KN-62

N-Arylpiperazine modified analogues of the P2X7 receptor KN-62 antagonist are potent inducers of apoptosis of human primary osteoclasts

Letizia Penolazzi1, Ercolina Bianchini1, Elisabetta Lambertini1, Pier Giovanni Baraldi2, Romeo Romagnoli2, Roberta Piva1 & Roberto Gambari3,4,*
1Department of Biochemistry and Molecular Biology, Molecular Biology section, University of Ferrara, 44100, Ferrara, Italy; 2Department of Pharmaceutical Sciences, University of Ferrara, 44100, Ferrara, Italy; 3Interdisciplinary Center for the Study of Inflammation (ICSI), University of Ferrara, 44100, Ferrara, Italy; 4ER-GenTech, University of Ferrara, 44100, Ferrara, Italy
Received 30 November 2004; accepted 25 August 2005
© 2005 National Science Council, Taipei

Key words: apoptosis, osteoclasts, P2X7 receptor

Summary

The P2X7 nucleotide receptor is an ATP-gated ion channel that plays an important role in bone cell function. Here, we investigated the effects of L-tyrosine derivatives 1–3 as potent P2X7 antagonists on human primary osteoclasts. We found that the level of expression of P2X7 receptor increased after treat- ment with the derivatives 1–3, together with the induction of high levels of apoptosis. This effect is associated with activation of caspase-3 and inhibition of expression of IL-6. Interestingly, no pro-apoptotic effect of compounds 1–3 was found on human osteoblasts. Our results suggest that the development of specific P2X7 receptor antagonists may be considered a useful tool to modulate apoptosis of human osteoclasts. Since bone loss due to osteoclast-mediated resorption represents one of the major unsolved problem in osteopenic disorders, the identification of molecules able to induce apoptosis of osteoclasts is of great interest for the development of novel therapeutic strategies.

Introduction

The P2X7 receptor, a ion channel plasma membrane receptor belonging to the P2X purinoreceptors family, is considered to be a potential target for pharmacological intervention, since it is involved in several biological processes, including cytokine release, NO generation, killing of intracellular pathogens, cytotoxicity [1]. The only known phys- iologically relevant ligand for P2X7 receptor is extracellular ATP, that induces an increase of cationic permeability and plasma depolarization [1].

It is well known that ATP stimulates bone resorption by osteoclasts (OCs) [2], which are large, multinucleated, terminally differentiated cells formed by the fusion of mononuclear hemopoietic precursors [3, 4]. Their function is the resorption of bone, which is an essential part of the growth, modelling and remodelling of the skeleton.Gartland et al. demonstrated that human oste- oclasts express P2X7 receptors in vitro and in vivo, and that these receptors are functional in vitro, as assessed by pore-formation studies [5]. More importantly, blockade of the P2X7 receptor with the antagonist oxidized ATP or a blocking mono- clonal antibody significantly inhibits the fusion of osteoclast precursors to form multinucleated osteoclasts [6]. Accordingly, recent published observations demonstrated that P2X7 receptors are also involved in differentiation of osteoclasts [7, 8].

As far as association with other pathways, Korcoc et al. demonstrated, in a recent paper, that benzoyl–benzoyl-ATP (BzATP, a P2X7 agonist) induced nuclear translocation of nuclear factor kappa B (NF-jB) in treated osteoclasts [9]. Their findings strongly suggest that P2X7 nucleotide receptors are responsible for the activation of NF-jB in osteoclasts. Therefore, nucleotides released at sites of inflammation or in response to mechanical stimuli, may act through NF-jB to regulate osteoclast formation and activity. Accordingly, the coordinated expression of P2X7 receptor and NF-jB in osteoclasts and other cellular systems has been recently reported [9–11].

Interestingly, NF-jB is known as a powerful anti-apoptotic molecule [12]; this was clearly sustained by the evidence that transcription factor molecules against NF-jB induce apoptosis of human OCs at very high levels. This was obtained using transcription actor decoy molecules based on oligonucleotides (ODN) and PNA–DNA–PNA chimeras mimicking NF-jB binding sites [13, 14]. Therefore, we were interested in determining whether inhibition of P2X7 receptor activity could be coupled with induction of apoptosis of human primary osteoclasts. To this aim, we studied a recent published class of L-tyrosine derivatives acting as potent and selective P2X7 antagonists [15, 16], in order to determine whether they exert biological effects on human primary osteoclasts.

Material and methods

Culture of human primary OCs

Human OCs were prepared as reported by Matsu- zaki et al. [17] with slight modifications. Briefly, peripheral blood was collected from healthy nor- mal volunteers after informed consent. Mononu- clear cells (PBMCs) were prepared from diluted peripheral blood (1:2 in Hanks Balanced Salt Solution), which was layered over Histopaque 1077 (Sigma, St. Louis, MO, USA) solution, centrifuged (400 g), washed and resuspended in D-MEM/10% FCS. 3 106 PBMCs/cm2 were plated in 24-well plates or in chamber slides and allowed to settled for 2 h; wells were then rinsed to remove non-adherent cells. Monocytes were main- tained at 37 °C, in 5% CO2, in medium supple- mented with 10% FCS and cultured for 14 days in the presence of human M-CSF (25 ng/ml), RANKL (30 ng/ml) and 10–7 M PTH. Culture media were replenished with fresh media every 3–4 days. Cells were used for the described experiments when mature multinuclear cells were predominant in the cultures [13].

Tartrate-resistant acid phosphatase (TRAP) staining

TRAP staining of the cells was performed as reported by Villanova et al. [18]. Cells were fixed in 3% paraformaldehyde with 0.1 M cacodylic buffer, pH 7.2 (0.1 M sodium cacodylate, 0.0025% CaCl2) for 15 min, exstensively washed in the same buffer, and stained for TRAP (Acid Phosphatase Kit n. 386 – Sigma, St. Louis, MO, USA). After washing with distilled water and drying, mature TRAP positive multinucleated cells containing more than three nuclei were counted as osteoclasts.

Measurement of apoptosis

After 14 days of cell culture and 36 h of treatment, the cells were rinsed two times with PBS solution and fixed for 25 min in 4% paraformaldehyde at room temperature. Apoptotic cells were detected by the DeadEnd Colorimetric Apoptosis Detection System (Promega) according to the manufacturer’s instructions. Measurement of apoptosis was calcu- lated as a percentage of apoptotic nuclei (dark brown nuclei) versus total nuclei of multinucleated TRAP positive cells, evaluated in three independent experiments.

Immunocytochemistry analysis

Immunocytochemistry analysis was performed employing the streptavidin-biotin method using Ultraystain Polyvalent-HRP Immunostaining Kit. OCs cells grown in multichamber slides were fixed in 100% cold methanol, and permeabilized with (v/ v) Triton X-100 (Sigma) in TBS (Tris-buffered saline). Cells were incubated in 3% H2O2 and blocked with Super Block reagent (Ultraystain Polyvalent-HRP Immunostaining Kit). After the reaction, rabbit polyclonal antibodies of human origin (Santa Cruz Biotech) against MMP9, IL-6, and caspase 3 (2 mg/ml) were used accordingly to the manufacturer’s protocols, at 1:500 (MMP9), 1:800 (IL-6) and 1:800 (caspase 3) dilutions. The rabbit polyclonal anti-P2X7 receptor serum was kindly provided by Dr Gary Buell (Serono Pharmaceutical Research Institute, Geneva, Switzerland) and was used at 1:100 dilution. Incu- bation was carried out at 4° for 16 h. Cells were then incubated at room temperature with anti- polyvalent biotinylated antibody (Ultraystain Poly- valent-HRP Immunostaining Kit). After rinsing in TBS, streptavidin HRP (Ultraystain Polyvalent- HRP Immunostaining Kit) was applied, followed by the addition of substrate–chromogen mix (AEC Cromogeno kit). After washing, cells were mounted in glycerol/TBS 9:1 and observed using a Leitz microscope.

Cytotoxicity studies

The cytoxicity analysis was determined on in vitro cultured human OCs cells. PBMCs were plated in 96-well plates and, after 14 days, OCs were incu- bated with the P2X7 blocking agents for 3 days. Determinations of viable cells were performed after colorimetric assay with MTT (thiazolyl blue). The assay, based on the conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells [19], provides a quantitative determination of viable cells. After 72 h of treatments in triplicate, 25 ll of MTT was added to each well of cells, and the plate was incubated for 2 h at 37 °C. The medium was removed, and the MTT crystals were solubilized with 50% DMF: spectrophotometric absorbance of each sample was then measured at 570 nm.

Statistical analysis

Data are presented as the mean±SEM from at least three independent experiments. Statistical analysis was performed by one-way analysis of variance followed by the Student’s t-test. A p value <0.05 was considered statistically significant. Results Analogues of the P2X7 receptor KN-62 antagonist The chemical structures of the analogues of KN-62 employed in this study (compounds 1–3) are shown in Figure 1. The synthesis of these mole- cules has been reported in detail elsewhere [15]. Antagonist activity of L-tyrosine derivatives 1, 2 and 3 was tested on monocyte-derived human macrophages, a cell type well known for its high level of expression of this receptor. When ATP-dependent Ca2+ influx across the plasma membrane was considered, IC50 values were found to be 5.8±0.6, 18.5±0.4 and 1.3±0.1 nM for compounds 1, 2 and 3, respectively. The chemical structures of compounds 1–3 are depicted in Figure 1. Primary cultures of human osteoclasts in the presence of P2X7 receptor antagonists In the experimental system used, human primary OCs were cultured in complete D-MEM plus MCSF, PTH and RANKL with or without the P2X7 blocking agents 1, 2 and 3. Osteoclasts formation was analyzed by counting tartrate-acid resistant acid phosphatase (TRAP)-positive cells and their metalloproteinase-9 (MMP-9) expression after 14 days. As shown in Figure 2, all multinu- cleated cells exhibited an OCs phenotype. Figure 1. Chemical structures of compounds 1–3. The biological effects of the P2X7 receptor blocking agents were studied on OCs from three normal donors. In the experiment depicted in Figure 3, OCs were treated for 72 h with 5, 50, 100, 200, 500 and 1000 lM 1, 2 and 3. The viability of the cells was examined by colorimetric MTT assay [19]. Cytotoxic effects were observed only when compounds 1 and 2 were added at 500 lM, while compound 3 failed to induce cytotoxicity at this concentration (IC50 higher than 1000 lM). Since the concentrations for determining possible effects on apoptosis were to be chosen in a wide range between concentrations inhibiting ATP-dependent Ca2+ influx and con- centrations leading to cytotoxicity, we decided not to exceed 200 lM when the biological assays on apoptosis were performed. No cytotoxic effects of compounds 1–3 were indeed found at 200 lM. P2X7 receptor antagonists induce osteoclast apoptosis We verified the effects of the three P2X7 receptor antagonists on OCs apoptosis using these compounds at 50, 100 and 200 lM. As well established, apoptosis is characterized by internu- cleosomal degradation of genomic DNA. DNA cleavage and free 3¢-OH groups generated by cellular endonucleases in apoptotic cells can be determined using in situ TUNEL assay [20]. A dark brown DAB signal indicates positive stain- ing, while shades of blue-green to greenish tan indicate a non-reactive cell. In Figure 4b the results of a representative experiment are reported, demonstrating that 200 lM of the compounds 1–3 induce apoptosis of OCs, whereas, as expected, no apoptosis was detected in untreated cells. The results from four independent experiments are shown in Figure 4a, which indicates that all the compounds induced an appreciable level of apop- tosis after 36 h treatment even when added at 50 lM. Times of exposure shorter than 36 h (16 and 24 h) were also tested without obtaining significative differences from untreated cells (data not shown). Activation of caspase-3 and inhibition of IL-6 in apoptotic OCs We then examined by immunocytochemistry the effect of the three P2X7 receptor antagonists on the expression of caspase-3 and IL-6. The expression of caspase-3 was analyzed because this protein is coded by a well known apoptosis-related gene [21]. IL-6 expression is also relevant, since this gene is downregulated during OCs apoptosis [13]. As shown in Figure 5, compounds 1–3 were all able to strongly increase caspase-3 expression in OCs. In addition, as found during induction of apopto- sis by inhibitors of NF-jB activity [13], apoptosis induced by compounds 1–3 is correlated with a decrease of expression of IL-6 [6]. To further characterize the biological activity of compounds 1–3, immunocytochemistry was also used to verify the expression of P2X7 receptor in untreated osteoclasts or in osteoclasts undergo- ing the process of apoptosis after treatment with compounds 1–3. As shown in Figure 5, the positive immunostaining for P2X7 receptor observed in the untreated multinucleated cells was substantially increased in osteoclasts treated with the P2X7 receptor antagonists. Figure 2. Primary osteoclasts were fixed and stained for TRAP to enumerate the attached TRAP-positive multinucleated cells and characterized for MMP-9 expression by immunocytochemical analysis. Figure 3. Effect of different concentrations (5–1000 lM) of P2X7 antagonists (compounds 1,2,3) on cell survival of human primary osteoclasts obtained by MTT colorimetric assay. Results are expressed as the percentage of surviving cells and are the mean±SEM of three independent experiments. The viability of controls only treated with vehicle (DMSO) has been set as 100%. Discussion The role of P2X7 receptor in the balance between osteoblasts and osteoclasts is controversial. Some authors reported that P2X7 receptor is involved in pre-fusion of osteoclasts [1]. However, in vivo model systems giving evidence for defective bone parameters in knockout mice lacking P2X7 recep- tor genes do not exhibit lacking of the total amounts of OCs [22]. The aim of the present work was to determine the effects of antagonists of the P2X7 receptor on biological functions of differentiated human primary OCs. This would make possible, on one hand, the screening of analogues displaying selectivity; on the other, it will be possible to inhibit P2X7 receptor downstream signals includ- ing specific controlled genes, in order to find out effects comparable to those of N-arylpiperazine modified analogues of the P2X7 receptor KN-62 antagonist, employed in the present study. The major result of our study is the finding that the employed analogues of KN-62 antagonists induce high levels of apoptosis in human primary OCs; this effect is associated with activation of caspase-3, inhibition of IL-6 production and increase of P2X7 receptor expression. These effects are expected in this experimental model system, as recently shown in two reports from our laboratory, employing transcription factor decoy molecules inhibiting the biological activity of NF-jB [13, 14]. In this respect, it is interesting to note that the P2X7 receptor activity is associated with activation of NF-jB. Figure 4. Detection of apoptosis by TUNEL assay in human primary osteoclasts. (a) Cells, cultured as described, were treated with different concentration (50–100–200 lM) of 1, 2, 3 for 36 h. The means percentage±SEM of TUNEL-positive nuclei are re- ported in the graphs and in the corresponding table. * Indicates statistically significant differences between the indicated treated cells (p<0.05). (b) The presence of apoptotic OCs after treatment with 200 lM of 1, 2 and 3 for 36 h, is shown. Brown colour reaction (arrows in panels) indicates cells that underwent apoptosis. Cells were photographed at the 20 magnification (inserts show higher magnification). Figure 5. Differential expression of caspase-3, IL-6 and P2X7 receptor in human primary osteoclasts analyzed by immunocyto- chemistry with specific antibody after incubation for 36 h with 200 lM of 1, 2 and 3. (–), without primary antibody. Cells were photographed at the 20 magnification. Similar results were obtained performing the same experiment with compounds 1–3 used at 100 lM. The results here described do not explain the cascade of molecular events leading to induction of apoptosis of OCs treated with the N-arylpiper- azine modified analogues of the P2X7 receptor KN-62 antagonist. Further experiments should be undertaken to fully explain this phenomenon and correlate it with a specific role of P2X7 receptor in osteoclasts. These compounds have been described to inhibit ATP-dependent Ca2+ influx across the plasma membrane [16]. In addition, it is interesting to note that the expression of the P2X7 receptor clearly changes following treatment of OCs with these compounds (Figure 5). Nevertheless, our studies may contribute in the design of novel therapeutic strategies for modula- tion of osteoclasts functions. In this respect, in order to propose inducers of apoptosis as possible therapeutic agents in osteopenic diseases, it is very important to verify their activity on osteoblasts. We have therefore started a project aimed at the analysis of the effects of compouds 1–3 on primary osteoblasts, obtaining preliminary data that dem- onstrate that no apoptosis is induced by the P2X7 receptor antagonists on osteoblasts (Penolazzi L. et al., manuscript in preparation). Acknowledgements This research was supported by grants from MI- UR FIRB-2002, MIUR COFIN-2002, Associazione Italiana Ricerca contro il Cancro (AIRC)- 2003 and 2004, Associazione Veneta per la Lotta alla Talassemia (AVLT), Fondazione Italiana per la Fibrosi Cistica, Spinner Project ‘‘GenTech-for- Thal’’ (Regione Emilia Romagna, Italy) and Fondazione Cassa di Risparmio di Padova e Rovigo. E.L. is a recipient of a fellowship from ‘‘Fondazione Cassa di Risparmio di Cento.’’ We thank Prof Francesco di Virgilio (Ferrara University) for providing the rabbit polyclonal anti-P2X7 receptor serum. References 1. North R.A., Molecular physiology of P2X receptors. Physiol. Rev. 82: 1013–1067, 2002. 2. Buckley K.A., Hipskind R.A., Gartland A., Bowler W.B. and Gallagher J.A., Adenosine triphosphate stimulates human osteoclast activity via upregulation of osteoblast- expressed receptor activator of nuclear factor-kappaB ligand. Bone 31: 582–590, 2002. 3. 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