Activation of p42mapk in human umbilical vein endothelial cells by interleukin-1alpha and tumor necrosis factor-alpha

Michael J. May, Caroline P. D. Wheeler-Jones, Rebecca A. Houliston, and Jeremy D. Pearson

Vascular Biology Research Centre, Biomedical Sciences Division, King's College London, Kensington, London W8 7AH, United Kingdom

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Work from this and other laboratories has identified a role for protein tyrosine kinases in interleukin-1alpha (IL-1alpha )- and tumor necrosis factor-alpha (TNF-alpha )-induced responses in endothelial cells. In this study, we show that activation of human umbilical vein endothelial cells (HUVEC) by IL-1alpha leads to increased tyrosine phosphorylation of several proteins including one with a molecular mass of ~42 kDa. This protein was identified as p42mapk by Western blot analysis. Tyrosine phosphorylation and catalytic activation of p42mapk by IL-1alpha was transient, reaching maximal levels after 30 min and returning to basal levels by 120-300 min. Activation of p42mapk in HUVEC was also observed in response to TNF-alpha or to the protein kinase C (PKC)-activating phorbol ester phorbol 12-myristate 13-acetate (PMA). Pretreatment of HUVEC with IL-1alpha or TNF-alpha prevented reactivation of p42mapk by either cytokine but did not affect subsequent activation in response to PMA. Activation of p42mapk by PMA was significantly reduced by the PKC inhibitor Ro-31-8220 and completely inhibited by the protein tyrosine kinase inhibitor genistein. Genistein, but not Ro-31-8220, attenuated IL-1alpha - and TNF-alpha -induced p42mapk activation. Taken together, the results of this study demonstrate 1) that p42mapk is transiently activated in HUVEC by IL-1alpha and TNF-alpha , 2) that this activation is PKC independent, and 3) that a genistein-inhibitable tyrosine kinase may be an upstream regulator of cytokine-induced p42mapk activation in human endothelium.

cytokines; signal transduction; protein kinases; phosphatases

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE PROINFLAMMATORY CYTOKINES interleukin-1 (IL-1) and tumor necrosis factor (TNF)-alpha induce a range of identical responses in vascular endothelial cells (EC) (44), including induction or upregulation of leukocyte adhesion molecule expression (11); secretion of inflammatory mediators such as nitric oxide (NO), prostacyclin, and platelet-activating factor (9, 10, 19); and regulation of pro- and antithrombotic functions (4, 42, 48). A major response to IL-1 and TNF-alpha in EC is rapid activation of the transcription factor NFkappa B, which is involved in the regulation of many of the genes responsible for the above effects (12), but the nature of the postreceptor signals evoked by these cytokines remains unclear.

In many different cell types, IL-1 and TNF-alpha promote phosphorylation on serine or threonine (Ser/Thr) of a number of intracellular substrates including the 27-kDa small heat shock protein (20), the epidermal growth factor (EGF) receptor (6), talin (45), the cap-binding protein (21), and unidentified 65- and 74-kDa proteins (53). Recently, Ser/Thr kinase activity has been found associated with the IL-1 receptor in T cells (36) and the p60 TNF-alpha receptor in U-937 cells (14, 58), although the downstream mode of action of these kinases remains poorly defined. One non-receptor-associated Ser/Thr kinase activated by IL-1 and TNF-alpha is protein kinase C (PKC) (41, 50, 51), and in EC, some responses to these cytokines can be mimicked by treatment of cells with the PKC-activating phorbol ester phorbol 12-myristate 13-acetate (PMA) (31). The role of PKC as an effector molecule induced by IL-1 or TNF-alpha in EC is questionable, however, because downregulation of PKC activity by long-term treatment with PMA does not affect responses to either cytokine (40, 48). In EC, IL-1 and TNF-alpha have also been shown to activate the separate Ser/Thr kinase protein kinase A (PKA), via elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) (17, 56). In addition, IL-1 and TNF-alpha induce the hydrolysis of sphingomyelin to ceramide (50), leading to activation of a 97-kDa Ser/Thr-specific ceramide-dependent kinase (32).

In addition to activation of Ser/Thr kinases, IL-1 and TNF-alpha have been shown to increase tyrosine phosphorylation in several cell types including melanoma cells (24), U-937 cells (23), and EC (8, 63). Furthermore, responses to IL-1 such as activation of NFkappa B in melanoma cells (24) and NO synthesis in smooth muscle cells (34) can be attenuated by the selective inhibitors of protein tyrosine kinases (PTK) genistein (2) and herbimycin A (57). In EC, use of these inhibitors has suggested a role for PTK in TNF-alpha -induced plasminogen activator inhibitor-1 production (59), IL-1-induced endothelin production (26), cyclooxygenase activity (8), and leukocyte adhesion molecule expression and function induced by both cytokines (1, 37, 63).

Recent studies have shown that members of the mitogen-activated protein kinase (MAPK) family, including p44mapk and p42mapk (ERK1 and ERK2, respectively), are activated by IL-1 and TNF-alpha in a variety of different cell types (60, 61, 66-68). MAPKs are Ser/Thr-specific kinases that are activated by phosphorylation on residues Thr-183 and Tyr-185 by members of a family of dual-specificity kinases named MAPK/ERK kinases (MEKs; Refs. 13, 52, 67, 69). Upstream activators of MEK include the kinase c-Raf-1 (29), which is activated via ligand engagement of receptors with intrinsic tyrosine kinase activity (33). In contrast, activation of MAPKs by IL-1 and TNF-alpha appears to require a MEK kinase (MEKK1) that is distinct from c-Raf-1 (5, 66). In EC, activation of p42mapk has been observed as an intracellular response to shear stress (43) and a number of vasoactive agonists including 1-oleoyl-lysophosphatidic acid (LPA), bradykinin, histamine, and bacterial lipopolysaccharide (LPS) (3, 16, 38). Because many of the cellular responses of EC to LPS, IL-1, and TNF-alpha are similar (44), it is possible that elements of the intracellular signals evoked by LPS and the cytokines are shared. Therefore, we have investigated the effects of IL-1alpha and TNF-alpha on the activation of MAPK in human umbilical vein endothelial cells (HUVEC) to understand more fully the intracellular signaling responses to these cytokines in EC.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents. The human recombinant cytokines IL-1alpha and TNF-alpha were purchased from R & D Systems (Oxford, UK). The phorbol esters phorbol 12-myristate 13-acetate (PMA) and 4alpha -phorbol 12,13-didecanoate (4alpha -PDD) were from Sigma (Poole, UK). The antiphosphotyrosine monoclonal antibody PY20 and the anti-p42/p44mapk monoclonal antibody MK-12 were purchased from Affiniti Research Products (Nottingham, UK). The phosphospecific p42/p44mapk antibody was obtained from Promega (Southampton, UK). The peroxidase-conjugated goat anti-mouse and anti-rabbit immunoglobulin G were purchased from Pierce (Rockford, IL). The PKC inhibitor Ro-31-8220 was generously provided by Dr. Trevor Hallam (Roche Products), and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), genistein, and daidzein were from Calbiochem (La Jolla, CA). Reagents for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) were from Bio-Rad (Hemel Hempstead, Herts, UK) or National Diagnostics (Hessle, Hull, UK). Enhanced chemiluminescence (ECL) Western blot detection reagent, Hyperfilm-ECL film, and [gamma -32P]ATP were obtained from Amersham (Bucks, UK), and Rennaissance 4CN Plus was from Du Pont (Dreieich, Germany). The specific anti-p42mapk antibody (antiserum 122) was kindly provided by Prof. Chris Marshall (Institute of Cancer Research, London, UK). Culture media were purchased from Sigma or Life Technologies (Paisley, Scotland, UK). All other reagents were obtained from Sigma or BDH (Poole, Dorset, UK) at the highest available grade.

Cell culture. Endothelial cells from human umbilical veins were isolated as previously described (18). Primary cultures of HUVEC were grown in medium 199 (Sigma) supplemented with 10% (vol/vol) fetal calf serum, 10% (vol/vol) newborn calf serum, 4 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 20 mM NaHCO3 at 37°C in 5% CO2-95% air atmosphere in 25-mm2 tissue-culture flasks (Becton Dickinson, Plymouth, UK) that had been precoated with 1% gelatin. When confluent (1 × 106 cells/flask), primary cultures were trypsinized using phosphate-buffered saline (PBS) containing 0.1% trypsin-0.025% EDTA, plated into 75-cm2 tissue-culture flasks (Becton Dickinson), and grown in the above medium containing 90 mg/ml heparin sodium and 20 mg/ml endothelial cell growth factor (ECGF). For experiments, confluent first passage HUVEC (3 × 106 cells/flask) were plated onto 60-mm tissue-culture dishes (Nunc, Roskilde, Denmark) at a cell density of 5 × 104/ml in growth factor-containing medium and grown to confluence (1 × 106 cells/dish). All experiments were performed using second passage HUVEC up to 72 h after reaching confluence. Cytokine-induced p42mapk activation (and adhesion molecule expression) were quantitatively similar over degrees of confluence ranging between 70 and 100% (unpublished observations).

Immunoblot analysis of tyrosine-phosphorylated proteins and p42mapk. Confluent HUVEC monolayers in 60-mm dishes were serum deprived and ECGF deprived for 12-16 h before experimentation in medium 199 containing 5 mM glutamine. Experiments were terminated by aspiration of cell supernatant followed by washing twice with ice-cold PBS containing 200 mM sodium orthovanadate. Cells were then lysed in buffer containing 63.5 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 6.8), 10% glycerol, 2% SDS, 5% beta -mercaptoethanol, 1 mM sodium orthovanadate, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and 50 µg/ml leupeptin, after which lysates were boiled for 10 min and then centrifuged at 10,000 g for 10 min. Proteins in cell lysates were separated by SDS-PAGE (10%) and electrotransferred onto Immobilon-P membrane (Millipore, Bedford, MA). The molecular weight markers used were either SeeBlue prestained standards (Novel Experimental Technology, San Diego, CA) or Rainbow colored protein markers (Amersham Life Sciences, Bucks, UK). Membranes were blocked for 4 h in TBST [50 mM Tris, 150 mM NaCl, 0.02% (vol/vol) Tween-20, pH 7.4] containing 3% (wt/vol) bovine serum albumin (BSA; Advanced Protein Products, Brierley Hill, UK) and subsequently probed overnight with either antiphosphotyrosine (PY20; 1 µg/ml), anti-p42/p44mapk (MK-12; 1:25,000 dilution), or antiphosphospecific p42/p44mapk (1:5,000 dilution) in TBST/0.3% (wt/vol) BSA. Blots were then washed in TBST (8 × 15 min) and incubated in TBST/0.3% (wt/vol) BSA containing horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin G as appropriate (1:10,000 dilution; 1 h). After the blots were further washed (8 × 15 min) in TBST, immunoreactive bands were visualized using either ECL reagent or Renaissance 4CN Plus according to the manufacturer's instructions. Blots probed with PY20 that were to be further probed with MK-12 were stripped using a buffer containing 62.5 mM Tris · HCl (pH 6.7), 2% SDS, and 0.7% beta -mercaptoethanol at 50°C for 30 min.

Assay of MAPK activity. Confluent monolayers of HUVEC in 60-mm dishes were prepared for experiments as described above. After washing twice with ice-cold PBS containing 200 µM sodium orthovanadate, monolayers were incubated on ice for 5 min in 200 ml of buffer containing 10 mM Tris · HCl (pH 7.4), 150 mM NaCl, 2 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM AEBSF, 2 mM dithiothreitol, and 1 mM sodium orthovanadate. Cell layers were then scraped using a rubber policeman and transferred to microcentrifuge tubes (Beckman Instruments, Palo Alto, CA). Lysates were then sonicated (4 × 10 s; 4°C) followed by centrifugation (35,000 g) for 20 min at 4°C. The supernatant was stored at -70°C until assayed for MAPK activity using the BIOTRAK p42/p44mapk assay system (Amersham Life Sciences) according to the manufacturer's instructions. MAPK activity in 15 µl of cell lysates was measured as the amount of 32P incorporated per minute into a peptide based on the Thr-669 phosphorylation site of the EGF receptor.

Immunoprecipitation and immune complex kinase assay. Confluent, quiescent HUVEC in 60-mm dishes were washed and treated as described for the immunoblotting studies. Cells were lysed in ice-cold lysis buffer [20 mM Tris · HCl (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 50 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 500 µM sodium orthovanadate, 250 µM NaF, and 0.1% beta -mercaptoethanol], scraped into 1.5-ml microcentrifuge tubes, and sonicated (4 × 5 s; on ice). After centrifugation (9,000 g, 1 min, 4°C), lysates were immunoprecipitated with 3 µl of rabbit anti-ERK2 antiserum (122) for 3 h at 4°C with constant rotation. Immune complexes were captured with protein A/G-agarose (3 h, 4°C) and washed three times in lysis buffer followed by a further wash in lysis buffer supplemented with 20 mM MgCl2. Immune complex kinase activity was assayed using 5 µCi [gamma -32P]ATP (50 µM), 20 mM MgCl2, and 1 mg/ml myelin basic protein (MBP) for 20 min at 30°C. Reactions were terminated by the addition of 25 µl of 2× sample buffer, and MBP phosphorylation was visualized by SDS-PAGE (12%) followed by autoradiography.

Statistical analysis. Student's t-test was used to compare means of groups of data. P < 0.05 was considered statistically significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

IL-1alpha and TNF-alpha activate p42mapk in HUVEC. Stimulation of quiescent HUVEC with 100 U/ml IL-1alpha caused increased tyrosine phosphorylation of a 42-kDa protein that was detectable after 15 min, maximal at 30 min, and returned to basal levels after 180 min (Fig. 1A). When antiphosphotyrosine immunoblots were reprobed using an antibody that recognized both p42mapk and p44mapk, only one band migrating at 42 kDa was observed (data not shown). The 42-kDa tyrosine-phosphorylated protein was confirmed as p42mapk by gel mobility-shift analysis (Fig. 1B), which revealed an upper, electrophoretically slower band corresponding to phosphorylated p42mapk. The intensity of the upper band in control samples varied between experiments but never exceeded 15% of the intensity of the lower band when measured by densitometry (data not shown). In response to IL-1alpha , the intensity of the upper band increased time dependently, reaching maximal levels at 30 min. Thereafter, the intensity decreased and reached basal levels within times ranging from 120 to 300 min in separate experiments.


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Fig. 1.   Interleukin-1alpha (IL-1alpha )-induced protein tyrosine phosphorylation and activation of p42mapk in human umbilical vein endothelial cells (HUVEC). A: immunoblot of lysates from quiescent HUVEC treated for times indicated with 100 U/ml IL-1alpha and probed with an antiphosphotyrosine antibody. Positions of major tyrosine- phosphorylated proteins are indicated by numbered arrows (1-6), and position of p42 is also shown. B and D: HUVEC were exposed to IL-1alpha (100 U/ml; B) for times indicated or to various concentrations of IL-1alpha for 30 min (D). Cell lysates were analyzed by immunoblotting with an anti-p42/p44mapk antibody. Mobility shift of phosphorylated (top arrow) compared with nonphosphorylated (bottom arrow) p42mapk is indicated. The 44-kDa mitogen-activated protein (MAP) kinase was not detected by this antibody. C: catalytic p42/p44mapk activity measured in HUVEC lysates prepared from cells treated with 100 U/ml IL-1alpha for times indicated. Enzyme activity was assayed using an in vitro p42/p44mapk kinase assay according to manufacturer's instructions. Data represent mean pmol 32P incorporated/min into a specific MAP kinase substrate from triplicate samples ± SD. Results presented in each panel are representative of at least 2 similar experiments.

Cell lysates from IL-1alpha -treated HUVEC were also tested for MAPK catalytic activity using an in vitro kinase assay. As shown in Fig. 1C, MAPK activity was rapidly induced by 100 U/ml IL-1alpha , reaching maximal levels (4.43 ± 0.06 pmol phosphate/min) at 30 min and returning to baseline (0.81 ± 0.06 pmol phosphate/min) after 120 min. Activation of p42mapk by IL-1alpha was dose dependent, being detectable in the presence of 1 U/ml and maximal at 10 U/ml (Fig. 1D). In all further experiments using IL-1alpha , HUVEC were treated with 100 U/ml IL-1alpha for 30 min to ensure maximal activation of p42mapk.

The ability of TNF-alpha to activate p42mapk in HUVEC was also examined by gel mobility-shift analysis. When HUVEC were treated for a range of times with 100 U/ml TNF-alpha , activated p42mapk was detectable at 5 min, peaked at 15 min, and then decreased, reaching basal levels at times ranging from 60 to 300 min (Fig. 2A). TNF-alpha dose-dependently activated p42mapk, with effects observed at 0.1 U/ml and maximal at 100 U/ml (Fig. 2B). Similar effects of TNF-alpha on p42mapk catalytic activity were detected using the in vitro kinase assay, and the kinetics of TNF-alpha -induced p42mapk activation coincided with the tyrosine phosphorylation of a 42-kDa protein detected using the antiphosphotyrosine antibody (data not shown).


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Fig. 2.   Kinetics of tumor necrosis factor-alpha (TNF-alpha )-induced p42mapk activation. HUVEC were stimulated for a range of times up to 120 min with 100 U/ml TNF-alpha (A) or for 15 min with a range of concentrations (0-100 U/ml) of TNF-alpha (B). Samples prepared from lysates of these cells were immunoblotted and probed using anti-p42/p44mapk. Positions of phosphorylated and nonphosphorylated forms of p42mapk are indicated by top and bottom arrowheads, respectively. Blots are representative of results obtained from at least 3 separate experiments.

To confirm that the cytokine-induced increases in p42mapk activation were immunochemically linked to a MAPK, p42mapk activity was measured after immunoprecipitation of p42mapk from whole cell lysates. Phosphorylation of MBP was detectable in p42mapk immunoprecipitates prepared from resting HUVEC (Fig. 3A), and subsequent exposure to maximal concentrations of either IL-1alpha , TNF-alpha , or PMA enhanced MBP phosphorylation above basal to levels commensurate with the effects of these agonists on p42mapk activation measured by gel-shift analysis (PMA >=  IL-1alpha  > TNF-alpha ). Similar results were obtained in further immunoblotting studies using an antibody recognizing the dually phosphorylated, active forms of p42 and p44mapk (Fig. 3B). Inflammatory cytokines increased the phosphorylation of p42mapk and p44mapk, with maximal phosphorylation observed after 30- or 15-min exposure to IL-1alpha or TNF-alpha , respectively.


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Fig. 3.   Effects of IL-1alpha , TNF-alpha , and phorbol 12-myristate 13-acetate (PMA) on phosphorylation and activation of p42mapk in HUVEC. A: HUVEC were exposed to vehicle alone (C; 30 min), IL-1alpha (I; 100 U/ml, 30 min), TNF-alpha (T; 100 U/ml, 15 min), or PMA (P; 100 nM, 15 min). Cell lysates were prepared and p42mapk was immunoprecipitated as described in MATERIALS AND METHODS. Immunoprecipitated MAP kinase was assayed using myelin basic protein (MBP) as substrate; MBP phosphorylation was analyzed by SDS-PAGE followed by autoradiography. Data shown are from a single experiment representative of 3 with similar results. B: HUVEC were challenged with vehicle alone or with IL-1alpha (100 U/ml) or TNF-alpha (100 U/ml) for times indicated. Cell lysates were prepared as described in MATERIALS AND METHODS and were subjected to immunoblotting using an antiserum recognizing active, dually phosphorylated forms of p42 and p44mapk. Arrows denote approximate molecular masses in kDa. Blot is representative of 2 individual experiments with similar results.

In addition to p42mapk, IL-1alpha time dependently increased the tyrosine phosphorylation of a number of other proteins (Fig. 1A), including three proteins (labeled 1-3) with molecular masses approximating 130, 110, and 98 kDa, respectively. Increased phosphorylation of these proteins was maximal at 30-60 min and remained elevated up to 120 min. Tyrosine phosphorylation of at least three other proteins of ~90, 74, and 67 kDa (labeled 4-6, respectively) was transiently increased by IL-1alpha , in each case reaching maximal levels of phosphorylation at 30-60 min and decreasing toward control levels thereafter. Activation of HUVEC with TNF-alpha (100 U/ml) resulted in a similar time-dependent pattern of protein tyrosine phosphorylation as IL-1alpha (data not shown).

Activation of p42mapk by PMA. The PKC-activating phorbol ester PMA (100 nM) also induced tyrosine phosphorylation of a 42-kDa protein that was detectable after 5 min and remained phosphorylated at 120 min (Fig. 4). PMA also time-dependently increased tyrosine phosphorylation of at least six other proteins in HUVEC, whereas the inactive PMA analog 4alpha -PDD (100 nM) did not affect tyrosine phosphorylation of any protein at either 15 or 120 min (data not shown). As depicted in Fig. 4, mobility-shift analysis identified the 42-kDa protein as p42mapk, and the appearance of the upper band corresponded with the time course of increased tyrosine phosphorylation as assessed by antiphosphotyrosine immunoblotting (data not shown). Activation of p42mapk was dose dependent, with maximal effects at 30 min observed in the presence of 100 nM PMA; no activation of p42mapk was observed in response to 100 nM 4alpha -PDD at 15 or 120 min (Fig. 4).


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Fig. 4.   Time course of PMA-induced p42mapk activation. HUVEC were treated with either 100 nM PMA for a range of times up to 120 min or with 100 nM 4alpha -phorbol 12,13-didecanoate (alpha -PDD) for 15 and 120 min. Cell lysates were then immunoblotted and probed with anti-p42/p44mapk antiserum. Top and bottom arrowheads refer to phosphorylated and nonphosphorylated forms of p42mapk. These results are representative of 3 separate experiments.

Effects of cytokine pretreatment on subsequent p42mapk activation. To investigate the effects of cytokine pretreatment on subsequent activation of p42mapk in HUVEC, cells were incubated for 4 h with either medium alone, IL-1alpha (100 U/ml), or TNF-alpha (100 U/ml) and then for a further 30 min with either IL-1alpha (100 U/ml) or PMA (100 nM) or for 15 min with TNF-alpha (100 U/ml) (Fig. 5A). After pretreatment with medium alone, similar levels of activated p42mapk were observed in response to IL-1alpha , TNF-alpha , and PMA. In contrast, pretreatment for 4 h with either IL-1alpha or TNF-alpha prevented reactivation of p42mapk by either cytokine when compared with medium (control)-treated cells. Pretreatment with either cytokine did not affect the ability of PMA to reactivate p42mapk. Similar results were observed when HUVEC were treated for times ranging from 2 to 8 h with cytokines (data not shown). We also investigated the recovery of reactivation of p42mapk in IL-1alpha -treated cells by incubating HUVEC for 2 h with IL-1alpha (100 U/ml) followed by removal of cytokine-containing medium and replacement with IL-1alpha -free medium. HUVEC were then treated for 30 min with 100 U/ml IL-1alpha at 2, 4, and 6 h after removal of the original IL-1alpha -containing medium. As shown in Fig. 5B, addition of fresh IL-1alpha immediately after removal of the original IL-1alpha did not lead to activation of p42mapk. In contrast, 2 h after removal of IL-1alpha , subsequent incubation with IL-1alpha caused partial reactivation of p42mapk. Maximal reactivation was observed 4 h after removal, but this did not reach the level of activation observed in medium-treated cells.


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Fig. 5.   Effects of cytokine pretreatment on subsequent cytokine- and PMA-induced p42mapk activation in HUVEC. A: cells were pretreated for 4 h with either medium alone, IL-1alpha , or TNF-alpha (100 U/ml each) and then incubated for a further 30 min with either medium alone (C), IL-1alpha (I; 100 U/ml), or PMA (P; 100 nM) or for 15 min with TNF-alpha (T; 100 U/ml). B: HUVEC were treated for 2 h with either medium alone (Med) or IL-1alpha (100 U/ml), which was then removed and replaced with IL-1alpha -free medium (IFM). Cells were then reexposed for 30 min to either medium alone (C) or to IL-1alpha (I; 100 U/ml) at 0, 2, 4, and 6 h after removal of IL-1alpha -containing medium. Blots were probed using an anti-p42/p44mapk antibody, and phosphorylated and nonphosphorylated forms of p42mapk are indicated by top and bottom arrowheads, respectively. Results in each panel are representative of 3 separate experiments.

Effects of protein kinase inhibitors on activation of p42mapk by PMA, IL-1alpha , and TNF-alpha . The effects of protein kinase inhibitors on PMA- and IL-1alpha -activated p42mapk are shown in Fig. 6. Pretreatment of HUVEC for 15 min with the selective PKC inhibitor Ro-31-8220 (1 µM) caused a marked inhibition of p42mapk phosphorylation in response to 100 nM PMA (Fig. 6A, top) but did not affect phosphorylation in response to 100 U/ml IL-1alpha (Fig. 6B, top). PMA-induced catalytic activity of p42mapk was also reduced by 1 µM Ro-31-8220 from 1.20 ± 0.01 to 0.38 ± 0.01 pmol phosphate/min (87 ± 1% inhibition; Fig. 6A, bottom), whereas activity induced by IL-1alpha was enhanced by 157 ± 1% from 1.95 ± 0.02 to 3.06 ± 0.10 pmol phosphate/min by 1 µM Ro-31-8220 (Fig. 6B, bottom). Phosphorylation of p42mapk in response to PMA was completely inhibited by pretreatment of HUVEC for 15 min with the PTK inhibitor genistein (100 mM) and partially inhibited by the less active analog daidzein (100 µM; Fig. 6A, top). Daidzein reduced PMA-induced p42mapk activity to 0.71 ± 0.05 pmol phosphate/min (52 ± 1% inhibition), but activity was reduced by genistein to 0.16 ± 0.01 pmol phosphate/min (111 ± 1% inhibition), which was lower than activity in either control cell lysates (0.26 ± 0.01 pmol phosphate/min) or from cells treated with 100 µM 4alpha -PDD (0.19 ± 0.01 pmol phosphate/min; Fig. 6A, bottom). Phosphorylation of p42mapk induced by IL-1alpha (100 U/ml) was unaffected by 100 µM daidzein but was significantly reduced by 100 µM genistein (Fig. 6B, top). Activity in response to IL-1alpha was reduced to 1.15 ± 0.04 pmol phosphate/min by genistein (48 ± 2% inhibition) but was not affected by 100 µM daidzein (1.83 ± 0.04 pmol phosphate/min; Fig. 6B, bottom). None of the inhibitors was cytotoxic for HUVEC at the times and concentrations used when measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as previously described (37).


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Fig. 6.   Effects of protein kinase inhibitors on PMA- and IL-1alpha -induced activation of p42mapk in HUVEC. Cells were incubated for 15 min with either medium alone, Ro-31-8220 (R; 1 µM), genistein (G; 100 µM), or daidzein (D; 100 µM) and then for a further 30 min with either medium alone (C), 4alpha -PDD (alpha P; 100 nM), or PMA (P; 100 nM) (A) or with medium alone (C) or IL-1alpha (I; 100 U/ml) (B) in continued presence of inhibitors. Lysates from samples were prepared for either immunoblot mobility-shift analysis using anti-p42/p44mapk (top panels) or for use in in vitro p42/p44mapk assay employing a peptide substrate as described in MATERIALS AND METHODS (bottom panels). Top and bottom arrows in top panels show positions of phosphorylated and nonphosphorylated forms of p42mapk, respectively. Each figure is representative of at least 2 similar experiments, and data in bottom panels are given as means of triplicate observations ± SD. (** P < 0.01; *** P < 0.001 vs. control).

Mobility-shift analysis was also used to determine the effects of PKC and PTK inhibitors on TNF-alpha -activated p42mapk in HUVEC. As shown in Fig. 7A, Ro-31-8220 did not affect TNF-alpha -activated p42mapk at either 0.1 or 1 µM, concentrations which significantly reduced PMA-induced activation (Fig. 6A). Another PKC inhibitor, H-7, which is also an inhibitor of the cAMP-dependent protein kinase (PKA; Ref. 22), failed to inhibit TNF-alpha -induced activation of p42mapk (Fig. 7A). Similarly, H-7 did not modify p42mapk activation induced by either IL-1alpha or PMA (data not shown). We have previously shown that at the concentrations employed in the present study (25 and 50 µM), H-7 completely inhibited IL-1alpha -, TNF-alpha -, or PMA-induced expression of the leukocyte adhesion molecule E-selectin by HUVEC (37). In contrast to the lack of effects of the PKC inhibitors, pretreatment of HUVEC for 15 min with genistein (100 µM) inhibited TNF-alpha -induced p42mapk activation (Fig. 7B); at the same concentration, daidzein was without effect on activation.


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Fig. 7.   Effects of protein kinase inhibitors on TNF-alpha -induced p42mapk activation in HUVEC. HUVEC were treated for 15 min with either medium alone, Ro-31-8220 (R), H-7 (H), genistein (G), or daidzein (D) at indicated concentrations and then for a further 15 min with either medium alone (C) or TNF-alpha (T; 100 U/ml). After SDS-PAGE and Western blotting, blots were probed using an anti-p42/p44mapk. Top and bottom arrowheads indicate positions of phosphorylated and nonphosphorylated forms of p42mapk. Each panel is representative of at least 3 separate experiments.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this report, we have shown that stimulation of HUVEC with the proinflammatory cytokines IL-1alpha or TNF-alpha leads to rapid activation of the Ser/Thr kinase p42mapk as evidenced by increased tyrosine phosphorylation, reduced mobility on SDS-PAGE, enhanced EGF receptor peptide phosphorylation in cell lysates, and increased MBP phosphorylation in p42mapk immunoprecipitates. We have also shown that this activation can be inhibited by genistein, a selective inhibitor of PTKs (2). Previous studies have shown that responses by EC to IL-1alpha and TNF-alpha can be inhibited by genistein (8, 26, 59, 63), but to date, no specific intracellular signaling mechanism involving genistein-sensitive tyrosine phosphorylation in response to these cytokines has been identified in EC. The results of this study represent the first demonstration of such a mechanism, although the specific upstream, postreceptor events leading to p42mapk activation in HUVEC remain to be determined.

Activation of p42mapk by TNF-alpha was first reported by Kohno et al. (27), who described rapidly increased tyrosine phosphorylation of two proteins with molecular masses of 41 and 43 kDa in human fibroblasts. These were later identified as p42 and p44mapk (60, 61), and subsequent workers demonstrated their activation by TNF-alpha in mouse macrophages (66, 68) and by IL-1alpha in various human and mouse cell lines (7). Activation of p42mapk after stimulation of receptor tyrosine kinases and G protein-coupled receptors occurs via phosphorylation of the dual-specific kinase MEK by the Ser/Thr kinase c-Raf-1 (29, 33), whereas activation by TNF-alpha in mouse macrophages is via phosphorylation of the MEK-1 isoform of MEK by the c-Raf-1-independent MEK kinase MEKK1 (66, 67). In addition to p42 and p44mapk, IL-1alpha and TNF-alpha activate jun NH2-terminal kinase (JNK) and p38mapk, which are distantly related to p42 and p44mapk (5, 30, 46, 54). Activation of JNK by IL-1alpha and TNF-alpha is also MEKK1 dependent but involves activation of a JNK kinase (JNKK) named MAPK kinase (MKK)4 in place of MEK (5, 15, 65). Several recent studies have demonstrated the cytokine-induced activation of JNK and p38mapk in endothelial cells (25, 39, 47), but further study will be required to identify which, if any, of these upstream mechanisms operate in HUVEC, leading to activation of p42mapk by IL-1alpha and TNF-alpha .

The results of our study demonstrate that cytokine-induced p42mapk activation in HUVEC requires an upstream tyrosine kinase, but the nature and location of this genistein-inhibitable kinase remains to be determined. Further evidence for this is the ability of peroxovanadate, a potent inhibitor of protein tyrosine phosphatases, to induce rapid activation of p42mapk in HUVEC (Wheeler-Jones, Houliston, and Pearson, unpublished data). It is possible that genistein directly inhibits tyrosine phosphorylation of p42mapk, although it has been suggested that the site of action of genistein is not MEK but before the interaction of c-Raf-1 with Ras-GTP (55). In the case of c-Raf-1-independent activation of p42mapk, genistein may inhibit an unidentified PTK upstream of MEKK1, and because MEKK1 has also been shown to bind to Ras-GTP (49), a genistein-sensitive PTK regulating Ras activation may be involved in cytokine-induced activation of p42mapk. However, inhibition of PMA-activated p42mapk in HUVEC by genistein suggests direct inhibition of MEK, as PKC is known to phosphorylate and activate c-Raf-1 (28). Because c-Raf-1 activates MEK directly, the only kinase capable of phosphorylating p42mapk on tyrosine in the pathway evoked by PMA is MEK itself. Identification of the MEK isoforms activated by cytokines and PMA in HUVEC and their relative succeptibilities to genistein will be required before any firm conclusions can be made about the nature of the genistein-inhibitable kinase.

Activation of p42mapk in response to PMA indicates a potential role for PKC in cytokine-induced activation; however, we do not believe that PKC is involved for the following reasons. First, the time courses of activation of p42mapk in response to cytokines and PMA were significantly different, suggesting activation of distinct signaling mechanisms by each stimulant. Second, treatment of HUVEC for 4 h with IL-1alpha and TNF-alpha did not affect subsequent activation of p42mapk by PMA but did prevent reactivation by each cytokine. If cytokines and PMA activated the same signaling mechanism in HUVEC, downregulation of this pathway leading to cross-inhibition of reactivation by separate cytokines would be expected to prevent reactivation by PMA. The long time course of p42mapk activation by PMA prevented detection of any effects of PKC downregulation by long-term PMA incubation (data not shown). Finally, PMA-induced activation of p42mapk was inhibited by the selective PKC inhibitor Ro-31-8220, whereas Ro-31-8220 failed to inhibit activation in response to either IL-1alpha or TNF-alpha . Although we observed increased catalytic activity of p42mapk by IL-1alpha in Ro-31-8220-pretreated cells, which may have been via inhibition of a PKC-activated MAPK phosphatase (62), no increased phosphorylation of p42mapk was detected by mobility-shift analysis, suggesting that the increased activity was because of a nonspecific effect of the inhibitor. Interestingly, the nonspecific Ser/Thr kinase inhibitor H-7, which we (37) and others (31, 48) have shown to inhibit adhesion molecule expression in response to IL-1alpha and TNF-alpha in HUVEC, failed to inhibit activation of p42mapk by either cytokine. It therefore seems that the H-7-dependent kinase required for activation of HUVEC lies on a distinct signaling pathway from that leading to p42mapk activation, for example, by being located upstream of NFkappa B activation that is required for adhesion molecule expression (12, 39, 47).

As discussed above, the different time courses of p42mapk activation in response to cytokines and PMA suggest involvement of separate signaling pathways. Furthermore, the marked differences between cytokine- and PMA-induced p42mapk activation may underlie differences in the outcome of endothelial activation by these separate stimuli. Previous studies of PC12 neuronal cells have shown that separate cellular responses to tyrosine kinase receptor activation including differentiation and proliferation are determined by the duration of MAPK activation (35). We also observed differences in the time taken to reach maximal activation in response to the individual cytokines. Maximal phosphorylation in response to TNF-alpha occurred after 15 min, but IL-1alpha -induced activation was slower, reaching a maximum at 30 min. Although the initial events induced by the cytokines leading to p42mapk activation are unknown, serine-specific kinase activities associated with the IL-1 type 1 receptor (36) and the p60 TNF-alpha receptor (14, 58) have been reported. It may be that the different time courses of p42mapk activation in HUVEC are because of differences in activation of these or other receptor-associated kinases. It is interesting to note that, like p42mapk activation by IL-1alpha and TNF-alpha in HUVEC, the TNF-alpha -receptor- and IL-1alpha -associated kinases are not inhibited by H-7 (36, 58). However, although the initial signaling processes evoked by separate cytokines may be different, cross-inhibition of p42mapk activation by pretreatment with cytokines suggests that later events induced by IL-1alpha and TNF-alpha leading to p42mapk activation overlap.

The anti-p42/p44mapk antibody employed for the mobility-shift analyses in this and an earlier study in HUVEC (64) detected only the p42 isoform of MAPK, whereas we have detected both MAPK isoforms in rat aortic smooth muscle cells (Houliston, Pearson, and Wheeler-Jones, unpublished data). This finding is in agreement with the recent reports of Pearce et al. (43) and McLees et al. (38), who demonstrated the presence of only p42mapk in HUVEC and the human endothelial cell hybrid line EAhy 926, respectively, using different antisera. Immunoblot analysis using an antiphosphotyrosine antibody also demonstrated phosphorylation of only one protein of 42 kDa in response to cytokines and no other proteins of a similar molecular mass (Fig. 1A). However, an antiserum raised against the active, dually phosphorylated forms of p42 and p44mapk detected both forms of MAPK in HUVEC (Fig. 3B) and, moreover, showed that treatment with either IL-1alpha or TNF-alpha enhanced the phosphorylation state of both p42 and p44mapk. Interestingly, Arditi et al. (3) recently demonstrated activation of both p42 and p44mapk in bovine and human brain microvascular ECs in response to LPS, which together with our results shows a diversity in expression of MAPK isoforms in endothelial cells derived from separate vascular beds and also indicates an antibody-dependent variation in the ability to detect p42/p44 MAP kinases and their activation.

In conclusion, we have demonstrated the activation of a tyrosine phosphorylation-dependent signaling pathway in human ECs in reponse to IL-1alpha and TNF-alpha . The precise nature of the upstream kinases leading to activation of p42mapk, along with the downstream effects of p42mapk activation, remains unknown, but further study of this pathway will be crucial for a full understanding of the effects of these cytokines on endothelial cells.

    ACKNOWLEDGEMENTS

We thank the midwives and delivery staff at St. Mary's Hospital, London, for help in obtaining the umbilical cords. We also thank Dr. Trevor Hallam and Prof. Chris Marshall for the kind gifts of Ro-31-8220 and antiserum 122, respectively.

    FOOTNOTES

This work was supported by the Arthritis and Rheumatism Council, the British Heart Foundation, and Ono Pharmaceuticals. C. P. D. Wheeler-Jones is a British Heart Foundation Intermediate Research Fellow.

Present address and address for reprint requests: M. J. May, Section of Immunobiology, Yale University School of Medicine, Rm. FMB 409, 310 Cedar St., PO Box 208011, New Haven, CT 06520-8011.

Received 31 January 1997; accepted in final form 25 November 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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