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The atrial natriuretic peptide and cGMP: Novel activators of the heat shock response in rat livers
The Atrial Natriuretic Peptide and cGMP: Novel Activators of the Heat Shock Response in Rat Livers Alexandra K. Kiemer,1,2 Alexander L. Gerbes,1 Manfred Bilzer,1 and Angelika M. Vollmar2 Preischemic treatment with atrial natriuretic peptide (ANP) attenuates ischemia-reperfusion injury of the rat liver via cyclic guanosine monophosphate (cGMP). The attenuated activation of nuclear factor B (NF-B) seems to contribute to this effect. The aim of this study was to determine whether heat shock proteins are involved in these molecular pathways. Livers of male Sprague-Dawley rats were continuously perfused with Krebs-Henseleit (KH) buffer with or without ANP or 8-Br-cGMP. In different experiments livers were perfused with or without ANP for 20 minutes, kept in cold storage solution for 24 hours, and reperfused. Activation of heat shock transcription factor (HSF) (by electrophoretic mobility shift assay), heat shock protein 70 (HSP70), and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA (by reverse transcription polymerase chain reaction [RT-PCR]), as well as HSP70 (by Western blot) were investigated in freeze-clamped liver samples. During continuous perfusion ANP as well as 8-Br-cGMP activated HSF, HSP70 protein concentrations paralleled HSF-activation. ANP pretreated livers exhibited elevated HSF after 24 hours of ischemia and elevated HSP70 mRNA levels during reperfusion. ANP prevented the marked decrease of HSP70 protein during reperfusion. Coimmunoprecipitation studies showed increased binding of HSP70 to inhibitory factor B (IB) in ANP-treated livers. In conclusion, we showed the cGMP-mediated activation of HSF by ANP, which resulted in elevated HSP70 mRNA and protein concentrations and correlated with enhanced binding of HSP70 to IB. This could be an important mechanism of ANP-mediated prevention of hepatic preservation damage. (HEPATOLOGY 2002;35:88-94.)
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he “heat shock response” is elicited by a variety of stimuli, including thermal, chemical, and physical stress, as well as short-term ischemia. All organisms respond to such detrimental environmental factors by induction of a group of protective proteins termed heat shock proteins (HSP) (see elsewhere for reviews1-4). The HSPs are subdivided into multimember families based on the molecular weights of the proteins encoded (HSP90, HSP70, HSP60, and the small HSP family), of which HSP70 is one of the most extensively studied in mammalian cells.2 Studies have revealed that HSPs, in particular HSP70, not only confer thermotolerance, but also protect against oxygen radical toxicity and seem to play an important role in the protection against ischemia reperfusion injury (IRPI).3,4 Both ischemic5 or heat shock Abbreviations: HSP, heat shock protein; IRPI, ischemia reperfusion injuries; HSF, heat shock transcription factor; NF-B, nuclear factor B; TNF-␣, tumor necrosis factor ␣; ANP, atrial natriuretic peptide; cGMP, cyclic guanosine monophosphate; AP-1, activator protein 1; KH, Krebs-Henseleit solution; UW, University of Wisconsin solution; LDH, lactic dehydrogenase; GAPDH, glyceraldehyde phosphate dehydrogenase; RT-PCR, reverse transcription polymerase chain reaction; IB, inhibitory factor B. From the 1Department of Pharmacy, Center of Drug Research and 2Department of Medicine II, Klinikum Grosshadern, University of Munich, Munich, Germany. Received April 11, 2001; October 1, 2001. Supported by the Deutsche Forschungsgemeinschaft (DFG: Ge 576/14-1). Address reprint requests to: Alexandra K. Kiemer, Ph.D., Department of Pharmacy, Center of Drug Research, Butenandtstr. 5-13, 81377 Munich, Germany. E-mail: [email protected]; fax: (49) 89-2180-7170. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3501-0013$35.00/0 doi:10.1053/jhep.2002.30080 88
preconditioning 6-8 induce HSP70 in the liver and the induced HSP70 may contribute to the attenuation of IRPI. This is supported by findings that overexpression of HSP70 in transgenic mice increases the resistance of the heart to ischemic injury.9,10 HSPs are regulated primarily by changes in gene transcription controlled by a transcription factor termed heat shock factor (HSF).11,12 The activation process of HSF appears to involve HSF oligomerization from a monomeric to a trimeric state and is associated with HSF hyperphosphorylation.13 Activated HSF binds to a DNA consensus sequence in the HSP70 promotor termed the heat shock element,14 leading to the transcription of the HSP70 gene. HSPs seem to protect cells by facilitating the folding of nascent proteins and renaturation or refolding of partially denatured or unfolded proteins.15 In addition, HSPs can also restrict inflammatory response. Several reports have shown the capacity of HSPs to inhibit the translocation of the proinflammatory transcription factor NF-B and the subsequent expression of inflammatory enzymes16 and cytokines, such as tumor necrosis factor ␣ (TNF-␣).1,17-21 Recently we could show that hormonal preconditioning of livers with the atrial natriuretic peptide (ANP) attenuates reperfusion injury after both warm22 and cold23 ischemia of rat livers. The hepatoprotective action of this cardiovascular hormone is mediated via its guanylate-cyclase– coupled A-receptor. Protection conveyed by ANP as well as by the cyclic guanosine monophosphate (cGMP) analogue 8-Br-cGMP involves the attenuated activation of the proinflammatory transcription factors NF-B and activator
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protein 1 (AP-1) and the reduced expression of TNF-␣.24 To elucidate whether the anti-inflammatory action of ANP is related to HSPs, the aim of this study was to investigate a potential influence of ANP on the activation of the heat shock response.
Materials and Methods Materials ratANP 99-126 was purchased from Calbiochem/Novabiochem (Bad Soden, Germany). [␥32P]-ATP (3,000 Ci/mmol) and Hyperfilm MP films were from Amersham Pharmacia (Braunschweig, Germany); Complete was from Boehringer Ingelheim Bioproducts (Heidelberg, Germany). Polyclonal anti-HSP70 and anti-IB␣ rabbit antibodies were purchased from Santa Cruz biotechnology (Heidelberg, Germany). Cell culture medium, fetal calf serum, and penicillin/streptomycine were from Life Technologies (Karlsruhe, Germany). Control rabbit IgG was provided by Dr. Hagedorn, Institute of Pharmacology (Munich, Germany). Protein A-agarose and all other materials were from Sigma (Deisenhofen, Germany). Liver Perfusion Male Sprague-Dawley rats weighing 250 to 300 g were purchased from SAVO (Kisslegg, Germany) and housed in a climatized room with a 12-hour light-dark cycle. The animals had free access to chow (Standard-Diet, Altromin 1314 Lage, Germany) and water up to the time of the experiments. After anesthetizing the animals with pentobarbital (50 mg/kg body weight, intraperitoneally), the portal vein was cannulated and the livers were perfused in situ with hemoglobin-free and albumin-free, bicarbonatebuffered Krebs-Henseleit (KH) solution (pH 7.4, 37°C) gassed with 95% O2 and 5% CO2. The perfusion medium was pumped through the livers with a membrane pump at a constant flow rate of 3.0 to 3.5 mL ⫻ min⫺1 ⫻ g liver⫺1 in a nonrecirculating fashion. After 10 minutes controlling the stability of the system, ANP (200 nmol/L) or 8-Br-cGMP (50 mol/L) was added to the perfusate and perfusion was performed for up to 75 minutes. In a different set of experiments, after 30 minutes of perfusion with KH buffer, livers were perfused with 30 mL of cold (4°C) University of Wisconsin (UW) solution for 1 minute. Then the organs were kept in 150 mL UW solution at 4°C for 24 hours. Pretreatment with ANP or 8-Br-cGMP was performed by adding ANP or 8-Br-cGMP to the preischemic perfusate for 20 minutes until ischemia and to the storage solution at a final concentration of 200 nmol/L for ANP and 50 mol/L for 8-Br-cGMP. Following the period of ischemia, livers of each group were reperfused with KH buffer for 2 hours. At the indicated times, i.e., before ischemia, at the end of ischemia, and after 45 and 120 minutes of reperfusion livers were snapfrozen and stored at ⫺70°C until further analysis. The activity of lactic dehydrogenase (LDH) in perfusate was analyzed as a parameter of cell damage.23 Unless otherwise stated, 4 to 5 independent experiments were performed. All animals received humane care. The study was registered with the local animal welfare committee. Preparation of Cell Extracts and Gel Shift Assay Whole cell extracts were prepared from frozen liver sections as described previously.14 Briefly, tissue samples were homogenized
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in ice-cold buffer containing 20 mmol/L HEPES pH 7.9, 25% glycerol, 0.42 mol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L PMSF, and 0.5 mmol/L DTT with a dounce homogenizer, centrifuged at 100,000g for 10 minutes at 4°C and the supernatant was frozen at ⫺70°C. The protein concentration was estimated after the method of Lowry. Complementary singlestranded DNA oligonucleotides with a binding sequence of 5⬘CTAGAACGTTCTAGAAGCTTCGAG-3⬘ used to bind HSF (synthesized by MWG Biotech, Ebersberg, Germany) were annealed at 54°C. The DNA was 5⬘ end-labeled with [␥32P]-ATP (3000 Ci/mmol; Amersham, Braunschweig, Germany) using T4 polynucleotide kinase (Promega, Heidelberg, Germany). Equal amounts of protein were incubated in a 15 L reaction volume containing 10 mmol/L Tris-HCl pH 7.5, 50 mmol/L NaCl, 1 mmol/L EDTA, 5% glycerol, 5 ⫻ 104 cpm radiolabeled oligonucleotide probe, 0.5 mmol/L poly(dIdC), and 0.5 mmol/L DTT for 20 minutes at room temperature. Nucleoprotein-oligonucleotide complexes were resolved by electrophoresis in a 4.5% nondenaturing polyacrylamide gel in 0.25 ⫻ TBE at 100 V. The gel was autoradiographed with an intensifying screen at ⫺70°C overnight. Specificity of the DNA-protein complex was confirmed by competition with a 100-fold excess of unlabeled HSF binding sequences. Signals were detected by autoradiography. Analysis of mRNA in Liver Samples mRNA Extraction and cDNA Synthesis. Total RNA of liver samples was isolated by the guanidinium thiocyanate/cesium chloride method. mRNA purification and reverse transcription were performed as previously described in detail.25 Reverse Transcription Polymerase Chain Reaction. All oligonucleotides were obtained from MWG (Ebersberg, Germany) and were HPLC-purified. For the HSP70 mRNA amplification, the oligonucleotide sequences were as follows: sense, 5⬘-TGCTGACCAAGATGAAG-3⬘; antisense, 5⬘-AGAGTCGATCTCCAGGC-3⬘ according to Longo et al.26; and for rat glyceraldehyde phosphate dehydrogenase (GAPDH), used for normalization, they were as follows: sense, 5⬘-TCCCTCAAGATTGTCAGCAA-3⬘; antisense, 5⬘-AGATCCACAACGGATACATT-3⬘. Reverse transcription polymerase chain reaction (RT-PCR) was performed in principle as described previously.27 Aliquots of RTPCR products were submitted to agarose electrophoresis (2.5% agarose) and stained by ethidium bromide. Signal intensities were evaluated by densitometric analysis (EASY plus system, Herolab, Wiesloch, Germany). Inhibitory factor B Immunoprecipitation and Western Blot Analysis For determination of association of inhibitory factor B (IB) with HSP70, IB immunoprecipitation was performed followed by Western blot detection of HSP70 in the precipitates. Livers were homogenized in ice-cold lysis buffer (containing 137 mmol/L NaCl, 20 mmol/L Tris, 2 mmol/L EDTA, 10% glycerol, 2 mmol/L Na-pyrophosphate, 1% triton, 20 mmol/L Na--glycerophosphate, 10 mmol/L NaF, 1 mmol/L Na-ortho-vanadate, and 1 mmol/L PMSF supplemented with Complete) and protein concentrations were determined by the Pierce assay. Equal amounts of protein were incubated with 3 L of IB antiserum for 2 hours at
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4°C. A total of 15 L of protein A-agarose was added and immunoprecipitation was performed overnight with gentle rocking at 4°C. As controls for IB enrichment, two strategies were used: in one group of organs the primary IB antibody was omitted; in another group, control rabbit IgG was used instead of IB antibody. The agarose conjugates were washed 3 times with lysis buffer. Pelleted conjugates were then mixed with 35 L of sample buffer, heated at 95°C for 5 minutes, and electrophoresed on a 10% sodium dodecyl sulfate polyachrylamide gel electrophoresis (SDS-PAGE) resolving gel. Resolved proteins were electrophoretically transferred to a PVDF membrane (Millipore, Eschwege, Germany) and labeled with primary antibody to HSP70 followed by chemiluminescent detection (NEN Lifescience, Cologne, Germany) with a Kodak Image Station. To control for successful IB immunoprecipitation, blots were also labeled with IB antibody.
Results ANP Protects From Ischemia Reperfusion Injury Preischemic treatment of livers with ANP (200 nmol/L) was previously shown by us to significantly attenuate IRPI.23 To routinely check for tissue protection by ANP throughout this work, sinusoidal efflux rates of LDH were determined and always showed reduced postischemic LDH release in ANP-preconditioned organs compared with control livers (Fig. 1). Activation of HSF by ANP and 8-Br-cGMP The activation of the heat shock transcription factor represents the first step in the regulation of the cytoprotective HSPs. Therefore, an effect of ANP or the respective second messenger analog, 8-Br-cGMP, on HSF DNA binding activities was investigated. Livers were continuously perfused with the respective compounds and HSF activation was determined by electromobility shift assay. Both ANP (200 nmol/L) and 8-Br-cGMP (50 mol/L) markedly elevated HSF binding activities (Fig. 2A and B). With both compounds, the maximal activation was observed at a perfusion time of 30 minutes. After 75 minutes of perfusion, HSF activation in ANP- or 8-Br-cGMP–treated livers were only slightly elevated compared with controls. To control for the specificity of the signal,
Fig. 2. ANP and 8-Br-cGMP activate HSF in continuously perfused rat liver. Livers were continuously perfused through the portal vein with KH-buffer only or in the presence or absence of ANP (200 nmol/L) or 8-Br-cGMP (50 mol/L), which were added after 10 minutes of perfusion. Livers were snap frozen at 10 minutes (Co), 30 minutes, and 75 minutes of perfusion, respectively. Deep-frozen organs were homogenized and assayed for HSF binding activity as described in Materials and Methods. Data show a representative autoradiogram out of 4 independent experiments.
a 100-fold excess of unlabeled DNA-binding sequence was added to the reaction volume, which abrogated the signal (data not shown). HSP70 Protein Expression Parallels HSF Activation During Continuous Perfusion Since both ANP and 8-Br-cGMP activated HSF, the functional significance of this effect regarding HSP70 expression was addressed. Therefore, Western blots were performed, which showed elevated levels of HSP70 at 30 minutes of perfusion (Fig. 3). Preconditioning With ANP Leads to Elevated HSF DNA Binding Activity During Reperfusion To investigate a potential role for ANP-mediated activation of HSF during ischemia and reperfusion, postischemic HSF DNA binding activities were determined. At the end of ischemia, ANPpretreated organs showed markedly elevated HSF activities compared with untreated controls (Fig. 4). In the course of reperfusion, the activation of HSF in ANP-treated livers decreased (Fig. 4).
Fig. 1. ANP attenuates sinusoidal washout of LDH after cold liver preservation. Livers were perfused with KH buffer for 30 minutes in the presence or absence of ANP (200 nmol/L), which was added after 10 minutes of perfusion. After 24 hours of preservation in UW solution, livers were reperfused with KH buffer for 120 minutes. Co, preischemic LDH release.
Increased Postischemic Expression of HSP70 mRNA in ANP-Treated Livers To determine whether increased postischemic HSF DNA binding activity influenced HSP70 gene transcription, HSP70 mRNA
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Fig. 3. ANP and 8-Br-cGMP increase HSP70 in continuously perfused rat liver. Livers perfused either with KH-buffer alone or in the presence or absence of ANP (200 nmol/L) or 8-Br-cGMP (50 mol/L) were snap frozen at 30 minutes of perfusion. Western Blot with HSP70 antibody was performed as described in Materials and Methods. Data show a representative blot of 2 to 4 independent experiments. Bars show densitometric evaluation of signal intensities of all data representing mean ⫾ SEM.
levels were determined by semiquantitative RT-PCR. As shown in Fig. 5, ANP-preconditioning led to a marked increase in HSP70 mRNA levels at both 45 and 120 minutes of reperfusion compared with untreated controls. GAPDH served as control for equal amounts of cDNA used for amplification.
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Fig. 5. Increased postischemic expression of HSP70 mRNA in ANPpretreated livers. Livers were perfused with KH-buffer in the presence or absence of ANP (200 nmol/L) for 30 minutes, stored in UW-solution (4°C, 24 hours ⫾ ANP 200 nmol/L), and reperfused with KH-buffer for 120 minutes. mRNA was isolated from liver samples snap-frozen at the indicated times and RT-PCR for the detection of HSP70 (upper panel) and GAPDH (lower panel) mRNA was performed as described in Materials and Methods. Data show one representative agarose gel stained with ethidium bromide of 2 independent experiments.
levels markedly decrease in the course of reperfusion, whereas ANP-pretreated organs showed constant HSP70 protein levels.
ANP Preconditioning Prevents the Decrease of HSP70 Protein During Reperfusion To investigate whether elevated HSP70 mRNA levels influenced HSP70 protein levels during reperfusion, Western blots were performed. As shown in Fig. 6A, ANP-preconditioned livers showed significantly elevated HSP70 levels compared with untreated control. Densitometric analysis (Fig. 6B) shows that HSP70
Elevated Binding of HSP70 to IB in ANP-Pretreated Livers ANP inhibits the activation of proinflammatory transcription factors, such as NF-B and AP-1 via cGMP.24 The inhibition of these factors during reperfusion was suggested to be responsible for the attenuation of IRPI in the rat liver by preconditioning with ANP. HSP70 was previously shown to inhibit the activation of NF-B by interacting with the inhibitory protein IB. Therefore,
Fig. 4. Elevated postischemic HSF binding acivity in livers preconditioned with ANP. Livers were perfused for 30 minutes with KH-buffer in the presence or absence of ANP (200 nmol/L), stored in cold (4°C) UW-solution (⫾ANP 200 nmol/L) for 24 hours, and reperfused with KH-buffer for 120 minutes. HSF binding activity was assessed in snap-frozen liver samples as described in Materials and Methods. Data show one representative of 2 experiments.
Fig. 6. ANP preconditioning prevents postischemic decrease of HSP70 protein. Livers were perfused with KH-buffer in the presence or absence of ANP (200 nmol/L) for 30 minutes, stored in UW-solution (4°C, 24 hours ⫾ ANP 200 nmol/L), and reperfused with KH-buffer for 120 minutes. Western blot for the detection of HSP70 was performed from liver samples snapfrozen at the indicated times as described in Materials and Methods. The upper panel shows one representative blot of 2 independent experiments. The lower panel shows densitometric evaluation of the Western blot shown in the upper one.
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we investigated whether elevated HSP70 levels induced by preconditioning of livers with ANP, interacted with IB.28,29 IB was immunoprecipitated and an interaction of IB with HSP70 by the consecutive detection of HSP70 was determined in immunoprecipitates by Western blot. First, control experiments were performed showing the specificity of the immunoprecipitation: either the IB antibody was omitted in the precipitation reaction or normal rabbit IgG instead of anti IB antibody was used. In both cases (Fig. 7A and B) no HSP70 signals could be detected. When IB was detected on Western blots after immunoprecipitation with IB antibody, an equal efficiency of immunoprecipitation was shown (Fig. 7C). When Western blots were performed with HSP70 antibody, ANP-pretreated livers showed markedly elevated amounts of HSP70 in IB immunoprecipitates compared with untreated livers (Fig. 7D). Densitometric evaluation showed this effect only at a reperfusion time of 45 minutes but not at 120 minutes of reperfusion (Fig. 7E).
Fig. 7. ANP and 8-Br-cGMP augments interaction of HSP70 and IB during reperfusion. Livers were perfused for 30 minutes with KH-buffer in the presence or absence of ANP (200 nmol/L), stored in cold (4°C) UW-solution (⫾ANP 200 nmol/L) for 24 hours, and reperfused with KH-buffer for 45 or 120 minutes. Immunoprecipitation in liver homogenates with protein A-agarose and IB antibody was performed as described in Materials and Methods followed by Western blot. (A) IB Western blot was performed after immunoprecipitation with IB antibody. (B) Detection of HSP70 in IB immunoprecipitates: Western blot of IB immunoprecipitates was performed with HSP70 antibody. Data show one representative blot out of 4 to 5 independent experiments. (C) Immunoprecipitation experiments were performed in the absence of IB antibody and the following Western blot was performed with HSP70 antibody. Data show one blot with 2 independent samples. (D) Immunoprecipitation was performed with control rabbit IgG instead of IB antibody, followed by HSP70 Western blot analysis. Data show 2 independent experiments. (E) Densitometric evaluation of HSP70 signal intensities form blots performed as described in (C). Data show mean ⫾ SEM of 4 to 5 independent experiments.
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Discussion This study shows that preconditioning of rat livers with ANP induces a heat shock response contributing to the protective effect of this peptide in IRPI. ANP augments HSP70 expression via activation of HSF. Interestingly, ANP preconditioned livers show increased binding of HSP70 to IB. It is hypothesized that this may be the mechanism by which ANP exerts its known decrease of NF-B activation during reperfusion.24 HSPs are considered pivotal for preconditioning by heat shock or hyperthermia. Their protective role was conclusively shown by the protection from cardiac ischemia/reperfusion in transgenic mice overexpressing HSP70.9,10 HSP70 is regulated transcriptionally by the heat shock transcription factor, HSF.12 HSF is normally present in the cytosol in an inactive, monomeric form. On activation HSF forms a trimer that enters the nucleus to bind to the heat shock element located on the promoter region of heat shock genes initiating transcription. Compounds that either direct toxic30 or oxidant31 potential imitate the heat shock response by activating HSF and corresponding transcription of HSPs. However, as yet a suitable pharmacologic induction of the heat shock response in the liver without major side effects has not been reported. Thus, the finding that the peptide hormone ANP as well as its second messenger cGMP activate the heat shock response is novel and may be of clinical relevance. We have shown previously22 that in order to protect against IRPI ANP must be administered (1) before ischemia and (2) for a period of more than 10 minutes. Together with our observation that ANP activation of HSF occurs at 30 minutes of perfusion, this suggests a role of HSF activation in ANP-mediated hepatoprotection. It is worth noting that in control livers HSF binding activity increased during reperfusion, but not at the end of ischemia. In this respect our data are at variance with other observations: Activation of HSF in the liver in the course of ischemia and reperfusion was first described by Tacchini et al.32 All data available on HSF activation in the liver refer to warm ischemia,32-34 where the activation of HSF was induced after a time of ischemia as short as 15 minutes34 with a decrease in DNA binding activity during reperfusion.33 These divergent results most likely are caused by different mechanisms of HSF activation depending on whether warm or cold ischemia is performed. The increased HSF DNA binding activity induced by ANP resulted in increased HSP70 gene expression as was shown by an ANP-mediated increase in HSP70 mRNA and HSP70 protein expression. Interestingly, HSF activation does not always lead to elevated expression of HSP70 mRNA and protein as was shown for HSF activation caused by arsenite, cadmium, or salicylate.35,36 By activating the synthesis of HSP70, ANP pretreatment is able to prevent the decrease of HSP70 protein levels occuring during reperfusion of untreated livers. Although HSP70 is often referred to as the “inducible” heat shock protein,4 it is known to be constitutively expressed in the liver.37 An up-regulation of HSP70 during reperfusion was observed mainly after warm ischemia,38 thereby representing a different pathophysiologic setting. Schutz et al., performing cold ischemia in a pig model, determined HSP70 mRNA expression only without investigating HSP70 protein concentrations.39 Our data
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confirm the up-regulation of HSP70 mRNA levels in the course of reperfusion. Moreover, we show an even further increase of HSP70 mRNA expression in ANP-treated organs. The cGMP-dependent induction of the heat shock response is shown here for the first time. A role of HSP70-mediated cytoprotection induced by cyclic nucleotides, however, has been reported before: Takano et al. showed that another cyclic nucleotide, dibutyryl cAMP, induced the expression of HSP70 in the liver, and thereby protected it from TNF-␣-induced hepatocyte apoptosis.40 Another important observation of our study is that HSP70 binds to the NF-B-regulatory protein IB in ANP-pretreated organs. This leads us to suggest that ANP-mediated activation of the heat shock response is related to the ANP-mediated inhibition of the NF-B system. We previously showed that preconditioning with ANP reduces NF-B DNA binding activity of reperfused livers after 24 hours of ischemia. Furthermore, nuclear translocation of NF-B was attenuated by ANP.24 Our data are in agreement with observations in different cell types, such as hepatocytes,41 astrocytes, microglial cells,21 and islets,16 where HSPs inhibited iNOS expression and NF-B nuclear translocation. HSPs may prevent NF-B activation by either inhibiting IB degradation, inducing IB expression, or both.28 The experiments performed here do not allow discrimination between these two possibilities. However, NF-B activation during reperfusion seems to be independent of IB degradation as shown by us24 and others.42 Regardless of the exact mechanisms, NF-B sequestered in the cytoplasm by HSP is unable to initiate mRNA transcription in the nucleus. HSPs were further shown to decrease the DNA binding activity of AP-1 transcription factor,43 and Gabai et al. showed that heat shock prevents the activation of stress-activated Jun N-terminal kinase, which is essential for regulating the activation of AP-1.44 These observations may suggest that the enhanced HSP70 expression exerted by ANP may also be related to the inhibitory action of ANP on AP-1 activity in reperfused rat liver.24 Taken together, our data suggest that, by mimicking the heat shock response, ANP may lead to selective inhibitory effects on the expression of proinflammatory genes in the course of ischemia and reperfusion. Our current observation points to a complex interplay between the heat shock response and the IB/NF-B pathway, two gene programs that play critical roles during liver injury. We have shown in this report that ANP represents a novel way to induce the heat shock response in the liver. This seems to inhibit the injury induced by ischemia and reperfusion. Thus, our data support the contention that hormonal preconditioning by ANP is a promising approach for preventing ischemia reperfusion injury via the induction of protective mechanisms.
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Acknowledgment: The authors thank Tobias Gerwig for performing liver perfusions. The excellent technical assistance of Ursula Ru¨berg and Raima Yas¸ar is gratefully acknowledged. They thank Dr. Hagedorn, Institute of Pharmacology, Toxicology, and Pharmacy (Munich, Germany) for providing rabbit control IgG.
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21. Feinstein DL, Galea E, Aquino DA, Li GC, Xu H, Reis DJ. Heat shock protein 70 suppresses astroglial-inducible nitric-oxide synthase expression by decreasing NFkappaB activation. J Biol Chem 1996; 271:17724-17732. 22. Bilzer M, Witthaut R, Paumgartner G, Gerbes AL. Prevention of ischemia/reperfusion injury in the rat liver by atrial natriuretic peptide. Gastroenterology 1994;106:143-151. 23. Gerbes AL, Vollmar AM, Kiemer AK, Bilzer M. The guanylate cyclase-coupled natriuretic peptide receptor: a new target for prevention of cold ischemia-reperfusion damage of the rat liver. HEPATOLOGY 1998;28:1309-1317. 24. Kiemer AK, Vollmar AM, Bilzer M, Gerwig T, Gerbes AL. Atrial Natriuretic Peptide reduces expression of TNF-␣ mRNA during reperfusion of the rat liver upon decreased activation of NF-kappaB and AP-1. J Hepatol 2000;33:236-246. 25. Vollmar AM, Schulz R. Gene expression and secretion of atrial natriuretic peptide by murine macrophages. J Clin Invest 1994;94:539-545. 26. Longo FM, Wang S, Narasimhan P, Zhang JS, Chen J, Massa SM, Sharp FR. cDNA cloning and expression of stress-inducible rat hsp70 in normal and injured rat brain. J Neurosci Res 1993;36:325-335. 27. Vollmar AM, Schmidt KN, Schulz R. Natriuretic peptide receptors on rat thymocytes: inhibition of proliferation by atrial natriuretic peptide. Endocrinology 1996;137:1706-1713. 28. Wong HR, Ryan M, Wispe JR. Stress response decreases NF-kappaB nuclear translocation and increases I-kappaBalpha expression in A549 cells. J Clin Invest 1997;99:2423-2428. 29. Uchinami H, Yamamoto Y, Yonezawa K, Kume M. Heat shock preconditioning suppresses the translocation of NF-kappaB to the nucleus during ischemia-reperfusion injury of the rat livers by protecting IkappaB degradation. HEPATOLOGY 1999;30(Suppl):227A. 30. Schiaffonati L, Tiberio L. Gene expression in liver after toxic injury: analysis of heat shock response and oxidative stress-inducible genes. Liver 1997;17:183-191. 31. Tacchini L, Pogliaghi G, Radice L, Anzon E, Bernelli-Zazzera A. Differential activation of heat-shock and oxidation-specific stress genes in chemically induced oxidative stress. Biochem J 1995;309: 453-459. 32. Tacchini L, Schiaffonati L, Pappalardo C, Gatti S, Bernelli-Zazzera A. Expression of HSP 70, immediate-early response and heme oxygenase genes in ischemic-reperfused rat liver. Lab Invest 1993;68: 465-471.
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33. Tacchini L, Radice L, Pogliaghi G, Bernelli-Zazzera A. Differential activation of heat shock and nuclear factor kappaB transcription factors in postischemic reperfused rat liver. HEPATOLOGY 1997;26:186191. 34. Tacchini L, Radice L, Bernelli-Zazzera A. Differential activation of some transcription factors during rat liver ischemia, reperfusion, and heat shock. J Cell Physiol 1999;180:255-262. 35. Liu RY, Corry PM, Lee YJ. Potential involvement of a constitutive heat shock element binding factor in the regulation of chemical stressinduced hsp70 gene expression. Mol Cell Biochem 1995;144:27-34. 36. Jurivich DA, Sistonen L, Kroes RA, Morimoto RI. Effect of sodium salicylate on the human heat shock response. Science 1992;255: 1243-1245. 37. Witzmann F, Clack J, Fultz C, Jarnot B. Two-dimensional electrophoretic mapping of hepatic and renal stress proteins. Electrophoresis 1995;16:451-459. 38. Broughan TA, Jin GF, Papaconstantinou J. Early gene response to hepatic ischemia reperfusion. J Surg Res 1996;63:98-104. 39. Schutz E, Wieland E, Heine L, Hensel A, Schmiedl A, Armstrong VW, Richter J. Acceleration of hepatocellular energy by idebenone during early reperfusion after cold preservation ameliorates heat shock protein 70 gene expression in a pig liver model. Transplantation 1997;64:901-907. 40. Takano M, Arai T, Mokuno Y, Nishimura H, Nimura Y, Yoshikai Y. Dibutyryl cyclic adenosine monophosphate protects mice against tumor necrosis factor-alpha-induced hepatocyte apoptosis accompanied by increased heat shock protein 70 expression. Cell Stress Chaperones 1998;3:109-117. 41. de Vera ME, Kim YM, Wong HR, Wang Q, Billiar TR, Geller Da. Heat shock response inhibits cytokine-inducible nitric oxide synthase expression in rat hepatocytes. HEPATOLOGY 1996;24:1238-1245. 42. Zwacka RM, Zhang Y, Zhou W, Halldorson J, Engelhardt JF. Ischemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and nuclear factor kappaB independently of IkappaB degradation. HEPATOLOGY 1998;28:1022-1030. 43. Carter DA. Modulation of cellular AP-1 DNA binding activity by heat shock proteins. FEBS Lett 1997;416:81-85. 44. Gabai VL, Meriin AB, Mosser DD, Caron AW, Rits S, Shifrin VI, Sherman MV. Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J Biol Chem 1997;272:1803318037.
T
he “heat shock response” is elicited by a variety of stimuli, including thermal, chemical, and physical stress, as well as short-term ischemia. All organisms respond to such detrimental environmental factors by induction of a group of protective proteins termed heat shock proteins (HSP) (see elsewhere for reviews1-4). The HSPs are subdivided into multimember families based on the molecular weights of the proteins encoded (HSP90, HSP70, HSP60, and the small HSP family), of which HSP70 is one of the most extensively studied in mammalian cells.2 Studies have revealed that HSPs, in particular HSP70, not only confer thermotolerance, but also protect against oxygen radical toxicity and seem to play an important role in the protection against ischemia reperfusion injury (IRPI).3,4 Both ischemic5 or heat shock Abbreviations: HSP, heat shock protein; IRPI, ischemia reperfusion injuries; HSF, heat shock transcription factor; NF-B, nuclear factor B; TNF-␣, tumor necrosis factor ␣; ANP, atrial natriuretic peptide; cGMP, cyclic guanosine monophosphate; AP-1, activator protein 1; KH, Krebs-Henseleit solution; UW, University of Wisconsin solution; LDH, lactic dehydrogenase; GAPDH, glyceraldehyde phosphate dehydrogenase; RT-PCR, reverse transcription polymerase chain reaction; IB, inhibitory factor B. From the 1Department of Pharmacy, Center of Drug Research and 2Department of Medicine II, Klinikum Grosshadern, University of Munich, Munich, Germany. Received April 11, 2001; October 1, 2001. Supported by the Deutsche Forschungsgemeinschaft (DFG: Ge 576/14-1). Address reprint requests to: Alexandra K. Kiemer, Ph.D., Department of Pharmacy, Center of Drug Research, Butenandtstr. 5-13, 81377 Munich, Germany. E-mail: [email protected]; fax: (49) 89-2180-7170. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3501-0013$35.00/0 doi:10.1053/jhep.2002.30080 88
preconditioning 6-8 induce HSP70 in the liver and the induced HSP70 may contribute to the attenuation of IRPI. This is supported by findings that overexpression of HSP70 in transgenic mice increases the resistance of the heart to ischemic injury.9,10 HSPs are regulated primarily by changes in gene transcription controlled by a transcription factor termed heat shock factor (HSF).11,12 The activation process of HSF appears to involve HSF oligomerization from a monomeric to a trimeric state and is associated with HSF hyperphosphorylation.13 Activated HSF binds to a DNA consensus sequence in the HSP70 promotor termed the heat shock element,14 leading to the transcription of the HSP70 gene. HSPs seem to protect cells by facilitating the folding of nascent proteins and renaturation or refolding of partially denatured or unfolded proteins.15 In addition, HSPs can also restrict inflammatory response. Several reports have shown the capacity of HSPs to inhibit the translocation of the proinflammatory transcription factor NF-B and the subsequent expression of inflammatory enzymes16 and cytokines, such as tumor necrosis factor ␣ (TNF-␣).1,17-21 Recently we could show that hormonal preconditioning of livers with the atrial natriuretic peptide (ANP) attenuates reperfusion injury after both warm22 and cold23 ischemia of rat livers. The hepatoprotective action of this cardiovascular hormone is mediated via its guanylate-cyclase– coupled A-receptor. Protection conveyed by ANP as well as by the cyclic guanosine monophosphate (cGMP) analogue 8-Br-cGMP involves the attenuated activation of the proinflammatory transcription factors NF-B and activator
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protein 1 (AP-1) and the reduced expression of TNF-␣.24 To elucidate whether the anti-inflammatory action of ANP is related to HSPs, the aim of this study was to investigate a potential influence of ANP on the activation of the heat shock response.
Materials and Methods Materials ratANP 99-126 was purchased from Calbiochem/Novabiochem (Bad Soden, Germany). [␥32P]-ATP (3,000 Ci/mmol) and Hyperfilm MP films were from Amersham Pharmacia (Braunschweig, Germany); Complete was from Boehringer Ingelheim Bioproducts (Heidelberg, Germany). Polyclonal anti-HSP70 and anti-IB␣ rabbit antibodies were purchased from Santa Cruz biotechnology (Heidelberg, Germany). Cell culture medium, fetal calf serum, and penicillin/streptomycine were from Life Technologies (Karlsruhe, Germany). Control rabbit IgG was provided by Dr. Hagedorn, Institute of Pharmacology (Munich, Germany). Protein A-agarose and all other materials were from Sigma (Deisenhofen, Germany). Liver Perfusion Male Sprague-Dawley rats weighing 250 to 300 g were purchased from SAVO (Kisslegg, Germany) and housed in a climatized room with a 12-hour light-dark cycle. The animals had free access to chow (Standard-Diet, Altromin 1314 Lage, Germany) and water up to the time of the experiments. After anesthetizing the animals with pentobarbital (50 mg/kg body weight, intraperitoneally), the portal vein was cannulated and the livers were perfused in situ with hemoglobin-free and albumin-free, bicarbonatebuffered Krebs-Henseleit (KH) solution (pH 7.4, 37°C) gassed with 95% O2 and 5% CO2. The perfusion medium was pumped through the livers with a membrane pump at a constant flow rate of 3.0 to 3.5 mL ⫻ min⫺1 ⫻ g liver⫺1 in a nonrecirculating fashion. After 10 minutes controlling the stability of the system, ANP (200 nmol/L) or 8-Br-cGMP (50 mol/L) was added to the perfusate and perfusion was performed for up to 75 minutes. In a different set of experiments, after 30 minutes of perfusion with KH buffer, livers were perfused with 30 mL of cold (4°C) University of Wisconsin (UW) solution for 1 minute. Then the organs were kept in 150 mL UW solution at 4°C for 24 hours. Pretreatment with ANP or 8-Br-cGMP was performed by adding ANP or 8-Br-cGMP to the preischemic perfusate for 20 minutes until ischemia and to the storage solution at a final concentration of 200 nmol/L for ANP and 50 mol/L for 8-Br-cGMP. Following the period of ischemia, livers of each group were reperfused with KH buffer for 2 hours. At the indicated times, i.e., before ischemia, at the end of ischemia, and after 45 and 120 minutes of reperfusion livers were snapfrozen and stored at ⫺70°C until further analysis. The activity of lactic dehydrogenase (LDH) in perfusate was analyzed as a parameter of cell damage.23 Unless otherwise stated, 4 to 5 independent experiments were performed. All animals received humane care. The study was registered with the local animal welfare committee. Preparation of Cell Extracts and Gel Shift Assay Whole cell extracts were prepared from frozen liver sections as described previously.14 Briefly, tissue samples were homogenized
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in ice-cold buffer containing 20 mmol/L HEPES pH 7.9, 25% glycerol, 0.42 mol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L PMSF, and 0.5 mmol/L DTT with a dounce homogenizer, centrifuged at 100,000g for 10 minutes at 4°C and the supernatant was frozen at ⫺70°C. The protein concentration was estimated after the method of Lowry. Complementary singlestranded DNA oligonucleotides with a binding sequence of 5⬘CTAGAACGTTCTAGAAGCTTCGAG-3⬘ used to bind HSF (synthesized by MWG Biotech, Ebersberg, Germany) were annealed at 54°C. The DNA was 5⬘ end-labeled with [␥32P]-ATP (3000 Ci/mmol; Amersham, Braunschweig, Germany) using T4 polynucleotide kinase (Promega, Heidelberg, Germany). Equal amounts of protein were incubated in a 15 L reaction volume containing 10 mmol/L Tris-HCl pH 7.5, 50 mmol/L NaCl, 1 mmol/L EDTA, 5% glycerol, 5 ⫻ 104 cpm radiolabeled oligonucleotide probe, 0.5 mmol/L poly(dIdC), and 0.5 mmol/L DTT for 20 minutes at room temperature. Nucleoprotein-oligonucleotide complexes were resolved by electrophoresis in a 4.5% nondenaturing polyacrylamide gel in 0.25 ⫻ TBE at 100 V. The gel was autoradiographed with an intensifying screen at ⫺70°C overnight. Specificity of the DNA-protein complex was confirmed by competition with a 100-fold excess of unlabeled HSF binding sequences. Signals were detected by autoradiography. Analysis of mRNA in Liver Samples mRNA Extraction and cDNA Synthesis. Total RNA of liver samples was isolated by the guanidinium thiocyanate/cesium chloride method. mRNA purification and reverse transcription were performed as previously described in detail.25 Reverse Transcription Polymerase Chain Reaction. All oligonucleotides were obtained from MWG (Ebersberg, Germany) and were HPLC-purified. For the HSP70 mRNA amplification, the oligonucleotide sequences were as follows: sense, 5⬘-TGCTGACCAAGATGAAG-3⬘; antisense, 5⬘-AGAGTCGATCTCCAGGC-3⬘ according to Longo et al.26; and for rat glyceraldehyde phosphate dehydrogenase (GAPDH), used for normalization, they were as follows: sense, 5⬘-TCCCTCAAGATTGTCAGCAA-3⬘; antisense, 5⬘-AGATCCACAACGGATACATT-3⬘. Reverse transcription polymerase chain reaction (RT-PCR) was performed in principle as described previously.27 Aliquots of RTPCR products were submitted to agarose electrophoresis (2.5% agarose) and stained by ethidium bromide. Signal intensities were evaluated by densitometric analysis (EASY plus system, Herolab, Wiesloch, Germany). Inhibitory factor B Immunoprecipitation and Western Blot Analysis For determination of association of inhibitory factor B (IB) with HSP70, IB immunoprecipitation was performed followed by Western blot detection of HSP70 in the precipitates. Livers were homogenized in ice-cold lysis buffer (containing 137 mmol/L NaCl, 20 mmol/L Tris, 2 mmol/L EDTA, 10% glycerol, 2 mmol/L Na-pyrophosphate, 1% triton, 20 mmol/L Na--glycerophosphate, 10 mmol/L NaF, 1 mmol/L Na-ortho-vanadate, and 1 mmol/L PMSF supplemented with Complete) and protein concentrations were determined by the Pierce assay. Equal amounts of protein were incubated with 3 L of IB antiserum for 2 hours at
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4°C. A total of 15 L of protein A-agarose was added and immunoprecipitation was performed overnight with gentle rocking at 4°C. As controls for IB enrichment, two strategies were used: in one group of organs the primary IB antibody was omitted; in another group, control rabbit IgG was used instead of IB antibody. The agarose conjugates were washed 3 times with lysis buffer. Pelleted conjugates were then mixed with 35 L of sample buffer, heated at 95°C for 5 minutes, and electrophoresed on a 10% sodium dodecyl sulfate polyachrylamide gel electrophoresis (SDS-PAGE) resolving gel. Resolved proteins were electrophoretically transferred to a PVDF membrane (Millipore, Eschwege, Germany) and labeled with primary antibody to HSP70 followed by chemiluminescent detection (NEN Lifescience, Cologne, Germany) with a Kodak Image Station. To control for successful IB immunoprecipitation, blots were also labeled with IB antibody.
Results ANP Protects From Ischemia Reperfusion Injury Preischemic treatment of livers with ANP (200 nmol/L) was previously shown by us to significantly attenuate IRPI.23 To routinely check for tissue protection by ANP throughout this work, sinusoidal efflux rates of LDH were determined and always showed reduced postischemic LDH release in ANP-preconditioned organs compared with control livers (Fig. 1). Activation of HSF by ANP and 8-Br-cGMP The activation of the heat shock transcription factor represents the first step in the regulation of the cytoprotective HSPs. Therefore, an effect of ANP or the respective second messenger analog, 8-Br-cGMP, on HSF DNA binding activities was investigated. Livers were continuously perfused with the respective compounds and HSF activation was determined by electromobility shift assay. Both ANP (200 nmol/L) and 8-Br-cGMP (50 mol/L) markedly elevated HSF binding activities (Fig. 2A and B). With both compounds, the maximal activation was observed at a perfusion time of 30 minutes. After 75 minutes of perfusion, HSF activation in ANP- or 8-Br-cGMP–treated livers were only slightly elevated compared with controls. To control for the specificity of the signal,
Fig. 2. ANP and 8-Br-cGMP activate HSF in continuously perfused rat liver. Livers were continuously perfused through the portal vein with KH-buffer only or in the presence or absence of ANP (200 nmol/L) or 8-Br-cGMP (50 mol/L), which were added after 10 minutes of perfusion. Livers were snap frozen at 10 minutes (Co), 30 minutes, and 75 minutes of perfusion, respectively. Deep-frozen organs were homogenized and assayed for HSF binding activity as described in Materials and Methods. Data show a representative autoradiogram out of 4 independent experiments.
a 100-fold excess of unlabeled DNA-binding sequence was added to the reaction volume, which abrogated the signal (data not shown). HSP70 Protein Expression Parallels HSF Activation During Continuous Perfusion Since both ANP and 8-Br-cGMP activated HSF, the functional significance of this effect regarding HSP70 expression was addressed. Therefore, Western blots were performed, which showed elevated levels of HSP70 at 30 minutes of perfusion (Fig. 3). Preconditioning With ANP Leads to Elevated HSF DNA Binding Activity During Reperfusion To investigate a potential role for ANP-mediated activation of HSF during ischemia and reperfusion, postischemic HSF DNA binding activities were determined. At the end of ischemia, ANPpretreated organs showed markedly elevated HSF activities compared with untreated controls (Fig. 4). In the course of reperfusion, the activation of HSF in ANP-treated livers decreased (Fig. 4).
Fig. 1. ANP attenuates sinusoidal washout of LDH after cold liver preservation. Livers were perfused with KH buffer for 30 minutes in the presence or absence of ANP (200 nmol/L), which was added after 10 minutes of perfusion. After 24 hours of preservation in UW solution, livers were reperfused with KH buffer for 120 minutes. Co, preischemic LDH release.
Increased Postischemic Expression of HSP70 mRNA in ANP-Treated Livers To determine whether increased postischemic HSF DNA binding activity influenced HSP70 gene transcription, HSP70 mRNA
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Fig. 3. ANP and 8-Br-cGMP increase HSP70 in continuously perfused rat liver. Livers perfused either with KH-buffer alone or in the presence or absence of ANP (200 nmol/L) or 8-Br-cGMP (50 mol/L) were snap frozen at 30 minutes of perfusion. Western Blot with HSP70 antibody was performed as described in Materials and Methods. Data show a representative blot of 2 to 4 independent experiments. Bars show densitometric evaluation of signal intensities of all data representing mean ⫾ SEM.
levels were determined by semiquantitative RT-PCR. As shown in Fig. 5, ANP-preconditioning led to a marked increase in HSP70 mRNA levels at both 45 and 120 minutes of reperfusion compared with untreated controls. GAPDH served as control for equal amounts of cDNA used for amplification.
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Fig. 5. Increased postischemic expression of HSP70 mRNA in ANPpretreated livers. Livers were perfused with KH-buffer in the presence or absence of ANP (200 nmol/L) for 30 minutes, stored in UW-solution (4°C, 24 hours ⫾ ANP 200 nmol/L), and reperfused with KH-buffer for 120 minutes. mRNA was isolated from liver samples snap-frozen at the indicated times and RT-PCR for the detection of HSP70 (upper panel) and GAPDH (lower panel) mRNA was performed as described in Materials and Methods. Data show one representative agarose gel stained with ethidium bromide of 2 independent experiments.
levels markedly decrease in the course of reperfusion, whereas ANP-pretreated organs showed constant HSP70 protein levels.
ANP Preconditioning Prevents the Decrease of HSP70 Protein During Reperfusion To investigate whether elevated HSP70 mRNA levels influenced HSP70 protein levels during reperfusion, Western blots were performed. As shown in Fig. 6A, ANP-preconditioned livers showed significantly elevated HSP70 levels compared with untreated control. Densitometric analysis (Fig. 6B) shows that HSP70
Elevated Binding of HSP70 to IB in ANP-Pretreated Livers ANP inhibits the activation of proinflammatory transcription factors, such as NF-B and AP-1 via cGMP.24 The inhibition of these factors during reperfusion was suggested to be responsible for the attenuation of IRPI in the rat liver by preconditioning with ANP. HSP70 was previously shown to inhibit the activation of NF-B by interacting with the inhibitory protein IB. Therefore,
Fig. 4. Elevated postischemic HSF binding acivity in livers preconditioned with ANP. Livers were perfused for 30 minutes with KH-buffer in the presence or absence of ANP (200 nmol/L), stored in cold (4°C) UW-solution (⫾ANP 200 nmol/L) for 24 hours, and reperfused with KH-buffer for 120 minutes. HSF binding activity was assessed in snap-frozen liver samples as described in Materials and Methods. Data show one representative of 2 experiments.
Fig. 6. ANP preconditioning prevents postischemic decrease of HSP70 protein. Livers were perfused with KH-buffer in the presence or absence of ANP (200 nmol/L) for 30 minutes, stored in UW-solution (4°C, 24 hours ⫾ ANP 200 nmol/L), and reperfused with KH-buffer for 120 minutes. Western blot for the detection of HSP70 was performed from liver samples snapfrozen at the indicated times as described in Materials and Methods. The upper panel shows one representative blot of 2 independent experiments. The lower panel shows densitometric evaluation of the Western blot shown in the upper one.
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we investigated whether elevated HSP70 levels induced by preconditioning of livers with ANP, interacted with IB.28,29 IB was immunoprecipitated and an interaction of IB with HSP70 by the consecutive detection of HSP70 was determined in immunoprecipitates by Western blot. First, control experiments were performed showing the specificity of the immunoprecipitation: either the IB antibody was omitted in the precipitation reaction or normal rabbit IgG instead of anti IB antibody was used. In both cases (Fig. 7A and B) no HSP70 signals could be detected. When IB was detected on Western blots after immunoprecipitation with IB antibody, an equal efficiency of immunoprecipitation was shown (Fig. 7C). When Western blots were performed with HSP70 antibody, ANP-pretreated livers showed markedly elevated amounts of HSP70 in IB immunoprecipitates compared with untreated livers (Fig. 7D). Densitometric evaluation showed this effect only at a reperfusion time of 45 minutes but not at 120 minutes of reperfusion (Fig. 7E).
Fig. 7. ANP and 8-Br-cGMP augments interaction of HSP70 and IB during reperfusion. Livers were perfused for 30 minutes with KH-buffer in the presence or absence of ANP (200 nmol/L), stored in cold (4°C) UW-solution (⫾ANP 200 nmol/L) for 24 hours, and reperfused with KH-buffer for 45 or 120 minutes. Immunoprecipitation in liver homogenates with protein A-agarose and IB antibody was performed as described in Materials and Methods followed by Western blot. (A) IB Western blot was performed after immunoprecipitation with IB antibody. (B) Detection of HSP70 in IB immunoprecipitates: Western blot of IB immunoprecipitates was performed with HSP70 antibody. Data show one representative blot out of 4 to 5 independent experiments. (C) Immunoprecipitation experiments were performed in the absence of IB antibody and the following Western blot was performed with HSP70 antibody. Data show one blot with 2 independent samples. (D) Immunoprecipitation was performed with control rabbit IgG instead of IB antibody, followed by HSP70 Western blot analysis. Data show 2 independent experiments. (E) Densitometric evaluation of HSP70 signal intensities form blots performed as described in (C). Data show mean ⫾ SEM of 4 to 5 independent experiments.
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Discussion This study shows that preconditioning of rat livers with ANP induces a heat shock response contributing to the protective effect of this peptide in IRPI. ANP augments HSP70 expression via activation of HSF. Interestingly, ANP preconditioned livers show increased binding of HSP70 to IB. It is hypothesized that this may be the mechanism by which ANP exerts its known decrease of NF-B activation during reperfusion.24 HSPs are considered pivotal for preconditioning by heat shock or hyperthermia. Their protective role was conclusively shown by the protection from cardiac ischemia/reperfusion in transgenic mice overexpressing HSP70.9,10 HSP70 is regulated transcriptionally by the heat shock transcription factor, HSF.12 HSF is normally present in the cytosol in an inactive, monomeric form. On activation HSF forms a trimer that enters the nucleus to bind to the heat shock element located on the promoter region of heat shock genes initiating transcription. Compounds that either direct toxic30 or oxidant31 potential imitate the heat shock response by activating HSF and corresponding transcription of HSPs. However, as yet a suitable pharmacologic induction of the heat shock response in the liver without major side effects has not been reported. Thus, the finding that the peptide hormone ANP as well as its second messenger cGMP activate the heat shock response is novel and may be of clinical relevance. We have shown previously22 that in order to protect against IRPI ANP must be administered (1) before ischemia and (2) for a period of more than 10 minutes. Together with our observation that ANP activation of HSF occurs at 30 minutes of perfusion, this suggests a role of HSF activation in ANP-mediated hepatoprotection. It is worth noting that in control livers HSF binding activity increased during reperfusion, but not at the end of ischemia. In this respect our data are at variance with other observations: Activation of HSF in the liver in the course of ischemia and reperfusion was first described by Tacchini et al.32 All data available on HSF activation in the liver refer to warm ischemia,32-34 where the activation of HSF was induced after a time of ischemia as short as 15 minutes34 with a decrease in DNA binding activity during reperfusion.33 These divergent results most likely are caused by different mechanisms of HSF activation depending on whether warm or cold ischemia is performed. The increased HSF DNA binding activity induced by ANP resulted in increased HSP70 gene expression as was shown by an ANP-mediated increase in HSP70 mRNA and HSP70 protein expression. Interestingly, HSF activation does not always lead to elevated expression of HSP70 mRNA and protein as was shown for HSF activation caused by arsenite, cadmium, or salicylate.35,36 By activating the synthesis of HSP70, ANP pretreatment is able to prevent the decrease of HSP70 protein levels occuring during reperfusion of untreated livers. Although HSP70 is often referred to as the “inducible” heat shock protein,4 it is known to be constitutively expressed in the liver.37 An up-regulation of HSP70 during reperfusion was observed mainly after warm ischemia,38 thereby representing a different pathophysiologic setting. Schutz et al., performing cold ischemia in a pig model, determined HSP70 mRNA expression only without investigating HSP70 protein concentrations.39 Our data
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confirm the up-regulation of HSP70 mRNA levels in the course of reperfusion. Moreover, we show an even further increase of HSP70 mRNA expression in ANP-treated organs. The cGMP-dependent induction of the heat shock response is shown here for the first time. A role of HSP70-mediated cytoprotection induced by cyclic nucleotides, however, has been reported before: Takano et al. showed that another cyclic nucleotide, dibutyryl cAMP, induced the expression of HSP70 in the liver, and thereby protected it from TNF-␣-induced hepatocyte apoptosis.40 Another important observation of our study is that HSP70 binds to the NF-B-regulatory protein IB in ANP-pretreated organs. This leads us to suggest that ANP-mediated activation of the heat shock response is related to the ANP-mediated inhibition of the NF-B system. We previously showed that preconditioning with ANP reduces NF-B DNA binding activity of reperfused livers after 24 hours of ischemia. Furthermore, nuclear translocation of NF-B was attenuated by ANP.24 Our data are in agreement with observations in different cell types, such as hepatocytes,41 astrocytes, microglial cells,21 and islets,16 where HSPs inhibited iNOS expression and NF-B nuclear translocation. HSPs may prevent NF-B activation by either inhibiting IB degradation, inducing IB expression, or both.28 The experiments performed here do not allow discrimination between these two possibilities. However, NF-B activation during reperfusion seems to be independent of IB degradation as shown by us24 and others.42 Regardless of the exact mechanisms, NF-B sequestered in the cytoplasm by HSP is unable to initiate mRNA transcription in the nucleus. HSPs were further shown to decrease the DNA binding activity of AP-1 transcription factor,43 and Gabai et al. showed that heat shock prevents the activation of stress-activated Jun N-terminal kinase, which is essential for regulating the activation of AP-1.44 These observations may suggest that the enhanced HSP70 expression exerted by ANP may also be related to the inhibitory action of ANP on AP-1 activity in reperfused rat liver.24 Taken together, our data suggest that, by mimicking the heat shock response, ANP may lead to selective inhibitory effects on the expression of proinflammatory genes in the course of ischemia and reperfusion. Our current observation points to a complex interplay between the heat shock response and the IB/NF-B pathway, two gene programs that play critical roles during liver injury. We have shown in this report that ANP represents a novel way to induce the heat shock response in the liver. This seems to inhibit the injury induced by ischemia and reperfusion. Thus, our data support the contention that hormonal preconditioning by ANP is a promising approach for preventing ischemia reperfusion injury via the induction of protective mechanisms.
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12. 13. 14.
15. 16.
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Acknowledgment: The authors thank Tobias Gerwig for performing liver perfusions. The excellent technical assistance of Ursula Ru¨berg and Raima Yas¸ar is gratefully acknowledged. They thank Dr. Hagedorn, Institute of Pharmacology, Toxicology, and Pharmacy (Munich, Germany) for providing rabbit control IgG.
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