Danièle Caruelle

Mitogenic properties of a new endothelial cell growth factor related to pleiotrophin

A growth factor was isolated from a neutral pH extract of adult bovine brain. Purification of this polypeptide was achieved by a three step procedure including cationic exchange, heparin-Sepharose affinity and Mono S chromatography. This heparin binding protein had a molecular weight of 18,000 as assessed by silver-stained SDS-PAGE and was not immunologically and structurally related to acidic or basic FGF. Freshly purified protein had a maximal mitogenic effect on bovine brain capillary cells at a concentration of 100 pM. Microsequencing revealed an unique amino-terminal sequence homologous to heparin-binding growth-associated molecule (HB-GAM), a neuronal maturation protein, to pleiotrophin (PTN), a fibroblast cell growth factor and to one form of the putative protein product of the MK gene, a retinoic acid induced-gene.

Increased Immunodetection of Acidic Fibroblast Growth Factor in Bladder Cancer, Detectable in Urine

Acidic fibroblast growth factor is a regulatory peptide involved in cell proliferation, differentiation and motility. We used a polyclonal antiserum raised against purified native bovine acidic fibroblast growth factor, with no cross-reactivity for basic fibroblast growth factor to detect acidic fibroblast growth factor in tissue extracts and urine samples by means of a competitive enzyme immunoassay. Histochemical analysis was also performed on 10 specimens of normal urothelium and 50 of bladder cancer. Acidic fibroblast growth factor immunoreactive material was found in normal urothelium (1.77 +/- 2 ng./gm. tissue) and was increased more than 10-fold in patients with transitional cell carcinoma of the bladder (20.36 +/- 12 ng./gm. tissue). Immunohistochemical analysis localized immunoreactivity in the epithelial compartment of bladder tumors. Acidic fibroblast growth factor was assayed in urine from 579 individuals comprising a control group (114) and patients with benign prostatic hypertrophy (133), carcinoma of the prostate (96) or transitional cell carcinoma of the bladder (236). There was a significant difference in the frequency of urinary acidic fibroblast growth factor detection among the patients with invasive transitional cell carcinoma, the control group (p < 0.001) and the patients with prostatic disease (p < 0.01). The sensitivity was 72% and the specificity was 91%. Furthermore, the frequency of acidic fibroblast growth factor detection by enzyme immunoassay in the urine and the intensity of immunostaining was correlated with the stage of the disease. These data strongly suggest that acidic fibroblast growth factor is a potential marker for bladder tumors that may be of use in the noninvasive followup of patients with bladder cancer. We present a simple and reliable enzyme immunoassay for the detection of acidic fibroblast growth factor in voided urine that might be useful to quantitate this marker.

Development and testing of radio and enzyme immunoassays for acidic fibroblast growth factor (aFGF)

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor from bovine brain stimulate growth in a variety of tissues in several species. Despite the 55% amino acid sequence homology of the two forms of FGF, a specific immunoassay of aFGF has been developed using a polyclonal antibody raised in a rabbit. Two immunoassays were compared: a radioimmunoassay (RIA) using 125I aFGF and an enzyme immunoassay (EIA) using aFGF coupled to the tetrameric form of acetylcholinesterase (aFGF-AchE) as tracer. With EIA, the detection limit was 1.5 ng/ml, versus 2.2 ng/ml with RIA, while the dose at 50% was 5.9 ng/ml for EIA and 9.6 ng/ml for RIA. Using a modified EIA procedure where aFGF-AchE was added 2 h after the other reagents, the dose at 50% binding was 1.5 ng/ml. Examples of the performance of both immunoassays are presented for various brain extracts of different species including human. The aFGF content obtained by these methods correlates (CR = 0.987) with the values obtained by biological assay.

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 β-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.

Involvement of heparin affin regulatory peptide in human prostate cancer

Heparin affin regulatory peptide (HARP) composes, together with midkine (MK), a new family of heparin‐binding growth/differentiation factors. Recently, HARP was incriminated in cancer progression, as an angiogenic factor and as a tumor growth factor. In this study, we analyzed the possible involvement of HARP in human prostate cancer (Pca).

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.

Inhibition of the mitogenic, angiogenic and tumorigenic activities of pleiotrophin by a synthetic peptide corresponding to its C-thrombospondin repeat-I domain

Pleiotrophin (PTN), is a heparin‐dependent growth factor involved in angiogenesis and tumor growth. PTN contains a thrombospondin repeat‐I (TSR‐I) motif in its two β‐sheet domains that are involved in its binding to heparin and its neurite outgrowth activity. Based on the importance of the binding of PTN to heparin in its dimerization and biological activities, we have designed two synthetic peptides, P(13–39) and P(65–97) corresponding to a part of the N‐terminal and C‐terminal TSR‐I motif of PTN, respectively. P(65–97) inhibited the mitogenic, tumorigenic and angiogenic activities of PTN, as well as the mitogenic and an angiogenic activity of fibroblast growth factor‐2 (FGF‐2). However, P(65–97) had no effect on the mitogenic activity of epidermal growth factor, which does not bind heparin. P(65–97) but not P(13–39) inhibited the binding of PTN and to a lesser extent of FGF‐2 to heparin using an immunoassay and an optical biosensor assay and bound directly to heparin with a Kd of 120 nM. These findings suggest that P(65–97), containing amino acids 65–97 of the TSR‐I motif of the C‐terminal domain of PTN, inhibits the activities of PTN and FGF‐2 by virtue of its ability to bind heparin very effectively and so compete with the growth factors for their polysaccharide co‐receptor. J. Cell. Physiol. 214:250–259, 2008.

Pleiotrophin mediates the neurotrophic effect of cyclic AMP on dopaminergic neurons: analysis of suppression-subtracted cDNA libraries and confirmation in vitro.

To better understand the particular vulnerability of mesencephalic dopaminergic neurons to toxins or gene mutations causing parkinsonism, we have taken advantage of a primary cell culture system in which these neurons die selectively. Antimitotic agents, such as cytosine arabinoside or cAMP, prevent the death of the neurons by arresting astrocyte proliferation. To identify factors implicated in either the death of the dopaminergic neurons or in the neuroprotective effect of cAMP, we constructed cDNA libraries enriched by subtractive hybridization and suppressive PCR in transcripts that are preferentially expressed in either control or cAMP-treated cultures. Differentially expressed transcripts were identified by hybridization of the enriched cDNAs with a commercially available cDNA expression array. The proteoglycan receptors syndecan-3 and the receptor protein tyrosine phosphatase zeta/beta were found among the transcripts preferentially expressed under control conditions, and their ligand, the cytokine pleiotrophin, was highly represented in the cDNA libraries for both conditions. Since pleiotrophin is expressed during embryonic and perinatal neural development and following lesions in the adult brain, we investigated its role in our cell culture model. Pleiotrophin was not responsible for the death of dopaminergic neurons under control conditions, or for their survival in cAMP-treated cultures. It was, however, implicated in the initial and cAMP-dependent enhancement of the differentiation of the dopaminergic neurons in our cultures. In addition, our experiments have provided evidence for a cAMP-dependent regulatory pathway leading to protease activation, and the identification of pleiotrophin as a target of this pathway.

Immunoassay for measuring the heparin-binding growth factors HARP and MK in biological fluids

ABSTRACT Heparin-affin regulatory peptide (HARP) and Midkine (MK) belong to a family of growth/differentiation factors that have a high affinity for heparin. The involvement of these molecules in various proliferative diseases prompted us to develop an assay for measuring the concentrations of these factors in biological fluids and culture media. This report describes an immunoassay that uses only commercially available materials, based on the high affinity of certain molecules for heparin. It consists of adsorbing heparin-BSA covalent complexes to microtiter plate wells and to quantify the heparin bound HARP or MK by using appropriate antibody. The method is specific and measures concentrations ranging from 40–1200 pg/mL HARP and from 25–1200 pg/mL MK and various parameters are investigated. The within-assay coefficient of variation was less than 5% for both assays. The method was checked by measuring the concentrations of these growth factors in the sera of healthy humans and in patients with cancer. As previously reported, we confirmed that the serum concentrations of MK are higher in patients with tumours (n = 139) than in controls (n = 19). The synthesis of HARP and MK by various cells in culture was also analysed.

Cellular Distribution of the Angiogenic Factor Heparin Affin Regulatory Peptide (HARP) mRNA and Protein in the Human Mammary Gland

The heparin affin regulatory peptide (HARP) growth factor, also known as pleiotrophin, is a developmentally regulated protein that displays biological functions during cell growth and differentiation. To study the physiological role of this protein, we investigated the cellular distribution of HARP mRNA and protein in the resting human mammary gland. In situ hybridization histochemistry revealed that HARP mRNA was localized in alveolar myoepithelial cells, whereas alveolar epithelial cells were negative. In the stroma, HARP mRNA was localized in endothelial cells and smooth muscle cells of blood vessels. Interestingly, HARP protein and mRNA were not always co-localized. HARP protein immunocytochemistry staining was observed in an area including both alveolar myoepithelial and epithelial cells, although epithelial cells do not express HARP transcript. In contrast, the distribution of HARP protein is parallel to that of HARP mRNA in endothelial and vascular smooth muscle cells. In the light of these results, the putative role of HARP in controlling the proliferation and/or differentiation of the different mammary cell types is proposed and discussed.