Tanja Kirchberger

Christelle Moreau

Gerd K Wagner

Ralf Fliegert

Cornelia C Siebrands

Merle Nebel

Frederike Schmid

Angelika Harneit

Francesca Odoardi

Alexander Flügel

Barry V L Potter

Andreas H Guse
email: guse@uke.uni-hamburg.de

8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist

Keywords: cADPR, cADPR analogue, Ca 2+ release, Ca 2+ entry, TRPM2, T cell activation

Ca 2+ ] i , free cytosolic Ca 2+ concentration; cADPR, cyclic adenosine 5'diphosphoribose; ADPRC, ADP-ribosyl cyclase; TBAHP, tetrabutylammonium dihydrogen phosphate; 8-Br-N1-cIDPR, 8-bromo-cyclic inosine 5'-diphosphoribose; 8-Br-IDPR, 8-bromo inosine 5'

INTRODUCTION

Cyclic ADP-ribose (cADPR) is a universal Ca 2+ mobilizing second messenger (reviewed in [START_REF] De Flora | Autocrine and paracrine calcium signaling by the CD38/NAD + /cyclic ADP-ribose system[END_REF][START_REF] Guse | Biochemistry, biology, and pharmacology of cyclic adenosine diphosphoribose (cADPR)[END_REF][START_REF] Guse | Second messenger function and the structure-activity relationship of cyclic adenosine diphosphoribose (cADPR)[END_REF][START_REF] Lee | Multiplicity of Ca 2+ messengers and Ca 2+ stores: a perspective from cyclic ADP-ribose and NAADP[END_REF]). Receptor-mediated formation has been demonstrated for several cell types and multiple ligands/receptors (reviewed in [START_REF] De Flora | Autocrine and paracrine calcium signaling by the CD38/NAD + /cyclic ADP-ribose system[END_REF][START_REF] Guse | Biochemistry, biology, and pharmacology of cyclic adenosine diphosphoribose (cADPR)[END_REF][START_REF] Guse | Second messenger function and the structure-activity relationship of cyclic adenosine diphosphoribose (cADPR)[END_REF][START_REF] Lee | Multiplicity of Ca 2+ messengers and Ca 2+ stores: a perspective from cyclic ADP-ribose and NAADP[END_REF]). Formation of cADPR in eukaryotic cells proceeds via ADP-ribosyl cyclase among which the multifunctional ectoenzyme CD38 is best characterized (reviewed in [START_REF] Lee | Structure and enzymatic functions of human CD38[END_REF]). Originally, cADPR was discovered as endogenous nucleotide to release Ca 2+ from intracellular stores of sea urchin eggs [START_REF] Clapper | Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate[END_REF][START_REF] Lee | Structural determination of a cyclic metabolite of NAD + with intracellular Ca 2+ -mobilizing activity[END_REF]. However, involvement of cADPR in Ca 2+ entry was discovered in 1997 in human T-lymphocytes [START_REF] Guse | Ca 2+ entry induced by cyclic ADP-ribose in intact T-lymphocytes[END_REF].

Ca 2+ entry in T-lymphocytes is a process essential for proliferation, since a continuously elevated level of the free cytosolic and nuclear Ca 2+ concentration ([Ca 2+ ] i ) is necessary for continuous activation of Ca 2+ /calmodulin-dependent protein phosphatase calcineurin [START_REF] Fruman | Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A[END_REF]. The latter dephosphorylates nuclear factor of activated T cells (NF-AT) thereby allowing translocation of NF-AT into the nucleus [10,[START_REF] Liu | Calcineurin-dependent nuclear translocation of a murine transcription factor NFATx: molecular cloning and functional characterization[END_REF].

As mechanism for Ca 2+ entry in T cells, capacitative Ca 2+ entry [12] has long been postulated (reviewed in [13]). With the discovery of key proteins involved, e.g. Orai1/CRACM1 [14,[START_REF] Feske | A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function[END_REF] and Stim 1 [START_REF] Zhang | STIM1 is a Ca 2+ sensor that activates CRAC channels and migrates from the Ca 2+ store to the plasma membrane[END_REF], it became clear that Stim1, by sensing decreasing luminal Ca 2+ concentration upon store depletion, would activate Ca 2+ entry involving Orai1/CRACM1 as channels or as part of a larger channel complex. In T-lymphocytes we have demonstrated a crucial role for cADPR in Ca 2+ entry, since blockade of cADPR action resulted in reduced Ca 2+ signalling, especially in the sustained phase [START_REF] Guse | Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose[END_REF]. Importantly, subsequent signalling events, e.g. CD25 and MHC-II expression and proliferation were likewise inhibited by cADPR antagonism [START_REF] Guse | Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose[END_REF].

Here, we characterize the Ca 2+ entry mechanism activated by the novel cADPR agonist, 8-Br-N1-cIDPR [18]. The advantages of this molecule over natural cADPR are its ability to permeate membranes and its metabolic stability. Indeed, we recently showed that substitution of the amino/imino-group at C6 by an oxo-group, thus converting the bond between N1 and C1´´ at the northern ribose into part of a much more chemically stable, amide system, produces a compound that is also biologically stable. A c c e p t e d M a n u s c r i p t EXPERIMENTAL Drugs and materials 8-Br-NHD and cADPR were obtained from Biolog (Bremen, Germany). 8-Br-N1-cIDPR was either synthesized and purified by some of the authors as described below (see also Fig. 1) or, at later stages, obtained from Biolog (Bremen, Germany). ADP-ribosyl cyclase from Aplysia californica, 8-Br-cADPR and GdCl 3 were purchased from Sigma-Aldrich (München, Germany). Ruthenium red, SKF96365, trichloroacetic acid (TCA), diethyl ether, methanol (LiChrosolv) and trifluoroacetic acid (TFA) were procured from Calbiochem/Merck (Darmstadt, Germany). Rat CD3 antibody, goat antimouse IgM and Lipofectamine 2000 were obtained from Caltag Laboratories/Invitrogen (Carlsbad, USA). Q-Sepharose Fast Flow and G10 Sephadex were obtained from Amersham Bioscience (Freiburg, Germany). FuGENE 6 was procured from Roche Applied Science (Indianapolis, USA). All other chemicals used were of the highest purity grade and purchased from Calbiochem/Merck, Fluka (Buchs, Switzerland) or Sigma-Aldrich. Culture medium reagents were supplied by Sigma-Aldrich, GIBCO-Invitrogen (Auckland, New Zealand) or Biochrom (Berlin, Germany).

Cell culture

Jurkat T-lymphocytes (subclone JMP) were cultured as described previously [START_REF] Guse | Intracellular Ca 2+ pools in Jurkat T-lymphocytes[END_REF] at 37°C in the presence of 5 % CO 2 in RPMI 1640 medium containing Glutamax I and HEPES (25 mM) and supplemented with 7.5 % (v/v) newborn calf serum (NCS), 100 units/ml penicillin and 100 µg/ml streptomycin.

MBP-specific T cell clones were established from lymph node preparations of pre-immunized Lewis rats. Stimulation, expansion and culture of specific rat T cells were conducted under conditions as described [START_REF] Ben-Nun | The rapid isolation of clonable antigenspecific T lymphocyte lines capable of mediating autoimmune encephalomyelitis[END_REF] . For determination of Ca 2+ signalling, frozen rat T cells were thawed und cultured in DMEM with Glutamax I, 4.5 g/l glucose, 100 units/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 10 % (v/v) horse serum, 0.004 % (v/v) 2-mercaptoethanol, 1 % (v/v) non-essential amino acids, 0.04 g/l asparagine at 37 °C in the presence of 10 % CO 2 . Resting state T cells were used in all experiments.

HEK293 cells were cultured at 37°C in the presence of 5 % CO 2 in DMEM medium containing Glutamax I and supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin.

Synthesis and purification of 8-Br-N1-cIDPR

8-Br-N1-cIDPR was synthesized as prevouisly described [START_REF] Wagner | First enzymatic synthesis of an N1-cyclised cADPR (cyclic-ADP ribose) analogue with a hypoxanthine partial structure: discovery of a membrane permeant cADPR agonist[END_REF]. 100 µmol 8-Br-NHD were incubated with 40 µg A.californica ADP-ribosyl cyclase in 25 mM HEPES pH7.4 for 15 h at room temperature under continuous stirring in the dark. ADP-ribosyl cyclase was removed by stepwise centrifugation for 1 to 2 h at 4000 g and 4°C by use of centrifugal filter devices type Centriprep YM-10 (10 kDa MW cutoff; Millipore). Synthesis of 8-Br-N1-cIDPR was confirmed by RP-HPLC. Purification was carried out as previously described [START_REF] Wagner | First enzymatic synthesis of an N1-cyclised cADPR (cyclic-ADP ribose) analogue with a hypoxanthine partial structure: discovery of a membrane permeant cADPR agonist[END_REF] or by another strategy in two steps by anion exchange chromatography and gelfiltration to remove the byproducts 8-Br-IDPR and nicotinamide. For anion exchange chromatography Q-Sepharose FF was washed 4 times with water, 4 times with 500 mM NaCl and 5 times with purified Milli-Q water (Millipore Waters, Eschborn, Germany). 0.5 ml Q-Sepharose FF were packed in plastic filtration tubes (Supelco, Bellefonte, USA) and washed with 10 ml purified water. The aqueous solution of 8-Br-N1-cIDPR was diluted 5-times with purified water to reduce ionic strength and applied to the column in 3 ml aliquots. The column was washed 2 times with 5 ml purified water. Elution of 8-Br-N1-cIDPR was carried out by subsequent addition of 1.5 ml NaCl solution portions of 20, 40, 60, 80, 100, 120, 140,160,180, 200 or 250 mM. The eluates were collected and analyzed by RP-HPLC. Eluates contained only 8-Br-N1-cIDPR (40 to 100 mM NaCl) were combined. Purified 8-Br-N1-cIDPR was desalted by gelfiltration on Sephadex G10. For gelfiltration, 30 g Sephadex G10 was suspended in 100 ml water, heated for 1 h at 90 °C and washed 4 times with water. A suspension with 75 % (w/v) Sephadex G10 was prepared, degassed, packed in a glass column (1.5 x 30 cm) and compacted for 16 h by gravity flow of water. The solution of 8-Br-N1-cIDPR was chromatographed with water as eluent. Eluates were collected in a volume of 1 to 5 ml, analyzed by RP-HPLC, combined and lyophilized. Final characterization of purity and identity was done on RP-HPLC (see Fig. 1).

Determination of membrane permeability of 8-Br-N1-cIDPR

Jurkat T cells (2 to 2.5 x10 A c c e p t e d M a n u s c r i p t HEPES, pH 7.4, resuspended in 1 ml buffer A and kept at room temperature for 10 min. 3 µmol 8-Br-N1-cIDPR were added, cells were incubated for 5 min at RT, placed on an ice-salt bath for 3 min, harvested by centrifugation at 4°C and washed once with 5 ml ice cold buffer A. Determination of 8-Br-N1-cIDPR was performed in a similar way as previously described for endogenous ADPR [START_REF] Gasser | Determination of intracellular concentrations of the TRPM2 agonist ADP-ribose by reversed-phase HPLC[END_REF]. Briefly, cells were lysed in trichloroacetic acid, and cell debris was removed. The samples were divided into identical halves, to one of which 50 nmol 8-Br-N1-cIDPR was added to identifiy the 8-Br-N1-cIDPR peak by HPLC and to calculate the recovery during the extraction. Protein precipitates were removed, and the supernatants were neutralized by extraction with diethyl ether. The samples were then purified by solid phase extraction. For solid phase extraction plastic filtration tubes (Supelco, Bellefonte, USA) were packed with Q-Sepharose FF and were cleaned in place with 150 mM TFA. Columns were equilibrated with 10 mM Tris-HCl, pH 8.0. The cellular extracts were diluted with 10 mM Tris-HCl, pH 8.0 and applied to the columns. The columns were washed first with 10 mM Tris-HCl, pH 8.0 followed by 600 µl 40 mM TFA. For the elution of 8-Br-N1-cIDPR, 1 ml of a 80 mM TFA solution was applied. Samples were neutralized by NaOH, filtered through 0.2 µm filters (Sartorius, Göttingen, Germany) and analyzed by RP-HPLC as described below. As a control the non-brominated dinucleotide nicotinamide hypoxanthine dinucleotide (NHD) was used in parallel.

Reversed Phase-HPLC

RP-HPLC analysis of nucleotides was performed on a 250x4.6mm Multohyp BDS C18-5µ column (CS Chromatographie Service, Langerwehe, Germany) equipped with a 17x4.6mm guard column filled with the same column material or with a 4.0x3.0 guard cartridge containing a C18 (ODS) filter element (Phenomenex, Aschaffenburg, Germany). The separation was performed as described previously [START_REF] Schweitzer | Assay for ADP-ribosyl cyclase by reversephase high-performance liquid chromatography[END_REF] at a flow rate of 1 ml/min with RP-HPLC buffer (20 mM KH 2 PO 4 , 5 mM tetrabutylammonium dihydrogen phosphate, pH 6) containing increasing amounts of methanol. The gradient used for separation was usually (% methanol) 0 min (6.5), 3.5 min (7.5), 5.5 min ( 16), 8 min (25), 18 min (6.5) and 27 min (6.5). For separation of 8-Br-N1-cIDPR in cell extracts the following gradient was used (% methanol) 0 min (6.5), 1.5 (6.5), 3.5 min (7.5), 5.5 min (16), 6.5 min ( 16), 14 min (32.5), 16 min (32.5), 18 min (6.5) and 27 min (6.5). Nucleotides were detected using an UV detector (HPLC detector 432, Kontron Instruments, Neufahrn, Germany) or a DAD (photo diode array detector 1200 series, Agilent technologies) at 250 nm or 260 nm for hypoxanthine or at 270 nm for adenine based nucleotides, respectively . Integration of peaks was performed with the data-acquisition system MT2 from Kontron Instruments or ChemStation from Agilent technologies (Rev. B.02.01). Quantification was performed with external standards.

Transient Transfection of HEK293 cells

A cDNA containing the total ORF of human TRPM2 was amplified from human RNA by one-step RT-PCR using Titan one-Tube RT-PCR system (Roche, Mannheim, Germany) according to the manufacturer's instructions (forward primer including EcoRI site: 5'-ggaattcatggagccctcagccctg-3'; reverse primer including a SalI site: 5'-tactgtcgactcagtagtgagcccc-3'). The resulting amplicon was cloned into the EcoRI and SalI sites of the multiple cloning site of pIRES2-EGFP (Clontech, Heidelberg, Germany). This vector was termed pIRES2-EGFP-TRPM2. For transfection, HEK293 cells were seeded in 35 mm dishes, cultured to a density of 50 to 80 % of confluency and were transfected by addition of transfection mixture consisting of 100 µl OptiMEM, 3 µl FuGENE or 3 µl Lipofectamine 2000 and 1 µg plasmid DNA (pIRES2-EGFP or pIRES2-EGFP-TRPM2) to the cells . For transfection with FuGENE transfection mixture was added directly to the cell culture medium, whereas transfection with Lipofectamine 2000 was carried out in OptiMEM. For Ca 2+ -imaging experiments, transfected cells were seeded in 8-well slides (ibidi, Martinsried, Germany) coated with poly-L-lysine (70-150 kDa) at low cell density.

Ratiometric Ca 2+ imaging

Jurkat T cells were loaded with Fura-2/AM as described [START_REF] Guse | Intracellular Ca 2+ pools in Jurkat T-lymphocytes[END_REF] and kept in the dark at room temperature until use. Primary rat T cells were loaded with 12 µM Fura2/AM and 2 mM probenicid for 30 min at 37°C in the dark and washed twice with buffer A and kept in the dark at 15 -18 °C until use. HEK293 cells were loaded in 8-well slides with 4 µM Fura 2/AM in HEK293 cell media for 30 min at 37°C in the dark and washed twice with buffer A.

For Ca 2+ imaging of Jurkat T cells or rat T cells thin glass coverslips (0.1 mm) were coated first with BSA (5 mg/ml) and subsequently with poly-L-lysine (0.06 -0.1 mg/ml). Silicon grease was used to seal small chamber slides consisting of a rubber O-ring on the glass coverslip. Then, 40 -60 µl buffer A and 40 µl T cell suspension (1 -2 × 10 6 cells/ml) suspended in the same buffer were added into the small chamber. Where the calcium free/calcium re-addtion protocol was used, 0.2 x 10 6 Jurkat T cells were washed twice in nominal Ca 2+ -free buffer A and added to the small chamber. The time span the cells were held under Ca 2+ free conditions was kept as short as possible to avoid emptying of the ER Ca 2+ pool; such emptying due to prolonged exposure to Ca 2+ free conditions resulted in very low or undetectable Ca 2+ signals upon cADPR microinjections in a previous study [START_REF] Guse | Ca 2+ entry induced by cyclic ADP-ribose in intact T-lymphocytes[END_REF]. The loaded coverslip or 8-well slide was mounted on the stage of a fluorescence microscope (Leica DMIRE2). In some experiments Jurkat T cells were preincubated with GdCl 3 (25 µM), SKF 96365 (30 µM) or 8-Br-cADPR (500 µM) in buffer A. In experiments with GdCl 3 Na 2 HPO 4 was not included in buffer A to prevent precipitation of GdPO 4 .

Ratiometric Ca 2+ imaging was performed as described recently [START_REF] Kunerth | Analysis of subcellular calcium signals in T-lymphocytes[END_REF]. We used an Improvision imaging system (Tübingen, Germany) built around the Leica microscope at 40-or 100-fold magnification. Illumination at 340 and 380 nm was carried out using a monochromator system (Polychrom IV, TILL Photonics, Gräfelfing, Germany). Images were taken with a gray-scale CCD-camera (type C 4742-95-12ER; Hamamatsu, Enfield, United Kingdom) operated in 8-bit mode. The spatial resolution was 512 x 640 pixels. Camera exposure times were 12 -30 ms (at 340 nm) and 4 -10 ms (at 380 nm) at a 3:1 ratio. The acquisition rate was adjusted to ~14 ratios per minute. Raw data images were stored on a hard disk and ratio images (340/380 nm) were constructed pixel by pixel. Confocal Ca 2+ images were obtained by off-line deconvolution (no-neighbor algorithm) using the volume deconvolution module of the Openlab software as described recently for 3T3 fibroblasts [START_REF] Bruzzone | Spatio-temporal propagation of Ca 2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors[END_REF]. The deconvoluted images were used to construct ratio images (340/380 nm) pixel by pixel. Finally, ratio values were converted into Ca 2+ concentrations by external calibration [START_REF] Kunerth | Analysis of subcellular calcium signals in T-lymphocytes[END_REF]. Data processing was performed using Openlab software, version 3.0.8 or 1.7.8 (Improvision, Tübingen, Germany).

Microinjection

Microinjections were carried out as described [START_REF] Guse | Ca 2+ entry induced by cyclic ADP-ribose in intact T-lymphocytes[END_REF]. We used an Eppendorf system (transjector type 5171, micromanipulator type 5246 or Femtojet (Eppendorf AG, Hamburg, Germany) with Femtotips I or II as pipettes. 8-Br-N1-cIDPR or cADPR were diluted to their final concentration in intracellular buffer (20 mM HEPES, 110 mM KCl, 10 mM NaCl, pH 7.2), filtered (0.2 µm) and centifuged (15700 g, 10 min, 4°C) before use. Injections were made using the semiautomatic mode of the system with following instrumental settings: injection pressure 60 to 80 hPa, compensatory pressure 25 to 40 hPa, injection time 0.5 sec and velocity of the pipette 700 µm/sec.

Electrophysiology

Membrane currents were recorded in the whole cell configuration of the patch clamp technique [26]. An EPC10 USB patch clamp amplifier was used in conjunction with the PULSE stimulation and data acquisition software (HEKA Elektronik, Lamprecht, Germany). Data were sampled at 5 kHz and compensated for both fast and slow capacity transients. Series resistance was compensated by 70%. All experiments were performed at room temperature with Jurkat T cells attached to tissue culture dishes before the experiment. The pipette solution contained 140 mM KCl, 1 mM MgCl 2 , 8 mM NaCl and 10 mM HEPES adjusted to pH 7.2 with KOH. In some experiments, the pipette solution additionally contained ADPR (30, 100, 300 or 1000 µM), cADPR (30, 100, 300 or 1000 µM), 8-Br-N1-cIDPR (300 µM), ADPR (30 µM) plus either cADPR (5 µM or 100 µM) or 8-Br-N1-cIDPR (300 µM). The external solution contained 140 mM NaCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 5 mM KCl, 10 mM HEPES, and 5 mM glucose adjusted to pH 7.4 with NaOH. The cells were held at -60 mV and current-voltage relations were obtained every 5 s using 250-ms voltage ramps from -85 to -65 mV.

RESULTS

8-Br-N1-cIDPR was prepared by quantitative conversion of 8-Br-NHD to 8-Br-N1-cIDPR using Aplysia californica ADP-ribosyl cyclase (Fig. 1). Comparison of the products after a 15 h incubation period with standards clearly showed that the main product was 8-Br-N1-cIDPR. However, a small portion of 8-Br-NHD was converted to the linear product 8-Br-IDPR (Fig. 1d). This small contamination of 8-Br-IDPR was quantitatively removed by anion exchange chromatography using Sepharose Q fast flow followed by desalting of purified 8-Br-N1-cIDPR on Sephadex 10 fast flow. The final product showed a purity of almost 100% on HPLC (Fig. 1e). Though we had reported earlier in a preliminary communication that 8-Br-N1-cIDPR is membranepermeant, initially the biological activity of 8-Br-N1-cIDPR was assessed in combined microinjection and Ca 2+ imaging experiments to obtain a proper concentration-response relationship without any potential additional effects due to membrane permeancy and/or pumping out of the compound by multiple drug resistance transporters. While microinjection of intracellular buffer into intact Fura2loaded Jurkat T cells had little effect (Fig. 2 a), the natural cADPR showed a robust response when microinjected at 100 µM (Fig. 2 b). It must be noted here that in such microinjection experiments only about 1 to 1.5 % of the cell volume is injected [START_REF] Guse | Ca 2+ entry induced by cyclic ADP-ribose in intact T-lymphocytes[END_REF] resulting in an effective intracellular concentration far below the pipette concentration. At 100 µM 8-Br-N1-cIDPR evoked Ca 2+ signalling only in some cells (Fig. 2 c), whereas at pipette concentrations ≥ 1 mM of 8-Br-N1-cIDPR, robust Ca 2+ reponses were recorded consisting of a rapid and high Ca 2+ peak within tens of seconds followed by a lower, but sustained plateau phase (Fig. 2 d to f). The effect was saturable and showed an EC 50 of about 300 µM (Fig. 2g).

Importantly, the 8-substitution with bromine in 8-Br-N1-cIDPR resulted in a membrane-permeant molecule, as already determined in initial experiments [START_REF] Wagner | First enzymatic synthesis of an N1-cyclised cADPR (cyclic-ADP ribose) analogue with a hypoxanthine partial structure: discovery of a membrane permeant cADPR agonist[END_REF]. To check the membrane permeabilty of 8-Br-N1-cIDPR directly, Jurkat T cells were incubated with 8-Br-N1-cIDPR for 5 min; then, cellular nucleotides were extracted with trichloroacetic acid; and crude cell extracts were purified by solid phase extraction and analysed by RP-HPLC (Fig. 3 a,b) in a similar way as described for ADPR [START_REF] Gasser | Determination of intracellular concentrations of the TRPM2 agonist ADP-ribose by reversed-phase HPLC[END_REF]. In Jurkat T cells incubated with 8-Br-N1-cIDPR a cellular uptake of 0.16 fmol/cell was measured (Fig. 3). In contrast, cells incubated with the non-brominated dinucleotide NHD did not take up this molecule indicating that bromination is a prerequisite for membrane permeancy (data not shown). In separate experiments, we checked that NHD was not metabolized in the time period of the uptake experiments.

Addition of membrane permeant 8-Br-N1-cIDPR to intact Jurkat T cells induced a dose dependent Ca 2+ signal (Fig. 4). At concentrations ≥ 1 mM 8-Br-N1-cIDPR a rapid Ca 2+ response in these cells consisting of a Ca 2+ peak and a small but sustained plateau phase was observed (Fig. 4c), whereas no Ca 2+ signal was mediated by addition of the non brominated analogue N1-cIDPR (Fig. 4b) or buffer (Fig. 4a). Since 8-Br-N1-cIDPR at 1 mM induced robust and significant Ca 2+ signalling, this extracellular concentration was chosen for most further experiments.

To test the effect of 8-Br-N1-cIDPR also on primary T cells, rat myelin-basic protein (MBP)-specific T cells were loaded with Fura2/AM and subjected to Ca 2+ imaging (Fig. 5). Buffer was added as a negative control, while cells were stimulated via the T cell receptor/CD3 complex (anti CD3/IgM) as a positive control (Fig. 5 a,b). Though anti-CD3 antibody alone resulted in a robust Ca 2+ signal, crosslinking of the primary antibody on the surface of the cells further enhanced the stimulation (Fig. 5 b). Addition of 8-Br-N1-cIDPR resulted in Ca 2+ signals with different kinetics and amplitudes with regard to individual cells (Fig. 5 c). When compared to T cell receptor/CD3 stimulation, similar individual differences in kinetics were observed (compare Fig. 5b andc), whereas peak amplitudes were significantly lower for 8-Br-N1-cIDPR (Fig. 5d). This difference is not unexpected since in the first 10 minutes of T cell activation, at least three Ca 2+ release systems (nicotinic acid adenine dinucleotide phosphate (NAADP), D-myo-inositol 1,4,5-trisphosphate (IP 3 ) and cADPR; (reviewed in [START_REF] Guse | Second messenger function and the structure-activity relationship of cyclic adenosine diphosphoribose (cADPR)[END_REF][START_REF] Fliegert | Regulation of calcium signalling by adeninebased second messengers[END_REF]) are involved. However, 8-Br-N1-cIDPR mimicked the second phase of TCR/CD3 mediated Ca 2+ signalling once again underpinning the essential role of cADPR in sustained Ca 2+ signalling in T cells.

cADPR has been shown to act both on intracellular Ca 2+ release via RyR [START_REF] Guse | Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose[END_REF], but also to sensitize TRPM2 cation channel for its ligand ADPR [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF]29]. To analyze the mechanism of the cADPR analogue 8-Br-N1-cIDPR in more detail, a Ca 2+ free/Ca 2+ reintroduction protocol was used to separate potential Ca 2+ release and Ca 2+ entry evoked by 8-Br-N1-cIDPR (Fig. 6). Under Ca 2+ free conditions, a robust Ca 2+ release upon addition of 8-Br-N1-cIDPR was observed (Fig. 6 a,b). Re-addition of extracellular Ca 2+ resulted in a huge Ca 2+ entry overshoot followed by a small sustained plateau phase (Fig. 6 a,b). These data suggest that 8-Br-N1-cIDPR activates both Ca 2+ release and Ca 2+ entry.

Secondly, co-microinjections of 8-Br-N1-cIDPR with ruthenium red (RuRed) were carried out (Fig. 7). RuRed largely reduced the initial Ca 2+ peak (Fig. 7 c,d) indicating that this initial peak mainly depends on RyR.

Taken together, we showed that 8-Br-N1-cIDPR behaved similarly to what we currently know about cADPR. In the next sets of experiments we aimed to analyze in more detail the Ca 2+ entry pathway activated by 8-Br-N1-cIDPR. Thus, we used inhibitors of Ca 2+ entry, e.g. GdCl 3 and SKF 96365 [START_REF] Guse | Ca 2+ entry induced by cyclic ADP-ribose in intact T-lymphocytes[END_REF]30,[START_REF] Kerschbaum | Single-channel recording of a store-operated Ca 2+ channel in Jurkat T lymphocytes[END_REF]. In both cases robust Ca 2+ release was observed under Ca 2+ free conditions, while Ca 2+ entry was inhibited (Fig. 8 c,d,g,h). Interestingly, preincubation with Gd 3+ increased Ca 2+ release induced by 8-Br-N1-cIDPR significantly (Fig. 8 d). A potential explanation for this effect is the sensitivity of the plasma membrane Ca 2+ ATPase towards Gd 3+ [START_REF] Herscher | Pre-steady-state kinetic study of the mechanism of inhibition of the plasma membrane Ca(2+)-ATPase by lanthanum[END_REF]; inhibition of Ca 2+ extrusion in the absence of extracellular Ca 2+ would in fact result in an apparent increase of [Ca 2+ ] i .

In the presence of the cADPR antagonist 8-Br-cADPR, Ca 2+ release was partially, but not significantly inhibited (Fig. 8 k,l). Interestingly, there was full inhibition of Ca 2+ entry under such conditions. Likely, the incomplete inhibition of Ca 2+ release is due to the partial antagonist character of 8-Br-cADPR [START_REF] Zhang | 2'-deoxy cyclic adenosine 5'-diphosphate ribose derivatives: importance of the 2'hydroxyl motif for the antagonistic activity of 8-substituted cADPR derivatives[END_REF]. As discussed above for Ca 2+ entry, at least two mechanisms may be involved: either the diminished Ca 2+ release resulted in inhibition of capacitative Ca 2+ entry via Orai1/CRACM1 channels [START_REF] Kiselyov | Regulation of Ca 2+ -release-activated Ca 2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphatereceptor-deficient DT40 cells[END_REF] or direct activation of Ca 2+ entry by 8-Br-N1-cIDPR was inhibited by 8-Br-cADPR. Since Kolisek et al. [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF] recently showed that cADPR together with ADPR activates the unspecific cation channel TRPM2, we used HEK 293 cells transiently overexpressing TRPM2 or EGFP (as negative control) for further experiments. As positive control, Ca 2+ signalling induced by hydrogen peroxide was used since it was recently shown that hydrogen peroxide increased the open probability of TRPM2 [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF][START_REF] Beck | Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes[END_REF]. These results were confirmed (Fig. 9 a,d). In contrast, statistically significant effects between TRPM2 overexpressing cells or controls were not obtained when 8-Br-N1-cIDPR was used as activator of Ca 2+ signalling up to extracellular concentrations of 10 mM (Fig. 9 b,e,g).

In the next series of experiments direct activation of endogenously expressed TRPM2 in Jurkat T cells was analyzed by patch-clamp recordings in the whole-cell configuration. Infusion of ADPR (300 µM) at a membrane potential of -80 mV evoked a marked inward current (Fig. 10 a) characterized by an almost linear IV-relationship (Fig. 10 b) and a saturable concentration-response relationship with an EC 50 of 133 µM (Fig. 10 c). Infusion of cADPR alone did not activate the TRPM2 current except showing a minor effect at 1 mM cADPR pipette concentration (Fig. 10 c). However, due to the small contamination of 4% ADPR (Fig. 10 e) amounting to 40 µM ADPR (!) in the pipette, it is likely that this ADPR contamination, and not the cADPR in the pipette, induced the current. Comparing the amplitudes evoked by 30 µM ADPR and 1 mM cADPR (containing 40 µM ADPR) further substantiates this conclusion (Fig. 10 c). Infusion of combination of a low concentration of ADPR (30 µM) plus cADPR had a small modulatory effect when 100 µM cADPR was used (Fig. 10 d) indicating a comodulatory role of cADPR on TRPM2 activated by low concentrations of ADPR as described [START_REF] Beck | Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes[END_REF]. However, 8-Br-N1-cIDPR neither activated TRPM2 alone (Fig. 10 c) nor in combination with low ADPR (Fig. 10 d).

DISCUSSION

In this report we demonstrate that the membrane-permeant cADPR agonist 8-Br-N1-cIDPR evokes Ca 2+ signalling in both the T-lymphoma cell line Jurkat and in primary T cells. Ca 2+ signalling induced by 8-Br-N1-cIDPR consisted of both Ca 2+ release and Ca 2+ entry. While Ca 2+ release was sensitive to both the RyR blocker ruthenium red and the cADPR antagonist 8-Br-cADPR, Ca 2+ entry was inhibited by the non-specific Ca 2+ entry blockers Gd 3+ and SKF 96365 as well as by 8-Br-cADPR [10]. Activation of TRPM2 by 8-Br-N1-cIDPR was neither observed in heterologously expressed TRPM2 in HEK cells activated by H 2 O 2 nor in endogenously expressed TRPM2 in Jurkat T cells. Further, 8-Br-N1-cIDPR did not co-activate TRPM2 when infused together with a low concentration of ADPR. Stimulation of T cell Ca 2+ signalling via the T cell receptor/CD3 complex involves the subsequent formation of at least three second messengers. Within a few seconds NAADP is produced peaking around 10 to 20 seconds and returning to the baseline rapidly [START_REF] Gasser | Second messenger function of nicotinic acid adenine dinucleotide phosphate revealed by an improved enzymatic cycling assay[END_REF]. NAADP serves as initial Ca 2+ trigger to "ignite" the Ca 2+ signalling system of T cells (for review see [START_REF] Guse | NAADP: A Universal Ca 2+ Trigger[END_REF]). Next, IP 3 is produced transiently and initiates global Ca 2+ signalling within the first 15 to 20 min of T cell activation [38]. In contrast, cADPR is formed more slowly and continues to release Ca 2+ from internal stores as IP 3 levels fall [START_REF] Guse | Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose[END_REF]. Thus, sustained elevated cADPR levels and sustained Ca 2+ signalling by cADPR are essential components of T cell activation [START_REF] Guse | Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose[END_REF]. Since the intracellular Ca 2+ stores of T cells are low in capacity, sustained Ca 2+ signalling is only possible by recruitment of Ca 2+ entry. In theory, at least two mechanisms for Ca 2+ entry evoked by the sustained high cADPR levels are possible: either capacitative Ca 2+ entry [12] via Orai1/CRACM1 channels [14,[START_REF] Feske | A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function[END_REF] is activated by reduced store loading secondary to cADPR action, or cADPR acts as a co-activator of TRPM2 as described recently [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF]29].

As described for a number of membrane-permeant cADPR agonists recently, e.g. N1-ethoxymethyl-cIDPR [START_REF] Gu | Synthesis and biological evaluation of novel membrane-permeant cyclic ADP-ribose mimics: N1-[(5''-O-phosphorylethoxy)methyl]-5'-O-phosphorylinosine 5',5''-cyclicpyrophosphate (cIDPRE) and 8-substituted derivatives[END_REF] A c c e p t e d M a n u s c r i p t cADPR) [START_REF] Kudoh | Synthesis of stable and cell-type selective analogues of cyclic ADP-ribose, a Ca(2+)-mobilizing second messenger. Structure--activity relationship of the N1-ribose moiety[END_REF], 8-phenyl-cIDPR [START_REF] Moreau | Structural determinants for N1/N7 cyclization of nicotinamide hypoxanthine 5'-dinucleotide (NHD + ) derivatives by ADP-ribosyl cyclase from Aplysia californica: Ca 2+ -mobilizing activity of 8-substituted cyclic inosine 5'diphosphoribose analogues in T-lymphocytes[END_REF], N1-[(5"-O-phosphoryl)ethoxyethyl]-5-O-phosphoryladenosine 5´,5"cyclic pyrophosphate [START_REF] Xu | Synthesis and agonist activity of cyclic ADP-ribose analogues with substitution of the northern ribose by ether or alkane chains[END_REF], 8-Br-N1-cIDPR essentially mimicks the cADPR branch of T cell Ca 2+ signalling. However, for none of these cADPR analogues with agonist properties has the mechanism of Ca 2+ entry been elucidated. Since 8-bromo-nicotinamide hypoxanthine dinucleotide, the synthetic precursor of 8-Br-N1-cIDPR, can be easily synthesized in larger quantities and since 8-Br-N1-cIDPR can then be formed in high yield chemo-enzymatically [START_REF] Wagner | First enzymatic synthesis of an N1-cyclised cADPR (cyclic-ADP ribose) analogue with a hypoxanthine partial structure: discovery of a membrane permeant cADPR agonist[END_REF], we performed this detailed analysis of cADPR-mediated Ca 2+ entry using 8-Br-N1-cIDPR as cADPR agonist. The advantage of this strategy clearly is the precise activation of only the cADPR/RyR branch; the disadvantage is that any signalling crosstalk from other pathways triggered upon T cell activation that may modulate the cADPR/RyR branch is not involved here. TRPM2 channel opening was described to be regulated by ADPR with a crucial role of cADPR as coactivator [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF]29]. Further, co-activatory effects of cADPR towards TRPM2 are sensitive to the cADPR antagonist 8-Br-cADPR [START_REF] Beck | Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes[END_REF]. Since we observed that 8-Br-cADPR effectively inhibits Ca 2+ entry evoked by 8-Br-N1-cIDPR (Fig. 8 k,l), a contribution of TRPM2 appeared possible. To analyze this in more detail, TRPM2 overexpressing HEK 293 cells were challenged with 8-Br-N1-cIDPR; however, no significant differences to control cells were observed, though hydrogen peroxide as positive control evoked huge and robust TRPM2 activation. Moreover, also the direct analysis of TRPM2 currents in patch clamp experiments did not reveal any evidence for activation of TRPM2 by 8-Br-N1-cIDPR. However, the previously published activation of TRPM2 by cADPR [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF][START_REF] Beck | Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes[END_REF] appears to be due to ADPR contamination. This is present in almost all commercially available cADPR preparations. These preparations may contain up to 20% of ADPR (!). The preparation used in our experiments was further purified to a purity of 96%; however, higher purities are very difficult to achieve due to the instability of cADPR in solution and during freeze/thaw cycles. In contrast, a small effect attributable to cADPR was observed when it was co-infused with a low concentration of ADPR. Replacement of cADPR by 8-Br-N1-cIDPR in such co-infusion experiments did not evoke TRPM2 currents suggesting that the coactivating effect of cADPR on TRPM2 cannot be mimicked by 8-Br-N1-cIDPR. Activation of Orai1/CRACM1 channels -though not discovered at that time and thus termed capacitative Ca 2+ entry channels -by cADPR via RyR mediated store depletion was described for DT 40 B lymphoma cells lacking expression of all 3 subtypes of IP 3 receptors [START_REF] Kiselyov | Regulation of Ca 2+ -release-activated Ca 2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphatereceptor-deficient DT40 cells[END_REF]. Since we did not find evidence for involvement of TRPM2, activation of capacitative Ca 2+ entry secondary to cADPR-or 8-Br-N1-cIDPR-mediated store depletion appears the most likely process involved. This conclusion is further supported by knock down of type 3 RyR in T cells resulting in significantly diminished sustained Ca 2+ signalling [START_REF] Schwarzmann | Knock-down of the type 3 ryanodine receptor impairs sustained Ca 2+ signaling via the T cell receptor/CD3 complex[END_REF]. Further, the sensitivity of 8-Br-N1-cIDPR mediated Ca 2+ entry to SKF96365 also is in accordance with activation of capacitative Ca 2+ entry by 8-Br-N1-cIDPR. Interestingly, complete inhibition of Ca 2+ entry was observed upon 8-Br-cADPR preincubation while Ca 2+ release was only partially inhibited. This obvious contradiction may be explained by the non-linear activation of capacitative Ca 2+ entry by increasing store depletion [START_REF] Parekh | The store-operated calcium current I(CRAC): nonlinear activation by InsP3 and dissociation from calcium release[END_REF]. Though this effect was explained partially by metabolism of IP 3 [START_REF] Parekh | The store-operated calcium current I(CRAC): nonlinear activation by InsP3 and dissociation from calcium release[END_REF], it is not unlikely that also the extent of store depletion is not linearly coupled to activation of capacitative Ca 2+ entry. Taken together, we provide a detailed analysis of mode of action of the synthetically easily accessible and membrane-permeant cADPR agonist 8-Br-N1-cIDPR. Since 8-Br-N1-cIDPR did not activate TRPM2, this cADPR analogue appears to be the first agonist with selectivity towards the targets RyR and TRPM2.

Figure 10 Activation of endogenous TRPM2 currents in Jurkat T cells by infusion of ADPR, cADPR and 8-Br-N1-cIDPR

All experiments were carried out with 2 mM extracellular Ca 2+ and with unbuffered intracellular Ca 2+ similar as described in [START_REF] Beck | Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes[END_REF]. Voltage ramps from -85 mV to + 65 mV were applied every 5 s.

Biochemical

  Journal Immediate Publication. Published on 03 Jun 2009 as manuscript BJ20082308 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308

Biochemical

  Journal Immediate Publication. Published on 03 Jun 2009 as manuscript BJ20082308 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 03 Jun 2009 as manuscript BJ20082308 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 03 Jun 2009 as manuscript BJ20082308 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308 A c c e p t e d M a n u s c r i p t

  , N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate [40], 2",3"-dideoxy-didehydro-cyclic ADP-carbocyclic ribose (2",3"-dideoxy-didehydro-Biochemical Journal Immediate Publication. Published on 03 Jun 2009 as manuscript BJ20082308 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308

  Figure 1 Synthesis of 8-Br-N1-cIDPR (a) RP-HPLC of standard 1: 8Br-NHD, 8-Br-N1-cIDPR (for reasons of clarity 8-Br-N1-cIDPR is abbreviated as 8Br-cIDPR in all figures). (b) RP-HPLC of standard 2: nicotinamide (NicAmid), 8Br-NHD and 8Br-IDPR; 8Br-IDPR was synthesized by incubation of 8Br-NHD with NADase of N. crassa. (c, d) 1 mM 8Br-NHD was incubated with 1 µg/µl ADP-ribosyl cyclase of A. californica in 25 mM HEPES pH 7.4 for 15 h at RT. Aliquots were taken at 0 and 15 h and analyzed by RP-HPLC. (e) RP-HPLC of purified 8-Br-N1-cIDPR.

Figure 2 Microinjection of 8 -

 28 Figure 2 Microinjection of 8-Br-N1-cIDPR in intact Jurkat T cells Jurkat T cells were loaded with Fura-2 and subjected to combined Ca 2+ imaging and microinjection. Time points of microinjection are indicated by arrows. Cells were microinjected with intracellular buffer (a; n=21) or 100 µM cADPR (b; n=20) as control and with 0.1 (c; n=6), 1 (d; n=7), 10 (e; n=10) or 100 mM (f; n=5) 8-Br-N1-cIDPR. Concentration-response curve of 8-Br-N1-cIDPR is shown in g. Data are mean ± S.E. (n= 5-21) of single tracings from time points 50 -100 s (Ca 2+ peak); * p < 0.01, ** p < 0.001 (t-test).

Figure 3 Membrane permeability of 8 -

 38 Figure 3 Membrane permeability of 8-Br-N1-cIDPR Jurkat T cells were incubated with 3 mM 8-Br-N1-cIDPR for 5 min at RT. Cellular nucleotides were extracted with trichloroacetic acid. Crude cell extracts were purified by solid phase extraction and analysed by RP-HPLC. Representative RP-HPLC analysis of absorbed 8-Br-N1-cIDPR in Jurkat T cells is shown (a, b black line). The peak of 8-Br-N1-cIDPR is indicated by an arrow. For control, cell extracts of cells not incubated with 8-Br-N1-cIDPR were analysed by HPLC (a, b grey line). Jurkat T cells incubated with 8-Br-N1-cIDPR took up 0.16 fmol/cell with a recovery of about 88%. Data are mean (n=2).

Figure 4 Extracellular addition of 8 -

 48 Figure 4 Extracellular addition of 8-Br-N1-cIDPR evoked Ca 2+ signalling in intact Jurkat T cells Jurkat T cells were loaded with Fura-2 and subjected to Ca 2+ imaging. The time points of addition of buffer (a), 1 mM N1-cIDPR (b) and 1 mM 8-Br-N1-cIDPR (c) are indicated by arrows. Characteristic tracings from a representative experiment are shown (a-c). Concentration-response curve of 8-Br-N1-cIDPR is shown in (d). Data represent mean ± S.E. (n= 21-109) of single tracings from time points 100 to 400 s (8-Br-N1-cIDPR mediated Ca 2+ peak).

Figure 5 Extracellular addition of 8 -

 58 Figure 5Extracellular addition of 8-Br-N1-cIDPR evoked Ca 2+ signalling in primary rat T cells Rat T cells were loaded with Fura-2 and subjected to Ca 2+ imaging. The time points of addition of buffer (a), anti-CD3 followed by a crosslinking IgM (40 µg/ml and 10 µg/ml; b) and 1 mM 8-Br-N1-cIDPR (c) are indicated by arrows. Characteristic tracings from a representative experiment are shown (a-c). Combined data are shown in d; these represent mean ± S.E. (n= 186-241) of single tracings from time points 100 -400 s (Ca 2+ peak). * p < 0.01, ** p < 0.001 (t-test).

Figure 6 8 -

 8 Figure 6 8-Br-N1-cIDPR activated both Ca 2+ release and Ca 2+ entry in intact Jurkat T cells (a, b) Jurkat T cells were loaded with Fura-2 and subjected to Ca 2+ imaging. (a) Cells were kept in a nominally Ca 2+ free buffer in the first part of experiment and 8-Br-N1-cIDPR was added as indicated. Then, CaCl 2 was re-added. (b) Buffer was added instead of 8-Br-N1-cIDPR as control. The time points of addition of buffer, 1 mM 8-Br-N1-cIDPR or 1 mM Ca 2+ are indicated by arrows. Characteristic tracings from a representative experiment are shown (a, b). Combined data are shown in (c); these represent mean ± S.E. (n= 362 to 404) of single tracings from time points 100 to 200 s (8-Br-N1-cIDPR mediated Ca 2+ release-peak) or 300 to 450 s (8-Br-N1-cIDPR mediated Ca 2+ entry-peak). ** p < 0.001 (t-test).

Figure 7 Effect of ruthenium red on 8 -

 78 Figure 7Effect of ruthenium red on 8-Br-N1-cIDPR mediated Ca 2+ signalling in intact Jurkat T cells Jurkat T cells were loaded with Fura-2 and subjected to combined Ca 2+ imaging and microinjections. Time points of microinjection are indicated by arrows. Cells were microinjected with intracellular buffer (a; n=26), 1 mM 8-Br-N1-cIDPR (b; n=21) or mixture of 10 µM ruthenium red and 1 mM 8-Br-N1-cIDPR (c; n=20). Combined data are shown in (d). Data represent mean ± S.E. of single tracings from time points 50 -150 s (Ca 2+ peak). * p < 0.01, ** p < 0.001 (t-test).

Figure 8

 8 Figure 8Effect of Gd 3+ , SKF and 8-Br-cADPR on Ca 2+ release and Ca 2+ entry mediated by 8-Br-N1-cIDPR in intact Jurkat T cells Jurkat T cells were loaded with Fura-2 and subjected to Ca 2+ imaging. (a,e,i) Negative control: cells were kept in a nominal Ca 2+ free buffer in the first part of experiment and buffer was added as indicated. Then, CaCl 2 was readded. (b,f,j) Positive control: 8-Br-N1-cIDPR was added instead of buffer. Cells were preincubated with 10 µM Gd 3+ (d), 30 µM SKF96365 (h) or 500 µM 8-Br-cADPR (l). Time points of addition of buffer, 1 mM 8-Br-N1-cIDPR or 1 mM Ca 2+ are indicated by arrows. Characteristic tracings from a representative experiment are shown (a-c, e-g, i-k). Combined data are shown in d,h,I; these data represent mean values ± S.E. (n= 86 to 138) of single tracings from time points 100 to 200 s (8-Br-N1-cIDPR mediated Ca 2+ release-peak), 300 to 450 s (8-Br-N1-cIDPR mediated Ca 2+ entry-peak).; * p < 0.01, ** p < 0.001 (t-test).

Figure 9 Effect of 8 -

 98 Figure 9 Effect of 8-Br-N1-cIDPR on Ca 2+ signalling in HEK293 cells overexpressing TRPM2 HEK293 cells transfected with pIRES2-EGFP-TRPM2 (TRPM2, a-c) and pIRES2-EGFP (EGFP/ control, d-f) were loaded with Fura-2 and subjected to Ca 2+ imaging. Time points of addition of a maximal concentration of H 2 O 2 (100 or 300 µM; a, d), 1 mM 8-Br-N1-cIDPR (b, e) and buffer (c, f) additions are indicated. Characteristic tracings of a representative experiment are shown (a to f). Concentration-response curve of 8-Br-N1-cIDPR in transfected HEK cells is shown in g (n=6 to 67). Data represent mean ± S.E. of single tracings from time points 100 to 600 s (Ca 2+ peak).

  (a) Representative membrane current over time recorded at -80 mV induced by perfusion with 300 µM ADPR is shown. (b) Representative current-voltage relationship derived from currents evoked by voltage ramps from -85 mV to +65 mV is displayed. (c) Concentration-response relationship for activation of TRPM2 currents by infusion of ADPR (open circle, n=6 to 8), cADPR (open triangle, n=5 to Biochemical Journal Immediate Publication. Published on 03 Jun 2009 as manuscript BJ20082308 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308 A c c e p t e d M a n u s c r i p t 8) and 300 µM 8-Br-N1-cIDPR (black circle, n=6). Data represent mean of maximum current at -80 mV ± S.E.. (d) TRPM2 currents at -80 mV after infusion of 30 µM ADPR (n=8), co-infusion of 5 µM cADPR/ 30 µM ADPR (n=9), 100 µM cADPR/ 30 µM ADPR (n=8) and 300 µM 8-Br-N1-cIDPR/ 30 µM ADPR (n=6). Data represent mean of maximum current at -80 mV ± S.E.. (e) RP-HPLC analysis of cADPR used in the infusion experiments. Biochemical Journal Immediate Publication. Published on 03 Jun 2009 as manuscript BJ20082308 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Biochemical Journal Immediate Publication. Published on 03 Jun 2009 as manuscript BJ20082308 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308 A c c e p t e d M a n u s c r i p t THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308 A c c e p t e d M a n u s c r i p t THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308 A c c e p t e d M a n u s c r i p t Biochemical Journal Immediate Publication. Published on 03 Jun 2009 as manuscript BJ20082308 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308 A c c e p t e d M a n u s c r i p t THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308 A c c e p t e d M a n u s c r i p t

  8 ) were harvested by centrifugation, washed once with buffer A containing 140 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 1 mM CaCl 2 , 1 mM NaH 2 PO 4 , 5.5 mM glucose, 20 mM

	Biochemical Journal Immediate Publication. Published on 03 Jun 2009 as manuscript BJ20082308
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082308

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements. We are grateful to the Deutsche Forschungsgemeinschaft for Grants GU 360/9-1 and 9-2 (to AHG.), the Gemeinnützige Hertie-Stiftung, (grant no. 1.01.1/07/005 to AHG and AF), and the Wellcome Trust for Project Grants 55709 (to BVLP) and 084068 (to BVLP and AHG), VIP support and a Biomedical Research Collaboration Grant 068065 (to BVLP and AHG).


Giuseppina Turturici

Fabiana Geraci

Maria E Candela

Giulio Cossu #

Giovanni Giudice

Gabriella Sconzo

Hsp70 is required for optimal cell proliferation in mouse A6 mesoangioblast stem cells

Keywords: MHC, major histocompatibility complex, HSE, heat shock element, CHBF, constitutive HSE-binding factor, RNAi, RNA interference Hsp70, mouse mesoangioblast, Ku factor, cell doubling time, shRNA

Mouse Hsp70 is preferentially induced by heat or stress stimuli. We previously found that Hsp70 is constitutively expressed in A6 mouse mesoangioblast stem cells, but its possible role in these cells and the control of its basal transcription remained unexplored. Here we report that in the absence of stress, Ku factor is able to bind the heat shock element (HSE) consensus sequence in vitro, and in vivo it is bound to the proximal hsp70 promoter. In addition, we show that constitutive hsp70 transcription depends on the cooperative interaction of different factors such as Sp1 and GAGA binding protein with Ku factor, which binds the HSE consensus sequence. We used mRNA interference assays to select knockdown cell clones. These cells were able to respond to heat stress by producing a large amount of Hsp70, and produced the same amount of Hsp70 as that synthesized by stressed A6 cells. However, severe Hsp70 knockdown cells had a longer duplication time, suggesting that constitutive Hsp70 expression has an effect on the rate of proliferation.

Introduction

Hsp70 (70kDa heat shock protein; synonyms: Hsp68; Hsp72; Hsp70A1; Hspa1b/Hspa1a; heat shock 70kDa protein 1B/1A; Hsp70.1/3; Hsp70-1/3) expression is induced in response to several types of cellular stress in all living organisms. This protein and its cognate form (Hsc70) are involved in several folding processes, such as refolding of aggregated proteins, prevention of aggregation, and folding and assembly of nascent polypeptides. They are also involved in translocation of protein through the intracellular membrane and in the interactions with signal transduction proteins [START_REF] Mayer | Hsp70 chaperones: cellular functions and molecular mechanism[END_REF]. Hsp70 also prevents the disruption by environmental stressors of normal cellular processes involving mitosis, meiosis, or differentiation [START_REF] Sarge | Regulation of hsp expression during rodent spermatogenesis[END_REF][START_REF] Luft | Heat shock factor 1-mediated thermotolerance prevents cell death and results in G2/M cell cycle arrest[END_REF]. Under stress, hsp70 transcriptional regulation involves a highly conserved cis-acting sequence, known as the heat shock element, HSE. HSE is the binding site of the heat shock transcription factor, HSF1 [START_REF] Pelham | A regulatory upstream promoter element in the Drosophila hsp 70 heatshock gene[END_REF][START_REF] Wu | Stress proteins in biology and medicine[END_REF], a positive regulatory factor in the stress response.

However, in a number of cases HSF1 activation is not sufficient for the induction of hsp70 gene expression [START_REF] Mathur | Deficient induction of human hsp70 heat shock gene transcription in Y79 retinoblastoma cells despite activation of heat shock factor 1[END_REF]. Furthermore, it has been observed that some human cell lines constitutively produce Hsp70 in the absence of stress, but no significant HSF1-binding activity was found, suggesting that constitutive Hsp70 expression in these cases is an HSF1-independent process [START_REF] Kingston | Heat-inducible human factor that binds to a human hsp70 promoter[END_REF][START_REF] Morimoto | Cells in stress: transcriptional activation of heat shock genes[END_REF]. In some mammalian cells it has been observed that a constitutive HSE-binding factor (CHBF) binds HSE in the absence of any stress [START_REF] Mosser | Coordinate changes in heat shock element-binding activity and HSP70 gene transcription rates in human cells[END_REF][START_REF] Hensold | DNA binding of heat shock factor to the heat shock element is insufficient for transcriptional activation in murine erythroleukemia cells[END_REF][START_REF] Mosser | In vitro activation of heat shock transcription factor DNA-binding by calcium and biochemical conditions that affect protein conformation[END_REF]. Purification of this factor demonstrated that it corresponds to Ku autoantigen [START_REF] Kim | A constitutive heat shock element-binding factor is immunologically identical to the Ku autoantigen[END_REF][START_REF] Yang | Modulation of thermal induction of hsp70 expression by Ku autoantigen or its individual subunits[END_REF], a heterodimer consisting of 86-kDa (Ku-80) and 70-kDa (Ku-70) polypeptides [START_REF] Mimori | Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies from patients with polymyositis-schleroderma overlap[END_REF]. Ku appears to be involved in many cellular processes such as DNA double-strand break repair, DNA replication, regulation of telomeric termini, and transcription regulation [START_REF] Tuteja | Ku autoantigen: a multifunctional DNA-binding protein[END_REF]. Under some types of stress, the Ku-70 subunit has been demonstrated to regulate hsp70 gene expression [START_REF] Yang | Modulation of thermal induction of hsp70 expression by Ku autoantigen or its individual subunits[END_REF][START_REF] Liu | Regulation of chemical stress-induced hsp70 gene expression in murine L929 cells[END_REF][START_REF] Liu | Dual control of heat shock response: involvement of a constitutive heat shock element-binding factor[END_REF].

Hsp70 overexpression is typical of several types of tumors, and strong evidence suggests that it plays a role in the control of cell cycling and growth. Under unstressed conditions, Hsp70 is expressed in some cases in proliferating cells during the G 1 /S and S phases of the cell cycle [START_REF] Milarski | Expression of human HSP70 during the synthetic phase of the cell cycle[END_REF][START_REF] Taira | Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein[END_REF]. Its role in A c c e p t e d M a n u s c r i p t proliferation has been demonstrated by overexpression and antisense administration assays [START_REF] Suzuki | Modulation of cell growth and mutation induction by introduction of the expression vector of human hsp70 gene[END_REF][START_REF] Wei | Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells[END_REF].

When studying the stress response in mouse A6 mesoangioblasts, vessel-derived stem/progenitor cells that can differentiate into different cell types of solid mesoderm such as muscle and bone [START_REF] Minasi | The mesoangioblast: a multipotent, self-renewing cell that originates from the aorta and differentiates into most mesodermal tissues[END_REF][START_REF] Cossu | Mesoangioblasts -vascular progenitors for extravascular mesodermal tissues[END_REF], we found that these stem cells constitutively synthesize inducible Hsp70 [START_REF] Geraci | Stress response in mesoangioblast stem cells[END_REF]. In the mouse, hsp70.1 and hsp70.3 are the major stress-inducible genes. They are located in the major histocompatibility complex (MHC) region of mouse chromosome 17 [START_REF] Hunt | Chromosomal localization of five murine HSP70 gene family members: Hsp70-1[END_REF][START_REF] Snoek | Three Hsp70 genes are located in the C4-H-2D region: possible candidates for the Orch-1 locus[END_REF], and encode proteins differing at only two amino acid residues, though they have completely different sequences in the 3' untranslated region (UTR) [START_REF] Dix | Inhibition of hsp70-1 and hsp70-3 expression disrupts preimplantation embryogenesis and heightens embryo sensitivity to arsenic[END_REF].

We also observed that in A6 cells, there are two classes of HSE-binding factors: one is constitutive, but the other is inducible by stress. We demonstrated that HSF1 is responsible for the stress response, but neither HSF1 nor HSF2 are involved in the basal HSE complex formation, which involves CHBF [START_REF] Geraci | Stress response in mesoangioblast stem cells[END_REF].

In the present study we investigated whether Ku-70 is involved in hsp70 basal transcription. We found using chromatin immunoprecipitation (ChIP) analysis that Ku factor is localized on the hsp70 proximal promoter. Our evidence also suggests that Ku factor has a prominent role in constitutive hsp70 gene transcription. Furthermore, to understand the possible role of Hsp70 constitutive expression in mesoangioblasts, we carried out stable RNA interference (RNAi) of hsp70, and demonstrated that a reduction (~50%) in Hsp70 level impairs cell proliferation, causing an increase in cell doubling time.

Experimental Cell culture, low oxygen tension, and heat shock treatment

Mouse A6 stem cells were grown on plates treated with collagen I in Dulbecco's modified Eagle medium (DMEM) (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum in a humidified 5% CO 2 atmosphere at 37°C. For experiments with lower oxygen tension, cells were grown as previously described [START_REF] Wright | Inexpensive low-oxygen incubators[END_REF]. For heat shock, plates were sealed with Parafilm and immersed for 1 h at 42°C in a water bath. Protein extracts were prepared as previously described [START_REF] Geraci | Stress response in mesoangioblast stem cells[END_REF]. Protein concentrations of extracts were determined using the Bradford microassay method (Sigma, St. Louis, MO) with bovine serum albumin as a standard.

Preparation of protein extracts

Western blot (immunoblot) analysis

Whole-cell extracts were subjected to 10% SDS-PAGE and transferred to an ECL-Hybond membrane (Amersham, Pittsburgh, PA) using a mini-electroblot (Biorad, Hercules, CA). After blocking for 2 h in 5% nonfat dry milk in TBST buffer (10 mM Tris-HCl, pH 7.5; 150 mM NaCl; and 0.2% Tween-20), the membranes were incubated overnight with rabbit polyclonal anti-Hsp70 (1:10,000 dilution; Calbiochem, San Diego, CA), mouse anti-actin (1:800 dilution; Neomarkers, Fremont, CA), rabbit anti-Ku-70 (1:200 dilution, Santa Cruz, Santa Cruz, CA). Alkaline phosphatase conjugated anti-rabbit and anti-mouse (1:5,000 dilution; Promega, Madison, WI) were used as secondary antibodies.

Gel mobility shift assay

Whole-cell extracts containing 45 µg of protein were incubated with a double-stranded -32 P-labeled HSE oligonucleotide (5'-GGGGTCAGAATATTCTAGAATATTCTAGAATGGGTCAGG-3'),

GAGA

(5'-GATCAGAGAGAGAGAGAGAAAAGAGAGAG-3') or GC (5'-TTCGATCGGGGCGGGGCGAG-3') in 30 µl of a solution containing 10 mM HEPES, pH 7.9; 10 mM KCl; 50 mM NaCl; 1.5 mM MgCl 2 ; and 10% glycerol. The samples were incubated at 25°C for 20 min. The HSE-containing complexes were analyzed on 4% non-denaturing polyacrylamide gels. For antibody perturbation experiments, two concentrations (1 and 3 g) of Ku antibody (AbD Serotec, Raleigh, NC) were incubated with the reaction mixture for 1 h at 25°C.

beta-galactosidase and chloramphenicol acetyltransferase (CAT) assays

A6 cells were cotransfected with phsplacZ [START_REF] Bevilacqua | Developmental action of an episomic hsp70 gene promoter in two-cell mouse embryos by transcription factor Sp1[END_REF] and with pCAT-control vector (Promega) into six-well plates. In total, 2 g of each plasmid was used for the transfection. Where indicated, phsplacZ with a mutation in the HSE, GAGA or GC consensus sequence was used. Plasmids were transfected into the

Flow cytometry

Cells were collected and washed with PBS. The pellets were treated with 0.1% C 6 H 5 Na 3 O 7 , 0.1% NP-40, 50 µg/ml propidium iodide, and 0.06 mg/ml RNase A for 4 h at room temperature in the dark. The cells were analyzed on a BD FACSCanto (Franklin Lakes, NJ) using FACSDiva Software. Analysis was performed using Modfit 3.2 software.

Proliferation assay

Cells were plated at a concentration of 4,000 cells/100 µl in 96-well multiwell plates. At 24, 48, and 72 h after plating, cells were incubated for 4 h at 37°C with 10 µM BrdU, and the assay was performed

Cell duplication time

To calculate cell duplication time we used Equation 1 below:

lnC lnC' t t' ln2
With C' = concentration at time t' (final); and C = concentration at time t (beginning). Cell concentration was calculated in the exponential growth phase.

Statistical analysis

Quantitative data are represented as the mean ± S.E.M., or the mean ± S.D. of three experiments. The statistical analyses were performed with GraphPad (InStat, Inc., San Mateo, CA) software (version 3.00) using Student´s t-test for two-group comparisons. Densitometry analysis was performed using ImageJ open-source software. Results were normalized to actin expression levels.

Results and discussion

A6 stem cells synthesize Hsp70 in low oxygen tension culture conditions

We previously found that A6 cells, a clone of mouse mesoangioblasts [START_REF] Minasi | The mesoangioblast: a multipotent, self-renewing cell that originates from the aorta and differentiates into most mesodermal tissues[END_REF], constitutively synthesize the inducible form of Hsp70 [START_REF] Geraci | Stress response in mesoangioblast stem cells[END_REF]. Immunofluorescence studies have also shown that Hsp70 and Hsc70 localize differently inside the cell; Hsp70 primarily associates with microfilaments and the cell cortex [START_REF] Turturici | Hsp70 localizes differently from chaperone Hsc70 in mouse mesoangioblasts under physiological growth conditions[END_REF].

Before investigating constitutive hsp70 gene regulation and a possible role for this protein, we tested whether constitutive synthesis of Hsp70 was due to hyperoxic stress. A6 cells were cultured in low oxygen tension, following the procedure described by Wright and Shay [START_REF] Wright | Inexpensive low-oxygen incubators[END_REF]. Cells cultured under unstressed laboratory conditions are exposed to a hyperoxic environment, because oxygen tension in A c c e p t e d M a n u s c r i p t most mammalian tissues ranges from 1% to 6%. Immunoblot assays of cell lysates demonstrated that constitutive Hsp70 synthesis in A6 cells occurs in low oxygen tension in the same amount as well as under 20% oxygen (Fig. 1, lane 2 and 1 respectively). This result indicates that high oxygen tension is not involved in Hsp70 basal expression and confirms our previous findings.

CHBF/Ku factor binds the HSE consensus sequence in vitro

Relatively little is known about the regulation of hsp70 genes at the transcriptional level in basal growth conditions. In A6 cells, we previously detected two distinct HSE-protein binding complexes using biochemical analysis: one, HSF1, was inducible by stress, but the other was constitutive, and supershift analysis showed that neither HSF1 nor HSF2 were involved in this HSE-complex formation [START_REF] Geraci | Stress response in mesoangioblast stem cells[END_REF]. We therefore hypothesized that in A6 stem cells, constitutive expression of Hsp70 might be due to CHBF, as previously described both in HeLa cells and in rat cells [START_REF] Mosser | Coordinate changes in heat shock element-binding activity and HSP70 gene transcription rates in human cells[END_REF][START_REF] Liu | Dual control of heat shock response: involvement of a constitutive heat shock element-binding factor[END_REF]. To test whether Ku might be the constitutive HSE binding factor, both in vitro and in vivo experiments were performed. First, the presence of Ku autoantigen in A6 cells was demonstrated by immunoblot assays with antibody against Ku-70 (Fig. 2A). Furthermore, DNA-binding perturbation assays with 32 P-labeled HSE-oligonucleotide and increasing amounts of anti-Ku-70 antibody were performed to test Ku DNA-binding ability. As the antibody interferes with the complex formation in a dose dependent manner (Fig. 2B lanes 3, 4), it is possible to conclude that Ku was the factor responsible for the constitutive DNA binding activity. The specificity of the complex was previously demonstrated by competition assays with an excess of the unlabeled HSE oligonucleotide [START_REF] Geraci | Stress response in mesoangioblast stem cells[END_REF]. These results indicated that Ku factor is present in A6 cells and is also able to bind the HSE consensus in vitro to form a constitutive complex, confirming our previous findings.

CHBF/Ku factor binds the hsp70 promoter in vivo

The in vivo relevance of the observed Ku binding activity was analyzed in the context of chromatin in A6 cells by chromatin immunoprecipitation (ChIP) of endogenous Ku. ChIP can directly detect the association of any protein with its in vivo binding site. Anti-Ku antibodies efficiently and specifically

CHBF/Ku is involved in hsp70 promoter regulation under basal conditions

We investigated whether transcription factors other than Ku are bound to the hsp70 gene promoter under non-stress conditions. The hsp70 proximal promoter contains, near the HSEs, two consensus sequences, for GAGA and the CG box. These two sequences are known to be involved in the basal transcription of many genes. DNA-binding assays showed that both elements form in vitro DNAprotein complexes and their specificity was demonstrated by competition assays (Fig. 3A and3B, respectively). To gain insight into the regulation of hsp70 expression, we analyzed the basal activity of the proximal promoter of the hsp70 gene using gene reporter assays. To this aim, A6 cells were transfected with a plasmid containing the beta-galactosidase reporter gene under the control of the hsp70 gene promoter with point mutations in the GAGA or GC consensus sequence. Beta-galactosidase activity was measured and transfection efficiency was monitored by co-transfection with a pCATcontrol vector. The beta-galactosidase values are reported in Fig. 4B. MutGCphsplacZ and mutGAGAphsplacZ showed lower basal-level activity of 65.7% and 75.0%, respectively, compared to the control vector phsplacZ. This result indicates partial involvement of these two factors in the A c c e p t e d M a n u s c r i p t regulation of the hsp70 gene promoter, which drives transcription of the reporter gene. We had previously found that transcription of the reporter gene under control of the hsp70 promoter with the HSE deleted ( HSEphsplacZ) dropped to 52% with respect to that of the phsplacZ control [START_REF] Geraci | Stress response in mesoangioblast stem cells[END_REF].

Together, these experiments show that the deletion of the HSE consensus sequence decreases the expression of the reporter gene much more than mutation of the GAGA and GC consensus sequences.

Previous studies of Hsp70 transcription under stress (i.e., IPTG, arsenite, sodium, salicylate) indicated that high HSF1 DNA-binding activity is sometimes insufficient for Hsp70 mRNA synthesis, and that dissociation of the HSE-Ku complex is necessary [START_REF] Hensold | DNA binding of heat shock factor to the heat shock element is insufficient for transcriptional activation in murine erythroleukemia cells[END_REF][START_REF] Liu | Regulation of chemical stress-induced hsp70 gene expression in murine L929 cells[END_REF][START_REF] Liu | Dual control of heat shock response: involvement of a constitutive heat shock element-binding factor[END_REF]. Based on the results presented here and our previous data [START_REF] Geraci | Stress response in mesoangioblast stem cells[END_REF], we suggest that CHBF/Ku factor is involved in basal transcription of hsp70 in cooperation with GC and GAGA box binding factors in the absence of stress.

hsp70 RNA interference assay (RNAi) and isolation of knockdown clones

To identify a possible role for basally expressed Hsp70, we knocked down both hsp70 mRNAs and studied the effects of silencing on A6 stem cell physiology. To choose between transient or stable RNA interference (RNAi) methods, we first studied the turnover of Hsp70 protein. After 1 h of shock at 42°C, A6 cells were labeled with 35 S-methionine, then chased with unlabeled methionine. Total cell lysates were prepared 3 h, 24 h, and 48 h after heat treatment. The twin Hsp70 proteins differ for only two amino acid residues, and they co-migrate as a single band on an electrophoresis gel [START_REF] Dix | Inhibition of hsp70-1 and hsp70-3 expression disrupts preimplantation embryogenesis and heightens embryo sensitivity to arsenic[END_REF]. The results of autoradiography indicated that the Hsp70 band was still evident 48 h after chase (Fig. 5 lane 3), showing that turnover of the protein is slow. For this reason, we used a vector containing a specific RNAi sequence targeting both hsp70.1/3 mRNAs for stable RNA silencing. A6 cells were transfected with a hsp70 shRNA vector or with a mock vector as negative control. Stable integrants were selected using puromycin 72 hours after the transfection, and after 15 days in the selective medium, resistant pools of mock-transfected cells and hsp70 shRNA (NM) cells were obtained. The level of Hsp70 protein of each cell clone was analyzed by western blot analysis and quantified using densitometric analysis (Fig. 6). The Hsp70 protein level in hsp70 knockdown cell clones was decreased to 16-50% that of A6 control cells. Negligible Hsp70 reduction was observed in mock-transfected cell clones (SH) (Fig. 6). Levels of hsp70.1 and hsp70.3 mRNAs of knockdown, mock-transfected, and A6 control cell clones were investigated using semi-quantitative RT-PCR. The results indicated a reduction of both hsp70.1 and hsp70.3 mRNAs in all knockdown cell clones although the reduction was variable. No reduction in hsp70 mRNA was observed in mock-transfected cell clones (data not shown).

Heat shock response in hsp70 knockdown A6 cells

We investigated whether a decreased level of Hsp70 in NM3 cells affects normal cell physiology. Hsp70 knockdown clones did not show alterations in cell morphology under unstressed growth conditions (data not shown). Moreover, to examine the influence of hsp70 knockdown on the ability of NM clones to respond to heat stress, we compared the Hsp70 level in NM3 and A6 cells before and after heat stress using western blot analysis. A6 and NM3 cells were subjected to hyperthermic treatment (42°C for 1 h) and then allowed to recover at 37°C. During recovery, NM3 cells synthesized a large amount of the protein, 8.6-fold greater than the basal level using densitometric analysis (Fig. 7). A 4.6-fold increase in protein level in stressed compared to unstressed A6 cells was observed (Fig. 7).

The result showed that the total amount of Hsp70 after heat stress was almost the same in A6 and NM3 cells. A similar result was obtained for all other NM cell clones subjected to heat stress (data not shown). Thus, NM cell clones, when subjected to stress, produce an amount of Hsp70 necessary to protect themselves from injury, suggesting tight regulation of Hsp70 expression under unstressed and stress conditions.

Decreased cell proliferation and longer doubling time in hsp70 knockdown A6 cells

Hsp70 function has been mainly ascribed to the protection of the cells, but there is growing evidence for its involvement in cell proliferation [START_REF] Barnes | Expression of inducible Hsp70 enhances the proliferation of MCF-7 breast cancer cells and protects against the cytotoxic effects of hyperthermia[END_REF]. For this reason we tested whether the suppression of Hsp70 elicits cell proliferation. The effect of hsp70 silencing on the cell cycle was determined using cell growth curves. Fig. 8 shows results for A6 cells, along with NM3, and SH2 cells representing knockdown and mock-transfected clones, respectively. A6 and SH2 cells expressing the same level of A6 cells and SH2 and NM3 clones showed similar cell-cycle phase distribution profiles as reported in supplementary data (Fig. S1 and table S1). The same result was obtained for all other NM cell clones (data not shown). This finding indicates that knockdown of hsp70 did not alter cell-cycle phase distribution profiles. We also measured cell duplication time and found that stable suppression of Hsp70 results in a marked decrease in the cell growth rate. A6 cells have a doubling time of 16 hours, as previously described for mouse mesoangioblasts [START_REF] Minasi | The mesoangioblast: a multipotent, self-renewing cell that originates from the aorta and differentiates into most mesodermal tissues[END_REF], but NM3, NM15 and NM17 cells exhibited a longer doubling time of about 25 hours, and mock-transfected shRNA cells did not exhibit a detectable change in growth rate. NM clones 3, 15, and 17 showed ~50% reduction in Hsp70 level. In contrast, NM clones 1, 5, and 8, which had levels of Hsp70 reduced by only 16% and 25%, showed a doubling time ranging between 16 and 17.7 hours, similar to that of A6 cells (Table I). The differences in population doubling time cannot be attributed to cell-cycle differences, as demonstrated by FACS analysis (data not shown). Flow cytometry can be used to estimate the length of G 1 , S and G 2 phases when certain conditions are met: the mean generation time for a cell population is known, the fraction of proliferating cells is high, the frequency of cell loss is low, and cells are reasonably homogeneous with respect to their cell cycle kinetics [START_REF] Merrill | Cell synchronization[END_REF]. The calculated cell-doubling times and cell-cycle analysis were used to provide an estimate of the duration of G 0 /G 1, S, and G 2 /M cell-cycle phases in A6, NM3, and SH2 cells. NM3 and SH2 are representative of ~50% knockdown clones and mock-transfected clones, respectively. Data presented in Table II indicates that cells expressing low levels of Hsp70 have a longer duration of the G 0 /G 1 phase compared to cells expressing a basal level of Hsp70 (14.9 vs. Together, these data indicate that a relevant reduction in Hsp70 level negatively influences cell proliferation, while a lower reduction has no effect, demonstrating that there is a correlation between the amount of basally expressed Hsp70 and cell proliferation.

hours).

Other previous work supports our data, confirming that Hsp70 is involved in both mitosis and meiosis.

Consistent with such a function, Hunt and co-workers [START_REF] Hunt | Genomic instability and enhanced radiosensitivity in Hsp70.1-and Hsp70.3-deficient mice[END_REF] demonstrated that the doubling time of MEF Hsp70.1/3 -/-cells obtained from knockout mice is longer than that of Hsp70.1/3 +/+ MEFs. MCF-7 breast cancer cells overexpressing Hsp70 [START_REF] Barnes | Expression of inducible Hsp70 enhances the proliferation of MCF-7 breast cancer cells and protects against the cytotoxic effects of hyperthermia[END_REF] have a shorter doubling time, providing further evidence of involvement of Hsp70 in the cell cycle. A role in meiosis was also demonstrated for Hsp70.2, a member of the Hsp70 family expressed specifically in mouse spermatogenic cells. Hsp70.2, expressed during the meiotic phase of spermatogenesis, is required for desynapsis of the synaptonemal complex and the completion of meiosis I in spermatocytes [START_REF] Dix | HSP70-2 is required for desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes[END_REF][START_REF] Eddy | Role of heat shock protein HSP70-2 in spermatogenesis[END_REF]. Constitutive Hsp70 involvement in mitosis and embryo cleavage was also demonstrated in P. lividus embryos, a model of embryonic development.

Soon after fertilization and during early cleavage, basally expressed Hsp70 is located on the mitotic apparatus, and blockage of its function impairs mitosis and embryo development [36,37].

In conclusion, we suggest that a basal level of Hsp70 expression may be required for optimal cell proliferation of mesoangioblast cells, which have a short doubling time.

Conclusions

In A6 stem cells, basally expressed Ku factor binds the HSE consensus sequence in the hsp70 proximal promoter and is positively involved in its basal transcription in the absence of stress. Hsp70 expressed under basal growth conditions influences the growth rate of A6 stem cells. A 50% reduction in the level of Hsp70 causes an increase in cell doubling time and prolongs the G 0 /G 1 phase of the cell cycle.

Biochemical

  Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309

Biochemical

  Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309 A c c e p t e d M a n u s c r i p t

  bring the final volume to 500 µl. To increase the volume, IP dilution buffer (20 mM Tris, 150 mM NaCl, 2 mM Na-EDTA, 0.01% SDS, and protease inhibitors, pH 8.1) was added, bringing the volume to 1 ml. Sonication was performed on ice with three 10-s pulses at 100% of maximum power with a VC 600 series Vibra Cell (Sonics Materials, Newtown, CT). Following sonication, samples were centrifuged (10,000 × g; 4°C, 10 min). Samples were precleared by overnight incubation at 4°C by addition of 100 µl of protein G-Sepharose to bring the final volume to 1 ml. To immunoprecipitate protein-DNA complexes, samples were incubated overnight at 4°C with 2 µg of antibody per 500 µg of sample. Antibodies used were: anti-Ku-70 (Santa Cruz), anti-HSF1 (AbCam, Cambridge, MA), and anti-Hsp27 (Neomarkers). Immune complexes were collected by incubation with 30 µl of protein G-Sepharose. Protein G-Sepharose was pre-treated for 2 days at 4°C with 1 mg/ml of BSA and 130 µg/µl of salmon sperm DNA. Following antibody binding and protein G-Sepharose absorption, protein G-Sepharose pellets were washed twice with buffer 1 (20 mM Tris; 50 mM NaCl; 2 mM EDTA; 0.1% SDS; and 1% Triton X-100, pH 8.1), once with buffer 2 (10 mM Tris; 0.25 M LiCl; 1 mM EDTA; 1% NP-40; and 1% deoxycholate, pH 8.1), and twice with TE (0.01 M Tris; and 1 mM EDTA, pH 8.0). Immune complexes were eluted twice at room temperature with 75 µl of elution buffer (0.1 M NaHCO 3 , and 1% SDS). RNase (3 µg/ml) and 0.3 M NaCl were added, and crosslinking was reversed by incubation for 4 h at 65°C. Samples were digested with 0.2 mg/ml proteinase K for 12 h at 45°C. Glycogen (5 µg) was added, and immunoprecipitated DNA was purified by extraction once each with phenol:chloroform and chloroform, followed by ethanol precipitation. Purified DNA was resuspended in 20 µl of water. Samples were analyzed by PCR. Oligonucleotides used for the amplification were sense 5'-TACTCTCTAGCGAACCCCAG-3', and antisense 5'-GACAAGCGAAGACAAGAGAA-3'. PCR reactions contained 20 µl of immunoprecipitated DNA, 0.5 µM of hsp70.1 and hsp70.3 primers, 0.5 U Taq DNA polymerase (Eppendorf, Westbury, NY), 0.2 mM dNTP mixture, and 1X Taq buffer (Eppendorf), in a total volume of 50 µl. Twenty-eight cycles of amplification were used.Metabolic labeling with 35 S-methionineBiochemical Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309A c c e p t e d M a n u s c r i p t Cells were incubated in methionine-free medium (Sigma) containing 10% fetal bovine serum and exposed to stress treatment. After the treatment, 20 µCi/ml of 35 S-L-methionine (1,000 Ci/mmol, Amersham) was added and cells were labeled for 3 h and then chased with unlabeled L-methionine.Samples were collected 0 h, 24 h, and 48 h after labeling. The incorporation of35 S-L-methionine into proteins was determined by trichloroacetic acid precipitation. Whole-cell extracts containing 30 g of protein were analyzed using 10% SDS-PAGE and fluorography.hsp70.1/3 RNA interference (RNAi)MISSION shRNA lentiviral vectors (Sigma), consisting of short hairpin sequences cloned into a lentiviral vector , were used. One shRNA specific for both hsp70.1 and hsp70.3 (phsp70.1/3) was chosen from an RNAi lentiviral library. The target sequence chosen is called 599, and the sequence of were transfected with vector 599 and selected for resistance to puromycin, and 20 clones with different levels of knockdown (between 15 and 55%) were obtained. For all subsequent experiments, we used clone A6-NM3, which exhibited 55% knockdown of the Hsp70 protein.

  Biochemical Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309A c c e p t e d M a n u s c r i p t using a cell proliferation ELISA kit (Roche), according to the manufacturer's instructions. The reaction products were quantified by measuring absorbance at 450 nm using a scanning multiwell spectrophotometer (ELISA reader; Celbio, Milan, Italy).

  Biochemical Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309

Biochemical

  Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309A c c e p t e d M a n u s c r i p t immunoprecipitated the proximal hsp70 promoter (Fig.2C lane 1), indicating that there is a stable association between the endogenous Ku factor and the hsp70 promoter in vivo. To confirm the specificity of the binding of Ku to the hsp70 promoter under unstressed growth conditions, we performed ChIP assays after heat shock. No DNA amplification was observed in samples treated with anti-Ku antibody (Fig.2C lane 2). On the contrary, as expected, DNA amplification was observed using anti-HSF1 antibodies under stress conditions, but no amplification occurred under unstressed growth conditions (Fig.2Clanes 4 and 3, respectively). Control sample treated with anti-Hsp27 or without anti-Ku antibody did not show DNA amplification (Fig.2Clanes 5 and 7, respectively). This result indicates that in A6 cells under physiological cell growth conditions, Ku is localized on the proximal promoter of the hsp70 gene, suggesting its involvement in regulation of constitutive hsp70 gene transcription.

Biochemical

  Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309

Biochemical

  Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309 A c c e p t e d M a n u s c r i p t Hsp70 exhibited similar growth curves, whereas NM3 cells displayed a reduced growth rate. The effect of hsp70 silencing on the cell cycle was determined by fluorescence-activated cell sorting (FACS) analysis of cell cycle phase distribution after propidium iodide staining of exponentially growing cells.

Biochemical

  Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309 A c c e p t e d M a n u s c r i p t

  This work was supported by a grant from MURST (the Italian Ministry of University and Scientific and Technological Research), a BdS Foundation grant, and a FSE PON 2006 grant to T.G. 36. Sconzo, G., Palla, F., Agueli, C., Spinelli, G., Giudice, G., Cascino, D. and Geraci, F. (1999) Constitutive hsp70 is essential to mitosis during early cleavage of Paracentrotus lividus embryos: the blockage of constitutive hsp70 impairs mitosis. Biochem Biophys Res Commun. 260, 143-149. 37. Agueli, C., Geraci, F., Giudice, G., Chimenti, L., Cascino, D. and Sconzo, G. (2001) A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed. Biochem J. 360, 413-419.

Figure 1

 1 Figure 1 Hsp70 synthesis under low oxygen tension

Figure 2

 2 Figure 2 Presence of Ku factor in A6 cells and in vivo and in vitro DNA binding

Figure 3

 3 Figure 3 GAGA and SP1 DNA-binding activity in vitro

Figure 4

 4 Figure 4 Basal activities of complete and mutated hsp70 proximal promoter

Figure 5

 5 Figure 5 Hsp70 half-life in A6 cells

Figure 6

 6 Figure 6 Use of shRNA to knock down Hsp70 protein expression

Figure 7

 7 Figure 7 Hsp70 level in A6 and NM3 cells after heat shock

Figure 8 4 )

 84 Figure 8 Effect of hsp70 knockdown on cell growth

Table II

 II Estimated duration (in hours) of G 0 /G 1, S, and G 2 /M, phases a

	Clone	G 0 /G 1	S	G 2 /M
	A6	7.5	6.9	1.6
	SH2	7.9	7.1	1.9
	NM3	14.9	7.2	2.8

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© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements

We thank Franco Mangia and Arturo Bevilacqua for the kind gift of the plasmid phsplacZ and its mutated forms. Biochemical Journal Immediate Publication. Published on 01 May 2009 as manuscript BJ20082309 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082309 A c c e p t e d M a n u s c r i p t