© 1997 American Heart Association, Inc.
Articles |
Oxidative Stress–Induced Actin Reorganization Mediated by the p38 Mitogen-Activated Protein Kinase/Heat Shock Protein 27 Pathway in Vascular Endothelial Cells
Centre de recherche en cancerologie de l'Universite Laval, L'Hotel-Dieu de Quebec (Canada).
Correspondence to Dr Jacques Huot, Le Centre de recherche en cancerologie de l'Universite Laval, L'Hotel-Dieu de Quebec, 11 Cote du Palais, Quebec, G1R-2J6, Canada. E-mail Jacques.Huot@phc.ulaval.ca
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Vascular endothelial cells are constantly in contact with oxyradicals and must be especially well equipped to resist their toxic effects and generate appropriate physiological responses. Despite the importance of oxyradicals in the physiopathology of the vascular endothelium, the mechanisms regulating the oxidative response of endothelial cells are poorly understood. In the present study, we observed that H2O2 in concentrations that induced severe fragmentation of F-actin in fibroblasts rather induced a reorganization of F-actin in primary cultures of human umbilical vein endothelial cells (HUVECs) that was characterized by the accumulation of stress fibers, the recruitment of vinculin to focal adhesions, and the loss of membrane ruffles. H2O2 also induced in these cells a strong (10- to 14-fold) activation of the p38 mitogen-activated protein (MAP) kinase, which resulted in activation of MAP kinase–activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). The MAP kinases extracellular-regulated kinase, and c-Jun N-terminal kinase/stress-activated protein kinase were only slightly increased by these treatments. Inhibiting p38 activity with the highly specific inhibitor SB203580 blocked the H2O2-induced endothelial microfilament responses. Moreover, fibroblasts acquired an endothelium-like SB203580-sensitive actin response when HSP27 concentration was increased by gene transfection to the same high level as found in HUVECs. The results indicate that activation of p38 MAP kinase in cells such as endothelial cells, which naturally express high level of HSP27, plays a central role in modulating microfilament responses to oxidative stress. Consequently, the p38 MAP kinase pathway may participate in the several oxyradical-activated functions of the endothelium that are associated with reorganization of microfilament network.
Key Words: oxidative stress • endothelial cell • p38 mitogen-activated protein kinase • heat shock protein 27 • F-actin
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Vascular endothelial cells are in constant contact with circulating reactive oxygen metabolites. These oxyradicals are used as agonist-like agents to generate specific physiological responses, such as an increase in neutrophil adhesion and the production of IL-1
and IL-6.1 2 Also, nitric oxide, an oxyradical released from endothelial cells in response to agents such as histamine, has a potent smooth muscle cell–relaxing property.3 In supraoptimal concentrations or when the endothelium is unable to efficiently deal with them, oxidants have been associated with increased cellular permeability and with various pathological conditions that affect the structural and/or functional integrity of the vascular endothelium, including atherosclerosis, organ ischemia–associated thrombosis, and inflammation.4 5 6 Despite the importance of oxyradical-associated physiological processes or pathological alterations, the mechanisms underlying and regulating the cellular responses to oxidative stress are poorly understood.
The MAP kinases ERK1-2, p38, and JNK/SAPK appear as central elements of three homologous pathways used by mammalian cells to transduce the messages generated by stressing agents or growth factors.7 The role of the MAP kinases in transducing the signal generated by stress is still unclear. ERK is involved in the regulation of transcriptional and translational activities and is essential for control of cell growth and differentiation.7 8 Activation of ERK by stressing agents may generate a growthlike response that facilitates the repair of stress-damaged constituents and the return to prestress equilibrium.9 Accordingly, ERK activation has been suggested to play a critical role in cellular survival following oxidative stress.10 JNK/SAPK and p38 MAP kinases are recognized as stress-sensitive kinases.11 12 13 JNK/SAPK was first demonstrated as a UV light–activated Jun kinase and, in this case, may trigger a beneficial stress response by activating activator protein 1–regulated genes coding for proteins that confer protection against stress.14 Activation of JNK/SAPK by inflammatory cytokines or stressing agents also leads to phosphorylation of Elk-1 and ATF-2 and then to an increase in the transcriptional activity of genes containing a serum response element or a cAMP response element, respectively.13 15 16 p38 activation by stress phosphorylates and activates the transcription factors CHOP, CREB, and ATF-1 and thereby may regulate the expression of a number of genes influencing growth and differentiation.17 18 p38 also phosphorylates ATF-2 in vitro and in vivo, and this phosphorylation is thought to regulate its transactivating activity.8 19 In monocytes, activation of p38 modulates the production of inflammatory cytokines in response to bacterial lipopolysaccharide endotoxin, and it mediates the TNF-–induced expression of IL-6 in murine L929 cells.20 21 Activation of p38 also leads to the activation of MAPKAP kinase-2/3, a serine protein kinase that phosphorylates HSP27 (Guay J, Lambert H, Huot J, Landry J, unpublished data, 1996, and references 12, 22, and 23).12 22 23 HSP27 has in vitro a phosphorylation-modulated inhibitory function on F-actin polymerization and, in vivo, influences actin dynamics in response to stress and growth factors.24 25 26 27 28
Several functions of endothelial cells are associated with microfilament reorganization. For example, F-actin reorganization accompanies the increased transendothelial permeability induced by mediators of inflammation4 29 and the activated cell migration during angiogenesis.30 The microfilament cytoskeleton is also reorganized in endothelial cells in response to shear stress.31 In vivo, the stress fibers of endothelial cells are oriented parallel to blood flow. A high amount of stress fibers is observed in cells exposed to high shear stress, suggesting that stress fibers contribute to maintain the integrity of the endothelium by enhancing cellular adhesion to the substratum.32 33 In many cell types, the integrity of the actin cytoskeleton is one of the earliest targets of the toxicity of oxyradicals and may well be a limiting factor in the ability of vascular endothelium cells to resist oxidative stress in pathological situations.
In the present study, we have investigated the response of the actin cytoskeleton to oxidative stress in primary cultures of HUVECs. We report that HUVEC microfilaments are extremely resistant to exposure to oxidants in concentrations that otherwise induce a total disruption of the F-actin cytoskeleton in fibroblastic cells. In HUVECs, H2O2 and menadione induced a marked reorganization of the microfilaments, causing the loss of membrane protrusions and the appearance of a dense stress-fiber network associated with the assembly of vinculin to focal adhesions. We further show that p38 is the major MAP kinase activated by oxidants in HUVECs and that activation of this pathway leads to HSP27 phosphorylation and is involved in the cytoskeletal reorganization induced in response to oxidative stress.
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Materials
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-32P]ATP (3000 Ci/mmol) and H3[32P]O4 (1000 mCi/mmol) were purchased from Dupont Canada Inc. TNF-, PMA, and bFGF were from Calbiochem; H2O2, menadione, and ECGS, from Sigma Chemical Co; and IL-1ß, from Boehringer-Mannheim. Recombinant Chinese hamster HSP27 was purified from Escherichia coli transformed with a plasmid containing the Chinese hamster HSP27 coding sequence. SB203580 and SKF106978 were gifts from SmithKline Beecham Pharmaceuticals; PD098059, from Parke-Davis. Chemicals for electrophoresis were purchased from Bio-Rad and Fisher.
Antibodies and Peptides
Hu27ab is a rabbit antiserum that specifically recognizes human HSP27.34 Anti–GST–MAPKAP kinase-2/3 is a rabbit polyclonal antibody raised in rabbit after injecting a GST fusion protein containing the 223 C-terminal amino acids of Chinese hamster MAPKAP kinase-2.27 This antibody immunoprecipitates both the p45 and p54 isoforms of human MAPKAP kinase-2, one of which may correspond to the recently described MAPKAP kinase-3.23 27 Anti-ERK2 is a rabbit polyclonal antibody raised against a synthetic peptide corresponding to the 14 carboxy-terminal amino acids of rat ERK2.27 Anti-p38 is a rabbit polyclonal antibody raised against the C-terminal sequence PPLQEEMES of murine p38. Epitope (LT)–tagged MAPKAP kinase-2 was generated by PCR using the human MAPKAP kinase-2 cDNA cloned by Zu et al.35 HA–tagged p38 was produced by PCR using the p38 sequence described by Han et al36 and J. Moscat, unpublished data, 1995. HA- and LT-tagged proteins were immunoprecipitated with KT337 and 12C5A monoclonal antibodies, respectively. Ha90-HSP27 peptide (RALNRQLSSGV) was synthesized using a solid-phase procedure and was purified by high-performance liquid chromatography.38 hVIN-1 is a human anti-vinculin monoclonal antibody purchased from Sigma.
Cells
HUVECs were isolated by collagenase digestion of umbilical veins from undamaged sections of fresh cords. Briefly, the umbilical vein was cannulated, washed with EBSS, and perfused for 10 minutes with collagenase (1 mg/mL) in EBSS at 37°C. After perfusion, the detached cells were collected, the vein was washed with medium 199, and the wash-off was pooled with the perfusate. The cells were washed by centrifugation and plated on gelatin-coated 75-cm2 culture dishes in medium 199 containing 20% heat-inactivated FBS, ECGS (60 µg/mL), glutamine, heparin, and antibiotics. Replicated cultures were obtained by trypsinization and were used at passages 
5. In certain experiments, HUVECs were made quiescent by incubating the cells for 16 hours in medium containing only 5% FBS. The identity of HUVECs as endothelial cells was confirmed by their polygonal morphology and by detecting their immunoreactivity for factor VIII–related antigens. BPAECs were obtained from the American Type Culture Collection and were maintained in MEM containing 10% FBS and nonessential amino acids. Hu27 cell lines B12 and V are Chinese hamster CCL39 transformants, which constitutively express 4.8 ng/µg of protein of human HSP27 or 3.3 ng/µg of protein of a nonphosphorylatable form of human HSP27, respectively. Clone 3 is a CCL39 transfectant that expresses only the selection gene, neo. The transfectants were maintained in DMEM containing NaHCO3 (2.2 g/L) and glucose (4.5 g/L) and were supplemented with 5% FBS.26 28 All the cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Transfection
Exponentially growing BPAECs in 175-cm2 (4 x106) tissue culture flasks were transfected with expression vectors specifying either HA-tagged p38 (6 µg/106 cells) or LT-tagged MAPKAP kinase-2 (10 µg/106 cells) or were cotransfected with both vectors. PUC19 DNA was used as filling DNA up to 30 µg/106 cells. Transfections were performed using the CaCl2 method as previously reported.34 Transfectants were trypsinized and expanded the day after transfection. Twenty-four hours later, they were exposed to H2O2.
In Vivo Phosphorylation
Phosphorylation of HSP27 was evaluated by labeling with H3[32P]O4. Briefly, cells were preincubated for 4 hours in phosphate-free medium containing 25 µCi/mL H3[32P]O4 (900 to 1100 mCi/mmol) and then were left untreated or treated as indicated. Immediately after treatments, the cells were lysed, and proteins were fractionated by SDS-PAGE. The radioactivity incorporated in HSP27 was evaluated by autoradiography. In other experiments, the cells were lysed in IEF buffer, and total proteins were fractionated by IEF.27 After blotting, HSP27 isoforms A, B, C, and D were revealed with an antibody specific for human HSP27.
Immunoprecipitation
After stimulation, cells were scraped and extracted in lysis buffer containing 20 mmol/L MOPS, pH 7.0, 10% glycerol, 80 mmol/L ß-glycerophosphate, 5 mmol/L EGTA, 0.5 mmol/L EDTA, 1 mmol/L Na3VO4, 5 mmol/L Na4P2O7, 50 mmol/L NaF, 1% Triton X-100, 1 mmol/L benzamidine, 1 mmol/L dithiothreitol, and 1 mmol/L PMSF. The extracts were vortexed and centrifuged at 17 000g for 12 minutes at 4°C. The clarified supernatants were immediately used for immunoprecipitation or were stored at -80°C. Further steps were performed at 4°C. The clarified supernatant was diluted four times in buffer I (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L MgCl2, 1 mmol/L Na3VO4, 1% Triton, and 1 mmol/L PMSF). Undiluted anti-p38, anti-ERK2, or anti–MAPKAP kinase-2/3 antibodies were added in limiting concentrations, and the mixtures were incubated for 1 hour. Ten to 15 µL of protein A–Sepharose (50% [vol/vol], Sigma) in buffer I was added, and the mixtures were incubated for 30 minutes. Samples were centrifuged for 15 seconds and washed three times with 300 µL of buffer I. Immunoprecipitates were directly used for kinase assays. The specificity of the immunoprecipitation by the anti-p38 antibody was determined by competition of the p38-immunoprecipitated activity with the immunogenic peptide.
Kinase Assays
Kinase activities from quiescent cells were assayed in immune complexes using appropriate substrates. MAPKAP kinase-2/3 activity was assayed using recombinant HSP27 or the HSP27 peptide Ha90 as substrate.27 38 The assays were performed in 25 µL of kinase buffer K containing 100 µmol/L ATP, 1 to 3 µCi of [32P]ATP (3000 Ci/mmol), 40 mmol/L p-nitrophenyl phosphate, 20 mmol/L MOPS, pH 7, 10% glycerol, 15 mmol/L MgCl2, 0.05% Triton X-100, 1 mmol/L dithiothreitol, 1 µmol/L leupeptin, 0.1 mmol/L PMSF, and 0.3 µg protein kinase A inhibitor. The kinase activity was assayed for 30 minutes at 30°C and was stopped by the addition of 10 µL SDS-PAGE loading buffer. Immunoprecipitated ERK2 and p38 were assayed analogously using MBP and ATF2-GST, respectively, as substrates.39 Kinase assay buffer K containing 10 mmol/L MgCl2 was used for ERK2, whereas the kinase assay medium for p38 contained 50 mmol/L HEPES, pH 7.4, 50 mmol/L ß-glycerophosphate, 50 mmol/L MgCl2, 0.2 mmol/L Na3VO4, ATF2-GST, and [-32P]ATP (3000 Ci/mmol). In the case of JNK/SAPK activity, the cell extract was adsorbed on GST-Jun beads, and the kinase was tested using the same GST–N-terminal Jun as substrate.11 Briefly, the GST-Jun fusion proteins bound to glutathione Sepharose beads were incubated for 30 minutes at 4°C with the extracts in buffer I. The beads were then pelleted, washed with buffer I, and incubated for 30 minutes at 30°C with 3 µCi [-32P]ATP (3000 Ci/mmol) in kinase buffer K containing 10 mmol/L MgCl2. The phosphorylated GST-Jun was boiled in SDS sample buffer to stop the reaction. The activity of the various kinases was quantified by measuring the incorporation of radioactivity into the specific substrate after SDS-PAGE. In the case of MAPKAP kinase-2/3, the activity was sometimes assayed using the Ha90-HSP27 peptide as substrate. In this assay, the activity of the kinase was evaluated by counting the radioactivity adsorbed on phosphocellulose p81 papers.38
Confocal Fluorescence Microscopy
F-actin detection was performed as previously reported.28 HUVECs or Hu27 cells were plated on gelatin-coated or fibronectin-coated slides, respectively. They were then fixed with 3.7% formaldehyde and permeabilized with 0.1% saponin in PBS, pH 7.5. F-actin was detected using FITC-conjugated phalloidin (33.3 µg/mL) diluted 1:50 in phosphate buffer. Vinculin was detected using the hVIN-1 monoclonal antibody. Vinculin antigen-antibody complexes were detected with biotin-labeled anti-mouse IgG and were revealed with Texas red–conjugated streptavidin. Cells were examined as previously reported by confocal microscopy with a Bio-Rad MRC-600 imaging system mounted on a Nikon Diaphot-TDM equipped with a x60 objective lens with a numerical aperture of 1.4.28 F-actin levels were determined by confocal microscopy using the Histogram Bio-Rad Comos software as previously described.25
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Oxidants Induce Microfilament Reorganization From Cortical Actin to Stress Fibers
Microfilaments are early targets of oxidants. Since vascular endothelial cells are constantly in contact with oxidants and since various functions of endothelial cells are associated with microfilament reorganization, we investigated the response of actin microfilaments to oxidative stress in primary cultures of HUVECs. Cells were exposed for varying periods of time to different concentrations of H2O2 or menadione. After exposure, the cells were fixed, stained for F-actin with FITC-conjugated phalloidin, and examined by confocal microscopy. In untreated HUVECs, F-actin was found mostly in cortical structures forming characteristic ruffles (Figs 1A
and 2A). The microfilament network of HUVECs was extremely resistant to both H2O2 and menadione, even in concentrations that produced a marked fragmentation of F-actin in fibroblasts.28 In HUVECs, H2O2 (250 µmol/L H2O2, 30 minutes) or menadione (100 µmol/L, 60 minutes) instead produced a dramatic reorganization of F-actin, rearranging cortical microfilaments into long stress fibers that traversed the cells (Fig 1B and 1C). Stress fiber formation was detected as early as after 5 minutes of exposure to 250 µmol/L H2O2 and reached a maximum by 15 minutes (Fig 2B and data not shown). The effect was also dose dependent, starting at 50 µmol/L H2O2 and being maximal at 100 µmol/L (Fig 2C and 2D). Similar results were obtained with the various primary cultures of HUVECs obtained from different cords whether they were cultivated exponentially or at quiescence (data not shown).
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p38 Is the Major MAP Kinase Activated by Oxidants
We characterized the responsiveness of the three MAP kinases, ERK, p38, and JNK/SAPK, to oxidants and compared the results with those obtained with other agonists of endothelial cells. HUVECs were exposed for 5 to 20 minutes to the oxidants H2O2 (1 mmol/L) or menadione (500 µmol/L), the cytokines TNF- (100 ng/mL) or IL-1ß (20 ng/mL), HOS (sorbitol, 0.3 mol/L), PMA (10 µmol/L), or bFGF (20 ng/mL). Extracts were prepared from these cultures, and the activities of the MAP kinases were assayed as described in "Materials and Methods." Results presented in Fig 3 show that p38 and, to a lesser degree, ERK were activated by H2O2 and menadione. JNK/SAPK was not or only very slightly activated by these oxidants. PMA and the growth factor bFGF activated only ERK. In contrast, all three MAP kinases were activated in response to HOS and the proinflammatory cytokines, TNF- and IL-1ß. A more detailed kinetics of the activation of the MAP kinases by H2O2 was determined (Figs 4 and 5A). The activation of p38 by H2O2 was biphasic, with two peaks, one at 5-minute and the other at 45-minute exposures. For both peaks, the activation reached 12- to 14-fold over the basal level. Activation of ERK was transient, reaching a peak of 4.5-fold activation after 5 minutes and then declining to near basal level after 45 minutes. JNK/SAPK was poorly activated by H2O2 over the periods of time investigated (between 2 and 60 minutes). It typically reached activation of 2- to 3-fold at 5 minutes compared with values of 20- to 30-fold obtained after treatment with TNF- or IL-1ß (data not shown).
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Oxidants Activate MAPKAP Kinase-2/3 via Activation of p38 MAP Kinase
p38 immunoprecipitated from extracts of HUVECs or BPAECs treated with H2O2, TNF-, and IL-1ß could efficiently reactivate semipurified MAPKAP kinase-2/3 in vitro (data not shown). Moreover, a close parallelism was found between the kinetics of activation of p38 and MAPKAP kinase-2/3 in vivo. This was particularly striking after stimulation with H2O2 ( Figs 4, 5A, and 5B
). As in the case of p38, the kinetics of activation of MAPKAP kinase-2/3 after H2O2 treatment was biphasic, with peaks at 5 and 45 minutes. This first demonstration of superimposable kinetics of activation of both p38 and MAPKAP kinase-2/3 in cells treated by H2O2 is a strong indication that p38 is a physiological activator of MAPKAP kinase-2/3 in response to oxidative stress. Similar correlations between activation of p38 and MAPKAP kinase-2/3 were obtained after treatment of the cells with TNF- and IL-1ß (Fig 5C through 5F).
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More direct evidence that activation of MAPKAP kinase-2 in response to oxidants resulted from the activation of p38 was obtained in transient transfection experiments that were carried out in BPAECs. These cells were chosen as a model of endothelial cells in this experiment since they can be more easily transfected than HUVECs. BPAECs were transfected with LT-tagged MAPKAP kinase-2 alone or together with HA-tagged p38 and, 48 hours after transfection, were stimulated by H2O2 (1 mmol/L for 20 minutes) (Fig 6). When transfected alone, LT-tagged MAPKAP kinase-2 activity was increased 
2-fold by treatment with H2O2 (lanes 3 and 4). Cotransfection with HA-tagged p38 resulted in a 2- to 3-fold increase in both the basal and stress-induced activities of LT-tagged MAPKAP kinase-2 (lanes 5 and 6). This p38-dependent enhancement of MAPKAP kinase-2 activities strongly suggested that p38 can activate MAPKAP kinase-2 in vivo. We evaluated next whether activation of p38 was a necessary condition for the activation of MAPKAP kinase-2/3 by H2O2 using SB203580, a pyridinyl imidazole derivative, which is a highly specific inhibitor of p38.20 40 Consistent with its direct action on p38, SB203580 (25 µmol/L for 1 hour) did not inhibit p38 activation but led to a total inhibition of MAPKAP kinase-2/3 activation by 5- or 45-minute H2O2 treatment (Fig 7A and data not shown). The SB203580-induced inhibition of MAPKAP kinase-2/3 activation by H2O2 was concentration dependent, with a maximal inhibition at 5 µmol/L (Fig 7B) and an IC50 lower than 1 µmol/L, a value similar to that found in vitro.40 This confirmed that p38 was a necessary upstream activator of MAPKAP kinase-2/3 in response to H2O2. In contrast, PD098059, an inhibitor of the ERK activator MEK-1,41 had no effect on the activation of MAPKAP kinase-2/3 by H2O2 even at 25 µmol/L, a concentration that had totally inhibited activation of ERK (Fig 7A and data not shown). SKF106978, an inactive analogue of SB203580, similarly had no effect on the activation of MAPKAP kinase-2/3 (Fig 7A).
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Oxidants Induce Phosphorylation of HSP27
In fibroblasts, activation of MAPKAP kinase-2/3 leads to phosphorylation of HSP27, and this confers actin dynamics–modulating properties to HSP27.26 27 This, coupled with the fact that unstimulated HUVECs express a high level of HSP27 (6 ng/µg of protein), suggested a role for HSP27 in modulating the actin-dependent functions of endothelial cells. Thus, we investigated whether HSP27 was phosphorylated in HUVECs exposed to oxidants. IEF electrophoresis revealed that HSP27 existed under four major isoforms, A, B, C, and D, which represent unphosphorylated, monophosphorylated, biphosphorylated, and triphosphorylated variants of the protein. In unstimulated HUVECs, HSP27 exists mostly under the nonphosphorylated A form. All activators of p38 (H2O2, HOS, TNF-, and IL-1ß) triggered phosphorylation of HSP27, causing a redistribution of the nonphosphorylated A form to the phosphorylated B, C, and D forms (Fig 8A). In SDS gels, phosphorylation of HSP27 is associated with the incorporation of 32P in a polypeptide band migrating at 27 kD.34 42 Pretreatments of the cells with SB203580, but not with SKF106978, completely blocked the H2O2-induced phosphorylation of that band (Fig 8B).
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p38 MAP Kinase Activation and HSP27 Phosphorylation Mediate H2O2-Induced Actin Reorganization
We next examined the role of p38 and HSP27 phosphorylation in modulating the H2O2-induced actin reorganization described in Figs 1 and 2. This was investigated by examining the effect of the p38 inhibitor SB203580 or its structural inactive analogue SKF106978 on the H2O2-induced stress fiber formation in HUVECs. Cells were treated for 1 hour with varying concentrations of SB203580 and then for 15 minutes with 250 µmol/L H2O2. After treatments, F-actin was stained, and the cells were examined by confocal microscopy (Fig 9). As described in Figs 1 and 2, H2O2 induced major changes in the microfilament arrangement that were characterized by the disappearance of membrane ruffles and the reorganization of F-actin from cortical microfilaments into transcytoplasmic stress fibers (Fig 9A and 9C). Pretreatments of the cells with SB203580 had no effect on H2O2-induced loss of membrane protrusions but inhibited the formation of stress fibers in a dose-dependent manner. The inhibitory effect of SB203580 started at 0.5 µmol/L and was maximal at 5 µmol/L (Fig 9E, 9G, and 9I). Stress fiber formation by H2O2 was associated with the assembly of vinculin into focal adhesions (Fig 9B and 9D), and this effect was also abolished in a dose-dependent manner by SB203580 (Fig 9F, 9H, and 9J). Interestingly, the concentrations of SB203580 that inhibited F-actin reorganization and recruitment of vinculin into focal adhesions were the same that inhibited the activation of MAPKAP kinase-2/3 by p38 (Fig 7B). SB203580 by itself had no effect on microfilament organization and focal adhesions even at concentrations up to 25 µmol/L (Fig 9K and 9L). SKF106978 did not affect either the basal distribution of F-actin or its response to H2O2 (data not shown). These results suggest that the actin reorganization and the assembly of focal adhesions triggered by H2O2 in HUVECs are mediated by activation of the p38 pathway.
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HUVECs express a high level of HSP27 (6 ng/mg total protein) compared with the low levels found in fibroblasts (1 to 2 ng/mg total protein), a cell type in which oxidative stress induced actin fragmentation instead of stress fiber formation.27 28 This and the finding that inhibiting p38 activity with SB203580 inhibits both HSP27 phosphorylation and the H2O2-induced stress fiber formation in HUVECs are correlative evidence suggesting that HSP27 might be the effector mediating actin reorganization in response to the oxidant-induced activation of p38. To more firmly attribute such a role to HSP27, we reasoned that overexpression of HSP27 in cells that normally expressed low levels of the protein should trigger in these cells the formation of stress fibers in a way similar to that found in endothelial cells. We thus characterized the microfilament response to H2O2 in CCL39-transfected fibroblasts expressing varying levels of human HSP27 compared with the response of a control cell line, which expresses only a normal level of endogenous Chinese hamster HSP27 (1.5 ng/µg). In untreated control cells (clone neo 3) as well as in the HSP27-overexpressing clones 6, B1, B2, and B12, F-actin was organized in cortical filaments, with some cells showing thin fibers traversing the cells. This is illustrated in panels A and C of Fig 10, which show the basal pattern of actin in control clone neo 3 and clone B12, an HSP27 transfectant that expresses the same amount of HSP27 as found in HUVECs (6.8 ng/µg protein). In control neo 3 cells, H2O2 produced a marked disorganization of the microfilament network that was characterized by fragmentation of F-actin, which formed patches concentrated around the nucleus (Fig 10B). In contrast to neo 3 cells, but as observed in HUVECs, H2O2 (800 µmol/L, 60 minutes) produced in B12 cells (Fig 10D) and other HSP27 transfectants (data not shown) a marked reorganization of F-actin, which was rearranged into stress fibers (Fig 10D and data not shown). Pretreating B12 cells with SB203580 totally annihilated the effect of HSP27 overexpression. SB203580 completely blocked the H2O2-induced F-actin rearrangement and sensitized the microfilament network of B12 cells, yielding (as in neo 3 cells) a marked fragmentation and patching of F-actin (Fig 10F). SB203580, in the absence of H2O2 treatment, had no effect on the microfilament organization (Fig 10E). As expected, overexpression of nonphosphorylatable HSP27 was ineffective in modulating stress fiber formation and preserving F-actin integrity in response to H2O2 (data not shown and Reference 28). These results are strong indications that the p38-dependent phosphorylation of HSP27 is involved in maintaining the integrity of F-actin in the presence of H2O2 and in mediating the H2O2-induced shift from cortical microfilaments to stress fibers both in fibroblasts, which artificially overexpress HSP27, and in HUVECs.
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In various cell types, including hepatocytes and fibroblasts, oxidative stress produces a severe disruption of the microfilament cytoskeleton characterized by the fragmentation and patching of F-actin.28 43 In contrast, we found, in the present study, that the microfilament network of HUVECs responds to H2O2 by reorganizing from a cortical localization in ruffles and submembranous bundles into long transcytoplasmic stress fibers, which are formed concomitantly with the assembly of vinculin into focal adhesions. The actin reorganization found in HUVECs in response to oxidants is much similar to that reported after the activation of vascular endothelial cells by agonists such as thrombin and TNF-
44 45 and may bear resemblance to shear stress–induced stress fiber formation and alignment parallel to the blood flow.46 In thrombin and shear stress–stimulated cells, the reorganization of the actin filament causes an increase in the adherence of the cells to the substratum and a retraction of the cytoplasm due to fiber contraction, and these effects may contribute to regulate the intercellular space and the endothelium permeability.45 46 47 In vivo, endothelial cells are constantly in contact with oxyradicals, and F-actin rearrangement in response to oxyradicals is likely to modulate important functions of the endothelium. For example, an oxidant-induced rearrangement of F-actin following release of oxyradicals during the neutrophil respiratory burst triggered by inflammation might contribute to modulate intercellular contacts, preparing the cells for leukocyte adhesion and subsequent transmigration.48 The failure of endothelial cells to efficiently deal with oxidative stress may cause pathologies such as atherosclerosis or ischemia-associated thrombosis.5 6 We found that p38 was the major MAP kinase activated by oxidative stress in HUVECs. Three lines of evidence have suggested that MAPKAP kinase-2/3 and HSP27 are effectors of p38 in these cells: (1) the biphasic activation kinetics of p38 and MAPKAP kinase-2/3 were strikingly superimposable; (2) overexpressing p38 enhanced the activation of MAPKAP kinase-2; and (3) inhibiting p38 activity by SB203580 abrogated both MAPKAP kinase-2/3 activation and HSP27 phosphorylation. That p38, via activation of MAPKAP kinase-2/3 and phosphorylation of HSP27, plays a pivotal role in modulating actin organization in vascular endothelial cells was evidenced by showing that inhibiting the transmission of the signals generated by p38 with SB203580 blocked the H2O2-induced reorganization of F-actin. This conclusion is strengthened by the findings that the same dose-effect relationships were found between the concentrations of SB203580 that inhibited the activity of p38 and those that blocked the reorganization of F-actin. In both cases, the effects started at 0.5 µmol/L and were maximal at 5 µmol/L SB203580. The role of HSP27 phosphorylation in mediating microfilament reorganization was supported by showing that transfected fibroblastic B12 cells, which expressed wild-type HSP27 in an amount comparable to that for HUVECs, responded similarly to endothelial cells to H2O2. In B12 transfectants, F-actin reorganized into transcytoplasmic cables after exposures to H2O2. However, the reorganization of actin did not occur in SB203580-treated B12 cells or in cells transfected with a phosphorylation mutant of HSP2727 28 (data not shown). These data strongly suggest that the p38 MAP kinase/HSP27 pathway modulates actin organization in response to oxidants and is consistent with previous studies suggesting that HSP27 might play a role in modulating actin dynamics.49 50 In vitro, purified HSP27 inhibits actin polymerization in a phosphorylation-dependent manner, and in vivo, HSP27 can modulate in a concentration- and phosphorylation-dependent manner the response of the actin filament to growth factor stimulation in fibroblasts.24 25 26
Regulation of actin polymerization and organization in mammalian cells is a highly complex process that involves a number of actin-binding proteins, including severing, sequestering, cross-linking, and membrane-anchoring proteins, all of which are under the regulation of various signal transduction pathways.50 In this context of competition between various effectors, the importance of the p38 MAP kinase/HSP27 pathway is expected to be related to the cellular concentrations of HSP27. In cells such as mouse NIH-3T3 cells, in which the amount of HSP27 is very low, activation of the p38 pathway would probably have little influence on the microfilament response. In contrast, in cells that express high levels of HSP27, the p38 MAP kinase pathway would have a more important role in modulating actin dynamics. This is supported by the observations of the present study, which show that activation of p38 by H2O2 modulates stress fiber formation in those CCL39 cells in which HSP27 has been artificially increased by gene transfection and also in HUVECs, a cell type that constitutively expresses a high amount of HSP27 (6 ng of HSP27/µg total protein). In addition to endothelial cells, high levels of expression of HSP27 are found in skeletal muscle cells and in some other tissues during development and growth and after stress in most tissues.49 Under these conditions, the p38 pathway may thus be a general pathway regulating actin organization–dependent processes.
Previous studies have shown that the activation of three Ras-related small GTPases of the Rho family, cdc42, rac1, and rhoA, tightly regulate the dynamics of actin filaments.51 The mechanisms by which the p38 MAP kinase/HSP27 pathway contributes to modulate actin reorganization in HUVECs and the relationship between the p38/HSP27 pathways and the Rho-mediated pathways are still obscure. In certain cell lines, rac1 and cdc42, but not rhoA, lie upstream from p38.52 Intriguingly, however, the actin organization found in HUVECs after H2O2 corresponds more closely to the phenotype observed in fibroblasts after microinjection of rhoA, which induces actin polymerization, stress fiber formation, and focal adhesion assembly.51 53 This raises the possibility that phosphorylation of HSP27 can modulate the rhoA response by interacting directly with actin or, alternatively, by interacting with some effectors of rhoA. In this context, we observed that the microfilament reorganization induced by H2O2 was accompanied by a slight (30%) increase in total level of F-actin and found that this increase was totally suppressed by inhibiting HSP27 phosphorylation with SB203580 (data not shown). Moreover, we found that SB203580 also inhibited the H2O2-induced recruitment of vinculin to focal adhesion. Whether formation of focal adhesion is a consequence of actin reorganization triggered by the p38 MAP kinase/HSP27 pathway or is directly modulated by p38 remains to be determined. Interestingly, HSP27 was previously purified as part of a turkey gizzard smooth muscle vinculin-rich fraction.54
In summary, we have shown that oxidative stress induces in vascular endothelial cells major changes in microfilament organization, rearranging from cortical ruffles to stress fibers, and that this rearrangement was modulated by the activation of p38 MAP kinase and phosphorylation of HSP27. These findings may be potentially important in understanding the physiopathology of endothelial cells submitted to oxidative stress, since these cells are heavily exposed to oxidants and since major functions of the endothelium, such as the barrier function and cell migration, are associated with actin reorganization. The observations that p38 was also very sensitive to activators of endothelial cells, such as TNF- and Il-1ß, suggest that this pathway also plays a role in modulating the response of the endothelium to these agonists.
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Acknowledgments |
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This study was supported by the Medical Research Council of Canada (grant MT-13177) and the Cancer Research Society Inc. We thank Drs Youli Zu, Jorge Moscat, and James Woodgett for providing the cDNA of MAPKAP kinase-2, the vector pcDNA3-HA-p38, and the plasmid GST–c-Jun, respectively. We also thank Drs John Grose and G.F. Walter for providing the anti-ERK2 and KT3 antibodies, respectively. We are grateful to Dr J.C. Lee (SmithKline Beecham, King of Prussia, Pa) for providing SB203580 and SKF106978. We also thank Claude Chamberland for his help in confocal microscopy, Diane Huot-Blais for her help in editing the manuscript, and the staff of the Pathology Laboratory of the Hopital St-Francois d'Assise, Quebec, for providing the umbilical cords.
Received September 20, 1996; accepted December 18, 1996.
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J. L. Martin-Ventura, V. Nicolas, X. Houard, L. M. Blanco-Colio, A. Leclercq, J. Egido, R. Vranckx, J.-B. Michel, and O. Meilhac Biological Significance of Decreased HSP27 in Human Atherosclerosis Arterioscler Thromb Vasc Biol, June 1, 2006; 26(6): 1337 - 1343. [Abstract] [Full Text] [PDF]
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J. Segarra, L. Balenci, T. Drenth, F. Maina, and F. Lamballe Combined Signaling through ERK, PI3K/AKT, and RAC1/p38 Is Required for Met-triggered Cortical Neuron Migration J. Biol. Chem., February 24, 2006; 281(8): 4771 - 4778. [Abstract] [Full Text] [PDF]
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K. A. Huey Regulation of HSP25 expression and phosphorylation in functionally overloaded rat plantaris and soleus muscles J Appl Physiol, February 1, 2006; 100(2): 451 - 456. [Abstract] [Full Text] [PDF]
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G. A. Knock, A. S. De Silva, V. A. Snetkov, R. Siow, G. D. Thomas, M. Shiraishi, M. P. Walsh, J. P. T. Ward, and P. I. Aaronson Modulation of PGF2{alpha}- and hypoxia-induced contraction of rat intrapulmonary artery by p38 MAPK inhibition: a nitric oxide-dependent mechanism Am J Physiol Lung Cell Mol Physiol, December 1, 2005; 289(6): L1039 - L1048. [Abstract] [Full Text] [PDF]
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H. Cai Hydrogen peroxide regulation of endothelial function: Origins, mechanisms, and consequences Cardiovasc Res, October 1, 2005; 68(1): 26 - 36. [Abstract] [Full Text] [PDF]
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Y.-J. Lee, D.-H. Lee, C.-K. Cho, S. Bae, G.-J. Jhon, S.-J. Lee, J.-W. Soh, and Y.-S. Lee HSP25 Inhibits Protein Kinase C{delta}-mediated Cell Death through Direct Interaction J. Biol. Chem., May 6, 2005; 280(18): 18108 - 18119. [Abstract] [Full Text] [PDF]
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M. Germann, E. Swain, L. Bergman, and J. T. Nickels Jr. Characterizing the Sphingolipid Signaling Pathway That Remediates Defects Associated with Loss of the Yeast Amphiphysin-like Orthologs, Rvs161p and Rvs167p J. Biol. Chem., February 11, 2005; 280(6): 4270 - 4278. [Abstract] [Full Text] [PDF]
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Q. Wang, M. Yerukhimovich, W. A. Gaarde, I. J. Popoff, and C. M. Doerschuk MKK3 and -6-dependent activation of p38{alpha} MAP kinase is required for cytoskeletal changes in pulmonary microvascular endothelial cells induced by ICAM-1 ligation Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L359 - L369. [Abstract] [Full Text] [PDF]
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L. Luo, D.-Q. Li, A. Doshi, W. Farley, R. M. Corrales, and S. C. Pflugfelder Experimental Dry Eye Stimulates Production of Inflammatory Cytokines and MMP-9 and Activates MAPK Signaling Pathways on the Ocular Surface Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4293 - 4301. [Abstract] [Full Text] [PDF]
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T. Borbiev, A. Birukova, F. Liu, S. Nurmukhambetova, W. T. Gerthoffer, J. G. N. Garcia, and A. D. Verin p38 MAP kinase-dependent regulation of endothelial cell permeability Am J Physiol Lung Cell Mol Physiol, November 1, 2004; 287(5): L911 - L918. [Abstract] [Full Text] [PDF]
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F. Le Boeuf, F. Houle, and J. Huot Regulation of Vascular Endothelial Growth Factor Receptor 2-mediated Phosphorylation of Focal Adhesion Kinase by Heat Shock Protein 90 and Src Kinase Activities J. Biol. Chem., September 10, 2004; 279(37): 39175 - 39185. [Abstract] [Full Text] [PDF]
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H. Ono, T. Ichiki, K. Fukuyama, N. Iino, S. Masuda, K. Egashira, and A. Takeshita cAMP-Response Element-Binding Protein Mediates Tumor Necrosis Factor-{alpha}-Induced Vascular Smooth Muscle Cell Migration Arterioscler Thromb Vasc Biol, September 1, 2004; 24(9): 1634 - 1639. [Abstract] [Full Text] [PDF]
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S. S. An, B. Fabry, M. Mellema, P. Bursac, W. T. Gerthoffer, U. S. Kayyali, M. Gaestel, S. A. Shore, and J. J. Fredberg Role of heat shock protein 27 in cytoskeletal remodeling of the airway smooth muscle cell J Appl Physiol, May 1, 2004; 96(5): 1701 - 1713. [Abstract] [Full Text] [PDF]
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P. V. Usatyuk and V. Natarajan Role of Mitogen-activated Protein Kinases in 4-Hydroxy-2-nonenal-induced Actin Remodeling and Barrier Function in Endothelial Cells J. Biol. Chem., March 19, 2004; 279(12): 11789 - 11797. [Abstract] [Full Text] [PDF]
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Y. Chen, R. Rajashree, Q. Liu, and P. Hofmann Acute p38 MAPK activation decreases force development in ventricular myocytes Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2578 - H2586. [Abstract] [Full Text] [PDF]
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S. M. Stamatovic, R. F. Keep, S. L. Kunkel, and A. V. Andjelkovic Potential role of MCP-1 in endothelial cell tight junction `opening': signaling via Rho and Rho kinase J. Cell Sci., November 15, 2003; 116(22): 4615 - 4628. [Abstract] [Full Text] [PDF]
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S. van Wetering, N. van den Berk, J. D. van Buul, F. P. J. Mul, I. Lommerse, R. Mous, J.-P. t. Klooster, J.-J. Zwaginga, and P. L. Hordijk VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration Am J Physiol Cell Physiol, August 1, 2003; 285(2): C343 - C352. [Abstract] [Full Text] [PDF]
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K. J. Cowan and K. B. Storey Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress J. Exp. Biol., April 1, 2003; 206(7): 1107 - 1115. [Abstract] [Full Text] [PDF]
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F. Houle, S. Rousseau, N. Morrice, M. Luc, S. Mongrain, C. E. Turner, S. Tanaka, P. Moreau, and J. Huot Extracellular Signal-regulated Kinase Mediates Phosphorylation of Tropomyosin-1 to Promote Cytoskeleton Remodeling in Response to Oxidative Stress: Impact on Membrane Blebbing Mol. Biol. Cell, April 1, 2003; 14(4): 1418 - 1432. [Abstract] [Full Text] [PDF]
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M. Zanetti, Z. S. Katusic, and T. O'Brien Adenoviral-mediated overexpression of catalase inhibits endothelial cell proliferation Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2620 - H2626. [Abstract] [Full Text] [PDF]
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U. S. Kayyali, C. M. Pennella, C. Trujillo, O. Villa, M. Gaestel, and P. M. Hassoun Cytoskeletal Changes in Hypoxic Pulmonary Endothelial Cells Are Dependent on MAPK-activated Protein Kinase MK2 J. Biol. Chem., November 1, 2002; 277(45): 42596 - 42602. [Abstract] [Full Text] [PDF]
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A. Kulisz, N. Chen, N. S. Chandel, Z. Shao, and P. T. Schumacker Mitochondrial ROS initiate phosphorylation of p38 MAP kinase during hypoxia in cardiomyocytes Am J Physiol Lung Cell Mol Physiol, June 1, 2002; 282(6): L1324 - L1329. [Abstract] [Full Text] [PDF]
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A. K. Kiemer, N. C. Weber, R. Furst, N. Bildner, S. Kulhanek-Heinze, and A. M. Vollmar Inhibition of p38 MAPK Activation via Induction of MKP-1: Atrial Natriuretic Peptide Reduces TNF-{alpha}-Induced Actin Polymerization and Endothelial Permeability Circ. Res., May 3, 2002; 90(8): 874 - 881. [Abstract] [Full Text] [PDF]
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P. L. Goldberg, D. E. MacNaughton, R. T. Clements, F. L. Minnear, and P. A. Vincent p38 MAPK activation by TGF-beta 1 increases MLC phosphorylation and endothelial monolayer permeability Am J Physiol Lung Cell Mol Physiol, January 1, 2002; 282(1): L146 - L154. [Abstract] [Full Text] [PDF]
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K. Suzuki, M. Hino, H. Kutsuna, F. Hato, C. Sakamoto, T. Takahashi, N. Tatsumi, and S. Kitagawa Selective Activation of p38 Mitogen-Activated Protein Kinase Cascade in Human Neutrophils Stimulated by IL-1{beta} J. Immunol., November 15, 2001; 167(10): 5940 - 5947. [Abstract] [Full Text] [PDF]
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L. H. E. H. Snoeckx, R. N. Cornelussen, F. A. Van Nieuwenhoven, R. S. Reneman, and G. J. Van der Vusse Heat Shock Proteins and Cardiovascular Pathophysiology Physiol Rev, October 1, 2001; 81(4): 1461 - 1497. [Abstract] [Full Text] [PDF]
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R. M. Fryer, H. H. Patel, A. K. Hsu, and G. J. Gross Stress-activated protein kinase phosphorylation during cardioprotection in the ischemic myocardium Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1184 - H1192. [Abstract] [Full Text] [PDF]
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T. Ishizuka, F. Okajima, M. Ishiwara, K. Iizuka, I. Ichimonji, T. Kawata, H. Tsukagoshi, K. Dobashi, T. Nakazawa, and M. Mori Sensitized Mast Cells Migrate Toward the Agen: A Response Regulated by p38 Mitogen-Activated Protein Kinase and Rho-Associated Coiled-Coil-Forming Protein Kinase J. Immunol., August 15, 2001; 167(4): 2298 - 2304. [Abstract] [Full Text] [PDF]
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B. C. Berk Vascular Smooth Muscle Growth: Autocrine Growth Mechanisms Physiol Rev, July 1, 2001; 81(3): 999 - 1030. [Abstract] [Full Text] [PDF]
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Q. Wang and C. M. Doerschuk The p38 Mitogen-Activated Protein Kinase Mediates Cytoskeletal Remodeling in Pulmonary Microvascular Endothelial Cells Upon Intracellular Adhesion Molecule-1 Ligation J. Immunol., June 1, 2001; 166(11): 6877 - 6884. [Abstract] [Full Text] [PDF]
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R. G. Deschesnes, J. Huot, K. Valerie, and J. Landry Involvement of p38 in Apoptosis-associated Membrane Blebbing and Nuclear Condensation Mol. Biol. Cell, June 1, 2001; 12(6): 1569 - 1582. [Abstract] [Full Text] [PDF]
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J. M. Kyriakis and J. Avruch Mammalian Mitogen-Activated Protein Kinase Signal Transduction Pathways Activated by Stress and Inflammation Physiol Rev, April 1, 2001; 81(2): 807 - 869. [Abstract] [Full Text] [PDF]
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H. Lum and K. A. Roebuck Oxidant stress and endothelial cell dysfunction Am J Physiol Cell Physiol, April 1, 2001; 280(4): C719 - C741. [Abstract] [Full Text] [PDF]
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T. Takahashi, F. Hato, T. Yamane, H. Fukumasu, K. Suzuki, S. Ogita, Y. Nishizawa, and S. Kitagawa Activation of Human Neutrophil by Cytokine-Activated Endothelial Cells Circ. Res., March 2, 2001; 88(4): 422 - 429. [Abstract] [Full Text] [PDF]
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S. Sanada, M. Kitakaze, P. J. Papst, K. Hatanaka, H. Asanuma, T. Aki, Y. Shinozaki, H. Ogita, K. Node, S. Takashima, et al. Role of Phasic Dynamism of p38 Mitogen-Activated Protein Kinase Activation in Ischemic Preconditioning of the Canine Heart Circ. Res., February 2, 2001; 88(2): 175 - 180. [Abstract] [Full Text] [PDF]
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N. Ohashi, A. Matsumori, Y. Furukawa, K. Ono, M. Okada, A. Iwasaki, T. Miyamoto, A. Nakano, and S. Sasayama Role of p38 Mitogen-Activated Protein Kinase in Neointimal Hyperplasia After Vascular Injury Arterioscler Thromb Vasc Biol, December 1, 2000; 20(12): 2521 - 2526. [Abstract] [Full Text] [PDF]
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J. P. Leger, F. M. Smith, and R. W. Currie Confocal Microscopic Localization of Constitutive and Heat Shock-Induced Proteins HSP70 and HSP27 in the Rat Heart Circulation, October 3, 2000; 102(14): 1703 - 1709. [Abstract] [Full Text] [PDF]
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K. K. Griendling, D. Sorescu, B. Lassegue, and M. Ushio-Fukai Modulation of Protein Kinase Activity and Gene Expression by Reactive Oxygen Species and Their Role in Vascular Physiology and Pathophysiology Arterioscler Thromb Vasc Biol, October 1, 2000; 20(10): 2175 - 2183. [Abstract] [Full Text] [PDF]
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S. Meloche, J. Landry, J. Huot, F. Houle, F. Marceau, and E. Giasson p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H741 - H751. [Abstract] [Full Text] [PDF]
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F.-X. Beck, W. Neuhofer, and E. Muller Molecular chaperones in the kidney: distribution, putative roles, and regulation Am J Physiol Renal Physiol, August 1, 2000; 279(2): F203 - F215. [Abstract] [Full Text] [PDF]
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C. G. Kevil, T. Oshima, B. Alexander, L. L. Coe, and J. S. Alexander H2O2-mediated permeability: role of MAPK and occludin Am J Physiol Cell Physiol, July 1, 2000; 279(1): C21 - C30. [Abstract] [Full Text] [PDF]
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D. Talmor, A. Applebaum, A. Rudich, Y. Shapira, and A. Tirosh Activation of Mitogen-Activated Protein Kinases in Human Heart During Cardiopulmonary Bypass Circ. Res., May 12, 2000; 86(9): 1004 - 1007. [Abstract] [Full Text] [PDF]
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R. Kacimi, J. Chentoufi, N. Honbo, C. S. Long, and J. S. Karliner Hypoxia differentially regulates stress proteins in cultured cardiomyocytes: Role of the p38 stress-activated kinase signaling cascade, and relation to cytoprotection Cardiovasc Res, April 1, 2000; 46(1): 139 - 150. [Abstract] [Full Text] [PDF]
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S. Rousseau, F. Houle, H. Kotanides, L. Witte, J. Waltenberger, J. Landry, and J. Huot Vascular Endothelial Growth Factor (VEGF)-driven Actin-based Motility Is Mediated by VEGFR2 and Requires Concerted Activation of Stress-activated Protein Kinase 2 (SAPK2/p38) and Geldanamycin-sensitive Phosphorylation of Focal Adhesion Kinase J. Biol. Chem., March 31, 2000; 275(14): 10661 - 10672. [Abstract] [Full Text] [PDF]
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J.-i. Abe, C. P. Baines, and B. C. Berk Role of Mitogen-Activated Protein Kinases in Ischemia and Reperfusion Injury : The Good and the Bad Circ. Res., March 31, 2000; 86(6): 607 - 609. [Full Text] [PDF]
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K. Kujime, S. Hashimoto, Y. Gon, K. Shimizu, and T. Horie p38 Mitogen-Activated Protein Kinase and c-Jun-NH2-Terminal Kinase Regulate RANTES Production by Influenza Virus-Infected Human Bronchial Epithelial Cells J. Immunol., March 15, 2000; 164(6): 3222 - 3228. [Abstract] [Full Text] [PDF]
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K. Breitschopf, J. Haendeler, P. Malchow, A. M. Zeiher, and S. Dimmeler Posttranslational Modification of Bcl-2 Facilitates Its Proteasome-Dependent Degradation: Molecular Characterization of the Involved Signaling Pathway Mol. Cell. Biol., March 1, 2000; 20(5): 1886 - 1896. [Abstract] [Full Text] [PDF]
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C. Schafer, P. Clapp, M. J. Welsh, R. Benndorf, and J. A. Williams HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells Am J Physiol Cell Physiol, December 1, 1999; 277(6): C1032 - C1043. [Abstract] [Full Text] [PDF]
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J. H. Park, N. Okayama, D. Gute, A. Krsmanovic, H. Battarbee, and J. S. Alexander Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton Am J Physiol Cell Physiol, December 1, 1999; 277(6): C1066 - C1074. [Abstract] [Full Text] [PDF]
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M. Wagner, I. Hermanns, F. Bittinger, and C. J. Kirkpatrick Induction of stress proteins in human endothelial cells by heavy metal ions and heat shock Am J Physiol Lung Cell Mol Physiol, November 1, 1999; 277(5): L1026 - L1033. [Abstract] [Full Text] [PDF]
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H. Massaeli, C. Hurtado, J. A. Austria, and G. N. Pierce Oxidized low-density lipoprotein induces cytoskeletal disorganization in smooth muscle cells Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H2017 - H2025. [Abstract] [Full Text] [PDF]
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C. Kunsch and R. M. Medford Oxidative Stress as a Regulator of Gene Expression in the Vasculature Circ. Res., October 15, 1999; 85(8): 753 - 766. [Abstract] [Full Text] [PDF]
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M. Rolli, A. Kotlyarov, K. M. Sakamoto, M. Gaestel, and A. Neininger Stress-induced Stimulation of Early Growth Response Gene-1 by p38/Stress-activated Protein Kinase 2 Is Mediated by a cAMP-responsive Promoter Element in a MAPKAP Kinase 2-independent Manner J. Biol. Chem., July 9, 1999; 274(28): 19559 - 19564. [Abstract] [Full Text] [PDF]
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R. M. A. van Gorp, J. L. V. Broers, C. P. M. Reutelingsperger, N. M. H. J. Bronnenberg, G. Hornstra, M. C. E. van Dam-Mieras, and J. W. M. Heemskerk Peroxide-induced membrane blebbing in endothelial cells associated with glutathione oxidation but not apoptosis Am J Physiol Cell Physiol, July 1, 1999; 277(1): C20 - C28. [Abstract] [Full Text] [PDF]
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