Identification of heparin affin regulatory peptide domains with potential role on angiogenesis

Heparin affin regulatory peptide (HARP) is a growth factor displaying high affinity for heparin. It is present in the extracellular matrix of many tissues, interacting with heparan sulfate and dermatan/chondroitin sulfate glycosaminoglycans. We have previously shown that HARP is implicated in the control of angiogenesis and its effects are mimicked, at least in part, by synthetic peptides that correspond to its N and C termini. In the present work, we show that HARP is cleaved by plasmin, leading to the production of five peptides that correspond to distinct domains of the molecule. Heparin, heparan sulfate and dermatan sulfate, at various HARP to glycosaminoglycan ratios, partially protect HARP from plasmin degradation. The molecules with higher affinity to HARP are the more protective, heparin being the most efficient. The peptides that are produced from cleavage of HARP by plasmin, affect in vivo and in vitro angiogenesis and modulate the angiogenic activity of vascular endothelial growth factor on human umbilical vein endothelial cells. Similar results were obtained in vitro with recombinant HARP peptides, identical to the peptides generated after treatment of HARP with plasmin. These results suggest that different regions of HARP may induce or inhibit angiogenesis.     

Characterization of heparin affin regulatory peptide signaling in human endothelial cells

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 beta/zeta (RPTPbeta/zeta), which is a receptor for HARP in neuronal cell types, is also expressed in HUVEC. We also document that HARP signaling through RPTPbeta/zeta 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, RPTPbeta/zeta suppression using small interfering RNA technology interrupts intracellular signals and HUVEC migration and tube formation induced by HARP. These results establish the role of RPTPbeta/zeta as a receptor of HARP in HUVEC and elucidate the HARP signaling pathway in endothelial cells.     

Dominant negative effectors of heparin affin regulatory peptide (HARP) angiogenic and transforming activities

Heparin affin regulatory peptide (HARP) is an heparin-binding growth factor, highly expressed in several primary human tumors and considered as a rate-limiting angiogenic factor in tumor growth, invasion, and metastasis. Implication of this protein in carcinogenesis is linked to its mitogenic, angiogenic, and transforming activities. Recently, we have demonstrated that the C-terminal residues 111-136 of HARP are required for its mitogenic and transforming activities (Bernard-Pierrot, I., Delbe, J., Caruelle, D., Barritault, D., Courty, J., and Milhiet, P. E. (2001) J. Biol. Chem. 276, 12228-12234). In this paper, HARP deleted of its last 26 amino acids was shown to act as a dominant negative effector for its mitogenic, angiogenic, transforming, and tumor-formation activities by heterodimerizing with the wild type protein. Similarly, the synthetic corresponding peptide P111-136 displayed in vitro inhibition of wild type HARP activities, but in this case, the inhibition was mainly explained by the competition of the peptide with HARP for the binding to the extracellular domain of the high affinity ALK receptor.     

The lysine-rich C-terminal tail of heparin affin regulatory peptide is required for mitogenic and tumor formation activities

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 beta-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.     

Hydrogen peroxide stimulates proliferation and migration of human prostate cancer cells through activation of activator protein-1 and up-regulation of the heparin affin regulatory peptide gene

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 HP-induced 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.     

Autophagy of bovine mammary epithelial cell induced by intracellular Staphylococcus aureus

Bovine mastitis is a common disease in the dairy industry that causes great economic losses. As the primary pathogen of contagious mastitis, Staphylococcus aureus (S. aureus) can invade bovine mammary epithelial cells, thus evading immune defenses and resulting in persistent infection. Recently, autophagy has been considered an important mechanism for host cells to clear intracellular pathogens. In the current study, autophagy caused by S. aureus was detected, and the correlation between autophagy and intracellular S. aureus survival was assessed. First, a model of intracellular S. aureus infection was established. Then, the autophagy of MAC-T cells was evaluated by confocal microscopy and western blot. Moreover, the activation of the PI3K-Akt-mTOR and ERK1/2 signaling pathways was determined by western blot. Finally, the relationship between intracellular bacteria and autophagy was analyzed by using autophagy regulators (3-methyladenine [3-MA], rapamycin [Rapa] and chloroquine [CQ]). The results showed that S. aureus caused obvious induction of autophagosome formation, transformation of LC3I/II, and degradation of p62/SQSTM1 in MAC-T cells; furthermore, the PI3K-Akt-mTOR and ERK1/2 signaling pathways were activated. The number of intracellular S. aureus increased significantly with autophagy activation by rapamycin, whereas the number decreased when the autophagy flux was inhibited by chloroquine. Therefore, this study indicated that intracellular S. aureus can induce autophagy and utilize it to survive in bovine mammary epithelial cells.     

Action of ultrasonic irradiation on the DNA of cultivated bovine epithelial lens cell

Cultivated epithelial lens cells have been submitted to a 20 kHz continuous ultrasonic irradiation. At relativity high intensity, destruction of the cells was observed. At lower intensities, where no cell destruction appeared, the molecular weight of the single strand DNA of these cells was monitored to determine whether breakage of DNA molecules was induced by the ultrasound. No breakage of the single strand DNA was observed for intensities below 0.1 and 0.4 W cm-2 with irradiation times of less than 20 minutes and 15 seconds, respectively. These intensities and their corresponding irradiation time are higher than those used in current diagnostic practice.     

Effect of epidermal chalone on vaginal epithelial cell proliferation stimulated by estradiol

Non-inbred and hybrid mice, line C57Bl and CBA in diestrus stage were subjected to ovariectomy and in 4-6 weeks they were given subcutaneously 17-beta-oestradiol (1 or 0.1 mkg per one animal). One hour before the animals were sacrificed, they were given 3H-thymidine intraperitoneally. It has been stated that 15-20 h after the estrogen administration the amount of DNA-synthesizing cells in the vaginal epithelium of these animals is 25 times as great as that in the control animals--castrated mice. When the epidermal chalone is administered locally or intraperitoneally (5 mg per one mouse), 10 min after the hormone injection, there is no inhibiting effect of the chalone on the proliferative processes in the vaginal epithelium stimulated with estrogen. When a single intraperitoneal injection of the epidermal chalone is given (5 mg per one mouse) 1 h before, or when it is given three times (2 mg per one mouse) 8, 4 or 1 h before the hormone is injected, there is a definite inhibiting effect in the proliferative processes. Their degree depends on how long the chalone was in contact with the cells.     

Effect of acrylamide on the cytoskeleton and apoptosis of bovine lens epithelial cells

Acrylamide, a known disrupter of intermediate filaments, has been used to produce the collapse of vimentin filaments in bovine lens epithelial (BEL) cells, and its potential modulation of staurosporine-induced apoptosis has been investigated. In BEL cells, short treatments with acrylamide caused the collapse of vimentin filaments and microtubules and the almost complete disappearance of stress fibers, with thick f-actin bundles remaining in the cell periphery. Actin organization was less affected in cells pretreated with colchicine and in spreading cells, suggesting that extended microtubules and vimentin filaments are required for acrylamide to produce its maximal effects. Acrylamide alone slightly increased apoptosis compared to controls. However, simultaneous exposure to acrylamide and staurosporine for 8h produced significantly less apoptosis than staurosporine alone, and preincubation with acrylamide followed by staurosporine markedly reduced apoptosis at 8 and 24h of treatment. Acrylamide seems therefore to have a dual effect on BEL cell survival.     

Early and late mitogenic events induced by FGF on bovine epithelial lens cells are not triggered by hydrolysis of polyphosphoinositides

Basic or acidic forms of FGF, a potent mitogen for Bovine Epithelial Lens cells caused a rapid and transient rise in cytoplasmic Ca2+ followed by an increase in intracellular pH of 0.4 units. When cells were labeled at equilibrium with [3H]-inositol, no significant breakdown of polyphosphoinositides (in the presence of 20 mM LiCl) could be detected in response to 10-100 ng/ml of FGF. Similarly, fetal calf serum efficiently reinitiated DNA synthesis in these cells with little stimulation of polyphosphoinositide hydrolysis. In contrast, prostaglandin F2 alpha and angiotensin II, two weak mitogens for BEL cells, were found potent agonists of polyphosphoinositide breakdown. These results strongly indicate that the mitogenic action of FGF is not coupled to phospholipase C activation, a conclusion consistent with the fact that the FGF-induced [Ca2+]i rise is strictly dependent upon external Ca2+.     

An eye-derived growth factor regulates epithelial cell proliferation in the cultured lens

Lenses in organ culture permit an analysis of factors acting on epithelial cell growth, while keeping the normal steric constraints of the cell population. By employing this technique with radioautography of epithelial whole mounts, we showed that the DNA synthesis found in the epithelia of cultured bovine lenses follows an organized spatial and temporal pattern during culture. Within the first 48 h, active cells were located at the preequatorial region ("germinative zone"), a distribution consistent with the in vivo spatial organization of multiplying cells. Starting at about 48 h, cells from the central region of the epithelium--a nonproliferating population--were triggered to synthesize DNA in the presence of eye-derived growth factor (EDGF). When cultured in serum-free medium, only a small fraction of the cells was labeled, but when a low serum concentration was present, this fraction reached 50% of the cell population. The stimulatory effect of EDGF required a lag period, but its effect reached a maximum exceeding that found for serum. However, the cells from the germinative region, having a cell density three- to four-fold higher than the central region, were not stimulated to proliferate. This occurred irrespective of the presence of EDGF or serum. If this growth-stimulatory activity derived from the retina were the actual factor controlling cell proliferation in the lens in vivo, then the results presented here would point to the presence of a regulatory mechanism similar to that known for some other hormones.     

Heparin affin regulatory peptide/pleiotrophin mediates fibroblast growth factor 2 stimulatory effects on human prostate cancer cells

Fibroblast growth factor 2 (FGF2) is a pleiotropic growth factor that has been implicated in prostate cancer formation and progression. In the present study we found that exogenous FGF2 significantly increased human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) or pleiotrophin seems to be an important mediator of FGF2 stimulatory effects, since the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, FGF2, through FGF receptors (FGFRs), significantly induced HARP expression and secretion by LNCaP cells and increased luciferase activity of a reporter gene vector carrying the full-length promoter of HARP gene. Using a combination of Western blot analyses, as well as genetic and pharmacological inhibitors, we found that activation of FGFR by FGF2 in LNCaP cells leads to NAD(P)H oxidase-dependent hydrogen peroxide production, phosphorylation of ERK1/2 and p38, activation of AP-1, increased expression and secretion of HARP, and, finally, increased cell proliferation and migration. These results establish the role and the mode of activity of FGF2 in LNCaP cells and support an interventional role of HARP in FGF2 effects, providing new insights on the interplay among growth factor pathways within prostate cancer cells.     

Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene.

Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.     

Control of lens epithelial cell survival

We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell- cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture.     

Inhibition of human endothelial cell proliferation by heparin and steroids

Previous works have reported the controversial effects of heparin and steroids on angiogenesis. In this study, we investigated the effect of these compounds on human endothelial cell (EC) growth in vitro. An antiproliferative heparin activity was found in low human serum concentrations (2%). When EC were exposed to heparin (10(-6) M), their proliferation index was reduced in the presence of endothelial cell growth factor added 6 hours or more later. These results suggest that there is an intracellular effect of heparin which reduces 3H-methylthymidine uptake. Hydrocortisone acetate and tetrahydroS induced inhibition of EC growth in a dose-dependent manner. Steroids inhibited proliferation of EC in culture medium in the presence or the absence of growth factor and in different human serum concentrations. These results suggest a possible synergistic antiangiogenic action of heparin plus steroids.     

Effect of actinomycin D on cell proliferation induced by phytohemagglutinin in human lymphocytes]

Actinomycin D at a dose inhibiting the synthesis of ribosomal RNA (0.15 MUG/ML) BLOCKS THE TRANSITION OF CELLS INTO ACTIVE PROLIFERATION DURING THE FIRST 6 HOURS AFTER PHYTOHAEMAGGLUTININ TREATMENT. The effect of actinomycin D becomes weaker as the cells proceed through the prereplicative period. 1 mug/ml actinomycin D blocks the synthesis of all RNSs. It has been found that actinomycin D at this concentration blocks the transition of cells into the proliferation during the entire prereplicative period.     

Turnover of proteoglycans by chick lens epithelial cells in cell culture and intact lens

Chick lens epithelial cells were cultured on plastic and type IV collagen substrata, and the confluent cultures were labeled continuously with [35S]sulfate for 20 h. Intact lenses were also labeled in the same way. 35S-Proteoglycans isolated from those cultures were compared for their molecular sizes and glycosaminoglycan compositions. The results have shown that: 1) Proteoglycans synthesized by cells on type IV collagen were significantly smaller than those by cells on plastic. 2) Proteoglycans of intact lens showed a broad distribution of molecular size and contained a high proportion of chondroitin sulfate in the medium fraction compared to those of the two cell cultures. In order to explain such differences between proteoglycans from cultures, label-chase experiments with [35S]sulfate were done for proteoglycans synthesized. 35S-Proteoglycans isolated at each chase time 0, 2.5, and 17 h) were compared and the following results were found: 1) The cell layers of both "plastic" and "type IV collagen" cultures contained glycosaminoglycan species predominantly at each chase time rather than proteoglycans. 2) Changes in the glycosaminoglycan compositions of medium fractions of cell cultures were observed during the chase period; in medium of the "plastic" culture, proteoheparan sulfate increased with chase time, whereas in medium of the "type IV collagen" culture, chondroitin sulfate glycosaminoglycan (not proteoglycan) increased with chase time. 3) In intact lens culture, lens capsule fraction at every chase time contained a proteoglycan unique in molecular size, which was not found in cell culture fractions. 4) All fractions from intact lens cultures contained a higher content of chondroitin sulfate at every chase time than the respective fractions from cell cultures. These results suggest that adhesion of the cells to type IV collagen or lens capsule influences the degradation and secretion of proteoglycans. In addition, they can account partially for the above-described differences in molecular sizes and glycosaminoglycan compositions between 35S-proteoglycans from various cultures continuously labeled with [35S]sulfate.