P2X7Rs are involved in cell death, growth and cellular signaling in primary human osteoblasts

    P2X7Rs are involved in cell death, growth and cellular signaling in primary human osteoblasts Ankita Agrawal, Zanne Henriksen, Susanne Syberg, Solveig Petersen, Derya Aslan, Marie Solgaard, Nis Nissen, Tommy Korsgaard Larsen, Peter Schwarz, Thomas H. Steinberg, Niklas Rye Jørgensen PII: DOI: Reference:

S8756-3282(16)30344-1 doi: 10.1016/j.bone.2016.11.011 BON 11183

To appear in:

Bone

Received date: Revised date: Accepted date:

8 August 2016 10 November 2016 11 November 2016

Please cite this article as: Agrawal Ankita, Henriksen Zanne, Syberg Susanne, Petersen Solveig, Aslan Derya, Solgaard Marie, Nissen Nis, Larsen Tommy Korsgaard, Schwarz Peter, Steinberg Thomas H., Jørgensen Niklas Rye, P2X7Rs are involved in cell death, growth and cellular signaling in primary human osteoblasts, Bone (2016), doi: 10.1016/j.bone.2016.11.011

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P2X7Rs are involved in cell death, growth and cellular signaling in primary human osteoblasts

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Ankita Agrawal1, Zanne Henriksen1, Susanne Syberg1, Solveig Petersen1, Derya Aslan1, Marie Solgaard1, Nis Nissen2, Tommy Korsgaard Larsen3, Peter Schwarz4,5, Thomas H. Steinberg6,

Research Centre for Ageing and Osteoporosis, Department of Clinical Biochemistry, Rigshospitalet,

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Niklas Rye Jørgensen1,7,

Denmark. 2Department of Orthopedic Surgery, Kolding Hospital, Kolding, Denmark. 3Department of

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Orthopedic Surgery, Copenhagen University Hospital Hvidovre, Denmark. 4Research Centre for Ageing and Osteoporosis, Department of Endocrinology, Rigshospitalet, Denmark. 5Faculty of Health Sciences, Copenhagen University, Copenhagen, Denmark.

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Department of Internal Medicine,

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Washington University School of Medicine, St. Louis, MO, USA. 7OPEN, Odense Patient data

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Explorative Network, Odense University Hospital/Institute of Clinical Research, University of

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Southern Denmark, Odense, Denmark

Running title: P2X7R and human osteoblasts

Address correspondence to: Niklas Rye Jørgensen

Professor, MD, PhD, DMSc Department of Clinical Biochemistry Rigshospitalet, Glostrup, Denmark Tel: +45 38 63 24 56 Fax: +45 38 63 49 38 E-mail: [email protected]

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ABSTRACT

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The ionotropic ATP-gated P2X7 receptor (P2X7R) is involved in the regulation of many physiological functions including bone metabolism. Several studies on osteoblasts from rodents and human

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osteoblast-like cell lines have addressed the expression and function of P2X7R on these bone- forming

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cells however; its role in human primary osteoblasts has not yet been reported. The aim of this study

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was to assess the expression of the P2X7R in bone marrow-derived stromal cells and in primary human trabecular osteoblasts and to determine the function in bone formation and cell signaling. We report

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that osteoblasts derived from human trabecular explants express a functional P2X7R capable of agonist-induced increase in intracellular calcium concentration and a positive permeability to

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fluorescent dyes. These osteoblasts are fully differentiated cells with alkaline phosphatase activity and

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the ability to form mineralized nodules. We show that the transcriptional regulation of osteoblastic

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markers can be modulated by P2X7R activity or blockade thereby influencing the differentiation, proliferation and bone matrix formation by these primary human osteoblasts. Finally, we demonstrate that the P2X7R is involved in propagation of mechanically-induced intercellular signaling in addition to the known mechanisms involving calcium signaling via P2Y2 receptors and gap junction.

Key words: Calcium signaling, P2X7R, osteoblast

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1. INTRODUCTION

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Purinergic signaling, via extracellular nucleotides or nucleosides and their corresponding cell surface receptors (purinoceptors), is involved in cellular growth, proliferation, differentiation and death,

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angiogenesis, atherosclerosis, regeneration and wound healing, pain, cancer and ageing [1-4]. It is

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widely recognized that these effects are mediated by one or more of the purinoceptors divided into two

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classes: P1 (nucleoside) receptors or P2 (nucleotide) receptors; the latter subdivided into P2X family of ligand-gated ion channels and P2Y family of G-protein-coupled receptors [5]. Currently, seven P2X 2, 4, 6, 11, 12, 13, 14)

mammalian receptor subtypes have been

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receptors (P2X1-7) and eight P2Y (P2Y1,

identified. The role of P2 receptors in bone has been extensively reviewed [6-10] and the expression

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and involvement of the P2X7 receptor (P2X7R) at various differential stages of bone cells has also

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been previously published [11]. Therefore, although it is established that a functional P2X7R is

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imperative for the formation and survival of the osteoclasts and osteoblasts, its role in osteogenesis remains controversial.

In rodents, the expression of P2X7R has been consistently described in vitro using bone-derived primary osteoblasts. In rat calvarial osteoblasts, the expression of P2X7R mRNA increases with differentiation of the cells along with a high P2X7R protein expression as confirmed by western blotting in differentiating cells [12, 13]. However, agonist application has confounding results. Panupinthu et al. reported that P2X7R activation caused increased osteoblast differentiation and matrix mineralization [14] whereas reports from another lab describe an agonist-induced inhibition of bone mineralization in osteoblasts obtained from the same source [13]. The latter is attributed to hydrolysis of endogenous ATP to produce pyrophosphate, a mineralization inhibitor [15]. In mouse osteoblasts, P2X7R activation induces reversible plasma membrane blebbing and promotes osteogenic responses

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such as potentiation of Wnt/beta-catenin transcriptional activity as well as production of lipid mediators or FosB-dependent COX-2 protein expression as reported using calvarial-derived primary osteoblasts

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and MC3T3-E1, a cell line established from C57BL/6 mouse calvaria [14, 16-19]. Moreover, P2X7Rs

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have been shown to mediate extracellular signal-regulated kinase (ERK) 1/2 activation by fluid shear

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stress in MC3T3-E1cell line [20]. Thus, P2X7Rs in osteoblastic cells couple to multiple signaling pathways, some of which are important in skeletal mechanotransduction. In vivo, targeted disruption of

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the gene encoding the P2X7R causes an increase in cortical bone in long bones of mice [21]. A second

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model, that develops an osteopenic phenotype with smaller bone diameters and reduced periosteal bone formation [22] as well as a considerably reduced anabolic response to mechanical stimulation [23], has

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also been described. Taken together, the studies recognize a positive role of P2X7R in

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mechanotransduction.

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In human osteoblast-like cells, the first studies found that P2X7Rs were only expressed by a subpopulation [24, 25], and that agonist application favored apoptosis following extensive membrane blebbing [24, 26]. Human MSC progenitors following either shockwave treatment or agonist stimulus to compromised cells have been reported to enhance osteoblastic differentiation and mineralization [27, 28] highlighting a P2X7R-mediated pro-osteogenic effect. Nucleotides are released from osteoblasts in response to mechanical stimuli [29] and the P2X7R subtype seems to be important in mediating mechanical stimuli in bone cells, as it is involved in the propagation of mechanically induced calcium signaling between human osteoblasts and osteoclasts and among osteoclasts [30], while the P2Y2 receptor and gap junctional communication are primarily involved in signaling in osteoblast cell lines [31], and in primary human bone marrow-derived osteoblasts [32, 33] with the predominant mechanism changing during osteoblast differentiation [34].

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The aim of this investigation was to study the expression of P2X7Rs in differentiated human primary

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osteoblasts and to determine its involvement in intercellular signaling among osteoblasts.

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2. MATERIALS AND METHODS

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2.1. Reagents Minimum Essential Medium, Earle’s without L-Glutamine and Phenol Red (MEM), GlutaMAX-I

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Supplement 200 mM (GlutaMAX), Penicillin / Streptomycin (P/S), trypsin/EDTA and Phosphate

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Buffered Saline (PBS) without Ca+2 and Mg+2 were obtained from Invitrogen (Thermo Fisher Scientific

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Inc.). AZ 11645373 (P2X7R antagonist) and A 740003 (P2X7R antagonist) were ordered from Tocris. Primers for the bone cell differentiation array were ordered from Eurofins. The fluorescent calcium

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indicator Fura-2/AM was purchased from Molecular Probes. 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'triphosphate tri(triethylammonium) salt (BzATP) were from Roche Diagnostics and Sigma. Lucifer

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Yellow, gap junction inhibitor 18- α-glycyrrhetinic acid (AGA), oxidized ATP (oATP, P2X7R

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indicated.

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antagonist), as well as the rest of the chemicals used, were purchased from Sigma unless otherwise

2.2.Osteoblast isolation and cell culture Bone marrow derived stromal cells (BMSC) were isolated from human bone marrow obtained from healthy volunteers, by puncture of the posterior iliac spine. All participants had read and signed informed consent, and the study was approved by the Danish Ethics Committee (approval # KF 01 311352). The marrow material was collected 1:1 in PBS+ containing 100 U/ml Heparin. The mononuclear fraction of the marrow was isolated on a Histopaque-1077 gradient by centrifugation at 300xg for 30 minutes at room temperature. The interface containing the mononuclear cell fraction was isolated and washed once in minimum essential medium (MEM) without phenol red containing 10% fetal bovine serum (FBS) by centrifugation at 200xg for 15 minutes at room temperature and plated in

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culture dishes. Cells were incubated in a humidified atmosphere of 5% CO2 at 37C for 24 hours after

culture

medium

(CM)

(MEM)

supplemented

with

10%

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which, the supernatant was aspirated to remove non-adherent cells. Adherent cells were then grown in heat-inactivated

FBS,

100U/ml

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penicillin/streptomycin and 2mM GlutaMAX-I Supplement) and maintained with medium change

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every 7 days. At confluence, usually after 6-8 weeks in culture, the cells were trypsinised to be used in experiments.

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Primary trabecular osteoblasts (ThOB) were derived from human trabecular bone obtained from

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orthopedic surgical procedures. Trabecular bone was separated from cortical bone and was cut into bone chips. These were washed thoroughly in PBS, digested with 1mg/mL collagenase for 2 hours in a

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shaking incubator at 37C and cultured for 3-4 weeks in the same medium as BMSC with medium

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change every 3-4 days. Cells were trypsinised at 80- 90 % confluency and seeded on coverslips,

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chamber slides or culture dishes and used for experiments. All assays were performed on first passage cells after primary plating. Due to the variation between cells from the different donors and in order to be able to better compare results, all data are expressed as percentage response relative to the untreated control.

2.3.Immunostaining Cells were cultured in glass chamber slides, washed in PBS before fixing in 3.7% paraformaldehyde for 25 minutes at room temperature. Cells were permeabilized with 0.2% Triton X-100 for 5 minutes at room temperature. Next, they were washed in PBS and incubated with the primary antibody against the human P2X7R (Sigma, cat. # P-8232), diluted 1:50 in 2% BSA overnight at 4°C. Cells were incubated with a Cy3-conjugated secondary antibody (Jackson, cat. # 111-165-144), diluted 1:50 in 2% BSA for

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60 minutes at room temperature. Slides were mounted and evaluated by fluorescence microscopy. For

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negative controls cells were incubated only with the secondary antibody.

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2.4. Dye uptake assay

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We assessed nucleotide-induced plasma membrane permeabilization by measuring uptake of the

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membrane-impermeant fluorescent dye Lucifer Yellow. ThOB were trypsinised and seeded onto glass chamber slides until approximately 80% confluence. Cells were washed with PBS and incubated in

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experimental buffer (PBS without Ca2+/Mg2+containing 10mM HEPES, pH 7.4) for 10 minutes at room temperature. Cells were incubated with the dye solution (experimental buffer with or without 300 µM

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BzATP, 500 µg/mL Lucifer Yellow and 100 µg/mL ethidium bromide or Hoechst 0.5 µg/mL (nucleic

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staining) was added to the cells for 15 minutes at 37C and subsequently washed thoroughly in

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experimental buffer. The culture chamber was removed and slide mounted with coverslips. Cells demonstrating diffuse cytoplasmic staining with Lucifer Yellow were identified by fluorescence microscopy and images recorded.

2.5.Proliferation assay ThOB were seeded in duplicates at a density of 10 x103 per 0.1 mL in a 96 well plate in culture medium and allowed to adhere at 37°C in a humidified atmosphere of 95% air and 5% CO2. After 24 hours, the medium was replaced with CM ± treatments. Cultures from the same donor was assessed for either 2, 4 or 6 days without any media change and 8 day assessment was performed with one media change at day 3. At the end of the culture period, proliferation was determined by adding The Cell Proliferation Reagent WST-1 (Roche Diagnostics) at a final dilution of 1:10 according to the

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manufacturer's instructions. The assay utilizes the activity of mitochondrial dehydrogenase enzymes to convert 4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) to

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formazan, an aqueous soluble dye, the amount of which correlates to the number of metabolically

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active cells. Absorbance of the formazan product produced by the cells was read at 450 nm using a

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Tecan Plate Reader. Absorbance at 650 nm was used as reference wavelength and was subtracted from those at the reading wavelength. Wells containing a same dilution of WST-1 in culture medium in the

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absence of cells was used to determine background absorbance.

2.6.Alkaline phosphatase assay

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ThOB from each donor were seeded at a density of 20 x103 in 24-well plates in 0.5 mL of CM and

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allowed to adhere at 37°C and 5 % CO2 for 24 hours. Media was replaced with CM ± treatments or

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osteogenic medium (OM) (CM supplemented with 50 μg/ml ascorbic acid and 10nM dexamethasone) ± treatments and replenished every 2–3 days. On day 14, cells were washed once in PBS before proceeding for determination of alkaline phosphatase (ALP). Cells were fixed and stained using the ALP kit (Sigma cat # 86C-1KT) as per the manufacturer’s instructions. Plates were scanned to obtain images and ALP staining analyzed by Image J. Phase contrast images were taken on stained cells at 10X magnification on a Primovert Inverted Microscope (Zeiss). To determine ALP activity, cells were washed in PBS, lysed in buffer (20 mM Tris, pH 8.0-8.3; 1 mM MgCl2; 0.2% Triton-X in nuclease-free water) and frozen at −80°C following completion of culture. Cell lysates were obtained after 3 freeze thaw cycles. ALP activity was measured using p-nitrophenyl phosphate (pNPP) as the chromogenic ALP substrate as described previously [35]. The absorbance was read at 405nm using a Tecan plate Reader. Values were expressed as percentage response relative to untreated control. DNA content was quantified using Quant-iT™ PicoGreen dsDNA Assay Kit (Invitrogen, Paisley, UK) according to the

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manufacturer's instructions. ALP activity and DNA content were measured on the same wells to

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determine the effect on cell numbers.

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2.7.Mineralization assay

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Bone chips were introduced into 24 well plates and cells were allowed to reach about 80% confluency

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at 37°C in a humidified atmosphere of 95% air and 5% CO2. Once confluent, chips were removed and cells were gently rinsed 3 times in warm PBS. Media was switched to OM ± treatments and replenished

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every 2- 3 days. To initiate mineralization (typically between days 8- 10), fresh OM supplemented with 10 mM β-Glycerophosphate was added to the cells. After 72 hours, cells were rinsed twice in PBS,

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fixed in 100% ethanol overnight. Cells were washed in PBS and incubated in 40 mM alizarin red S, pH

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4.2, for 90 mins on an orbital shaker at room temperature. The cells were washed extensively in 95%

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ethanol to remove excess stain and air-dried. Plates were scanned to determine the percentage of each well stained with alizarin red using Image J software (NIH: http://rsb.info.nih.gov/ij/) and phase contrast images were taken at 10X magnification on a Primovert Inverted Microscope (Zeiss).

2.8.Total RNA extraction and Semi-quantitative RT-PCR RNeasy minikit and RNase-free DNase set from Qiagen (Stockach, Germany) was used to extract total RNA from BMSC, ThOB and U-937. Five microgram total RNA was reverse transcribed using the Omniscript Reverse Transcriptase Kit from Qiagen. Primers specific for the human P2X7R (Forward5′-AGATCGTGGAGAATGGAGTG-3′ and Reverse - 5′-TTCTCGTGGTGTAGTTGTGG-3′) were obtained from Invitrogen. HotStarTaq DNA Polymerase kit from Qiagen and following cycling conditions were used for the PCR: activation at 95°C for 15 minutes, 35 cycles of denaturation at 94°C

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for 30 seconds, annealing at 55°C 30 seconds and extension at 72°C for 1 minute followed by final extension at 72°C for 10 minutes. This resulted in a 399 bp product, which was separated on a 2%

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agarose gel.

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To determine the expression of osteoblastic differentiation markers, semi-quantitative RT-PCR

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amplification was performed on total RNA isolated after 1 week (w1) and after 2 weeks (w2) from

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ThOB cultured in either culture medium (CM) or osteogenic medium (OM). Each preparation of total RNA (up to 4 µg) was used as a template for cDNA synthesis using the High Capacity cDNA Reverse

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Transcription Kit (Qiagen) following the manufacturer’s instructions. A no RT control was used in parallel with the samples to detect genomic contamination in samples. The primers used for the

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osteoblast markers have been previously published in [36]. HotStarTaq DNA Polymerase kit from

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Qiagen and following cycling conditions were used for the PCR: Initial heat activation at 95°C for 15

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minutes, 30 cycles each of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 1 minute. A final extension was performed at 72°C for 10 minutes. Bands were separated using a 20bp ladder on a 4% agarose gel and visualized under UV light.

2.9.Quantitative real time polymerase chain reaction (qPCR) To detection the effect of short-term P2X7R induced changes in osteoblast-specific genes; a comparative relative standard curve (ΔΔCT) method was selected for Real-Time PCR (OneStep Realtime PCR System, Applied Biosystems). TaqMan gene expression assays (Assay IDs in Table 1, Applied Biosystems) of alkaline phosphatase gene (ALPL), osteoblast transcription factor (runt-related gene 2) (RUNX2); collagen type-1 (COL1) and Receptor Activator for Nuclear Factor κ B Ligand (RANKL), osteocalcin (BGLAP), osteoprotegerin (OPG), caspase-1 (CASP1), caspase-3 (CASP3), and

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gap junction protein A1 (Connexin43) (GJA1) were used as target genes and GAPDH (Hs99999905_m1) was used as endogenous control. Manually prepared cDNA (2μl) and master mix

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(18 μl) were pipetted to each well of a 48-well plate (MicroAmpFastOptical Reaction Plate) to reach a

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final volume of 20 µL in each well, according to manufacturer’s instructions.

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2.10. Caspase-3/7 activity assay

ThOB from each donor were seeded in duplicates at a density of 10 x103 in 96-well plates in 0.1 mL of

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CM and allowed to adhere at 37°C and 5 % CO2 for 24 hours. The medium was replaced with CM ± treatments and after 3 hours, the caspase-3/7 activity was determined using Caspase-Glo 3/7 Assay

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(Promega) as per the manufacturer’s instructions. Briefly, caspase-3/7 detection reagent was added at

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1:1 ratio with the CM and cells were incubated at 25 °C for 1 hour. The generation of a “glow type”

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luminescent signal was measured by a luminometer and is directly proportional to the amount of caspase cleavage, or a reflection of the caspase-3 activity present. The background (containing CM and caspase-3/7 detection reagent) value was measured in a no cell sample and subtracted from each measurement.

2.11. Calcium imaging Measurement of [Ca2+]i was performed using the fluorescent calcium indicator Fura-2 as previously described in [31, 32]. Briefly, cells were cultured on 25 mm diameter # 1 glass coverslips for 2-3 days. Subsequently, cells were changed to serum-free medium and loaded with 5 µM Fura-2/AM for 30 minutes at 37ºC, and incubated an additional 20 minutes in medium without dye. Coverslips were affixed to a teflon chamber and mounted in a PDMI-2 perfusion micro incubator (Medical Systems

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Corp.,Greenvale, NY, USA) maintained at 37ºC with super fused CO2 on a Zeiss Axiovert 35 inverted microscope. Calcium waves were induced by stimulating a single cell with a borosilicate glass

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micropipette affixed to an Eppendorf 5171 micromanipulator system (Eppendorf Inc, Madison, WI,

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USA). Addition of specific agonists/antagonists of P2X7R and of gap junction coupling and

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visualization of intercellular calcium wave (ICW) was done in real-time. Imaging was performed with the Metamorph/Metafluor system (Universal Imaging, Westchester, PA, USA) with excitation

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wavelengths of 340 nm and 380 nm for acquiring ratio images of Fura-2. Probenecid, at a concentration

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of 1 mM, was added throughout to prevent leakage of the dye.

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2.12. Statistical analysis

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GraphPad Prism 7 software (GraphPad, La Jolla, CA, USA) was used for statistical comparisons

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between two groups by either a Student’s t-test with Welch’s correction for parametric data or MannWhitney for non- parametric data. Differences between three or more groups were evaluated by oneway analysis of variance followed by a Dunnett’s multiple comparisons test. Data are presented as mean  SEM.

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3. RESULTS

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3.1. Expression of P2X7Rs in ThOB and BMSCs

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ThOB and BMSCs were cultured in CM and the expression of RNA coding for the P2X7R gene in cells obtained from different donors was examined by semi quantitative RT-PCR (Figure 1A). Next, we

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wanted to detect the expression of the P2X7R protein by immunofluorescence in both ThOB and in

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BMSC (Figure 1B). To test whether P2X7Rs are functional in the two cell types, we stimulated cells loaded with the calcium indicator dye Fura-2 with the P2X7R agonist BzATP. In ThOB monolayers

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almost all cells in the field of view increased in [Ca2+]i (n=7) (Figure 1C) while in the BMSC monolayers no changes in [Ca2+]i could be detected (n=9), demonstrating the P2X7Rs are functional in

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ThOB but not in BMSC. In other cell types, prolonged stimulation of P2X7R with higher concentrations of ATP and BzATP induces opening of a P2X7R-related pore. Therefore, we wanted to

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investigate whether P2X7R activation in our cells induces pore formation. In ThOB, stimulation with BzATP induced uptake of the large dye Lucifer Yellow (Figure 1D) (n=5), demonstrating that P2X7R activation induces pore formation in the cells. In contrast, no dye uptake could be demonstrated in the BMSC

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Figure 1: Expression of P2X7R in human osteoblasts. A) Semi quantitative PCR of samples from 2 different donors shows a positive P2X7R expression (399bp product) in both the ThOB and BMSC. U937 (a monocyte cell line) was used as a positive control and no template RNA sample was used as a negative control. B) Immunostaining for P2X7R protein using a primary antibody shows P2X7R expression on the plasma membrane and in the nucleus in both the ThOB and BMSC. Scale bar 50µM and inset images are 100 X magnification. A negative control with the primary antibody eliminated was used to eliminate non-specific staining. C) BzATP (100 µM) clearly increased intracellular Ca2+ concentration, in ThOB cells indicating that P2X7Rs are functional in ThOB but not in BMSC. D) Dye

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uptake assay in ThOB shows positive pore formation following addition of BzATP (300 µM) resulting

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in influx of Lucifer Yellow into the cells but not in BMSC. Scale bar 50µM.

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3.2. P2X7R activation and inhibition reduces proliferation of ThOB

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The P2X7R has been shown to be involved in cell growth. Therefore, cell proliferation was determined in ThOB after treatment with either the P2X7R agonist BzATP in concentrations of 1, 10 or 100 µM,

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or P2X7R antagonists (oATP, AZ11645373, and A740003). The proliferation assay using WST-1

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measures the mitochondrial activity and the measurements are therefore proportional to the number of

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metabolically active cells present.

After two days of treatment of the cultures with BzATP, no significant effect on the number of cells

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was observed, compared to the untreated controls. Exposure to 1 μM BzATP significantly increased the proliferation at day 4 (110%) but treatment with 10 μM BzATP had no significant effect for the

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duration of the experiment. One hundred micromolar BzATP caused a reduction in proliferation at day

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6 (86%) and day 8 (76% compared to the untreated control (Figure 2A). oATP consistently reduced

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cell proliferation from day 2 (91%), and further at days 4, 6 and 8 (79%, 60% and 50% respectively) (Figure 2B). Two other P2X7R antagonists A740003 and AZ11645373, were tested and unexpectedly, there was an increase in cell proliferation at day 4 (119% and 115%, respectively). However, both A740003 and AZ11645373 resulted in a reduction in proliferation (91% and 82% by A740003; 94% and 82% by AZ11645373, respectively), when ThOBs were treated for days 6 and 8 relative to the untreated control (Figure 2B).

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Figure 2: Effect on proliferation. A) ThOB treated in duplicates in the presence of 1, 10 or 100 μM BzATP (A) or P2X7R antagonists (α); oATP at 300 μM, A740003 at 1 μM or AZ11645373 at 1 μM (B) were evaluated for proliferation at 2 day intervals. Cells containing 0% FBS in culture medium was used as a negative control. Data from 5-7 independent donors, represents mean ± SEM. a = p<0.05, b = p<0.01, c= p<0.001, d = p<0.0001 significance from the corresponding untreated control (100%) at each time point.

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3.3. P2X7R activation and inhibition reduces alkaline phosphatase activity

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To demonstrate the role of the P2X7R on osteoblast function, the effect of receptor activation and inhibition on the proportion of ALP positive cells was determined. The number of ALP positive cells

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was significantly reduced after 14 days exposure to BzATP at 10 μM when cells were cultured in CM

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(67%) and at 100 μM in CM (16%) and OM (34%) (Figures 3A - D). In addition, oATP reduced the

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ALP staining to almost undetectable levels (4.0% in CM and 2.9% in OM). Similarly a second antagonist, AZ11645373 caused a significant reduction in ALP staining after 14 days (9% in CM and

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28% in OM). Whilst A740003 was unaffective when ThOB were cultured in CM (96%), treatment

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suppressed ALP staining significantly in OM (57%) (Figures 3A - D)

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Next, we determined the cellular enzymatic ALP activity. After 14 days exposure to BzATP the ALP

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activity was significantly reduced at all concentrations when ThOB were cultured in CM and OM (Figure 3C). However, treatment with 100 μM BzATP also lead to a reduction in DNA content when ThOB were cultured in CM but not in OM (Figure 3D). When ThOB were cultured in the presence of P2X7R antagonists, there was a reduction in ALP activity despite the presence of differentiation factors (Figure 3C). DNA content was also reduced in cells exposed to P2X7R antagonists (Figure 3D). Thus, the suppressed osteoblast activity in these treatments was largely a function of reduced cell number rather than reduced activity per cell.

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Figure 3: Effect on ALP staining and activity. A) ThOB were cultured in culture medium (CM) and

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osteogenic medium (OM) to promote differentiation and treated with 1, 10 or 100 μM BzATP or

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antagonists 300 μM oATP, 1 μM A740003 or 1 μM AZ11645373. B) Cells were fixed and alkaline

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phosphatase activity (blue staining) was evaluated by light microscopy. In situ enzyme activity is shown for cultures, where wells are 15.6  mm across. C) Micrographs showing cell monolayers and

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alkaline phosphatase staining in cells in growth medium or osteogenic medium (D); scale bar is 100

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µm. E) For each condition in panel A, alkaline phosphatase is measured as signal at 450–490  nm in assays of replicate cultures and F) DNA content assessed by PicoGreen assay as a reflection of cell

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numbers. n = 6 independent donors, represents mean ± SEM. a = p<0.05, b = p<0.01, c= p<0.001, d =

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p<0.0001 significance from the corresponding untreated control (100%).

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3.4. P2X7R activation and inhibition reduces the formation of mineralized matrix

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To determine the effect of P2X7R activation on the formation of mineralized matrix, ThOB were cultured for 14 days in the presence of β-Glycerophosphate, ascorbic acid and dexamethasone supplied

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with increasing concentrations of the P2X7R agonist BzATP (1, 10, and 100 µM) or the P2X7R

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antagonists (oATP, A740003, AZ11645373). Mineralization, measured by Alizarin red staining after

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culture in osteogenic medium, determined by percentage of area stained with nodules, was reduced with BzATP treatment (43% in 10 μM and 37% in 100 μM) (p-value = 0.0311 one-way ANOVA) but

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this was not statistically significant after correcting for multiple comparisons. Also, antagonist treatments A740003 (30%) and 1 μM AZ11645373 (34%) significantly reduced matrix formation by

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ThOB (Figure 4).

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Figure 4: Effect on mineralization A) ThOB were cultured with 1, 10 or 100 μM BzATP or antagonists 300 μM oATP, 1 μM A740003 or 1 μM AZ11645373 in osteogenic medium and mineralization was assessed. B) Micrographs representative of the histochemical staining of mineral deposition (red) for each condition; scale bar is 100 µm. n = 4 independent donors, represents mean ± SEM. a = p<0.05 significance from untreated control (100%).

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3.5.Cells obtained from human trabecular bone express osteoblast- specific genes

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To demonstrate that cells obtained from human trabecular bone maintain osteoblast -specific marker expression for a time period relevant to the current study, ThOB were cultured in CM or OM for 7 or

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14 days, lysed, total RNA collected and reverse transcribed to determine gene expression. All donors

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maintained the expression of osteoblastic markers as determined by semi-quantitative RT-PCR at the

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two time points (Figure 5A).

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The role of P2X7Rs in regulating osteoblast activity and bone formation was determined by stimulating ThOB with either vehicle, increasing concentrations (10 and 100 µM) of BzATP or by blocking the

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receptor by 300 µM oATP and measuring the expression of a number of genes relevant for osteoblast differentiation and activity. Cells were stimulated for either one or three hours and RNA collected.

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Receptor activation by BzATP resulted in a change in expression of a number of genes after one and three hours (Figure 5B - E and Table 1).

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Figure 5 Gene expression profiles of osteoblastic markers A) Determined by semiquantitative RTPCR. Images are representative from three different donors, RNA prepared after 7 days (w1) and 14

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days (w2) from ThOB in either CM or OM. B) Relative expression of osteoblastic markers after

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treatment with BzATP, or oATP (α). An expression < 1.0 indicates a down-regulation of gene expression compared to the untreated control, while an expression > 1.0 indicates up-regulation. Data

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from 5-7 independent donors, represents mean ± SEM. a = p<0.05, b = p<0.01, c = p<0.001

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significance from the corresponding to the vehicle treated control at each time point.

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Table 1: The effect of P2X7R activation (BzATP) and blockade (α) on the expression of genes related to bone formation and osteoblast differentiation. Upregulated genes are highlighted in green

GENE

1 HOUR 0.936 ± 0.025 (0.0409)

0.775 ± 0.022 (0.0002)

RUNX2 Hs00231692_m1

1.136 ± 0.303 (0.8785)

COL1 Hs01076777_m1

0.780 ± 0.142 (0.3608)

Mean ± SEM (p- value)

BzATP 10µM Mean ± SEM (p- value)

BzATP 100µM Mean ± SEM (p- value)

α Mean ± SEM (p- value)

3 HOURS

0.552 ± 0.774 (0.035)

0.958 ± 0.024 (0.3776)

0.927 ± 0.211 (0.2664)

0.460 ± 0.053 (0.0021)

1.188 ± 0.173 (0.3895)

1.37 ± 0.095 (0.0053)

0.915 ± 0.115 (0.6084)

0.598 ± 0.098 (0.007)

0.600 ± 0.087 (0.0415)

0.777 ± 0.125 (0.3358)

1.028 ± 0.186 (0.7818)

0.941 ± 0.068 (0.7159)

1.041 ± 0.155 (0.8466)

0.983 ± 0.213 (0.3562)

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ALP Hs00758162_m1

α

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BzATP 100µM Mean ± SEM (p- value)

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BzATP 10µM Mean ± SEM (p- value)

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TREATMENT

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and downregulated genes in red

RANKL Hs001092186_m1

0.575 ± 0.089 (0.036)

0.946 ± 0.144 (0.8017)

0.974 ± 0.058 (0.8857)

0.982 ± 0.145 (0.9345)

0.871 ± 0.121 (0.5403)

0.628 ± 0.100 (0.007)

OPG Hs00900360_m1

0.591 ± 0.157 (0.0278)

0.907 ± 0.160 (0.381)

1.509 ± 0.148 (0.0112)

0.909 ± 0.0092 (0.4211)

0.837 ± 0.07 (0.0696)

1.778 ± 0.139 (0.0002)

CASP1 Hs00169146_m1

0.529 ± 0.065 (0.012)

0.53 ± 0.085 (0.0142)

0.584 ± 0.050 (0.0002)

1.082 ± 0.127 (0.717)

1.58 ± 0.379 (0.1978)

0.26 ± 0.094 (0.0002)

CASP3 Hs00234387_m1

0.698 ± 0.092 (0.0486)

0.334 ± 0.059 (0.0002)

0.975 ± 0.096 (0.8348)

0.902 ± 0.115 (0.5724)

0.592 ± 0.065 (0.0143)

1.514 ± 0.066 (0.001)

GJA1 Hs00748445_s1

0.61 ± 0.066 (0.0182)

0.564 ± 0.089 (0.0458)

1.641 ± 0.109 (<0.0001)

0.862 ± 0.118 (0.4148)

0.586 ± 0.08 (0.0115)

1.959 ± 0.09 (0.0002)

BGLAP Hs00609452_g1

1.03 ± 0.294

1.447 ± 0.308

0.902 ± 0.176

1.21 ± 0.263

1.862 ± 0.21

0.537 ± 0.065

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(0.1049)

(0.1717)

(0.4795)

(0.0041)

(0.011)

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(>0.9999)

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A P2X7R

Negative control

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ThOB

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BMSC

Figure 6: Effect on Caspase-3 activity A) Following cleavage of the Z-DEVD substrate (Z-DEVD-

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aminoluciferin) by Caspases, aminoluciferin (a substrate for luciferase) is released which results in generation of light by a proprietary, thermostable, recombinant luciferase (formulated within the assay

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reagent) (Promega). B) The luminescence was proportional to the amount of intracellular caspase

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activity in ThOB cultured with 100 μM BzATP or antagonists 300 μM oATP for 3 hours and which

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was quantified using a Luminometer. n = 8 independent donors, represents mean ± SEM. d = p<0.0001 significance from untreated control (100%).

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3.6.P2X7Rs are involved in intercellular calcium signaling among ThOB

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We have previously shown that osteoblasts can propagate ICW in response to mechanical stimulation by two mechanisms: 1) by nucleotide release and subsequent activation of purinergic P2Y2 receptors

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and 2) by propagation of a signal through gap junctions inducing activation of voltage-operated

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calcium channels in the neighboring cells [31-34]. To investigate whether ThOB are able to propagate

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calcium waves we therefore induced a calcium wave in a monolayer of ThOB. First, calcium levels were determined in resting cells followed by mechanical stimulation of one single

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cell. The stimulated cell increased in intracellular calcium and subsequently the calcium transient was propagated to the neighboring cells (Figure 7B). To investigate the mechanism for the wave

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propagation of the transient from cell to cell, oxidized ATP (oATP) was used to inhibit P2X7R function, UTP was used to activate and subsequently desensitize the P2Y2 receptors, and AGA was

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used to block gap junctional communication. Each of the inhibitors was tested either alone or in combination with each other, to determine the involvement of the inhibitors in the propagation of the calcium wave (Figure 7A- H). We demonstrated that P2X7Rs, in addition to gap junctional communication and P2Y receptors, are involved in ICW propagation among ThOB in contrast to BMSC were only P2Y receptors and gap junctions are involved [31, 32]. The average number of cells in ICW was plotted after activation of P2Y2 receptors (Figure 7I) and P2X7R blockade (Figure 7J). Each individual inhibitor reduced calcium wave propagation; however only, using all three inhibitors together, wave propagation was completely abolished (Figure 7K and L).

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B

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Figure 7: ICW in a monolayer of ThOB cells stained with the calcium-indicator dye, Fura-2. Sequence shows the different steps in the experiment, arrow shows the mechanically stimulated cell. A) Calcium

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levels in resting cells B) a basic calcium wave was initiated by stimulating one cell using a glass

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micropipette C) increase in the intracellular calcium levels following addition of agonist BzATP D) a

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limited ICW after addition of oATP E) No change in intracellular calcium levels after the addition of BzATP confirming the blockade of P2X7R F) UTP, via P2Y2 and P2Y4 receptors caused a huge

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[Ca2+]i increase in ThOB but also desensitized the receptors which was confirmed by G) a reduced

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calcium wave upon mechanical stimulus H) Gap junctional communication blocked by addition of AGA preventing propagation of calcium transients Scale bar shows [Ca2+]i. Graphical representation of

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individual experiments showing the number of cells in ICW after I) P2Y2 desensitization followed by

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gap junction inhibition J) P2X7R blockade K) P2X7R blockade followed by P2Y2 desensitization and

inhibition.

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gap junction inhibition and L) P2Y2 desensitization followed by P2X7R blockade and gap junction

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4. DISCUSSION

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The expression of P2X7Rs in human osteoblasts has been extensively studied. The very first evidence of P2X7R expression was demonstrated in MG-63 human osteosarcoma cells by RT-PCR [25]. Using

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another osteosarcoma cell line, Gartland et al., showed that P2X7R was expressed but only in a

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subgroup of Saos-2 cells [24] which was also confirmed in MG-63 cells [26]. This is consistent with

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studies using osteoblasts obtained from rodent bone explants where only a subpopulation of cells demonstrate a positive nucleotide response in vitro [16, 22]. Moreover, the expression of P2X7R is

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dependent on the differentiation stage of these rodent osteoblasts [12, 13]. Human P2X7R protein has been reported in mesenchymal stem cells [27, 28], which are cells of osteoblastic lineage but are

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progenitors to a more differentiated, mature osteoblast capable of bone formation. Our ThOB, derived

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from explant bone tissue, express the gene for P2X7R and show a positive protein expression as

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determined by immunofluorescence. Moreover, BzATP elicits an increase in [Ca2+]i as well as induces opening of membrane pores, typical of P2X7R activation, in ThOB. Interestingly, BMSC are positive for the P2X7R mRNA, show a positive but weaker P2X7R protein expression but BzATP failed to increase [Ca2+]i or induce dye uptake. Cells of similar osteoblastic lineage have been previously shown to be unresponsive to BzATP mediated calcium changes [31], in line with our findings that P2X7Rs are expressed but not functional in BMSC. These differences could be attributed to the differentiation state of these osteoblasts, as BMSC are uncommitted cells capable of differentiation into lineage of tissues such as bone, cartilage, adipocytes, and hematopoietic supporting tissues and are therefore, less differentiated than cells from bone explants (ThOB). Our ThOB derived in vitro show ALP activity and are capable of mineral deposition, properties of bona-fide osteoblasts, as well as positive expression of osteoblast related genes and hence a good model for exploring properties of human osteoblasts. To our

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knowledge, this is the first evidence demonstrating that P2X7R is expressed and functional in human

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primary osteoblasts. A previous study with MC3T3 cells treated with 100 μM ATP for 24 hours significantly induced DNA

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synthesis demonstrating a growth promoting effect of the stimulus on these human osteoblast-like cells

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[25]. Although the exact reason is unclear, we notice an increase in the number of proliferative cells

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when treated with 1 μM BzATP for 4 days. Interestingly, P2X7R activation, in high agonist concentration, inhibited the viability of ThOB which was also true for P2X7R blockade for 6 or more

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days in vitro (Figure 2). We know that ATP is the natural ligand of a number of not only P2X receptors but also P2Y receptors, it can be speculated that P2X7R mediated release of endogenous ATP, in low

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doses might be crucial for the survival of these cells, at least in vitro. Moreover, ALP activity was

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reduced whether P2X7R was activated or blocked in the human osteoblasts in our current study. One

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explanation is that agonist activation resulted in the endocytosis of the P2X7R by a lipid chaperone, caveolin-1, which a recent study showed is a regulatory mechanism for continual signaling [37]. This loss of protein could result in an attenuation of the agonist -ligand interaction and cause a reduced P2X7R activation in these human osteoblasts. Additionally, the BzATP-stimulated decline in ALP activity is accompanied by a reduction in cell numbers (as determined by the amount of double stranded DNA) when ThOB are cultured in normal cultured medium, but not when osteogenic supplements were added (Figure 2C and 2D). This suggests that ThOB committed to osteogenesis might be more resilient to the BzATP induced cell death. This is an important observation when comparing the data from the studies by Orriss et al, where BzATP-mediated P2X7R stimulation also inhibited ALP activity by 50% in rodent calvarial osteoblasts in vitro [13]. Interestingly, we observe an agonist induced reduction in ALP mRNA after 1 hour of treatment but the expression is restored at the

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3 hour time point (Figure 5B) similar to the unaltered ALP mRNA expression with BzATP as observed by Orriss et al. in their model. Finally, P2X7R activation and inhibition by specific antagonists

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significantly reduced the formation of mineralized matrix. This is in contrast to the observations in

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rodent calvarial osteoblasts, where BzATP-mediated P2X7R stimulation inhibited bone nodule

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formation and treatment with P2X7R antagonists abolished this inhibition [13]. The conflicting results could be due to a number of factors. Firstly, there could be species differences based on different

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pharmacological profiles of human and rodent P2X7Rs [38, 39]. Secondly, the different antagonists

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involve blockade of different P2X receptors and even for P2X7R differently. For instance, KN62 (potent antagonist for human P2X7R but inactive at rat P2X7R) and A438079 (a potent and selective

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antagonist at rat and human P2X7R) show different inhibition of bone nodule formation as has been

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previously reported [13]. Similarly, even though oATP is considered a relatively specific P2X7R

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antagonist, it does not competitively block agonist activation of P2X7R. It also exhibits inhibition of other P2X receptors so that the oATP effect we see could be the results of inhibition of more P2X receptors than just P2X7R – this is also supported by our results where more selective and potent inhibitors A740003 and AZ11645373 both inhibit matrix formation while oATP does not. This difference in the action of oATP, A740003 and AZ11645373 was also seen in the effects of these antagonists on ThOB proliferation and alkaline phosphatase activity (Figure 2 and 3). While there was generally an overall inhibition of the ThOB function, the pharmacologic effects of each antagonist is known to be dependent on the assay end point and the differences in incubation times required by the antagonists to inhibit the functional activation of the P2X7R [40]. The expression of 2 crucial markers of bone homeostasis was also altered with blocked P2X7R function. RANKL mRNA was significantly reduced but OPG mRNA was increased cumulating in a

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significant reduction in the RANKL/OPG ratio in these ThOB in vitro (Table 1). A reduced RANKL/OPG ratio is an indication of decreased bone resorption in vivo, an imperative signal for

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maintaining bone mass. However, disruption of the gene encoding the P2X7R includes the

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development of an osteopenic phenotype with reduced periosteal bone formation in mice persisting

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even after application of a mechanical stimulus thus demonstrating a positive role for this receptor in osteogenesis [22, 23], which is in line with the findings in the current study. Additionally, a number of

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functional single nucleotide polymorphisms that impair P2X7R function have also been associated with

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reduced bone mass and increased osteoporosis risk in various human cohorts [41-45], in addition to a predisposition to stress fracture [46] further strengthening the role of a fully functional P2X7R in

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maintaining bone strength. However, no studies have yet addressed the functional consequences of

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P2X7R polymorphisms on osteoblast activity and differentiation in vitro, which would be the ultimate

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demonstration of the effect of genetic mutations on osteoblast function. Other changes included a significant reduction in caspase- 1 mRNA within the first hour of BzATP addition as well as with receptor blockade. While P2X7R blockade continued to inhibit, BzATP caused an increase in caspase-1 mRNA at 3 hours (Table 1). Caspases, a family of intracellular cysteine proteases, are essential for the execution of apoptosis primarily through the activation of caspase-3. While, Caspase-1 activity has been implicated with P2X7R mediated apoptosis in human osteoclasts [42], BzATP has previously been unable to induce the active form of caspase-3 in MC3T3 osteoblast like cells [23]. Unexpectedly, addition of BzATP to ThOB reduced the caspase-3 gene expression which was also replicated in the activity of the enzyme (Figure 6). There was an upregulation of the mRNA in cells treated with the antagonist; however this was not seen in the enzymatic assay. It has been widely examined that activation of caspase-3 induces apoptosis in human osteoblastic cells but

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activation of effector caspases, which include caspase-3, is also required for osteoblast differentiation [47]. Reportedly, through a mechanism that promotes cell differentiation and does not lead to apoptotic

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cell death. It is likely that this P2X7R mediated attenuation of caspases supports the differentiated state

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of ThOB, as is also reflected by the loss in ALP staining/activity and mineral deposition by these

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primary human osteoblasts.

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Lastly, we found that P2X7Rs are partially responsible for the propagation of mechanically-induced intercellular calcium signals in monolayers of human ThOB. We have previously shown that autocrine

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activation of P2Y2 receptors [31, 32] and gap junction-dependent activation of voltage-operated calcium channels [33] are involved in the propagation of mechanically-induced calcium signals among

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osteoblasts and that the mechanism for the propagation of calcium waves among cells changes during

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the differentiation of cells [34]. Also, we showed that osteoblast-osteoclast signaling involves

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activation of P2X7Rs on osteoclasts [30]. However, the osteoblast models used in the previous studies did not involve mature osteoblasts. In the current study, we show that calcium waves are propagated by activation of purinergic receptors and via gap junctional communication in mature human osteoblasts. We found that P2X7Rs are involved in the propagation of the mechanically-induced calcium transient among ThOB, as calcium waves were reduced after mechanical stimulation following pharmacological blockade of P2X7R, in contrast to BMSC were only P2Y receptors and gap junctions are involved [31, 32]. This is interesting in relation to a study by Li et al, where they showed that osteoblasts from P2X7R null mice were able to release ATP upon mechanical stimulation by fluid flow, but had an attenuated PGE2 release capacity [23] and therefore showed a significantly reduced anabolic response to mechanical loading.

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Since a gap junction comprises of 2 hemi channels on adjacent cells, the hemi channels formed by connexin units, and connexin43 (Cx43)-mediated gap junctional communication are known to regulate

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bone matrix production in vitro [48] and required for normal bone formation in vivo [49]. We wanted to

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determine whether modulation of P2X7R in vitro alters the regulation of Cx43 gene (GJA1) in human

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osteoblasts. Interestingly, we found that BzATP activation significantly reduced the expression of GJA1 mRNA while antagonism of P2X7R resulted in an almost 2 fold increase in GJA1 mRNA

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expression (Table 1) in ThOB. It is likely that the transcriptional regulation of Cx43 gene compensates

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for the P2X7R mediated ATP secretion or ICW propagation and therefore, suggest that expression of Cx43 hemi channels, at least partly overcomes, any alteration in P2X7R mediated transport dynamics

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in human primary osteoblasts.

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In conclusion, this study demonstrates that only the most mature human osteoblasts derived from

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trabecular bone express functional P2X7Rs. These receptors are partially responsible for the response of osteoblasts to mechanical stimuli and for the communication of mechanically-induced signals to a larger number of cells. Also, P2X7R activation and inhibition can regulate the expression of osteoblast genes and bone formative activity of the cells and may therefore be an interesting novel target for modulating bone homeostasis.

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5. ACKNOWLEDGEMENTS The work was kindly supported by the European Commission under the 7th Framework Programme

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(proposal # 202231) performed as a collaborative project among the members of the ATPBone

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consortium (Copenhagen University, University College London, University of Maastricht, University

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of Ferrara, University of Liverpool, University of Sheffield and Université Libre de Bruxelles), and is a sub study under the main study “Fighting osteoporosis by blocking nucleotides: purinergic signaling in

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bone formation and homeostasis”.

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[49] Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH, Civitelli R. Connexin43 deficiency causes delayed ossification, craniofacial abnormalitites, and osteoblast dysfunction. J Cell Biol 2000;151:931-43.

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Highlights

Cells derived from human trabecular explants express a functional P2X7R.

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These primary osteoblasts have a positive alkaline phosphatase activity.

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They are fully differentiated with the ability to form mineralized nodules.

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P2X7R mediates transcriptional regulation of osteoblastic markers.

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P2X7R is involved in propagation of mechanically-induced intercellular signaling.

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