Pleiotrophin inhibits HIV infection by binding the cell surface-expressed nucleolin ´ ´ Elias A Said1, Jose Courty2, Josette Svab1, Jean Delbe2, Bernard Krust1 and Ara G Hovanessian1 ´ ` UPR 2228 CNRS, UFR Biomedicale des Saints-Peres, Paris, France ´ ´ ´ ´ Laboratoire de Recherche sur la Croissance Cellulaire, la Reparation et la Regeneration Tissulaires (CRRET), FRE CNRS 2412, ´ ´ Universite Paris Val de Marne, Creteil, France Keywords binding; HIV; pleiotrophin; receptor; surface nucleolin Correspondence E A Said, UPR 2228 CNRS, UFR ´ ` Biomedicale des Saints-Peres; 45 rue des ` Saint-Peres, 75270 Paris Cedex 06, France Fax: +33 142862042 Tel: +33 142864136 E-mail: elias.said@umontreal.ca (Received 11 May 2005, revised 30 June 2005, accepted 18 July 2005) doi:10.1111/j.1742-4658.2005.04870.x 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 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 pseudo- peptide HB-19 that binds specifically the C-terminal tail of nucleolin, a cell-surface-expressed protein identified to be implicated in HIV attachment [3–5] 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 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 tyrosine phosphatase 4646 FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS E A Said et al (PTN) that binds surface nucleolin inhibits HIV attachment to cells by its capacity to bind surface nucleolin as a low affinity receptor PTN is an 18-kDa protein which was first identified as a heparin-binding protein that progresses mitogenic activity in rat and mouse fibroblasts [10] The first purification was from bovine uterus and neonatal rat, brain, bone and kidney PTN is rich in basic amino acids especially lysine in both N- and C-terminal tails It was also named as heparin-binding-growth-associated molecule (HB-GAM) [11], heparin-binding neurite-promoting factor (HBNF) [12] or heparin affin regulatory peptide (HARP) [13] Biological functions of PTN are mitogenic, angiogenic and oncogenic activities, cell motility, differentiation, and synaptic plasticity [14] Elevated serum PTN levels have been detected in patients with testicular, pancreatic, colon, breast and other cancers [15–17] Consequently, the circulating levels of PTN have been proposed to serve as a tumor marker Interestingly, PTN is expressed in fracture healing [18] and its gene expression is also upregulated in newly forming blood vessels, in OX42-positive macrophages, and in invasion-independent pathways of blood-borne metastasis [14,19,20] PTN is also expressed in adults with inflammatory diseases, and proinflammatory cytokines enhance its expression [21,22] Finally, PTN inhibits infectivity of human herpes simplex viruses type and and human cytomegalovirus [23] Several cell surface components have been reported as potential receptors for PTN, such as the heparan sulfate proteoglycans of N-syndecan [24] and the chondroitin sulfate proteoglycan of receptor-type tyrosine phosphatase b ⁄ f (RPTP b ⁄ f) [25,26] In addition, anaplastic lymphoma kinase (ALK) has been reported to be a receptor that transduces PTN-mediated signals and the PTN-ALK axis can play a significant role during development and disease processes [27] PTN binds the extracellular domain of ALK with a Kd of 32 ± pm [27] PTN shows a striking structural homology with another heparin binding growth-associated factor called midkine, with whom it shares 45% sequence identity [14,28–31] Therefore, like PTN, the binding of midkine to heparan sulfate and chondroitin sulfate proteoglycans could be clearly demonstrated using purified and soluble components [32,33] Midkine binds also ALK with a high affinity and this binding is inhibited by PTN [34] We have previously demonstrated that midkine is a cytokine that binds the cell surface expressed nucleolin as a low affinity receptor Synthetic and recombinant preparations of midkine inhibited in a dose-dependent manner infection of cells FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS Nucleolin is a low affinity receptor of pleiotrophin by various HIV-1 isolates; this inhibition is due to the capacity of midkine to bind cells specifically and to prevent the attachment of HIV particles to cells [5,35] Nucleolin is a component of the cell surface which could act as a receptor for various ligands Indeed, on the cell surface nucleolin interacts with several molecules such as lipoproteins, J factor [50], and the alpha-1 chain of laminin [51] Cell surface-expressed nucleolin could also act as a receptor of viruses such as Coxsackie B [52] and Human Parainfluenza Virus type [53] Indeed, while nucleolin does not have a hydrophobic domain [54], it is expressed on the cell surface, and it represents 20% of the cytoplasmic portion of nucleolin [55] Our previous results showed that cytoplasmic nucleolin is found in small vesicles that appear to translocate nucleolin to the cell surface Translocation of nucleolin is markedly reduced at low temperature or in serum-free medium, whereas conventional inhibitors of intracellular glycoprotein transport have no effect Thus, translocation of nucleolin is the consequence of an active transport by a pathway which is independent of the endoplasmic reticulum ⁄ Golgi complex [55] Here, we show that PTN inhibits HIV infection by binding the cell-surface expressed nucleolin leading to the inhibition of HIV-attachment to the cell surface PTN binding to cells involves high and low affinitybinding sites even in cells that are deficient for the expression of both heparan and chondroitin sulfate proteoglycans The synthetic ligand of nucleolin, the HB-19 pseudopeptide, prevents the binding of PTN to the low affinity receptor, thus suggesting that such a receptor is the cell surface-expressed nucleolin Accordingly, by confocal immunofluorescence laser microscopy, we show that cell-surface bound PTN colocalizes with that of surface-expressed nucleolin Results Inhibition of HIV-infection by PTN We investigated the inhibitory effect of PTN on HIV infection by using the experimental model of HeLa P4 or HeLa P4C5 cells HIV entry and replication in these cells result in Tat-mediated transactivation of HIV LTR, leading to the expression of the LacZ gene Consequently, the b-galactosidase activity could be measured in cell extracts to monitor HIV entry The b-galactosidase expression in noninfected cells is considered as the background value in this experiment As we had shown previously, midkine inhibited HeLa P4 cells infection by HIV-1 LAI isolate with more than 90% inhibition at lm of midkine [8,35] 4647 Nucleolin is a low affinity receptor of pleiotrophin A E A Said et al HIV-1 Ba-L HIV-1 LAI Control Control AZT AZT MK µM 30 60 60 125 PTN 125 [nM] PTN 250 [nM] 500 250 500 1000 0.5 1.5 β-Galactosidase Activity (OD) 1.5 2.5 0.5 β-Galactosidase Activity (OD) B Control No HIV 25 50 PTN 100 [nM] 200 250 50 100 In this model, PTN inhibited the entry of the X4 HIV-1 LAI isolate in a dose-dependent manner with IC50 and IC90 values of 60 and 250 nm, respectively (Fig 1A, HIV-1 LAI) PTN also inhibited infection of HeLa P4C5 cells by the R5 HIV-1 Ba-L isolate in a dose-dependent manner with IC50 and IC90 values of 60 nm and 500 nm, respectively (Fig 1A, HIV-1 Ba-L) A similar inhibition profile was obtained with the infection of MT4 cells (data not shown) HeLa P4 cells preincubated with PTN at 20 °C for 45 and washed with medium to remove unbound PTN, resisted HIV-1 LAI infection However, incubation of HIV-1 LAI with PTN and centrifugation at 100 000 g to pellet the virus gave an HIV pellet that was still infectious (data not shown) These data indicate that the inhibitory effect of PTN is mediated through its action on target cells rather through a direct effect on virus particles The effect of PTN on the HIV attachment was monitored by measuring the concentration of the HIV major 4648 Fig Inhibition of HIV infection by PTN (A) HeLa P4 cells were treated (30 min, 37 °C) with midkine (MK) (1 lM) or PTN (60, 125, 250, 500, 1000 nM) HeLa P4C5 cells were treated (30 min, 37 °C) with PTN (30, 60, 125, 250, 500 nM) HeLa P4 and HeLa P4C5 cells were then infected with the HIV1 LAI or HIV-1 Ba-L isolate, respectively At 48 h postinfection, the b-galactosidase activity was measured in cell extracts directly to monitor HIV entry (the abscissa; OD, optical density) The histogram AZT represents the background b-galactosidase activity when HIV retrotranscription is inhibited (B) HeLa P4 cells were incubated (45 min, 20 °C) with PTN (25, 50, 100, 200, 250 nM) and the HIV-1 LAI isolate HIV attachment was monitored by measuring the concentration of the HIV major core protein p24 in cells extracts The histogram No HIV represents the background of p24 concentration in the absence of virus attachment The mean ± SD of triplicate samples is shown core protein p24 in the lysate of HeLa P4 cells that were incubated with HIV-1 LAI at room temperature in the presence of different concentrations of PTN PTN inhibited HIV-attachment in a dosedependent manner with more 50% and 90% inhibition at 50 and 250 nm, respectively (Fig 1B) These results demonstrate that the inhibition of HIV infection by PTN is due to its inhibitory effect on the attachment of HIV particles The inhibiting action of PTN on the HIV-1 infection is mediated through the b-sheet domains of PTN PTN consists of two b-sheet domains located between N- and C-terminal tails rich in lysine residues [44] In order to locate the domain of PTN responsible of the inhibitory effect on HIV infection, we tested the capacity of deletion constructs corresponding to various domains of PTN to inhibit infection of HeLa P4 cells FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS E A Said et al by HIV-1 LAI The peptide PTN Nt-tail corresponds to the N-terminal tail of PTN (residues 1–8), the peptide PTN Ct-tail corresponds to the C-terminal tail of PTN (residues 110–136), PTN (residues 9–110) corresponds to the b-sheet domains, PTN (residues 1–110) corresponds to the N-terminal tail and the two b-sheet domains, PTN (residues 9–136) corresponds to the C-terminal tail and the two b-sheet domains, PTN-Nf corresponds to the b-sheets on the N-terminal side (residues 9–59), and PTN-Cf corresponds to the b-sheets on the C-terminal side (residues 60–110) Whereas lysine-rich peptides corresponding to the N and C-terminal tails of PTN have no effect on HIV-1 infection, peptides containing the b-sheet domains [PTN (1–110) and PTN (9–136)], or peptides containing the b-sheets alone [PTN (9–110)] inhibit HIV infection by a dose-dependent manner, at an IC50 value of 200 and 250 nm for PTN (1–110), and PTN (9–136), respectively (Fig 2) The most potent inhibitory effect is observed with the peptide PTN (9–110) that inhibits HIV-1 LAI infection with an IC50 value of 30 nm Finally, PTN-Nf does not have an effect on HIV infection, whereas PTN-Cf inhibits the infection with an IC50 of 200 nm (Fig 2) These results suggest that the domains containing the b-sheets are the regions responsible for the inhibitory effect of PTN on HIV infection The presence of N or C-terminal tails with the two b-sheet domains (at residues 9–110) decreased the inhibitory effect of the two b-sheet domains without the respective tail (Fig 2, compare the results obtained with PTN 1–110 and PTN 9–136 with PTN 9–110) The presence of either one of the tails alone might affect the proper folding of such truncated PTN constructs and consequently affect the inhibitory effect on HIV infection Inhibition of HIV particles attachment by PTN requires a cell surface component other than heparan and chondroitin sulfate proteoglycans Described as a HB-GAM, PTN interacts with glycoaminoglycans such as heparan sulfate proteoglycans [25,26], which are also implicated in HIV attachment to the cell surface [7] In order to investigate whether the inhibitory effect of PTN on HIV attachment is due to its interaction with heparan or chondroitin sulfate proteoglycans, we used Chinese hamster ovary (CHO) wild-type cells (CHO K1) and mutant cells lines that are deficient in the expression of heparan sulfate (CHO 677), or both heparan ⁄ chondroitin sulfate proteoglycans (CHO 618) [38,39] Despite lacking proteoglycan expression, these mutant cell lines FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS Nucleolin is a low affinity receptor of pleiotrophin Control PTN 200 nM 100 nM PTN (1-110) 200 nM 500 nM 100 nM PTN (9-136) 200 nM 500 nM 100 nM PTN (9-110) 200 nM 500 nM 100 nM PTN Nt-tail 200 nM 500 nM 100 nM PTN Ct-tail 200 nM 500 nM 100 nM PTN-Nf 200 nM 500 nM 100 nM PTN-Cf 200 nM 500 nM 50 100 % of HIV infection 150 Fig The inhibitory of various PTN domains on HIV infection HeLa P4 cells were preincubated or not (30 min, 37 °C) with 200 nM of PTN, PTN (1–110), PTN (9–136), PTN (9–110), PTN Nt-tail (1–9), PTN Ct-tail (110–136), PTN-Nf, or PTN-Cf at 100, 200 and 500 nM concentrations Cells were then infected with HIV-1 LAI (90 min, 37 °C) The b-galactosidase activity was measured at 48 h postinfection The percentage HIV infection (abscissa) gives the proportion of b-galactosidase activity compared to infected cells without PTN (histogram Control) express similar levels of the cell-surface nucleolin [8] In these HIV attachment experiments, culture supernatants were removed from CHO K1, 677 and 618 cells pretreated with PTN or the nucleolin-binding HB-19 pseudopeptide, before adding the virus preparation on cells The fact that CHO K1 cells not express the HIV receptor CD4 or the coreceptors CCR5 and CXCR4, demonstrates that HIV attachment should mainly be mediated via the heparan ⁄ chondroitin sulfate proteoglycans and cell-surface expressed nucleolin 4649 Nucleolin is a low affinity receptor of pleiotrophin E A Said et al Control Role of heparan and chondroitin sulfate proteoglycans in PTN binding on the cell surface CHO K1 HB-19 µM PTN 0.5 µM Control CHO 677 HB-19 µM PTN 0.5 µM Control CHO 618 HB-19 µM PTN 0.5 µM 100 200 300 p24 [pg/ml] 400 500 Fig Attachment of HIV particles to CD4– CHO cell lines, expressing or not expressing heparan ⁄ chondroitin sulfate proteoglycans is inhibited by PTN and the nucleolin-binding HB-19 pseudopeptide CHO K1 cells (wild type), CHO 677 cells (deficient in heparan sulfate proteoglycan) or CHO 618 cells (deficient heparan ⁄ chondroitin sulfate proteoglycans) were treated (30 min, 20 °C) with HB-19 (1 lM) or PTN (500 nM) Both reagents were then removed from the culture before incubation of cells with the HIV-1 LAI isolate (45 min, 20 °C) HIV attachment was monitored by measuring the concentration of the HIV major core protein p24 in the cells lysate The mean ± SD of triplicate samples is shown [4] HB-19 was included in these binding experiments in order to estimate the contribution of nucleolin in the HIV attachment process Accordingly, HB-19 markedly inhibited HIV attachment at a concentration of lm in all CHO wild-type and mutant cell lines, thus indicating the capacity of surface expressed nucleolin to serve as a receptor for HIV binding independent of heparan and chondroitin sulfate proteoglycans (Fig 3) Interestingly, PTN at a concentration of 500 nm inhibited HIV attachment by about 70% to the surface of all CHO cell lines used in this assay (Fig 3) In similar HIV attachment assays, when PTN was not removed before addition of HIV, then more than 90% inhibition of HIV attachment was observed (data not shown) These results not rule out a potential role of heparan ⁄ chondroitin sulfate proteoglycans in the inhibitory activity of PTN, but suggest the implication of other cell surface component(s) It should be noted that HIV attachment in control CHO cell lines is decreased by 50 and 80% in CHO 677 and CHO 618 cells, respectively (Fig 3) This decrease is probably due to the lack of heparan sulfate and heparan ⁄ chondroitin sulfate proteoglycan expression, and illustrates the implication of such proteoglycans in the HIV attachment process The capacity of HB-19 to inhibit HIV attachment in the CHO wildtype cells is in accord with our previous results using various CD4 positive and HIV permissive cell lines; such results confirm once again that HIV attachment is coordinated by both proteoglycans and nucleolin [4] 4650 To evaluate the potential implication of the heparan and chondroitin sulfate proteoglycans in PTN binding, we used the three CHO cell lines previously described [8] These binding experiments were carried out at 20 °C to prevent PTN entry into cells (see Experimental procedures) We first investigated the specific and nonspecific binding of 125I-labeled PTN to the wildtype CHO K1 cells, which express heparan and chondroitin sulfate proteoglycans by washing cells at 300 and 150 mm NaCl, respectively In cells washed with 300 mm NaCl, 125I-labeled PTN specific binding occurs in a dose-dependent manner and reaches saturation at lm of 125I-labeled PTN (Fig 4A), whereas total binding does not reach a saturation limit Because of the considerable amount of nonspecific binding, cells were routinely washed at 300 mm NaCl in all the following experiments It is important to note that PTN specific binding resists drastic wash conditions such as normal or acidic culture medium (pH ¼ 4) containing (or not) m NaCl (not shown) Interestingly, the 125 I-labeled PTN binding profile (binding curve and saturation point) to heparan sulfate-deficient CHO 677 cells (not shown) and to both heparan and chondroitin sulfate proteoglycan-deficient CHO 618 cells was similar to that observed for the wild-type CHO K1 cells (Fig 4B) However, the levels of 125I-labeled PTN binding (amount of 125I-labeled PTN bound to cells) to the CHO 618 and 677 cells was lower than that to the wild-type CHO K1 cells These results indicate that under our experimental conditions, heparan and chondroitin sulfate proteoglycans may play a role in the overall binding of PTN to cells, even if the specific binding reaches saturation independently of their presence As high affinity binding sites for PTN have been reported in the literature [27], we investigated the presence of such sites on CHO cells Indeed, results show that in CHO K1 cell lines the specific binding of the 125 I-labeled PTN reaches saturation at nm (Fig 4C) A similar saturation curve was obtained in CHO 618 and HeLa cells (not shown) Taken together, these results suggest the presence of low affinity and high affinity binding sites of PTN on the cell surface Scatchard analysis of the 125I-labeled PTN binding using high and low concentrations confirmed the presence of low affinity and high affinity binding sites The estimated Kd value for PTN binding to the low affinity binding site on CHO K1 and 618 cells was 1.3 · 10)6 m (3.6 · 107 sites per cell) and 1.4 · 10)6 m (1.9 · 107 sites per cell), respectively (Table 1) The estimated Kd value for the binding of PTN to the high affinity binding site FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS E A Said et al Binding of I-labeled PTN A Nucleolin is a low affinity receptor of pleiotrophin CHO K1 cells 20000 18000 16000 14000 12000 10000 8000 5000 4000 2000 Cell lines Binding of I-labeled PTN 370 750 1500 3000 6000 CHO 618 cells 14000 12000 10000 8000 5000 4000 2000 185 Binding of I-labeled PTN C Kd ¼ (M) High High Low Low 0.049 0.049 1.3 1.4 K1 618 K1 618 · · · · 10)9 10)9 10)6 10)6 Receptors per cell 2.5 2.5 3.6 1.9 · · · · 105 105 107 107 on both CHO K1 and CHO 618 cells was 4.9 · 10)11 m (2.5 · 105 sites per cell) which is somewhat in accord with the value reported previously [27] The binding of PTN to the high affinity receptor reaches saturation at nm, a concentration that has no effect on HIV particle attachment to cells; the order of PTN concentrations required to inhibit HIV attachment to cells corresponds to the concentrations of PTN that are required for the interaction and the saturation of the low affinity binding site Consequently, the binding of PTN to the low affinity-binding site should be responsible for the inhibitory effect of PTN on HIV infection 370 750 1500 3000 6000 CHO K1 cells 1800 1600 1400 1200 1000 800 600 400 200 Affinity CHO CHO CHO CHO 185 B Table High affinity and low affinity Pleiotrophin receptors on CHO wild-type and proteoglycan-free cells Scatchard analysis of the binding data on CHO K1 and CHO 618 cells (heparan ⁄ chondroitin-proteoglycan-deficient cells) carried out as shown in Fig suggested the presence of high and low affinity binding sites for PTN The Kd values and the number of sites per cell are as indicated 0.5 2.5 Concentration of PTN [nM] Fig The binding of 125I-labeled PTN to CHO cell lines Binding at high concentrations of PTN is shown in (A) and (B) The nonspecific (squares) and specific (circles) binding of 125I-labeled PTN to cells was investigated using wild-type CHO K1 cells (A) and the heparan ⁄ chondroitin sulfate-deficient CHO 618 cells (B) using different concentrations of the 125I-labeled PTN (abscissa) C Binding of 125 I-labeled PTN to CHO K1 cells was carried out as in the sections A and B but at lower concentrations of the 125I-labeled PTN The specific binding was measured after washing cells in 300 mM NaCl (see Experimental procedures) The ordinate gives the values of measured c rays in counts per minute (c.p.m.) Each point represents the mean ± S.D of duplicate samples FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS PTN binding to the low affinity receptor is blocked by nucleolin-binding HB-19 pseudopeptide The different CHO cell lines were employed to investigate competition experiments for the low and high affinity binding sites Typical results are shown with the CHO K1 cells (Fig 5) The specificity of PTN binding to both binding sites was confirmed by the fact that unlabeled PTN completely inhibited 125I-labeled PTN binding to the low and high affinity binding sites (Fig 5) As expected, the nucleolin binding pseudopeptide HB-19 prevented 125I-labeled PTN binding to the low affinity but not to the high affinity site (Fig 5) A similar profile of inhibition was observed with MK for the binding of PTN to its low affinity binding site (Fig 5), whereas our previous results showed that PTN inhibited just 50% of 125I-labeled MK binding to the low affinity receptor [8], this might be due to the fact that MK interacts with such a binding site with an affinity higher than that of PTN Interestingly, it was shown that MK binding to its high affinity binding site, which was defined to be ALK also, is competed by PTN [34] Our results suggest that PTN and HB-19 share a common receptor, to which both MK and PTN bind with a low affinity Consequently, nucleolin should be the low affinity-binding site of PTN These observations along with the role of surface 4651 Nucleolin is a low affinity receptor of pleiotrophin A E A Said et al a I-PTN/PTN 120 % Binding of I-PTN 100 80 60 40 20 0 –9 –8 –7 –6 –5 PTN [log (M)] –4 B I-PTN/PTN I-PTN/HB-19 I-PTN 80 60 40 I-PTN/PTN 20 I-PTN/HB-19 0 –9 –8 –7 –6 –5 PTN [log (M)] –4 I-PTN 50 100 % Binding of I-labeled PTN c I-PTN/MK 120 100 80 60 40 20 0 –9 –8 –7 –6 –5 PTN [log (M)] –4 nucleolin in HIV attachment to cells point out that the inhibitory action of PTN on HIV infection could be the consequence of PTN binding to the cell-surface expressed nucleolin Internalization of PTN is independent of heparan and chondroitin sulfate expression but it is inhibited by the nucleolin-binding HB-19 pseudopeptide The use of specific anti-PTN antibodies in confocal laser immunofluorescence microscopy experiments, 4652 Low affinity % Binding of I-PTN 100 % Binding of I-PTN High affinity b I-PTN/HB-19 120 Fig The effect of the nucleolin binding molecules on the binding of PTN to the low and high affinity-binding site (A) Inhibitory effect of HB-19 on the binding of the 125 I-labeled PTN to the low affinity-binding site CHO K1 cells were incubated with the 125 I-labeled PTN (25 nM) in the presence of various concentrations of unlabeled PTN (a), HB-19 (b) or midkine (c) The cells were washed in culture medium containing 300 mM NaCl to monitor the specific binding The mean ± SD of duplicate samples is shown (B) HB-19 prevents the binding of the 125I-labeled PTN to the low but not the high affinity-binding site CHO K1 cells were incubated with nM for the high affinity binding site or 25 nM for the low affinity binding site with the 125I-labeled PTN in the presence of 100-fold higher concentrations of PTN, HB-19 or midkine (MK) as it is indicated Cells were washed in culture medium containing 300 mM NaCl to monitor for specific binding Each histogram represents the mean ± SD of duplicate samples The ordinate gives the percentage 125I-labeled PTN binding to cells in the presence of the different reagents demonstrated internalization of PTN in HeLa cells at 37 but not at 20 °C (not shown), thus indicating that PTN entry occurs by an active process In order to investigate the role of surface nucleolin in the PTN internalization process, PTN entry was monitored in the different CHO cell lines Because heparan sulfate proteoglycans are implicated in the internalization of FGF-2 [45], we also monitored entry of PTN in these same cells Our results show that PTN enters efficiently in the heparan sulfate-deficient CHO 677 and heparan ⁄ chondroitin sulfate-deficient CHO 618 cells as in the wild-type CHO K1 cells (Fig 6) In contrast, FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS E A Said et al Nucleolin is a low affinity receptor of pleiotrophin CHO K1 (HS, CS) CHO 677 (CS) CHO 618 A PTN B FGF-2 Fig Internalization of PTN does not require expression of heparan (HS) ⁄ chondroitin sulfate (CS) proteoglycans CHO wild-type K1, the heparan sulfate-deficient CHO 677, and the heparan ⁄ chondroitin-deficient CHO 618 cells were incubated (60 min, 37 °C) in fresh culture medium containing 10% (w ⁄ v) FBS and 200 nM PTN or FGF-2 Cells were then washed and fixed with 3.7% PFA and permeabilized with Triton X-100 The primary antibodies were goat anti-PTN and anti-FGF-2, which was revealed by FITC-conjugated rabbit anti-(goat IgG) Ig For each condition, a scan corresponding to a cross-section towards the middle of the cell monolayer is shown along with the respective phase contrast FGF-2 internalization occurs only in the CHO K1 cells thus confirming the requirement of proteoglycans in its entry process The internalization of 125I-labeled PTN was also monitored in CHO K1, 677 and 618 cell lines by treating cells with trypsin to eliminate cell surface bound PTN (not shown) These experiments demonstrated once again that internalization of PTN occurs at 37 °C and does not require heparan and chondroitin sulfate proteoglycans In accord with the inhibition of PTN binding to cells by the nucleolin-binding HB-19 pseudopeptide (Fig 5B), PTN entry was inhibited almost completely by HB-19 (Fig 7) It is of interest to note that a synthetic peptide composed of nine arginine residues [8] has no apparent effect on the binding and internalization of PTN (not shown), thus pointing out that the inhibitory effect of HB-19 is an specific event on the FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS binding of PTN to its low affinity binding site and not simply due to the basic nature of HB-19 These observations further confirm that PTN binding and internalization in cells is mediated by the cell-surface nucleolin Cross-linking of surface-bound PTN results in the clustering of surface nucleolin In general, the cross-linking of a ligand leads to the clustering or capping of its surface receptor Previously, we had reported that antibody-mediated cross-linking of ligands of nucleolin, such as the pseudopeptide HB-19, HIV virions, midkine and lactoferrin, results in clustering of surface nucleolin and its coaggregation with the specific ligand [5,8,9] Similarly, here we show that cross linking of cell surface bound PTN with 4653 Nucleolin is a low affinity receptor of pleiotrophin PTN Phase contrast PTN + HB-19 Phase contrast Fig Internalization of PTN is inhibited by the HB-19 pseudopeptide HeLa P4 cells in culture medium containing 10% (w ⁄ v) FBS were incubated (45 min, 37 °C) with PTN (200 nM) in the absence or presence of HB-19 (2 lM) Cells were then washed and fixed with 3.7% PFA and permeabilized with Triton X-100 The primary antibody was anti-PTN polyclonal antibody, which was revealed using FITC-conjugated rabbit anti-(goat IgG) Ig A scan corresponding to a cross-section toward the middle of the cell monolayer is shown anti-PTN Igs results patching of nucleolin at one pole of the cell, which coincided with the PTN signal (Fig 8) This observation is consistent with the surface nucleolin being a binding site of PTN Discussion Here we show for the first time that the growth factor PTN inhibits HIV-1 infection by its capacity to inhibit HIV attachment to the cell surface The b-sheet domains of PTN, especially those located on the C-terminal side, appear to be implicated in this inhibitory effect PTN binding to the cell surface is mediated by high and low affinity binding sites, the low affinity binding sites being nucleolin Following binding, PTN enters cells by an active process that is independent of heparan and chondroitin sulfate proteoglycans, but is inhibited by the nucleolin-binding pseudopeptide HB-19 Cross-linking of surface-bound PTN with a specific antibody results in the clustering of cell surfaceexpressed nucleolin and the colocalization of both PTN 4654 E A Said et al Nucleolin-TR Merge Pleiotrophin-FITC Phase contrast Fig PTN induced clustering of nucleolin in MT4 cells: colocalization of PTN with nucleolin at the surface of PTN treated cells MT4 cells were incubated with lM of PTN at 20 °C for 45 Cells were then washed before incubation at 20 °C for 45 in the presence of anti-PTN antibody to induce lateral clustering while inhibiting PTN entry At this stage, cells were first partially fixed with 0.25% PFA before the addition of the monoclonal antibody against nucleolin (mAb D3; 20 °C, 45 min) After washing, cells were fixed, and the primary anti-PTN Ig was revealed by FITC-conjugated rabbit anti-(goat IgG) Ig, whereas mAb D3 against nucleolin was revealed by Texas Red dye-conjugated horse anti-(mouse IgG) Ig A cross-section towards the middle of cells for each staining with the merge of the two colors in yellow are presented and nucleolin; thus confirming the interaction of PTN with surface-expressed nucleolin The interaction of PTN with surface nucleolin is in accord with a previous report showing that PTN binds nucleolin in solution [46] PTN inhibits HIV attachment to CD4+ permissive cells as well as to CD4– nonpermissive CHO cell lines that express (or not) heparan ⁄ chondroitin-sulfate proteoglycans In such CD4+ or CD4– cell lines HIV attachment is also inhibited by the HB-19 The demonstration that both PTN and HB-19 inhibit HIV attachment even in the absence of heparan ⁄ chondroitin sulfate proteoglycans (such as in CHO 618 cells), suggests that their inhibitory effect on virus attachment should be due to binding to the cell-surface-expressed nucleolin We have previously reported that the initial attachment of HIV particles to cells is coordinated on one hand by heparan ⁄ chondroitin sulfate proteoglycans FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS E A Said et al [6,48,49], and on the other hand by the surfaceexpressed nucleolin [4,5] Consequently, HIV attachment could be inhibited either by FGF-2 which uses heparan-sulfate proteoglycans as low affinity receptors [45], and by various specific ligands of nucleolin such as the HB-19 pseudopeptide, midkine, PTN, and lactoferrin [4,5,8,9,35] The capacity of HB-19 to inhibit HIV attachment to CHO 618 cells that are deficient in both heparan ⁄ chondroitin sulfate proteoglycans expression (the results herein) provides further evidence illustrating that surface nucleolin is implicated in the HIV attachment process Several groups have previously shown that PTN interacts with heparan ⁄ chondroitin sulfate proteoglycans [24–26,47] Accordingly in our experiments, although PTN binds specifically CHO cells deficient in the expression of heparan and chondroitin sulfate proteoglycans, the total amount of binding is much lower compared to wild-type CHO cells expressing both proteoglycans The latter therefore suggests that heparan and chondroitin sulfate proteoglycans are also implicated in the mechanism of PTN binding to cells This is somewhat analogous to the mechanism of HIV binding cells in which both heparan and chondroitin sulfate proteoglycans and nucleolin are implicated Accordingly, both HIV attachment and PTN binding to cells is decreased at a similar level of by 50 and 80% in CHO 677 and 618 cells, respectively In contrast to cell binding, nucleolin-mediated PTN entry appears to be independent of heparan and chondroitin sulfate proteoglycans Thus it is plausible that heparan and chondroitin sulfate proteoglycans might be necessary for the concentration of PTN on the cell surface for an efficient interaction with nucleolin The b-sheets located on the C-terminal side of PTN (amino acids 60–110) appear to be responsible for its inhibitory effect on HIV infection Accordingly, the construct representing the b-sheets located on the C-terminal side inhibits the HIV infection, whereas its counterpart on the N-terminal side has no apparent inhibitory effect Interestingly, the construct that contains both b-sheet domains is more active in the inhibition of HIV infection This latter could be due to conformational effects on the PTN structure that is optimal for the interaction with nucleolin Finally, the presence of the N- and C-terminal tails of PTN along with the b-sheet domains results in a decrease in the inhibitory effect of PTN on HIV infection, indeed this might affect the folding of such PTN constructs and consequently affect the inhibitory effect on HIV infection Little information about the conditions of PTN expression is available Nevertheless, its expression in FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS Nucleolin is a low affinity receptor of pleiotrophin inflammatory deceases was described [56–58] PTN is found at high concentration in the serum of patient suffering from pancreas, colon, testicular and breast cancer [59–61] Also PTN is expressed during fracture healing [62] Whereas, at low concentrations, PTN enhance the proliferation of the PBMCs (Achour et al 2001), PTN has not been detected in resting or activated T lymphocytes [35] Thus, this information does not allow us to form a clear idea about a potential role of PTN in in vivo HIV infection Thus the conditions of PTN expression and its role in in vivo HIV infection have to be studied Taken together, our results demonstrate that PTN uses the cell surface-expressed nucleolin as a low affinity cell surface receptor This binding and its internalization by an active process might be implicated in the mechanism of action of PTN as a mitogenic and growth regulatory factor Consequently, HB-19 that prevents PTN binding to surface nucleolin provides a potential inhibitor of PTN Experimental procedures Materials Recombinant human PTN (rh PTN) and human midkine (rh MK) produced in Escherichia coli were purchased from R & D systems Basic fibroblast growth factor (FGF-2) produced in E coli was from Sigma (St Louis, MO, USA) PTN was iodinated (2.2 · 103 lCiỈlmol)1) using the Bolton-Hunter reagent (PerkinElmer Life Sciences, ON, Canada) by a procedure as recommended by the manufacturer The HB-19 pseudopeptide was synthesized as described previously [4] Antibodies Goat anti-(human PTN) Ig, and anti-(human FGF-2) Ig were purchased from R & D systems The monoclonal antibody (mAb) D3 specific for human nucleolin was provided by J.S Deng, Veterans Affairs Medical Center, Pittsburgh, PA, USA [36] Cell lines and virus preparation The MT4 is a human T lymphocyte cell line that was propagated in RPMI 1640 (BioWhittaker, Verviers, Belgium) Human HeLa-CD4-LTR-LacZ cells expressing or not expressing CCR5 were referred to as HeLa P4-C5 and HeLa P4, respectively These HeLa cells (provided by P Charneau and O Schwartz, Institut Pasteur, Paris, France) were cultured in Dulbecco’s modified Eagles’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with G418 4655 Nucleolin is a low affinity receptor of pleiotrophin sulfate (500 lgỈmL)1) (Calbiochem-Novabiochem, San Diego, CA, USA) [37] Chinese hamster ovary cell lines were obtained from American Type Culture Collection: wild-type cells (CHO K1) and mutant cells defective in heparan sulfate proteoglycan expression (CHO 677) or heparan ⁄ chondroitin sulfate proteoglycans expression (CHO 618) [38,39] CHO cell lines were cultured in Ham’s F12K medium All cells were cultured with 10% (v ⁄ v) heat inactivated (56 °C, 30 min) fetal bovine serum (FBS) (Roche Molecular Biochemicals, Indianapolis, IN) and 50 international units ⁄ mL penicillin-streptomycin (Invitrogen) The HIV-1 LAI, and HIV-1 Ba-L isolates were propagated and purified as described previously [37] HIV infection of HeLa CD4+ cells HIV infection was monitored indirectly in HeLa-CD4LTR-LacZ cells containing the bacterial lacZ gene under the control of HIV-1 LTR The multiplicity of infection of the HIV-1 for infection of HeLa P4 and HeLa P4C5 cells was HIV-1 entry and replication result in trans-activation of HIV-1 LTR by the viral Tat protein, leading to the expression of b-galactosidase At 48 h postinfection, cell monolayers were lysed in a phosphate buffer containing Nonidet P-40 (Sigma) (1%; v ⁄ v) and assayed for b-galactosidase activity by measuring optical density at 570 nm [37] Expression of PTN constructs by CHO-K1 cells The human PTN cDNA was subcloned into the EcoRI site of the eucaryotic expression plasmid pcDNA3 (Invitrogen, Cergy Pontoise, France) The resulting plasmid named pcDNA3-HARP was used as template to generate pcDNA3-HARPD111-136 as described previously [40] Mutant plasmid pcDNA3-HARPD1-12 and pcDNA3HARPD1-12,D111-136 were generated using Quick-Change site-directed mutagenesis kit (Stratagene, Saint Quentin en Yvelines, France) Oligonucleotides were synthesized by MWG (Ebersberg, Germany) The presence of the mutations was confirmed by double stranded DNA sequencing Transfections of CHO-K1 cells with recombinant plasmids and purification of the resulting recombinant proteins from cells conditioned media were performed as described by [41] PTN Nt-tail, PTN Ct-tail peptides were generated as described in [40] HIV attachment on HeLa CD4+ and CHO cell lines HIV attachment was monitored in HeLa P4, CHO K1, CHO 677 and CHO 618 cell lines HIV attachment was measured after 45 at room temperature (20 °C) in order to block viral entry [42] and potential HIV endocytosis [43] by measuring the concentration of the HIV p24 4656 E A Said et al protein in cell extracts by p24 Core Profile enzyme-linked immunosorbent assay (DuPont, Boston, MA, USA) Cells were washed with culture medium containing 10% FBS to eliminate unbound HIV particles before cell extraction Assay of 125 I-labeled PTN Binding to cells HeLa P4 and CHO cells were plated at · 104 cells ⁄ well in 96-well plates Twenty-four hours later, binding experiments were performed after incubation of the cell monolayers for h at room temperature (20 °C) Cells were then incubated (30 at 20 °C) with different concentrations of 125I-labeled PTN before washing in culture medium containing 10% FBS For the total amount of binding (specific and nonspecific), cells were washed seven times with culture medium To characterize the specific binding measurements, cells were first washed eight times in culture medium followed by four washes with culture medium supplemented with 150 mm NaCl (thus bringing the final concentration of NaCl to 300 mm) Washed cells were extracted in 1% SDS, and the radioactivity was measured in an automatic c counter (LKB Wallac Clini Gamma 1272) To determine the concentration of NaCl in the culture medium necessary for the elimination of nonspecifically bound PTN, CHO K1 and CHO 618 cells were incubated for 30 at 20 °C with different concentrations of [125I]PTN (90, 185, 370, 750, 1500, and 3000 nm) before washing in culture medium containing increasing concentrations of NaCl Saturation of PTN binding was not observed when cells were washed at 150 and 200 mm NaCl, thus indicating that the values obtained correspond to the amount of total binding (specific and nonspecific) For cells washed at concentrations higher than 300 mm NaCl (such as 0.5; or m), the saturation curves were similar to those obtained with the 300 mm NaCl wash but at much lower values This latter pointed out that specifically bound PTN could be washed away at NaCl concentrations higher than 300 mm Confocal microscopy Laser-scanning confocal immunofluorescence microscopy (Leica TCS 4D) (Leica Lasertechnik, Heidelberg, Germany) was carried out by fixing cells either by paraformaldehyde (PFA; 3,7%) or PFA ⁄ Triton X-100 solution for membrane and cytoplasmic staining, respectively HeLa P4 or CHO K1, 677 and 618 cell lines were plated 24 h before the experiment in eight-well glass slides (LAB-TEK Brand, Nalge Nunc International, Naperville, IL, USA) Cells in suspension (MT4) were added to slides that were precoated with poly L-lysine at 30 lg (Sigma) and left for 15 before washing the attached cells with NaCl ⁄ Pi and proceeding with the experimental protocol For the colocalization experiments, MT4 cells in RPMI medium containing 10% FBS were incubated with PTN at 20 °C FEBS Journal 272 (2005) 4646–4659 ª 2005 FEBS E A Said et al for 45 before washing with RPMI medium containing 1% FBS Cells were then incubated at 20 °C for 45 in the presence of the anti-PTN polyclonal antibody (2 lgỈmL)1) to cross-link PTN adsorbed on the cell surface Cells were first washed in RPMI, 1% FBS and, second, with NaCl ⁄ Pi before fixation with 0.25% PFA Partial fixation was used at this stage to prevent further lateral movements of surface antigens [5] when cells were incubated (20 °C, 45 min) with mAb D3 against nucleolin After washing, cells were fixed with 3.7% PFA and washed again, and the primary anti-PTN antibody was revealed by FITC-conjugated rabbit anti-(goat IgG) Ig, whereas mAb D3 against nucleolin was revealed by Texas Red dyeconjugated horse 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and pharmacologic targeting of angiogenesis factors – the example of pleiotrophin Breast Cancer Res Treat 36, 157–168 60 Souttou B, Juhl H, Hackenbruck J, Rockseisen M, Klomp HJ, Raulais D, Vigny M & Wellstein A (1998) Relationship between serum concentrations of the growth factor pleiotrophin and pleiotrophin-positive tumors J Natl Cancer Inst 90, 1468–1473 61 Aigner A, Brachmann P, Beyer J, Jager R, Raulais D, Vigny M, Neubauer A, Heidenreich A, Weinknecht S, Czubayko F & Zugmaier G (2003) Marked increase of the growth factors pleiotrophin and fibroblast growth factor-2 in serum of testicular cancer patients Ann Oncol 14, 1525–1529 62 Petersen W, Wildemann B, Pufe T, Raschke M & Schmidmaier G (2004) The angiogenic peptide pleiotrophin (PTN ⁄ HB-GAM) is expressed in fracture healing: an immunohistochemical study in rats Arch Orthop Trauma Surg 124, 603–607 4659 ... PTN inhibits HIV infection by binding the cell- surface expressed nucleolin leading to the inhibition of HIV- attachment to the cell surface PTN binding to cells involves high and low affinitybinding... Inhibition of HIV- infection by PTN We investigated the inhibitory effect of PTN on HIV infection by using the experimental model of HeLa P4 or HeLa P4C5 cells HIV entry and replication in these cells... Fig The effect of the nucleolin binding molecules on the binding of PTN to the low and high affinity -binding site (A) Inhibitory effect of HB-19 on the binding of the 125 I-labeled PTN to the