Life Sciences 98 (2014) 88–95

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Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro Martin Roesslein a,⁎, Christian Froehlich a, Frank Jans b, Tobias Piegeler c,d, Ulrich Goebel a, Torsten Loop a a

Dept. of Anaesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany Dept. of Anaesthesiology and Critical Care Medicine, Ziekenhuis Oost-Limburg, Genk and Biomedical Research Institute, UHasselt, Diepenbeek, Belgium Institute of Anaesthesiology, University Hospital Zurich, Switzerland d Dept. of Anesthesiology, University of Illinois at Chicago, USA b c

a r t i c l e

i n f o

Article history: Received 5 September 2013 Accepted 7 January 2014 Chemical compound studied in this article: Dobutamine hydrochloride (PubChem CID: 65324) Keywords: Dobutamine Apoptosis Protection Heat shock response Heat shock protein 70

a b s t r a c t Aims: Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response. Main methods: Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile. Key findings: Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20–32] % vs. 12 [9–15] % for dobutamine 0.1 mM and 7 [5–12] % for dobutamine 0.5 mM, p b 0.001), cleavage of procaspase-3 as well as caspase-3-like activity (0.46 [0.40–0.48] vs. 0.32 [0.27–0.39] for Dobutamine 0.1 mM and 0.20 [0.19–0.23] for Dobutamine 0.5 mM, p b 0.01). Dobutamine induced DNA-binding of HSF-1 and mRNAexpression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32–36] % vs. 18 [16–24] %) and caspase-3-like activity (0.21 [0.19–0.23] vs. 0.16 [0.13–0.17], p b 0.05). Significance: Dobutamine protects from apoptotic cell death via the induction of Hsp70. © 2014 Elsevier Inc. All rights reserved.

Introduction Dobutamine is a synthetic catecholamine used as an inotropic drug for hemodynamic support in the treatment of congestive heart failure, cardiogenic and septic shock. Its primary mechanism is direct stimulation of β-adrenergic receptors, thereby increasing contractility and cardiac output (Ruffolo, 1987). Catecholamines including dobutamine seem to be involved in the process of pharmacological preconditioning describing the concept of eliciting intracellular processes by drugs that lead to the protection of cells and organs from subsequent stressors (Berger et al., 2000; Miura et al., 1998; Salvi, 2001; van der Woude et al., 2004; Yard et al., 2004). The exact molecular mechanisms of these effects, however, remain to be identified. Our previous study demonstrated that dobutamine

⁎ Corresponding author at: University Medical Center Department of Anaesthesiology and Critical Care Medicine Hugstetter Str. 55 D-79106 Freiburg, Germany. Tel./fax: +49 761 270 23 380/930. E-mail address: [email protected] (M. Roesslein). 0024-3205/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2014.01.005

inhibits the activation of nuclear factor (NF)-κB, a central regulator of the immune response, in human T lymphocytes (Loop et al., 2004). Inhibitors of NF-κB have been shown to induce a so-called heat shock response (HSR), a cellular defense system conferring cyto- and organ protection (Malhotra and Wong, 2002). The aim of this study was to investigate whether dobutamine exerts cytoprotective properties in a cell model of apoptosis induced by staurosporine. We further hypothesized that the protective effects of dobutamine may be mediated via an induction of the HSR or other possible mechanisms, namely a stimulation of β-adrenergic receptors, the activation of mitogen activated protein kinases, and anti-oxidative properties of dobutamine. Materials and methods Reagents Dobutamine hydrochloride was obtained from Tocris (Bristol, United Kingdom). All other reagents were purchased from Sigma

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(Deisenhofen, Germany) unless specified otherwise. Oxidation of the dobutamine molecules was performed by pre-incubation with H2O2 (100 μM, 30 min) and confirmed by flowcytometric analysis using a BD FACSCalibur® flow cytometer after incubation with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), (Life Technologies, Darmstadt, Germany) according to the manufacturer's recommendations. Cell culture of Jurkat T lymphoma cells Human Jurkat T lymphoma cells (ACC 282, DSMZ, Braunschweig, Germany) were maintained in RPMI 1640 medium (Gibco BRL, Karlsruhe, Germany), supplemented with 10% fetal calf serum, 1% Lglutamine and 50 mg/ml penicillin and streptomycin (all from Gibco BRL, Karlsruhe, Germany). The cells were grown at 37 °C, 5% CO2 in a humidified atmosphere. Jurkat cells in logarithmic growth phase were harvested by centrifugation and washed in phosphate-buffered-saline (PBS). Experimental protocol 106 cells/5 ml medium was pre-incubated with dobutamine (0.1 or 0.5 mM) or respective sympathomimetic tested for up to 4 h before the addition of staurosporine (2 μM, 2 h). Pharmacological inhibition of Hsp90 was achieved by incubation with 17-allylamino-17demethoxygeldanamycin (17-AAG, 2 μM) for 24 h prior to dobutamine incubation. Pharmacological inhibition of Hsp70 was achieved by incubation with N-formyl-3,4-methylenedioxy-benzylidene-gammabutyrolaetam (KNK437, 100 μM) for 24 h prior to dobutamine incubation. Protein extraction Nuclear and cytoplasmic proteins were prepared using a highly concentrated salt detergent buffer (Totex: 20 mM HEPES [pH 7.9], 350 mM NaCl, 20 vol-% glycerol, 1% Nonidet P40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol [DTT], 0.1% phenylmethylsulfonyl fluoride [PMSF] and 1% aprotinin). Cells were harvested by centrifugation and resuspended in 4 times the cell volume of the detergent buffer. The cell lysate was incubated for 30 min on ice and afterwards centrifuged at 13.000 g, at 4 °C for 5 min. Fluorescent staining and flowcytometric analysis After the incubation, 1 ml of cell suspension was harvested in 3 ml ice-cold PBS and centrifuged. The supernatant was discarded and the cells were resuspended in 50 μl staining solution for 20 min at room temperature in the dark. The staining solution consisted of 2% annexin V fluorescein isothiocyanate (FITC) and 1% propidium-iodide (PI) diluted in 1 × annexin binding buffer (Becton Dickinson Pharmingen, Heidelberg, Germany). In the combined apoptosis reactive oxygen species assay the cells were stained using 2% annexin V phycoerythrin (PE; BD Pharmingen, Heidelberg, Germany) and 1% 7-aminoactinomycin-D (7AAD; BD Pharmingen, Heidelberg, Germany) respectively. After resuspending with 450 μl annexin binding buffer, fluorescence intensity was measured of 1 × 104 cells per sample using a BD FACSCalibur® flow cytometer. Annexin V FITC positive cells were measured in Fl1, PI was detected in Fl3. In the combined apoptosis reactive oxygen species assay chloromethyl-dichlorodihydrofluorescein-diacetate (CM-H2DCFDA; Molecular Probes, Eugene, USA) was detected in Fl1, annexin V PE in Fl2 and 7AAD in Fl3. Electrophoretic mobility shift assay Cytoplasmic and nuclear protein extracts of 1 × 107 Jurkat cells were used for eletrophoretic mobility shift assays. Inhibitors of proteinases and phosphotases were added. For the bandshift assays a 32P-labeled

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HSF-1 oligonucleotide (5′-CTA GAA GCT TCT AGA AGC TTC TAG-3′, Promega, Madison, USA) was used. The kinase reaction consisted of 37 μl of purified water, 25 ng of HSF-1 nucleotides and 5 μl of kinase buffer, 5 μl of [γ-32P]dATP (GE Healthcare, Munich, Germany) and 1.5 μl of T4 kinase (PNK buffer and PNK T4 kinase; New England Biolabs, Schwalbach, Germany). The kinase reaction was incubated for 30 min at 37 °C. The protein content was measured with a Bradford assay (BioRad Laboratories, München, Germany) and 30 μg of protein extract was added to a 20 μl electrophoretic mobility shift assay mixture, containing 20 μg of bovine serum albumin, 2 μg of poly(dI-dC) (Roche Diagnostics, Mannheim, Germany), 2 μl of Buffer D + (20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25% Nonidet P40, 2 mM dithiothreitol and 0.1% PMSF), 4 μl 5 × Ficoll buffer (20% Ficoll 400, 100 mM HEPES, 300 mM KCl, 10 mM dithiothreitol and 0.1% phenylmethylsulfonyl fluoride), 3 μl of double distilled H20 and 1 μl of HSF-1 32P-labeled oligonucleotide. The samples were incubated at room temperature for 30 min and then loaded on a 4% acrylamide gel in 0.5 × Tris-borate-EDTA (900 mM Tris–HCl, 900 mM boric acid and 20 mM EDTA pH 8.0), 400 μl of ammonium persulfate and 40 μl of tetramethylethylenediamine. For supershift analysis, 1 μl of antibody HSF-1 (clone 10H8, SPA-950E; Assay Designs, Ann Arbor, USA) was added to the reaction together with the protein and incubated as described above. Gels were vacuum dried (Gel dryer 543, Bio-Rad, Hercules, CA) for 30 min and then exposed to X-ray film (Kodak, Stuttgart, Germany). Isolation of RNA and northern blot analysis Total RNA was extracted from approximately 1 × 107 cells according to the manufacturer's recommendation (TRIzol®, a monophasic onestep solution of phenol and guanidine isothiocyanate; Invitrogen, Darmstadt, Germany). Aliquots of 10 μg total RNA per lane were sizefractionated on a denaturating 1% agarose gel, transferred to a nylon membrane (Hybond-N, GE Healthcare, Munich, Germany) by capillary blotting in 20 × sodium saline citrate (3 mM NaCl and 0.3 M sodium citrate) and crosslinked to the membrane by UV irradiation. The membrane was pre-incubated for 30 min in hybridisation solution (ExpressHyb; Clontech, Palo Alto, USA) and incubated overnight at 68 °C with a 32P-labeled (Prime-It II labeling kit; Stratagene, La Jolla, USA) probe against hsp70. The blots were stripped and re-probed with 18S ribosomal cDNA fragment to confirm equal loading. The following specific primers were used: hsp70, 5′-CAG CGG CAG GCC ACC AAG GAC-3′ as upper primer and 5′-TGC ACC GCC GCC CCG TAG G-3′ as lower primer; hsp90, 5′-GCG AGT CGG ACG TGG TCC-3′ as upper primer and 5-CTG AGG GTT GGG GAT GAT GTC-3′ as lower primer; and 18 s, 5′-CGC CGC GCT CTA CCT TAC CTA CCT-3′ as upper primer and 5′-GAC CGC CCG CCC GCT CCC AAG AT-3′ as lower primer. SDS-polyacrylamide gel electrophoresis and western blotting Cytoplasmic and nuclear protein extracts of 1 × 107 cells per sample were boiled in Laemmli sample buffer and subjected to 10% SDSpolyacrylamide gel electrophoresis Proteins were transferred to Immobilon P membranes (Millipore Corporation, Eschborn, Germany). Equal protein loading was confirmed by stripping of the membranes and by incubating them with specific antibodies. The following primary antibodies were used for western blotting: Hsp70 (clone C92F3A-5, SPA-810; Assay Designs/BIOMOL; dilution 1:1000), heat shock cognate protein (Hsc)70 (polyclonal, SPA-816; Assay Designs/BIOMOL; dilution 1:1000), Hsp90 (monoclonal, ab13492; Abcam plc; dilution 1:3000), ßactin (polyclonal, No. 4967), Caspase-3 (polyclonal, No. 9662), Cleaved Caspase-3 (polyclonal, No. 9661), Cleaved PARP (polyclonal, No. 9541), Phospho-JNK (polyclonal, No. 9251), JNK (polyclonal, No. 9252), Phospho-ERK (polyclonal, No. 9101), ERK (monoclonal, No. 4695), Phospho-p38 (polyclonal, No. 9211), p38 (polyclonal, No. 9212), Phospho-AKT (No. 9271), AKT (No. 4685), CDK4 (No. 2906), (all Cell

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Signaling Technology Inc., Beverly; dilution 1:1000), and RAF-1 (No. SC133; Santa Cruz Biotechnology Inc., Dallas; dilution 1:1000). Nonspecific binding sites were blocked by immersing the membrane into blocking solution [20 mM Tris–HCl, pH 7.6, 0.1% Tween 20 with 5% (w/v) nonfat dry milk powder (Fluka, Buchs, Switzerland)]. Membranes were washed in 20 mM Tris–HCl, pH 7.6, plus 0.1% Tween 20, and were afterwards incubated in a recommended dilution of specific antibodies. Bound antibodies were detected by goat anti-rabbit/horseradish peroxidase-conjugated secondary antibody (7074; Cell Signaling Technology Inc., Boston, USA; dilution 1:2000). To investigate the activity of the MAP kinases p38 and JNK, nonradioactive MAP kinase activity assay kits (Cell Signaling Technology, Danvers, USA) were used and performed according to the manufacturer's instructions. The immune complexes were detected using enhanced chemiluminescence western blotting reagents (GE Healthcare, Munich, Germany) according to the manufacturer's instructions and exposed to enhanced chemiluminescence western blot films (GE Healthcare, Munich, Germany) for 15 s to 1 min. Measurement of caspase activity Whole cellular protein extracts of 1 × 107 cells per sample (10 μl) were mixed with 90 μl of assay buffer (100 mM HEPES, pH 7.5, 2 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride). The fluorogenic substrate for caspase-3 N-Ac-DEVD (1 μl, 60 μM per sample; Alexis Corporation, Gruenberg, Germany), was added and the fluorescence was measured at 30 °C for 30 min in a Microplate Spectra Max Gemini XS reader (Molecular Devices, Sunnyvale, USA) at 380/460 nm. Real-time polymerase chain reaction RNA was isolated using the RNeasy Mini Kit (Qiagen, Erkrath, Germany). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using cDNA reverse transcription kit (Applied Biosystems, Inc, Foster City, Calif) according to the manufacturer's protocol. The resulting cDNA was used in semiquantitative real-time polymerase chain reaction (PCR) analysis. Reactions were performed in duplicate for each sample on an ABI Prism 7000 (Applied Biosystems). All primer/probes and mixtures were purchased from Applied Biosystems; Taq Man probe human hsp70 (assay Hs00359163_s1). Parameters for quantitative PCR were as follows: 10 min at 95 °C, followed by 40 cycles of amplification for 15 s at 95 °C and 1 min at 60 °C. As endogenous control, GAPDH gene expression was measured for each probe using a VIC/MGB-labeled probe (human: 4326317E). The obtained data from GAPDH were used to standardize the sample variation in the amount of input cDNA by the [DELTA][DELTA]CT method. Statistical analysis

2002; Roesslein et al., 2008b), induced a strong increase of annexin Vpositive cells indicating cellular externalization of membrane-bound phosphatidylserine, a hallmark sign of apoptosis (Fig. 1). While dobutamine alone had no effect, pre-incubation with dobutamine lead to an inhibition of staurosporine-induced apoptosis as measured by the rate of annexin V-positive cells (28 [20–32] % vs. 12 [9–15] % for dobutamine 0.1 mM and 7 [5–12] % for dobutamine 0.5 mM, p b 0.001, Fig. 1). As shown in a representative experiment (Fig. 2A), incubation of Jurkat cells with staurosporine leads to a strong increase in caspase degradation into the active fragments p19 and p17, a critical step in the apoptotic process. This degradation was inhibited dose-dependently by dobutamine pre-incubation (Fig 2A, upper panel). In addition, the increase in cleaved Poly-(ADP-Ribose)-Polymerase (PARP) indicating the execution of apoptosis was also inhibited after pre-incubation with dobutamine (Fig. 2A, lower panel). Induction of apoptosis in Jurkat cells with staurosporine leads to a marked increase in caspase-3-like activity, (Fig. 2B). While incubation with dobutamine alone had no effect on caspase-3-like activity (Fig. 2B), pre-incubation with dobutamine significantly inhibited the staurosporine-induced caspase-3-like activity in a dose-dependent manner (0.46 [0.40–0.48] vs. 0.32 [0.27–0.39] for dobutamine 0.1 mM and 0.20 [0.19–0.23] for dobutamine 0.5 mM, p b 0.001, Fig. 2B).

Cytoprotection is not mediated by other catecholamines Unlike dobutamine, neither epinephrine (0.1–0.5 mM) nor norepinephrine (0.1–0.5 mM) was able to suppress the number of annexin V-positive cells after staurosporine-induced apoptosis (Fig. 3). Also, none of the sympathomimetics tested (dobutamine, epinephrine, formoterol, isoproterenol, phenylephrine, all 0.1 mM) was able to increase intracellular cAMP-levels indicating a lack of adrenergic receptor stimulation by these substances in our setting (data not shown).

Dobutamine activates mitogen-activated protein (MAP) kinases p38 and c-Jun-N-terminal kinase (JNK) Incubation of Jurkat cells with dobutamine increased the phosphorylation of p38 (Fig. 4A, upper panel) and its substrate ‘activating transcription factor 2’ (ATF-2, Fig. 4A, lower panel), and also induced the phosphorylation of JNK (Fig. 4B, upper panel), and its substrate c-Jun (Fig. 4B, lower panel) in a dose- and time-dependent manner displaying the activation of these two MAP kinases by dobutamine. In contrast, pre-incubation of Jurkat cells with dobutamine induced no phosphorylation of ‘extracellular signal-related kinase’ (ERK) (Fig. 4C, upper panel).

All data are given as means ± standard deviation (after proof of normality distribution) or median and 25/75% percentile. Differences in measured variables between the experimental conditions were assessed using a one-way analysis of variance followed by a Student– Newman–Keuls post-hoc test for multiple comparisons for caspase-3 activity data and a one-way analysis of variance on ranks followed by a nonparametric Student–Newman–Keuls test for multiple comparisons or Student's t test for flowcytometric data. Results were considered statistically significant at p b 0.05. The tests were performed using the SigmaStat software package (SPSS Inc., Chicago, IL, USA). Results Dobutamine pre-incubation protects Jurkat cells from staurosporine-induced apoptosis Incubation of Jurkat cells with staurosporine, a well-established inducer of apoptotic cell death (Chae et al., 2000; Feng and Kaplowitz,

Fig. 1. Effects of dobutamine on staurosporine-induced apoptosis. Flowcytometric analysis after FITC annexin-V and propidium iodide staining (n = 44; box plots show the median, 25/75th and 5/95th percentiles; *** = p b 0.001 staurosporine vs. dobutamine + staurosporine. The percentage of cells positive for both annexin V and propidium-iodide representing necrotic and end stage apoptotic cells, did not differ significantly between the treatment groups (data not shown).).

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Fig. 4. Effects of dobutamine on the activation of mitogen-activated protein (MAP) kinases. Representative western blot displaying the status of phosphorylation and activity of MAP kinases after incubation with dobutamine. (A): p38 MAP kinase; (B): c-Jun-Nterminal Kinase (JNK); (C): Extracellular signal-Related Kinase (ERK1/2). Equal loading was confirmed after reprobing with antibodies directed against the respective unphosphorylated protein. Positive controls were incubated with phorbol myristate acetate (15 ng/ml) and ionomycin (1 μg/ml) for 30 min. Fig. 2. Effects of dobutamine on (A) cleavage of caspase-3 and Poly (ADP-Ribose) Polymerase (PARP) and (B) activity of caspase-3 after staurosporine-induced apoptosis. (A): Representative western blot of caspase-3 and PARP cleavage. Equal loading was confirmed after reprobing with anti-ß-actin antibody. (B): Fluorogenic caspase-3-like activity assay. Data are expressed as relative fluorescence units [RFU] (n = 5; box plots show the median, 25/75th and 5/95th percentiles; * = p b 0.05 or ** = p b 0.001 staurosporine vs. dobutamine + staurosporine).

Dobutamine-mediated cytoprotection is independent of activation of p38 and JNK Specific pharmacological inhibition of p38 (SB202190 (Roesslein et al., 2008a)) and JNK (SP600125 (Bennett et al., 2001)) prior to incubation with dobutamine did not reverse the protective effects of dobutamine, indicating that p38 and JNK are not involved in the dobutamine-mediated cytoprotection (data not shown).

Anti-oxidative effects of dobutamine are not responsible for cytoprotection As expected, dobutamine acted as a potent scavenger of reactive oxidative species in our experimental setting, starting to be effective in concentrations as low as 1 μM (data not shown). To further investigate whether the anti-apoptotic effects of dobutamine are due to its antioxidative properties, we oxidized the dobutamine molecules using H2O2 (100 μM, 30 min), thereby obliterating their ROS-scavenging properties. However, oxidized dobutamine did not attenuate the observed cytoprotective effect at 0.5 mM (Fig. 5). Dobutamine induces a heat shock response (HSR) Dobutamine induced DNA-binding of the transcription factor ‘heat shock factor’ (HSF)-1 dose- and time-dependently as shown in a representative experiment (Fig. 6A). Increased DNA-binding activity was followed by dobutaminemediated induction of mRNA levels of hsp70 and hsp90 (Fig. 6B) and expression of Hsp70 and Hsp90 proteins (Fig. 6C). Dobutamine-induced cytoprotection is mediated via Hsp70

Fig. 3. Effects of adrenaline or noradrenaline on staurosporine-induced apoptosis. Flowcytometric analysis after FITC annexin-V and propidium iodide staining (n = 5; box plots show the median, 25/75th and 5/95th percentiles. The percentage of cells positive for both annexin V and propidium-iodide representing necrotic and end stage apoptotic cells, did not differ significantly between the treatment groups (data not shown).).

Specific pharmacological inhibition of Hsp90 (17-AAG, confirmed by down-regulation of Hsp90 client proteins PhosphoAKT, AKT, CDK4 and RAF-1 (Kurashina et al., 2009; Schulte and Neckers, 1998; Sreedhar et al., 2004) (Fig. 7A)) did not inhibit the dobutamine-mediated protection from apoptosis as measured by the number of annexin V-positive cells (Fig. 7B) and caspase-3-like activity (Fig. 7C). In contrast, inhibition of Hsp70 (KNK437, confirmed by downregulation of hsp70-mRNA (Yokota et al., 2000) (Fig. 8A)) before incubation with dobutamine leads to a significant increase in the number of annexin V-positive cells (33 [32-36] vs. 18 [16–24] %, p b 0.05, Fig. 8B) and caspase-3-like activity (0.21 [0.19–0.23] vs. 0.16 [0.13–

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were attenuated after pre-incubation with dobutamine. (2) The β-receptor does not seem to be responsible for the dobutaminemediated protection from apoptosis, since neither β-receptor agonism nor antagonism induced or attenuated this effect. (3) Dobutamine differentially induced MAP-kinase activation: while ERK was not affected, p38 and JNK were strongly induced. However, the dobutaminemediated cytoprotection seems to be independent of the activation of p38 or JNK, since specific pharmacological inhibition was not able to attenuate the effects of dobutamine. (4) The cytoprotection is also independent of the anti-oxidative properties of dobutamine. (5) In contrast, dobutamine-induced Hsp70 expression seems to be pivotal for the observed protection from apoptosis, because specific pharmacological inhibition significantly attenuates the dobutamine-mediated cytoprotection. Fig. 5. Effect of oxidized dobutamine on cytoprotection. Flowcytometric analysis after FITC annexin-V and propidium iodide staining (n = 7; box plots show the median and 25/75th percentiles; * = p b 0.05 staurosporine vs. dobutamine + staurosporine. The percentage of cells positive for both annexin V and propidium-iodide representing necrotic and end stage apoptotic cells, did not differ significantly between the treatments (data not shown).).

0.17], p b 0.05, Fig. 8C) indicating a key role for Hsp70 in the dobutamine-mediated cytoprotection. Discussion Our study supports the hypothesis that dobutamine protects cells from apoptosis, and that the induction of Hsp70 may be a molecular mechanism contributing to this effect by several lines of evidence: (1) Induction of caspase-3-like activity and apoptosis by staurosporine

Fig. 6. Effect of dobutamine on the heat shock response. Representative electrophoretic mobility shift assay using HSE oligonucleotides ( ) = HSF-1/HSE = specific HSF-1/DNA binding. (O) = non-specific binding to the probe (A). Representative northern blot for hsp70 and hsp90 mRNA (18 s = rRNA) (B). Representative western blot for Hsp70 and Hsp90 proteins. Equal loading was confirmed after reprobing with heat shock cognate protein (Hsc) 70 for Hsp70 and anti-β-actin antibody for Hsp90 (C).

(1) Dobutamine is a synthetic catecholamine whose hemodynamic and circulatory effects are mediated mainly via agonism on the β1-receptor, while it also possesses some α1- and β2-mimetic properties. Next to these well-described characteristics, several studies have identified additional cytoprotective properties of dobutamine in various cell types (Asimakis and Conti, 1995; Rensing et al., 2004; White et al., 2006; Yard et al., 2004). The molecular mechanisms of these effects, however, are not completely understood. We have demonstrated in this study that dobutamine applied before a pro-apoptotic stimulus inhibits apoptosis as measured by pivotal events in the execution of this kind of programmed cell death whose clinical implications become more and more evident (Hotchkiss et al., 2009). While the role of apoptosis in disease is complex and incompletely understood, decreased apoptosis in an acute scenario may provide additional protection against infection or immunity (Lydon and Martyn, 2003). The in vitro concentrations used in our experimental model were comparable to those obtained in the plasma of patients during the administration of dobutamine in a clinical setting (Knoll and Brandl, 1986). (2) Data on the role of the β-receptor in the protective effects of catecholamines are conflicting. Some studies attribute β-adrenergic stimulation to pre-conditioning effects in the case of ischemia/ reperfusion in the rat heart (Asimakis and Conti, 1995) and reduction of endothelial cell death during hypoxia/reoxygenation (Pottecher et al., 2006), while others showed protection against cold-preservation injury in the same cell type by dobutamine independent of receptor-stimulation (Yard et., 2004), which would be in agreement with the findings in our study. (3) The MAP kinase superfamily has been shown to play a major role in the regulation of cellular responses to internal and external stresses including apoptosis by phosphorylating other target proteins (Cowan and Storey, 2003). Catecholamines may have opposing effects on the activation of MAP kinases depending on the co-stimulus (Szelenyi et al., 2006). Since activation of MAP kinases is involved in the decision between both survival and death of cells depending on the intensity of the damage (Takekawa et al., 2011), it is not surprising that their role in apoptosis is also ambivalent with both pro- and anti-apoptotic effects depending on the type and duration of the harmful signal as well as the cell type (Davis, 2000). HSR and MAP kinase activation are both evolutionary conserved cellular response-mechanisms that seem finely regulated and interconnected: Hsps have been shown to regulate MAP kinase activation and MAP kinases to regulate the activation of Hsps (Dorion and Landry, 2002). For example, Hsp70 has been shown to mediate suppression of JNK (Gabai et al., 1998), while Hsp90 can associate with other members of the MAP kinase system (Helmbrecht et al., 2000). However, much remains to be learned concerning the mechanism of activation and the role of the MAP kinases in the heat shock response.

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Fig. 7. Effect of Hsp90 inhibition on dobutamine-mediated cytoprotection. Western blot analysis of Phopho-AKT, AKT, CDK4 and RAF after incubation with 17-AAG. Equal loading was confirmed after reprobing with anti-β-actin antibody (A). Flowcytometric analysis after FITC annexin-V and propidium iodide staining (B, n = 18) and fluorogenic caspase-3-like activity assay (C, n = 10) after Hsp90 inhibition with 17-AAG. Box plots show the median, 25/75th and 5/95th percentiles; * = p b 0.05 vs. staurosporine. (The percentage of cells positive for both annexin V and propidium-iodide representing necrotic and end stage apoptotic cells, did not differ significantly between the treatments (data not shown).).

We have demonstrated that dobutamine induces p38 and JNK but not ERK while counteracting apoptosis. However, since inhibition of p38 and JNK had no significant effect on the dobutamine-mediated cytoprotection, the activation of these kinases does not seem to be relevant for the observed protection in our setting. (4) Catecholamines have been shown to improve long-term graft survival after renal transplantation reducing the need for dialysis (Schnuelle et al., 2001, 2009, 1999), possibly by protecting endothelial cells against cold-storage induced injury by either scavenging of ROS or by inhibition of ROS production. This effect does not seem to be receptor-mediated, but depending on the drugs' own redox state (Yard et al., 2004). Although our results confirm that dobutamine acts as a ROS scavenger, this property was not responsible for the antiapoptotic effects in our model, since obliterating the antioxidative action of dobutamine (by prior oxidation of the dobutamine molecule itself) did not prevent its anti-apoptotic action. (5) The heat shock response (HSR) is a cellular defense system that is highly conserved throughout evolution and can be found in a wide spectrum of organisms ranging from prokaryotes to human beings (Malhotra and Wong, 2002; Welch, 1992). Next to increased environmental temperature, the HSR can be elicited by factors such as hypoxia, ischemia/reperfusion as well as inflammatory or oxidative stress (Chi and Karliner, 2004; Giffard and Yenari, 2004; Kiang, 2004). The induction of a HSR involves the activation and DNA-binding of the transcription factor HSF-1 and the expression of different Hsps. In the past, we reported that dobutamine is a potent and specific inhibitor of NF-κB, a critical transcription factor in the execution of

inflammatory processes (Loop et al., 2004). A number of links have been established between the HSR and the NF-κB pathway. The existing reciprocity between these two pathways is underlined by the fact that while Hsp70 and Hsp90 proteins regulate the function of the IKK complex, which is the major activator of the NF-κB complex (Salminen et al., 2008), various pharmacologic inhibitors of the NF-κB pathway, such as dexamethasone, acetylsalicylic acid and bimoclomol also act as inducers of the HSR (Malhotra and Wong, 2002). Our findings that dobutamine induces a HSR and protects from apoptotic cell death via Hsp70 in this T cell model is supported by reports about this protein as a key determinant in the regulation of apoptosis (Xanthoudakis and Nicholson, 2000): Hsp70, which functions as molecular chaperone and confers protection against various stressful stimuli in vitro (Wong et al., 1996, 1998) and in vivo such as ischemia/ reperfusion injury (Marber et al., 1995), has also been identified to inhibit apoptotic cell death by preventing mitochondrial cytochrome c release and activation of pro-caspases to caspases (Mosser et al., 2000; Xanthoudakis and Nicholson, 2000). While Hsp90 had no protective role in our study, it still needs to be determined whether Hsps other than Hsp70 are also involved in the dobutamine-mediated cytoprotection. The HSR may play an important immunomodulatory role during times of stress, and the ability to manipulate levels of Hsps may be advantageous in the clinical scenario when inflammatory enhancement or immunosuppression is desired. Jurkat cells belong to a human T lymphocyte cell line, commonly used to study T cell signaling and stress-mediated pathways (Abraham and Weiss, 2004). Obviously, since our results have been generated in a pure in vitro cell model setting, conclusions for the clinical application should only be drawn very cautiously. One conceivable application may be the use of dobutamine as an additive to cold preservation solutions where catecholamines have been shown to improve the

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Fig. 8. Effect of Hsp70 inhibition on dobutamine-mediated cytoprotection. Real-time PCR experiments using primers for hsp70 or GAPDH. Expression of hsp70 mRNA was normalized to GAPDH mRNA levels and presented as arbitrary units (n = 2) (A). Flowcytometric analysis after FITC annexin-V and propidium iodide staining (B, n = 10) and fluorogenic caspase-3-like activity assay (C, n = 10) after Hsp70 inhibition with KNK 437. Box plots show the median, 25/75th and 5/95th percentiles; * = p b 0.05. (The percentage of cells positive for both annexin V and propidium-iodide representing necrotic and end stage apoptotic cells, did not differ significantly between the treatments (data not shown).).

postischemic integrity of organ grafts in the context of transplantations (Benck et al., 2011; Koetting et al., 2010; Schnuelle et al.,2001, 2009). However, the verification of our findings in primary cells and/or an in vivo model does seem reasonable.

Conflict of interest statement The authors declare that there is no conflict of interest.

Role of the funding sources Conclusion Our data suggest that dobutamine exerts cytoprotection from subsequent apoptotic stress via the induction of heat shock protein 70.

The funding sources had no involvement in study design or in the collection, analysis and interpretation of data or in the writing of the report or in the decision to submit the article for publication.

M. Roesslein et al. / Life Sciences 98 (2014) 88–95

Acknowledgments This work was conducted at the Department of Anaesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany and the Biomedical Research Institute, UHasselt, Diepenbeek, Belgium. It was supported by the European Society of Anaesthesiology and departmental funding.

References Abraham RT, Weiss A. Jurkat T cells and development of the T-cell receptor signalling paradigm. Nat Rev Immunol 2004;4:301–8. Asimakis GK, Conti VR. Preconditioning with dobutamine in the isolated rat heart. Life Sci 1995;57:177–87. Benck U, Hoeger S, Brinkkoetter PT, Gottmann U, Doenmez D, Boesebeck D, et al. Effects of donor pre-treatment with dopamine on survival after heart transplantation: a cohort study of heart transplant recipients nested in a randomized controlled multicenter trial. J Am Coll Cardiol 2011;58:1768–77. Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, et al. Sp600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci U S A 2001;98:13681–6. Berger SP, Hunger M, Yard BA, Schnuelle P, Van Der Woude FJ. Dopamine induces the expression of heme oxygenase-1 by human endothelial cells in vitro. Kidney Int 2000;58:2314–9. Chae HJ, Kang JS, Byun JO, Han KS, Kim DU, Oh SM, et al. Molecular mechanism of staurosporine-induced apoptosis in osteoblasts. Pharmacol Res 2000;42:373–81. Chi NC, Karliner JS. Molecular determinants of responses to myocardial ischemia/reperfusion injury: focus on hypoxia-inducible and heat shock factors. Cardiovasc Res 2004;61: 437–47. Cowan KJ, Storey KB. Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress. J Exp Biol 2003;206: 1107–15. Davis RJ. Signal transduction by the JNK group of map kinases. Cell 2000;103:239–52. Dorion S, Landry J. Activation of the mitogen-activated protein kinase pathways by heat shock. Cell Stress Chaperones 2002;7:200–6. Feng G, Kaplowitz N. Mechanism of staurosporine-induced apoptosis in murine hepatocytes. Am J Physiol Gastrointest Liver Physiol 2002;282:G825–34. Gabai VL, Meriin AB, Yaglom JA, Volloch VZ, Sherman MY. Role of Hsp70 in regulation of stress-kinase JNK: implications in apoptosis and aging. FEBS Lett 1998;438: 1–4. Giffard RG, Yenari MA. Many mechanisms for Hsp70 protection from cerebral ischemia. J Neurosurg Anesthesiol 2004;16:53–61. Helmbrecht K, Zeise E, Rensing L. Chaperones in cell cycle regulation and mitogenic signal transduction: a review. Cell Prolif 2000;33:341–65. Hotchkiss RS, Strasser A, McDunn JE, Swanson PE. Cell death. N Engl J Med 2009;361: 1570–83. Kiang JG. Inducible heat shock protein 70 kD and inducible nitric oxide synthase in hemorrhage/resuscitation-induced injury. Cell Res 2004;14:450–9. Knoll R, Brandl M. Determination of dobutamine levels in human plasma — methodological and clinical aspects. Anasth Intensivther Notfallmed 1986;21:34–7. Koetting M, Stegemann J, Minor T. Dopamine as additive to cold preservation solution improves postischemic integrity of the liver. Transpl Int 2010;23:951–8. Kurashina R, Ohyashiki JH, Kobayashi C, Hamamura R, Zhang Y, Hirano T, et al. Anti-proliferative activity of heat shock protein (Hsp) 90 inhibitors via beta-catenin/ Tcf7l2 pathway in adult T cell leukemia cells. Cancer Lett 2009;284:62–70. Loop T, Bross T, Humar M, Hoetzel A, Schmidt R, Pahl HL, et al. Dobutamine inhibits phorbol-myristate-acetate-induced activation of nuclear factor-kappab in human T lymphocytes in vitro. Anesth Analg 2004;99(5):1508–15. Lydon A, Martyn JA. Apoptosis in critical illness. Int Anesthesiol Clin 2003;41:65–77. Malhotra V, Wong HR. Interactions between the heat shock response and the nuclear factor-kappab signaling pathway. Crit Care Med 2002;30:S89–95.

95

Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, Dillmann WH. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 1995;95:1446–56. Miura T, Muraoka S, Ogiso T. Antioxidant activity of adrenergic agents derived from catechol. Biochem Pharmacol 1998;55:2001–6. Mosser DD, Caron AW, Bourget L, Meriin AB, Sherman MY, Morimoto RI, et al. The chaperone function of Hsp70 is required for protection against stress-induced apoptosis. Mol Cell Biol 2000;20:7146–59. Pottecher J, Cheisson G, Huet O, Laplace C, Vicaut E, Mazoit JX, et al. Beta2-adrenergic agonist protects human endothelial cells from hypoxia/reoxygenation injury in vitro. Crit Care Med 2006;34:165–72. Rensing H, Bauer I, Kubulus D, Wolf B, Winning J, Ziegeler S, et al. Heme oxygenase-1 gene expression in pericentral hepatocytes through beta1-adrenoceptor stimulation. Shock 2004;21:376–87. Roesslein M, Frick M, Auwaerter V, Humar M, Goebel U, Schwer C, et al. Sevofluranemediated activation of P38-mitogen-activated stresskinase is independent of apoptosis in Jurkat T-cells. Anesth Analg 2008a;106:1150–60. [table of contents]. Roesslein M, Schibilsky D, Muller L, Goebel U, Schwer C, Humar M, et al. Thiopental protects human T lymphocytes from apoptosis in vitro via the expression of heat shock protein 70. J Pharmacol Exp Ther 2008b;325:217–25. Ruffolo Jr RR. The pharmacology of dobutamine. Am J Med Sci 1987;294:244–8. Salminen A, Paimela T, Suuronen T, Kaarniranta K. Innate immunity meets with cellular stress at the Ikk complex: regulation of the Ikk complex by Hsp70 and Hsp90. Immunol Lett 2008;117:9–15. Salvi S. Protecting the myocardium from ischemic injury: a critical role for alpha(1)adrenoreceptors? Chest 2001;119:1242–9. Schnuelle P, Lorenz D, Mueller A, Trede M, Van Der Woude FJ. Donor catecholamine use reduces acute allograft rejection and improves graft survival after cadaveric renal transplantation. Kidney Int 1999;56:738–46. Schnuelle P, Berger S, de Boer J, Persijn G, van der Woude FJ. Effects of catecholamine application to brain-dead donors on graft survival in solid organ transplantation. Transplantation 2001;72(3):455–63. Schnuelle P, Gottmann U, Hoeger S, Boesebeck D, Lauchart W, Weiss C, et al. Effects of donor pretreatment with dopamine on graft function after kidney transplantation: a randomized controlled trial. JAMA 2009;302(10):1067–75. Schulte TW, Neckers LM. The benzoquinone ansamycin 17-allylamino-17demethoxygeldanamycin binds to hsp90 and shares important biologic activities with geldanamycin. Cancer Chemother Pharmacol 1998;42:273–9. Sreedhar AS, Soti C, Csermely P. Inhibition of Hsp90: a new strategy for inhibiting protein kinases. Biochim Biophys Acta 2004;1697:233–42. Szelenyi J, Selmeczy Z, Brozik A, Medgyesi D, Magocsi M. Dual beta-adrenergic modulation in the immune system: stimulus-dependent effect of isoproterenol on MAPK activation and inflammatory mediator production in macrophages. Neurochem Int 2006;49:94–103. Takekawa M, Kubota Y, Nakamura T, Ichikawa K. Regulation of stress-activated MAP kinase pathways during cell fate decisions. Nagoya J Med Sci 2011;73:1–14. van der Woude FJ, Schnuelle P, Yard BA. Preconditioning strategies to limit graft immunogenicity and cold ischemic organ injury. J Invest Med 2004;52:323–9. Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev 1992;72:1063–81. White M, Ducharme A, Ibrahim R, Whittom L, Lavoie J, Guertin MC, et al. Increased systemic inflammation and oxidative stress in patients with worsening congestive heart failure: improvement after short-term inotropic support. Clin Sci 2006;110:483–9. Wong HR, Mannix RJ, Rusnak JM, Boota A, Zar H, Watkins SC, et al. The heat-shock response attenuates lipopolysaccharide-mediated apoptosis in cultured sheep pulmonary artery endothelial cells. Am J Respir Cell Mol Biol 1996;15:745–51. Wong HR, Menendez IY, Ryan MA, Denenberg AG, Wispe JR. Increased expression of heat shock protein-70 protects A549 cells against hyperoxia. Am J Physiol 1998;275:L836–41. Xanthoudakis S, Nicholson DW. Heat-shock proteins as death determinants. Nat Cell Biol 2000;2:E163–5. Yard B, Beck G, Schnuelle P, Braun C, Schaub M, Bechtler M, et al. Prevention of cold-preservation injury of cultured endothelial cells by catecholamines and related compounds. Am J Transplant 2004;4(1):22–30. Yokota S, Kitahara M, Nagata K. Benzylidene lactam compound, Knk437, a novel inhibitor of acquisition of thermotolerance and heat shock protein induction in human colon carcinoma cells. Cancer Res 2000;60:2942–8.