTN binding to cells and it is also implicated in PTN entry into cells by an active process.Abbreviations ALK, anaplastic lymphoma kinase; AZT, azidothymidine; CHO, Chinese hamster ovary; HARP, heparin affin regulatory peptide; HB-GAM, heparin-binding growth-associated molecule; HBNF, heparin-binding neurite-promoting factor; MK, midkine; PTN, pleiotrophin; RPTP, receptor-type t...

We have previously described the purification of a heparin binding growth factor from adult bovine brain named heparin affin regulatory peptide (HARP), which was identical to an uterus derived growth factor named pleiotrophin and to a developmentally regulated neurite promoting factor named heparin-binding growth associated molecule. However, for yet unclear reasons, the mitogenic activity of this purified polypeptide following isolation from animal tissue extracts is a subject of controversy, due to conflicting and irreproducible data when produced by recombinant DNA technologies in E. coli or insect cells. The purified protein was inactive in mitogenic assays but the natural molecule was active in assay of neurite outgrowth. In order to clarify these conflicting results and to obtain a recombinant protein free from other contaminating heparin-binding growth factors, we have cloned human cDNA encoding human HARP, engineered its expression in NIH 3T3 cells and characterised the resulting recombinant polypeptide. Purified recombinant HARP displayed mitogenic activity for capillary endothelial cells with half-maximal stimulation at approximately 1 ng/ml (55 pM) and induced angiogenesis in an in vitro model. Interestingly, while the NH2 terminal sequence of tissue purified HARP was NH2-GKKEKPEKK, the NH2 terminal sequence of the biologically active recombinant protein was NH2-AEAGKKEKPEKK, corresponding to a three amino acid extended form.

Recently, we have found that the skin secretions of the Amazonian tree frog Phyllomedusa bicolor contains molecules with antitumor and angiostatic activities and identified one of them as the antimicrobial peptide dermaseptin (Drs) B2. In the present study we further explored the in vitro and in vivo antitumor activity of this molecule and investigated its mechanism of action. We showed that Drs B2 inhibits the proliferation and colony formation of various human tumor cell types, and the proliferation and capillary formation of endothelial cells in vitro. Furthermore, Drs B2 inhibited tumor growth of the human prostate adenocarcinoma cell line PC3 in a xenograft model in vivo. Research on the mechanism of action of Drs B2 on tumor PC3 cells demonstrated a rapid increasing amount of cytosolic lactate dehydrogenase, no activation of caspase-3, and no changes in mitochondrial membrane potential. Confocal microscopy analysis revealed that Drs B2 can interact with the tumor cell surface, aggregate and penetrate the cells. These data together indicate that Drs B2 does not act by apoptosis but possibly by necrosis. In conclusion, Drs B2 could be considered as an interesting and promising pharmacological and therapeutic leader molecule for the treatment of cancer.

HARP seems to act as an important regulator of diverse biological activities in human prostate cancer cells.

Heparin affin regulatory peptide (HARP) is an heparinbinding molecule involved in the regulation of cell proliferation and differentiation. Here, we report that HARP inhibited the biological activity induced by the 165-amino-acid form of vascular endothelial growth factor (VEGF 165 ) on human umbilical vein endothelial cells. Endothelial-cell proliferation induced by VEGF 165 showed about 50% inhibition in the presence of HARP in a concentration of 3 nM. In similar range of concentrations, HARP blocked tube formation induced by VEGF 165 in three-dimensional angiogenesis assay. In vivo studies showed that HARP inhibited the VEGF 165 -induced Matrigelt infiltration of endothelial cells. We then investigated the mechanisms of this inhibition and shown that HARP inhibited the binding of 125 I-VEGF 165 to the VEGF receptors of endothelial cells. Additional studies using VEGF soluble receptors indicated that binding of 125 I-VEGF 165 to kinase insert domain-containing receptor and neuropilin receptor was inhibited by HARP, but conversely the binding of 125 I-VEGF 165 to fms-like tyrosine kinase I receptor was unaffected. A competitive affinity-binding assay demonstrated that HARP interacted directly with VEGF 165 with a dissociation coefficient of 1.38 nM. Binding assay using deletion mutants of HARP revealed that the thrombospondin type-1 repeats domains were involved in this interaction. These data demonstrate for the first time that the angiogenic factor HARP can also negatively regulates the angiogenic activity of VEGF 165 .

Heparin affin regulatory peptide (HARP) is a 18-kDa heparin-binding polypeptide that is highly expressed in developing tissues and in several primary human tumors. It seems to play a key role in cellular growth and differentiation. In vitro, HARP displays mitogenic, angiogenic, and neurite outgrowth activities. It is a secreted protein that is organized in two ␤-sheet domains, each domain containing a cluster of basic residues. To assess determinants involved in the biological activities of HARP, C-terminally truncated proteins were produced in Chinese hamster ovary-K1 cells and tested for their mitogenic, tumor formation in nude mice and neurite outgrowth activities. Our data clearly indicate that the residues 111-136 of the lysine-rich C-terminal domain are involved in the mitogenic and tumor formation activities of HARP. Correlatively, no signal transduction was detected using the corresponding mutant, suggesting the absence of HARP binding to its high affinity receptor. However, this C-terminal domain of HARP is not involved in the neurite outgrowth activity. We also demonstrate that HARP signal peptide cleavage could led to two maturated forms that are both but differentially mitogenic.

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