Toponomics Analysis of Drug-Induced Changes in Arachidonic Acid

Aug 24, 2009 - Thus, we show for the first time that monitoring protein network profiles by topological proteomic analysis is a useful tool to identif...
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Toponomics Analysis of Drug-Induced Changes in Arachidonic Acid-Dependent Signaling Pathways during Spinal Nociceptive Processing Bona Linke,† Sandra Pierre,† Ovidiu Coste,† Carlo Angioni,† Wiebke Becker,† Thorsten Ju ¨ rgen Maier,‡ Dieter Steinhilber,‡ Claus Wittpoth,§ Gerd Geisslinger,† and Klaus Scholich*,† Pharmazentrum Frankfurt, ZAFES, Institute of Clinical Pharmacology, Klinikum der Goethe-Universita¨t Frankfurt, Germany, Institute of Pharmaceutical Chemistry, Goethe-Universita¨t Frankfurt, Germany, and MelTec GmbH&Co KG, Magdeburg, Germany Received February 9, 2009

Abstract: Multi-Epitope-Ligand-Carthography (MELC) allows consecutive immunohistochemical visualization of up to 100 proteins on the same tissue sample. Subsequent biomathematical analysis of these images allows a quantitative description of changes in protein networks. We used the MELC technology to study the effect of the nonopioid analgesic drug dipyrone on protein network profiles associated with arachidonic acid-dependent signaling pathways. MELC analysis with 31 different fluorescence-labeled tags was used to compare the effect of dipyrone on protein networks in spinal cords of mice with zymosan-induced hyperalgesia, a common model for inflammatory pain. We found that the number of motifs which describe the colocalization of 5-lipoxygenase (5LO) or 12-LO with other proteins increased disproportionally after dipyrone treatment. Activation of 5-LO and 12LO induces their translocation to membrane compartments which was also reflected by MELC results. Although no changes in 5-LO or 12-LO expression were seen by Western blot analysis or by immunohistochemistry in spinal cords of dipyrone-treated mice, the activation of both enzymes was verified by determining LO-products. Spinal amounts of 5(S)-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, which are generated by 5-LO and 12-LO, respectively, were significantly increased in spinal cords of dipyrone-treated animals. In primary spinal cord neurons, dipyrone selectively and dose-dependently increased 5(S)-(HETE) and 12(S)-HETE synthesis. Thus, we show for the first time that monitoring protein network profiles by topological proteomic analysis is a useful tool to identify mechanisms of drug actions. * To whom correspondence should be addressed. Klaus Scholich Pharmazentrum Frankfurt, ZAFES, Institute of Clinical Pharmacology, Klinikum der Goethe-Universita¨t Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt, Germany. Tel.: 49-69-6301-83103. Fax: 49-69-6301-83378. E-mail: scholich@ em.uni-frankfurt.de. † Institute of Clinical Pharmacology, Klinikum der Goethe-Universita¨t Frankfurt. ‡ Institute of Pharmaceutical Chemistry, Goethe-Universita¨t Frankfurt. § MelTec GmbH&Co KG. 10.1021/pr900106v CCC: $40.75

 2009 American Chemical Society

Keywords: Toponome • protein networks • immunohistochemistry • dipyrone • lipoxygenase • arachidonic acid • pain • spinal cord

Introduction Multi-Epitope-Ligand-Carthography (MELC) is a novel imaging technology which utilizes biomathematical tools to identify and quantify protein complexes after consecutive immunohistochemical visualization of up to 100 proteins on the same sample.1,2 The detection of targets is achieved by using fluorescence-labeled tag libraries that may comprise antibodies, lectins, proteins or toxins. After application and visualization of each tag, the dye is bleached and relabeling of the same sample with the next tag occurs. The different colocalization combinations of the tags are described as “combinatorial molecular phenotypes (CMP)” and are used to describe statistical changes between different treatment groups.3-6 On the basis of this statistical analysis, it is possible to quantitatively describe changes in the composition and distribution of protein complexes in an intact tissue sample. Thereby new insights in the protein interactions and protein network organization, referred to as the toponome,1,2 are generated. The advantage of the MELC system over other common proteomic tools is based on the fact that analysis of protein networks occurs in samples with an intact cell or tissue structure. The usefulness of this technology to describe qualitative changes regarding immune cells during inflammatory skin and bowel diseases as well as colorectal cancer has already been demonstrated.3-5 More recently, we showed that the quantitative analysis of CMPs in the MELC system allows the identification of changes on the subcellular level in tissue samples. Using an animal model for acute and inflammatory pain, we detected and verified rare and intermittent activity-dependent changes at spinal cords synapses.6 Here, we aimed to investigate whether the MELC technology can be used as screening tool to study the influence of drug treatment on protein networks in an in vivo setting using dipyrone as example. Dipyrone is a potent nonopioid analgesic and antipyretic drug that has been used clinically for more than 100 years. Over the last 20 years, several groups have demonstrated that dipyrone inhibits in vitro7,8 and in vivo9,10 the activity of cyclooxygenases (COX). COX converts arachidonic Journal of Proteome Research 2009, 8, 4851–4859 4851 Published on Web 08/24/2009

technical notes acid to PGH2 which serves as substrate for terminal synthases that generate, for example, prostaglandin E2 (PGE2), which is an important mediator in pain and inflammation.11 However, COX-inhibition does not sufficiently explain the clinical effects of dipyrone, since COX-inhibition after application of high doses of dipyrone is seen only in the plasma but not in the central nervous system.10,12 To date, alternative mechanisms of dipyrone actions remain elusive. Since dipyrone inhibits COX activity through an iron-dependent mechanism resulting in the sequestration of radicals that are necessary to initiate the catalytic cycle of COX,13 other iron-containing enzymes might also be affected in their activity and contribute to the clinical effects of dipyrone. We performed MELC immunofluorescence microscopy on spinal cords slices with 31 fluorescence-labeled tags recognizing proteins related to the arachidonic acid metabolism and signaling. Dipyrone-induced alterations in the toponome were monitored during zymosan-induced inflammation, a common model for inflammatory pain. Batch analysis of motifs which describe differences between untreated and dipyrone-treated animals identified an unproportional increase in the number of motifs containing 5-lipoxygenase (LO) and 12-LO. The activation of both enzymes is connected with a translocation from the cytoplasm to membranes suggesting that the increased motif number reflects their translocation/activation. Indeed, dipyrone increased dose-dependently 5-LO and 12LO products in vivo as well as in a cellular model. Thus, these data show for the first time the usefulness of monitoring protein networks using the MELC system as a novel tool that uses intact tissues to unravel mechanisms of action of drugs.

Materials and Methods Animals. BL6 mice (20-25 g) were supplied by Charles River Wiga GmbH (Sulzfeld, Germany). In all experiments, the ethics guidelines for investigations in conscious animals were obeyed and the procedures were approved by the local Ethics Committee. Sample Preparation. Twenty microliters of zymosan (12.5 mg/mL in phosphate buffered saline) was injected subcutaneously into the dorsal surface of one hindpaw of adult mice and lumbar spinal cords (L4-L5) were excised after 24 h. Dipyrone (0.2 g/kg ip) was given immediately before zymosan injection and 21 h after the injection. Spinal cords were snap-frozen in liquid nitrogen and stored at -80 °C. After the spinal cords were embedded in Tissue-Tek (Sakura Finetek, Leiden, Belgium), cryosections of 10 µm thickness were sliced using the cryotome Frigotom 2800 (-30 °C; Leica, Wetzlar, Germany) and applied on silane-coated coverslips. After fixation of the tissue with acetone for 10 s at room temperature, the coverslips were stored at -20 °C. In preparation for the MELC procedure, the tissue was fixed once again with acetone for 10 min at -20 °C. Afterward, the sample was rehydrated with Dulbecco’s PBS (PAA Laboratories, Pasching, Austria) and nonspecific signals were blocked with normal goat serum (PAA Laboratories, diluted 1/15 with PBS) for 30 min at room temperature. After the sample were rinsed five times with PBS, analysis of the tissue with the MELC technique was started. MELC Library. We used a MELC library of 31 fluorescence tags (Table 1 and Table S1 in Supporting Information). Table S1 gives a complete list of the abbreviations used. The appropriate working dilutions, incubation time (15 min), and positions within the MELC run had been established and validated in the course of systematic experiments based on 4852

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Linke et al. conventional immunohistochemistry and MELC calibration runs.1,2 EP1 and EP2 antibodies were from Morath et al.14 and the CRP-2 antibody was from Schmidtko et al.15 Data Acquisition by Toponome Imaging Cycler MultiEpitope Readout. For each condition (untreated as well as 24 h after zymosan injection with or without dipyrone treatment), 3 animals were prepared and at least two spinal cord slices for each animal were used. The MELC technology has been described previously.1,2 Briefly, a slide with the spinal cord slice was placed on the stage of an inverted wide-field fluorescence microscope (Leica DM IRE2; ×63 oil lens NA 1.32). By a robotic process, first the slices were incubated for 15 min with predetermined fluorescence tags and rinsed with wash solution. Afterward, the phase contrast and fluorescence signals were imaged by a cooled charge-coupled device camera (Apogee KX4; Apogee Instruments, Roseville, CA, 2× binning results in images of 1024 × 1024 pixels; final pixel size was 286 × 286 nm2). To delete the specific signal of the given tag before addition of the next, a bleaching step was performed. A postbleaching image was recorded and subtracted from the following fluorescence tag image during the data analysis. Performance of the first three MELC cycles regarding intrinsic fluorescence and unspecific tag binding was controlled with PBS and fluorochrome-labeled mouse IgG. Pipetting, recording of all image data, and coordinating all system components were controlled by software developed by MelTec (Magdeburg, Germany). Three visual fields were recorded simultaneously in each MELC run. Data Analysis. With the use of the corresponding phase contrast images, fluorescence images produced by each tag were aligned pixel-wise. The alignment reached a resolution of (1 pixel. Images were corrected for illumination faults using flat-field correction. Postbleaching images were subtracted from the following fluorescence tag images. Finally, cases of section artifacts were excluded as invalid by a mask-setting process. Preprocessed image data were subjected to binarization. The thresholds automatically generated by the system were validated and adjusted manually for each fluorescence signal. The expression of a protein was set to the value of zero for a signal below the threshold and to 1 for a signal above the threshold in projection to a pixel. Superimposed binarized images composed a matrix of CMPs which represented a binary (yes/no) code of n epitope expression in relation to each pixel (286 × 268 nm2) of a visual field (1024 × 1024 pixels). Thus, this MELC approach detected a theoretical maximum of as high as 2n different CMPs. Further analysis dealt with CMP motifs characterizing corresponding pixels. These CMP motifs are defined as pixel-related code of one/zero/wildcard ciphering. We used TopoMiner software packages (MelTec, Magdeburg, Germany) to search for CMP motifs, whose overall frequency differs significantly in two different sample groups using the Wilcoxon rank-sum test. In detail, TopoMiner calculated the relative frequency of CMP motifs in relation to the number of all valid pixels of the observed visual field or to the frequency of predefined CMP motifs (base motifs). The search through the space of motifs was performed in a sequentially ordered strategy: all single epitopes, combinations of two, three, four, and so on (n ) epitopes) were searched. Because of the highly organized system, the CMP-motifs are strongly dependent upon each other. For example, a CMP-motif of two proteins which shows a significant difference between two groups may have a large number of CMP-motifs which consists of one extra protein which also show the difference between two groups.

technical notes

Toponomics of Drug-Induced Changes in the Spinal Cord Table 1. MELC Antibody and Fluorescence Tag Library antibody/tag

Cyclooxygenase-1 (COX-1) Cyclooxygenase-2 (COX-2) Cytosolic PGE2 synthase (cPGES) Microsomal PGE2 synthase-1 (mPGES-1) 5-Lipoxygenase (5-LO) 12-Lipoxygenase (12-LO) 15-Lipoxygenase (15-LO) PGD2 synthase (PGD2S)

source

Arachidonic Acid Metabolism rabbit rabbit rabbit rabbit rabbit rabbit sheep goat Eicosanoid Receptors rabbit rabbit rabbit rabbit goat goat goat

E-prostanoid receptor-1 (EP1) E-prostanoid-2 (EP2) F-prostanoid receptor (FP) D-prostanoid receptor-1 (DP) TWIK-related K+ channel-1 (TREK-1) TWIK-related K+ channel-1(TREK-2) TWIK-related AA-activated K+channel (TRAAK) Isolectin B4 (IB4) Calcitonin gene related protein (CGRP) Neuronal nuclei (NeuN) Synapsin I Syntaxin1A Cysteine rich protein-2 (CRP-2) Glutamate receptor 2 (GluR2-3) NMDA receptor 2 (NR2B) Post synaptic density protein 95 (PSD95) shank Cellular src (c-src)

Synaptic/Neuronal Markers lectin rabbit mouse rabbit rabbit rabbit rabbit rabbit mouse goat rabbit

Glial fibrillar acid protein (GFAP) Binding immunoglobulin protein (BiP) 7-Aminoactinomycin (7-AAD) ATP synthase lysosome-associated membrane protein (Lamp1)

Cellular Markers mouse rabbit

These CMP motifs are called “Child”-motifs and the related two protein motif “Parent” motif. The Topominer software package implements an algorithm which analyzes this relationship between parent and child motifs in order to reduce the number of resulting CMP-motifs. Because of the large number of possible motifs, and of restricted computational possibilities, the search depth was limited to n ) 5 and to a significance level of P e 0.005 in the Wilcoxon rank-sum test. Preparation of Tissue Extracts. Lumbal spinal cord (L4-L5) of adult mice were taken, homogenized, sonicated in Tris buffer (pH 7.4) and used for Western blots. To determine Calcitonin Gene-related Peptide (CGRP) concentrations, the homogenates were subjected to centrifugation at 10 000g. CGRP concentrations in murine spinal cord homogenates were determined using the CGRP (human) EIA Kit from Cayman Chemical (Ann Arbor, MI). Primary Spinal Cord Cells. Primary spinal cord cells were prepared from embryos of pregnant rats 16 days postcoitus as described previously.16 Stimulation of neuronal cells was done between day 5 and 6 after preparation. A total of 1 µg/mL lipopolysaccharide (LPS) from Escherichia coli (Sigma-Aldrich, St. Louis, MO) was added for 16 h. Where indicated, 2 µM calcium-ionophore A23187 and increasing concentrations of

mouse rabbit

distributor

order number

Cayman Cayman Cayman Cayman Cayman Cayman Cayman Santa Cruz

160109 160126 160150 160140 160402 160304 160704 sc-14825

Morath et al.14 Morath et al.14 Brenneis et al.17 Cayman Santa Cruz Santa Cruz Santa Cruz

101640 sc-11557 sc-11559 sc-11324

Sigma Chemicon Chemicon MelTec Synaptic Systems Schmidtko et al.15 Upstate Upstate Antibodies Inc. Santa Cruz MelTec

L2140 AB5920 MAB377 mel-752 110 302 06-307 06-600 73-028 sc-23543 mel-687

Dianova BioReagents Calbiochem Upstate MelTec

DLN-07655 PA1-014 129935 05-709 mel-712

dipyrone (a friendly gift from Sanofi-Aventis, Frankfurt, Germany) were given for 4 h to the cells. Liquid Chromatography Tandem Mass Spectroscopy (LC-MS/MS) Detection of Eicosanoids. The LC/MS-MS system comprised an API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Darmstadt, Germany), equipped with a Turbo-V-source operating in negative ESI mode, an Agilent 1100 binary HPLC pump and degasser (Agilent, Waldbronn, Germany), and an HTC Pal autosampler (Chromtech, Idstein, Germany) fitted with a 25 µL LEAP syringe (Axel Semrau GmbH, Sprockho¨vel, Germany). Sample extraction and measurements were performed as published previously17 and is described in detail in the Supplementary Methods. Expression and Purification of 5-LO from E. coli. 5-LO was expressed in E. coli Bl21 (DE3) cells and transformed with pT35LO, and purification of 5-LO was performed as described previously.18 In brief, E. coli were harvested and lysed in 50 mM triethanolamine/HCl, pH 8.0, 5 mM EDTA, soybean trypsin inhibitor (60 µg mL-1), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT and lysozyme (1 mg/mL), homogenized by sonication (6 × 6 s) and centrifuged at 10 000g for 15 min followed by centrifugation at 100 000g for 70 min at 4 °C. The supernatant was then applied to an ATP-agarose column (Sigma A2767; Deisenhofen, Germany), and the column was Journal of Proteome Research • Vol. 8, No. 10, 2009 4853

technical notes

Linke et al. 19

eluted as described previously. Partially purified 5-LO was immediately used for in vitro activity assays. Determination of Product Formation of Recombinant 5-LO. For determination of the activity of recombinant 5-LO, partially purified 5-LO (0.5 µg) was added to 1 mL of a 5-LO reaction mix (PBS, pH 7.4, 1 mM EDTA, 1 mM ATP). After incubation with the test compounds or vehicle (DMSO) for 15 min at 4 °C, samples were prewarmed for 30 s at 37 °C and 2 mM CaCl2 and 20 µM AA were added. The reaction was stopped after 10 min by the addition of 1 mL of ice-cold methanol and 5-LO products formed including 5-HETE, trans-LTB4 and epitrans LTB4 were analyzed by HPLC as described previously.20 Arachidonic acid (AA), BWA4C and dipyrone were purchased from Sigma Aldrich (Munich, Germany).

Results Here, we aimed to evaluate the MELC technology for its use to identify drug mechanisms by monitoring drug-induced toponomic changes. As example we chose the analgesic drug dipyrone that is known to affect arachidonic acid-dependent signaling pathways.13 First, we established a library of 31 fluorescence-tags that recognize proteins involved in arachidonic acid metabolism as well as cell and tissue markers by classical immunohistochemistry and in several independent MELC runs (Table 1, Supplementary Data S1 and S2). We used zymosan-induced hyperalgesia, a commonly used model for inflammatory pain that depends on sensitization processes in the spinal cord, and compared spinal cords from untreated mice with spinal cords that were removed 24 h after zymosan injection in one hind paw. Additionally, zymosan-treated mice were included in the analysis which received dipyrone (0.2 g/kg ip) 24 and 3 h before removal of the lumbar potion (L4-5) of the spinal cord. The expression of the 31 fluorescence-tags was mapped in the three groups (untreated and 24 h after zymosan injection with or without dipyrone treatment) using 2 slices from 3 different animals per group. Statistical evaluation was performed for all possible colocalization combinations (named combinatorial molecular phenotype (CMP)) that contained, out of computational reasons, a maximum of 5 different proteins. The statistical calculation was set to identify all CMPs that either increase or decrease in their relative frequency between two of the treatment groups with a statistical significance of P e 0.005 using the Wilcoxon rank-sum test. We identified 564 CMP motifs that were distinct between animals without treatment and animals undergoing the zymosan-treatment. A total of 2230 CMP motifs distinguished the spinal cords of untreated mice and mice undergoing concomitant dipyrone and zymosan-treatment (Table 2). Since current methods and resources limit the validation of all motifs by behavioral, cell biological or biochemical methods, we investigated the possibility to use a batch analysis of the motifs to extract information about drug actions. Therefore, we calculated for each protein the percentage of motifs containing this protein in each comparison group (control vs zymosan, control vs dipyrone/zymosan and zymosan vs dipyrone/zymosan). For most of the proteins, the percentage of motifs was fairly constant (Table 2). However, the frequencies of colocalization motifs of 6 proteins increased or decreased by more than 10%. These proteins were the arachidonic acid-metabolizing enzymes 5-LO and 12-LO, the arachidonic acid-gated K+-channel TRAAK, the neurotransmitter CGRP, the structural protein GFAP and the synaptic vesicle protein Syntaxin 1A. 4854

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Table 2. Percentage of CMPs Containing Selected Proteinsa tag

Total CMP number COX-1 cPGES COX-2 mPGES-1 5-Lipoxygenase 12-Lipoxygenase 15-Lipoxygenase TREK-1 TREK-2 TRAAK IB4 CGRP Shank Syntaxin1A NeuN GFAP

control vs control vs zymosan vs zymosan dipyrone/zymosan dipyrone/zymosan

546 (100%) 2.2 7.6 3.6 2.4 4.7 4.5 6.0 2.3 0.3 16.6 0.9 1.2 9.1 32.4 4.3 15.2

2230 (100%) 6.9 6.8 5.4 4.3 20.3 10.0 7.7 1.1 1.9 30.2 0.1 14.5 10.5 14.6 6.6 0.8

2482 (100%) 7.2 3.2 6.7 2.5 19.6 14.1 8.9 3.7 0.8 12.1 0.8 18.5 14.3 11.6 3.5 4.1

a Lumbar spinal cords were taken from untreated animals and 24 h after zymosan injection in one hind paw. Spinal cords of control mice, 24 h after zymosan injection and dipyrone-treated mice 24 h after zymosan injection were compared. The given values are the percentage of all combinatorial molecular phenotypes (CMP) which contain a certain protein (Wilcoxon, P < 0.005).

Table 3. Total Number of Selected CMPsa tag

Total number of CMPs 5-Lipoxygenase increaseb decreasec 12-Lipoxygenase increase decrease TRAAK increase decrease CGRP increase decrease Syntaxin1A increase decrease GFAP increase decrease

control vs control vs zymosan vs zymosan dipyrone/zymosan dipyrone/zymosan

546

2230

2482

15 11

411 42

459 29

5 20

195 28

342 8

40 50

615 58

229 52

5 2

14 309

8 453

1 170

181 152

285 3

81 2

6 12

7 95

a Lumbar spinal cords were taken from untreated animals and 24 h after zymosan injection in one hind paw. The given values are the total number of all combinatorial molecular phenotypes (CMP) within one group (Wilcoxon, P < 0.005). b Number of CMPs showing an increased relative frequency after treatment. c Number of CMPs showing a decreased relative frequency after treatment.

A more detailed analysis on whether the relative frequency of the motifs increases or decreases after dipyrone treatment showed a differentiated picture. In case of 5-LO, 12-LO, TRAAK and Syntaxin 1a, a dramatic increase in the number of motifs with increased relative frequencies after dipyrone-treatment was seen (Table 3). In contrast, CGRP showed an increase in the number of motifs with decreased relative frequencies after dipyrone treatment. Finally, the number of motifs containing GFAP showed an overall decrease (Table 3). Since a change of relative CMP-frequencies might be due to an altered protein expression, we tested in the next step whether the expression of any of these six proteins was changed

Toponomics of Drug-Induced Changes in the Spinal Cord

Figure 1. Dipyrone-treatment does not alter the expression of 5-LO, 12-LO, TRAAK, CGRP, Syntaxin 1A or GFAP. (A) The relative frequency of the fluorescent signals from 5-LO, 12-LO, TRAAK, CGRP, Syntaxin 1A and GFAP immunostaining is shown for spinal cords from untreated, zymosan-treated and concomitant zymosan/dipyrone treated mice. The data are shown as average of 8-10 MELC runs. Statistical analyses were performed using the Wilcoxon rank-sum test. (B) Representative Western blots for 5-LO, 12-LO, TRAAK, Syntaxin 1A and GFAP expression with spinal cord lysates (30 µg) from the three treatment groups. ERK was used as loading control. (C) ELISA determination of CGRP amounts in spinal cord lysates from the three treatment groups.

by one of the treatments. However, neither the relative frequencies of the immunohistological signals of the six proteins (Figure 1A) nor the expression of these proteins as determined by Western blot analysis (Figure 1B) or, in case of CGRP, by ELISA (Figure 2C) varied significantly between spinal cords from animals from the three groups (untreated, zymosantreated, zymosan/dipyrone treated). To investigate whether the MELC data are indicative of an altered protein function, we chose to study exemplarily the effect of dipyrone on 5-LO and 12-LO activities in more detail. 5-LO and 12-LO were chosen since 5-LO and 12-LO activities can easily be determined by monitoring their specific products. Since 15-LO distinguished itself from the other two lipoxygenases in the MELC analysis by not showing increased CMP numbers, it was included in all subsequent experiments as negative control. Since 5-LO and 12-LO are known to translocate from cytoplasmic to membrane compartments upon their activation,21-23 we investigated whether such a translocation is detected by the MELC system. Therefore, we calculated the relative frequencies of the colocalization of the three lipoxygenases with 5 markers (COX-2, BiP, 7-AAD, LAMP-1, and ATPsynthase) for different membrane compartments. COX-2 is found at the nuclear envelope, endoplasmic reticulum (ER), nucleus, caveolae and mitochondria; the Binding-immunoglobulin-Protein (BiP) is localized at the rough ER, 7-aminoactinomycin (7-AAD) stains nuclei, lysosome-associated membrane protein-1 (LAMP-1) lysosomes and ATP-synthase mitochondria. Out of these 5 membrane compartment markers, COX-2 and BiP showed an enhanced colocalization frequency with 5-LO

technical notes

Figure 2. Dipyrone induces the translocation of 5-LO and 12-LO to membrane compartments and their activity in spinal cords. (A) Boxplot analysis shows an increased frequency for the colocalization of 5-LO with COX-2 and BiP in zymosan-treated mice after concomitant dipyrone treatment. (B) Boxplot analysis shows an increased frequency for the colocalization of 12-LO with BiP and 7AAD in zymosan-treated mice after concomitant dipyrone treatment. (C) Boxplot analysis shows no increased frequencies for the colocalization of 15-LO with COX-2, BiP or 7AAD in zymosan-treated mice after concomitant dipyrone treatment. (D-F) 5(S)-HETE (D), 12(S)-HETE (E) and 15-(S)HETE (C) concentrations in spinal cords of zymosan-treated mice after concomitant dipyrone treatment. The data are shown as average of 8-10 MELC runs. Statistical analyses for all panels were performed using the Wilcoxon rank-sum test. The respective significance levels are depicted within the graphs.

outside of lamina I (labeled by calcitonin gene-related protein (CGRP) antibodies) after dipyrone treatment (Figure 2A). Similarly, 12-LO showed an increased colocalization frequency with BiP and 7-AAD (Figure 2B). No significant changes in the colocalization frequencies with 15-LO were observed (Figure 2C). The MELC data suggest a translocation and, therefore, possible activation of 5-LO and 12-LO but not for 15-LO. Therefore, we determined the products of 5-LO and 12-LO in spinal cords of animals 24 h after zymosan-injection in one hind paw in presence and absence of the concomitant dipyrone treatment. 5(S)-HETE, a 5-LO-dependent metabolite of arachidonic acid, 12(S)-HETE, a 12-LO-dependent metabolite of arachidonic acid, and 15(S)-HETE, a product of 15-LO, were detectable in the spinal cords. However, only 5(S)- and 12(S)HETE amounts, but not 15(S)-HETE, increased significantly in the dipyrone treatment suggesting an activation of 5-LO and 12-LO (Figure 2D-F). Visualization of the localization of the motifs showed that colocalization of 5-LO with COX-2 or BiP Journal of Proteome Research • Vol. 8, No. 10, 2009 4855

technical notes

Linke et al.

Figure 3. Visualization of colocalization patterns of 5-LO and 12-LO with membrane compartments markers in spinal cords. Flourescence images of the independent tags and the localization (red) of the indicated motifs are shown for one representative MELC run. White arrows depict areas where the selected motifs for either 5-LO (5-LO with COX-2 or BiP) or 12-LO (12-LO with 7-AAD or BiP), colocalize.

as well as 12-LO with BiP or 7-AAD occurs only to a small part in the same areas (Figure 3, white arrows). Next, we used primary spinal cord cultures to study the effect of dipyrone on 5-LO and 12-LO activities in more detail. First, we showed that in accordance with the known inhibition of cyclooxygenases by dipyrone,13 dipyrone inhibited in these cultures dose-dependently the PGE2-synthesis in the primary spinal cord cells (Figure 4A). A similar dose-dependency was seen for the dipyrone-induced increase of 5(S)-HETE and 12(S)HETE in cells that were costimulated with dipyrone and LPS or the calcium-ionophore A23187 (Figure 4B,C). In line with the in vivo data, we observed an increased production of 5(S)HETE and 12(S)-HETE. Notably, the concentrations of 15(S)HETE that are generated by 15-LO did not change after dipyrone treatment supporting the MELC data that suggested an activation of 5-LO and 12-LO, but not of 15-LO (see Table 2, Figure 2). Since 5-LO, 12-LO and COX enzymes all use arachidonic acid as substrate and dipyrone can inhibit COX activity,13 arachidonic acid might be more available for other arachidonic acidmetabolizing enzymes such as the lipoxygenases. However, inhibition of COX activity by indomethacin or naproxen (Figure 4D) was not sufficient to increase the 5-LO and 12-LO products in primary spinal cord cultures (Figure 4E). Furthermore, we found that dipyrone inhibited partially purified 5-LO with a comparable potency as seen for COX-inhibition (Figure 4A,F). Taken together, these data suggest that high dipyrone concentrations inhibit cyclopxygenases as well as lipoxygenases. Since, additionally, earlier publications showed that COX-inhibition by dipyrone does not occur in the spinal cord in vivo,12 it seems that spinal dipyrone-concentrations are in vivo too low to inhibit either enzyme and that the increased lipoxygenase products are not a secondary effect of COX inhibition.

Discussion With the MELC-technology, we obtained over 2000 colocalization combinations (named combinatorial molecular phenotype (CMPs)) which describe a multitude of possible dipyrone4856

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induced protein-protein interactions in spinal cords of adult mice. Out of this number, the influence of dipyrone on 5-LO and 12-LO activities were subjected to further validation. We chose to focus on these enzymes, since CMPs containing these two proteins showed a disproportional increase in their relative frequencies after dipyrone treatment and since activation of both enzymes can easily be followed by determining their products. In accordance with the MELC data, dipyrone treatment of adult animals raised the concentrations of products from both 5-LO and 12-LO but not 15-LO in spinal cords and primary spinal cord cultures. The observed changes in the frequencies of CMPs containing a selected protein (i.e., 5-LO) as shown in Table 2 may have two reasons. On one hand, the expression of the protein may be upregulated. However, since neither the frequencies of the fluorescence signals for the discussed targets changed after zymosan treatment nor any changes in the Western blots were observed, this possibility seems unlikely. On the other hand, CMPs basically describe the colocalization of proteins. Therefore, the number of CMPs can also increase if the protein (i.e., 5-LO) translocates upon stimulation and, therefore, increases the number of colocalized interaction partners. In other cases, such as GFAP, a protein that is not very likely to translocate, the increased CMP numbers might be due to other proteins that are themselves translocating to colocalize/interact with GFAP. In case of syntaxin or TRAAK, translocation or internalization processes might explain the increase number of CMPs. In accordance with the second hypothesis, the MELC data suggested a translocation of 5-LO and 12-LO to membrane compartments after dipyrone treatment. Indeed, it is known that, upon stimulation, 5-LO translocates from the cytoplasm to the nuclear membrane. In resting cells, 5-LO is located either in the cytosol (e.g., in neutrophils, eosinophils or peritoneal macrophages) or in a nuclear compartment associated with chromatin (e.g., in alveolar macrophages, Langerhans cells or rat basophilic leukemia cells).23 A similar translocation after its activation has also been described for 12-LO, although much less is known about its target compartments.21,22 As a result of

Toponomics of Drug-Induced Changes in the Spinal Cord

Figure 4. Dipyrone increases 5-LO and 12-LO activity in primary spinal cord cells. (A) Primary spinal cord cells were incubated for 24 h with LPS (1 µg/mL) and the indicated dipyrone concentrations. PGE2-concentrations were determined in the medium. Data are shown as average of 3 independent experiments each determined in duplicates. Statistical analyses for panels A-F were performed using the Student’s t test: *P < 0.05, **P < 0.01, **P < 0.001. (B) Same as panel A except that 5(S)-, 12(S)-, and 15(S)-HETE concentrations were determined in the medium by LC-MS/MS. A representative of 4 independent experiments is shown. (C) Primary spinal cord cells were incubated with the calcium-ionophore A23187 (2 µM) and the indicated dipyrone concentrations for 4 h prior 5(S)-, 12(S)-, and 15(S)-HETE determination in the medium. A representative of 3 independent experiments is shown. (D and E) PGE2-concentrations (panel D) and 5(S)-, 12(S)-, and 15(S)-HETE concentrations (E) of primary spinal cord cells incubated for 24 h with LPS (1 µg/mL) and 1 µM indomethacin or 10 µM naproxen. Data are shown as average of 3 independent experiments. (F) Partially purified 5-LO (0.5 µg) was incubated with the indicated concentrations of dipyrone or the LO inhibitor BWA4C (1 µM) or vehicle (DMSO) for 15 min at 4 °C, samples were prewarmed for 30 s at 37 °C and 2 mM CaCl2, and 20 µM AA were added. 5-LO products were analyzed by HPLC. Data are shown as average of 3-4 independent experiments.

the translocation of 5-LO and 12-LO, new colocalization partners for both proteins emerge and the number of potential motifs containing 5-LO and 12-LO multiply. Accordingly, the MELC-system detected a high number of motifs containing 5-LO and 12-LO that show increased relative frequencies. The increased synthesis of 5-LO and 12-LO products in presence of dipyrone can be explained by different mecha-

technical notes nisms. First, a direct activation of 5-LO and 12-LO could be possible. However, the in vitro experiments using partially purified 5-LO rule out a direct activation. Second, since 5-LO, 12-LO and COX enzymes all use arachidonic acid as substrate, and since dipyrone can inhibit COX activity,13 the common substrate arachidonic acid might be more available for other arachidonic acid-metabolizing enzymes such as the lipoxygenases. A similar redirection in the arachidonic acid metabolism has been described for the deletion of prostaglandin E2 synthases.17,24,25 Here, the COX-derived endoperoxide intermediate PGH2 serves as a common substrate for several prostanoid synthases. In cells and tissues which were deficient for the PGE2 synthase mPGES-1, a redirection from PGE2 to other prostanoids was observed.17,24,25 However, since no inhibition of COX activity by dipyrone is observed in the spinal cord12 and COX inhibition in primary spinal cord cells did not increase 5- or 12-HETE synthesis, it seems unlikely that this is the mechanism which causes the dipyrone-induced 5- and 12HETE concentrations. An alternative activation mechanism could be based on the redox status of the cells. Since catalysis by 5-LO requires oxidation of Fe2+ to the active Fe3+ state by lipid hydroperoxides, the redox status is an important parameter of cellular 5-LO activity. Conditions that promote lipid peroxidation, such as formation of reactive oxygen species by phorbol 12-myristate 13-acetate, addition of peroxides, and depletion of glutathione, activate 5-LO, whereas reduction of peroxides suppresses 5-LO activity.23 Although less is known about the activation of 12LO, it seems that this enzyme is also activated in presence of reactive oxygene species.26 Indeed, dipyrone was shown to reduce Fe3+ in vivo13 thereby causing an increased generation of reactive oxygen species (ROS).27-29 Dipyrone treatment increases lipid peroxidation in RAW 264.7 cells and a single dose of dipyrone is sufficient to increase the expression of superoxide dismutase and peroxiredoxin V, two enzymes which are necessary to neutralize superoxide and other ROS species, in the spinal cord of adult rats.13 The fact that dipyrone activates 5-LO and 12-LO is on the first view surprising, since 5-LO and 12-LO products are known to promote hyperalgesia and inflammation,30 which led to the development of selective inhibitors and their use in clinical trials.31 However, some recent reports indicate also an antiinflammatory and antinociceptive role of 5-LO and 12-LO products. Although the exact mechanisms are unknown, the lipoxygenases were implicated in the analgesic effects of the phospholipase A2 neurotoxin crotoxin and of opioids.32,33 Furthermore, it was shown that 5-LO and 12-LO are generating resolvins and lipoxins, respectively,34-36 which seem to play a crucial role in the termination of inflammation.37,38 In this context, a recent report demonstrated that pioglitazone and atorvastatin induce 5-LO activity by a site specific phosphorylation that allows the interaction with COX-2 to produce the antiinflammatory 15-epi-lipoxin A4.39 However, how far the here described effect of dipyrone on lipoxygenase activity accounts for its clinical effects needs to be clarified in future studies. We show here that the MELC-technology is a useful tool to screen tissues for drug-induced changes in protein networks, which are not easily detectable by other common cell biological and protonomic methods. In most cases, drug-induced changes can be expected to be detectable by the MELC-technology, since in general, drugs aim to influence protein interactions in a direct or indirect manner. For example, inactivation of an Journal of Proteome Research • Vol. 8, No. 10, 2009 4857

technical notes enzyme by a drug will prevent its translocation, its interaction with substrate proteins (i.e., protein kinases or ubiquitin ligases), or, in case of receptors, antagonists, can be expected to alter downstream-signaling events which then can be detected using phosphorylation-specific antibodies. However, the appropriate antibody-targets have to be chosen carefully for the analysis to cover these different possibilities and to exclude that activation or inactivation of pathways fails to be detected in this quantitative immunohistological approach. Thus, toponomic analysis allows the identification of signaling pathways and “off-target” effects of drugs using tissues of treated animals or humans.

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Acknowledgment. The work was supported by the DFG grants SCHO817-1 and -2, GE695 and the LOEWE Lipid Signaling Forschungszentrum Frankfurt (LIFF). We would like to thank Lars Philipsen and Alexis Beyer, MelTec GmbH& Co KG, Magdeburg, for their advice and the technical support with the MELC technology.

Supporting Information Available: Supplementary methods: LC-MS/MS method for detection of prostanoids and HETEs; Table S1: order and concentrations of the tags used in MELC runs. This material is available free of charge via the Internet at http://pubs.acs.org.

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