Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 85
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A similar biphasic activation of ERK1/2 has been reported for mitogens such as fibroblast growth factor [13]. Recently, the delayedERK1/2 activation has been reported to be mediated by different mechanismsthan the early ERK1/2 activation and to be critical for cell cycle progression andcell proliferation [13, 14].One logical mechanism for endothelial cells and VSMC to respond to ROSwould be to produce autocrine/paracrine signals that enhance cell survival orstimulate pathways that protect cells from the damaging effects of ROS. VSMCare particularly likely to secrete protective factors that also promote cell survivalbased on previous studies that demonstrate secretion of a number of growth factors from VSMC in response to various stimuli. These growth factors includeadrenomedullin, endothelin, epiregulin, FGF, Gas6, PDGF, and TGF-b [15–20].For example, Gas6 is secreted from rat VSMC after stimulation by Ang II andProteomic and Genomic Analysis of Cardiovascular Disease.Edited by Jennifer E.
van Eyk, Michael J. DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-30596-330818 Vascular SOXF and associated proteinsthrombin, and exhibits growth factor activity [21, 22]. Epiregulin, an epidermalgrowth factor (EGF)-related growth factor, is a potent VSMC-secreted mitogenwhose expression is regulated by Ang II, endothelin-1 and thrombin [23]. However, no factors have been identified as mediators of VSMC proliferation in response to ROS. In this paper we describe purification of factors secreted fromVSMC exposed to oxidative stress that stimulate ERK1/2. These activities, termedSOXF for secreted oxidative stress induced factors are potentially important physiologic mediators of the vessel wall response to ROS.18.2Identification of SOXF by Tandem MS18.2.1Secreted Factors Are Involved in Regulation of ERK1/2 Activation by Oxidative StressTo generate oxidative stress VSMC were exposed to LY83583, which generates O–2[4].
Production of O–2 in VSMC to exposed to 1 lmol/L LY83583 was measured bylucigenin chemiluminescence as described previously [4]. LY83583-induced generation of O–2 peaked at 15 min and returned to baseline by 120 min. Tiron (10lmol/L), a membrane-permeant nonenzymatic O–2 scavenger, completely abolishedLY83583-induced O–2. Exposure of VSMC to LY83583 stimulated ERK1/2 activitywith an initial peak at 10 min that paralleled O–2 production and a second peak at120 min, a time when O–2 production was minimal.
ERK1/2 activity at 120 minwas greater than activity at 10 min, and was nearly equivalent to that observedwith PDGF or with xanthine plus xanthine oxidase.18.2.2A Trypsin-sensitive Secreted Oxidative Stress-induced Factor (SOXF)Is Released in Response to LY83583To identify the presence of SOXF from VSMC stimulated by LY83583, the abilityof conditioned medium to stimulate ERK1/2 was assayed. Conditioned mediumwas prepared and then transferred to growth-arrested VSMC for 10 min.
ERK1/2activity, measured with a phospho-ERK1/2 specific antibody, demonstrated a significant increase with conditioned medium from cells treated with LY83583, butnot control (Fig. 18.1). The activity was sensitive to trypsin (5 lg/ml for 30 min),and was inhibited by heating to 100 8C. To determine whether a few proteins comprised the majority of SOXF, we analyzed the proteins released into the mediumin response to LY83583. Cells were labeled with [35S]methionine for 4 h and thenexposed to 1 lmol/L LY83583 for 120 min. Conditioned medium was harvested,concentrated and proteins analyzed by 5–15% SDS-PAGE.
Approximately 35 protein bands were detected by autoradiography suggesting that multiple proteinswere released in response to LY83583.Ctl-CM18.2 Identification of SOXF by Tandem MSFig. 18.1 Secreted factors are involved in regulation of ERK1/2 activation by oxidativestress. Growth-arrested vascular smooth muscle cells (VSMC) were exposed to 1 lMLY83583 for the indicated times (a), or treatedwith conditioned medium from LY83583-stimulated VSMC (LY-CM), control medium fromHBSS-incubated cells (Ctl-CM) for 10 min, or1 lM LY83583 for 2 h (b), or with concentrated LY-CM for the indicated times (c).
Celllysates were prepared and analyzed for ERK1/2 activity or total ERK1/2 proteins by Westernblot using phospho-specific ERK1/2 (pERK1/2) antibody, or ERK1 plus ERK2 antibody. Theresults were quantified by densitometry ofautoradiograms using NIH Image 1.49. Results were normalized to control (time = 0min) which was arbitrarily set to 1.0 (c). Results are representative or mean ± SD of 3 experiments.18.2.3Purification of SOXF and Identification of SOXF Candidate Proteinsby Mass SpectrometryTo purify SOXF we used a sequential chromatographic approach that involved SPSepharose, heparin-Sepharose, phenyl Sepharose, and S-200 gel filtration chroma-30931018 Vascular SOXF and associated proteinsFig.
18.2 Coomassie stain of the 0.15 M ammonium sulfate fraction from phenyl-Sepharose (SOXF). Fraction III from phenyl Sepharose chromatography containing ~25 lg protein was subjected to 12% SDS-PAGE. Proteins were stained with 0.1% Coomassie Brilliant Blue 250 in 40% methanol and 1% aceticacid, destained in 50% methanol. Approximate molecular weights were determined bylogarithmic plot of migration of molecularweight markers.
SOXF* indicates putativeSOXF containing fraction from phenyl-Sepharose column.tography [24]. For each step, activity was assayed for ERK1/2 stimulation by phospho-ERK1/2 Western blot using growth arrested VSMC. On S-200 chromatography, SOXF activity eluted in three peaks at approximate molecular weights (in order of relative ERK1/2 stimulating activity) of 80–100 kD > 20–30 kD > 45–65 kD.Proteins were identified by SDS-PAGE and staining with Coomassie Blue(Fig. 18.2).
The proteins were digested by trypsin and the resulting peptides wereanalyzed by an electrospray triple quadrupole mass spectrometer (Finnigan-MATTSQ 7000). The collision induced dissociation spectra generated were used toidentify the proteins from which the peptide originated by database searchingusing the Sequest software program [25, 26]. Unambiguous identification of proteins in the 90 kD band revealed heat shock protein 90-a (HSP90-a). Unambiguous identification of proteins in the 20 kD band revealed cyclophilin B and cyclophilin A.
No proteins were identified in the 56 kD and 27 kD bands. Other proteins identified included ezrin and moesin.18.2.4LY83583 Stimulates Release of HSP90-a Specifically from VSMCTo prove that proteins identified as putative SOXFs were released specifically fromLY83583 treated VSMC, a conditioned medium experiment was analyzed.
Toprove specificity we compared the relative protein abundance of candidate SOXFproteins in the total cell lysate and in conditioned medium, before and afterLY83583 stimulation. Because HSP90-a was the largest SOXF identified, and18.2 Identification of SOXF by Tandem MStherefore least likely to be released non-specifically, we studied its abundance.Conditioned media from control and LY83583 treated cells were concentrated.SDS-PAGE followed by Western blot analysis for HSP90 (with an antibody that recognizes both HSP90-a and HSP90-b). The abundance of HSP90 was comparedto two intracellular proteins of similar molecular weights; PKC-n (with an antibody which also recognizes PKC-a), and c-Raf-1.
After treatment with LY83583 for2 hr, there was a 10-fold increase in HSP90-a present in the conditioned medium,but no detectable HSP90-b, PKC or c-Raf-1. These results suggest that regulatedsecretion of HSP90-a occurred in response to LY83583.18.2.5Human Recombinant HSP90-a and Cyclophilin A (CyPA) Activate ERK1/2To provide further evidence that HSP90-a and CyPA are SOXF, human recombinant HSP90-a (hrHSP90-a) and hrCyPA were studied. The preparations used forthese studies were highly purified as shown by silver stain analysis, which revealed that > 95% of total protein migrated at appropriate molecular weights of90-kDa, and 18 kDa, respectively.
Both CyPA and hrHSP90-a stimulated ERK1/2activity in VSMC in a concentration-dependent manner [24, 27].18.2.6Human Recombinant Cyclophilin A Stimulates VSMC Growthand Protects VSMC against ApoptosisTo determine the physiological significance of SOXF, we studied the effects ofhrCyPA on VSMC DNA synthesis. 10 nmol/L hrCyPA significantly stimulatedDNA synthesis in VSMC (2-fold increase versus 0.1% serum) assayed by[3H]thymidine incorporation [27].
Thus, CyPA has growth promoting effects onVSMC which may contributes significantly to the growth promoting activity ofROS in VSMC. To further determine whether CyPA prevents VSMC apoptosis, weused sodium nitroprusside (SNP), which was shown to induce VSMC apoptosis[28, 29]. Incubating VSMC with 1 mmol/L SNP for 24 hours decreased cell viability to 19.4% of control, measured with a modified MTT assay.
Addition of 10 nMhrCyPA in the presence of 1 mmol/L SNP blocked apoptosis, with cell viability returning to 47% of control [27]. In response to 0.5 mmol/L SNP for 24 hours, 10%of VSMCs were apoptotic as measured by nuclear morphology after DAPI staining, consistent with previous reports [28, 29]. Addition of hrCyPA significantly inhibited apoptosis induced by 0.5 mmol/L SNP with a decrease of 55%. These results indicated that SOXF have significant physiological effects on VSMC.31131218 Vascular SOXF and associated proteins18.3Identification of SOXF-associated Proteins by MALDI-TOF MS18.3.1Identification of SOXF-associated Proteins by MALDI-TOF MSTo understand the mechanisms responsible for secretion of CyPA and activationof VSMC by SOXF, we studied SOXF-associated proteins by MALDI-TOF MS(Matrix-assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry).In brief, VSMC cell lysates were incubated with recombinant GST-CyPA wild type(WT), GST-CyPA R55A (isomerase inactive mutant), or GST (negative control) inbinding buffer [30].
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