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Published ahead of print on July 1, 2004, doi:10.1165/rcmb.2003-0384OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 423-431, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0384OC

Regulation of c-Jun N-terminal Kinase and p38 Kinase Pathways in Endothelial Cells

Raj Wadgaonkar, Jacqueline W. Pierce, Kaumudi Somnay, Rachel L. Damico, Michael T. Crow, Tucker Collins and Joe G. N. Garcia

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, Maryland; and Children's Hospital and Harvard Medical School, Boston, Massachusetts

Address correspondence to: Raj Wadgaonkar, Ph.D., Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: raj.wadgaonkar{at}downstate.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The rapid and transient induction of E-selectin gene expression by inflammatory tumor necrosis factor (TNF)–{alpha} in endothelial cells is mediated by signaling pathways which involve c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) kinase pathways. To explore this regulation, we first observed that in the continuous presence of cytokine TNF, activation of JNK-1 in both nuclear and cytoplasmic compartments peaked at 15–30 min, with activity returning to uninduced levels by 60 min. Phosphorylation of both the p38 kinase and its molecular target, the nuclear transcription factor, activating transcription factor–2, were transient after TNF-{alpha} or interleukin (IL)-1ß induction. However, cycloheximide treatment prolonged the TNF-{alpha}–induced JNK-1 kinase activity beyond 60 min, suggesting that protein synthesis is required to limit this signaling cascade. We investigated the possible role of the dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-2 in limiting cytokine-induced MAPK signaling. Maximum induction of MKP-1 mRNA and nuclear protein levels by TNF-{alpha} or IL-1ß were noted at 60 min and their expression correlated with the termination of JNK kinase activity, whereas nuclear levels of MKP-2 were not significantly affected by treatment with TNF-{alpha} or IL-1ß. Transient overexpression of MKP-1 demonstrated significant specific inhibition of E-selectin promoter activity consistent with a regulatory role for dual-specificity phosphatases. Inhibition of MKP-1 expression through the use of small interfering RNAs prolonged the cytokine-induced p38 and JNK kinase phosphorylation. Our results suggest that endogenous inhibitors of the MAPK cascade, such as the dual-specificity phosphatases like MKP-1 may be important for the postinduction repression of MAPK activity and E-selectin transcription in endothelial cells. Thus, these inhibitors may play an important role in limiting the inflammatory effects of TNF-{alpha} and IL-1ß.

Abbreviations: activating transcription factor, ATF • cAMP response element, CRE • extracellular signal-regulated kinase, ERK • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • green fluorescence protein, GFP • human umbilical vein endothelial cells, HUVEC • human VH-1 homologue, hVH • c-Jun N-terminal kinase, JNK • mitogen-activated protein kinase, MAPK • MAPK phosphatase, MKP • nuclear factor–{kappa}B, NF-{kappa}B • scrambled RNA sequence, Scr-RNA • sodium dodecyl sulfate, SDS • serine, Ser • silencing or inhibitory RNA, SiRNA • threonine, Thr • thymidine kinase, TK • tumor necrosis factor, TNF • ultraviolet, UV • vaccinia virus phosphatase, VH-1


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The recruitment of leukocytes from the circulation into the extravascular space is critical for inflammatory responses and repair of tissue injury (reviewed in Refs. 1 and 2). The endothelial–leukocyte adhesion molecule E-selectin gene is transcriptionally silent in quiescent endothelial cells but becomes dramatically induced at sites of inflammation by the inflammatory cytokines tumor necrosis factor (TNF)–{alpha} or interleukin (IL)-1ß (3, 4), resulting in increased leukocyte adhesion. Transcription of E-selectin in endothelium peaks in 1–2 h and returns to baseline levels by 12 h after induction. The postinduction repression of E-selectin transcription is crucial in limiting cytokine responsiveness of the endothelium and appears to correlate with a transition from neutrophil to mononuclear cell recruitment during inflammatory processes (reviewed in Ref. 5). The mechanism by which the transcription of this gene is negatively regulated is not completely understood; however, protein synthesis is required for transcriptional repression, suggesting that labile protein inhibitors regulate the rate of E-selectin mRNA synthesis (6–8).

Previous studies of the E-selectin promoter identified several promoter elements necessary for TNF-{alpha} responsiveness, including binding sites for nuclear factor–{kappa}B (NF-{kappa}B) and an element similar to the cAMP response element (CRE)/activating transcription factor (ATF) element (reviewed in Ref. 9) (3, 10). This CRE/ATF-like sequence binds to heterodimers of the nuclear transcription factors, ATF-2 and c-Jun (6, 11). ATF-2 is necessary for E-selectin expression because ATF-2 homozygous null mice are defective in E-selectin induction (12). We have previously demonstrated a role of the CRE/ATF element and its associated transcriptional activators within the E-selectin promoter region as signaling target for the stress-activated kinase c-Jun N-terminal kinase (JNK) and p38 kinases (13).

In response to TNF-{alpha} or IL-1ß, the JNK/p38 protein kinases are activated through a signaling cascade in which the upstream activator, mitogen-activated protein kinase (MAPK) kinase kinase, phosphorylates and activates MAPK kinase 3, 4, and 6, which in turn phosphorylate and activate the JNK and p38 MAPKs (for review see Ref. 14). JNK and p38 kinases phosphorylate c-Jun (Ser 63 and 73) and ATF-2 (Thr 69 and 71) increasing the transactivating properties of these transcription factors (15–17). In endothelial cells, TNF-{alpha} stimulation results in transient activation of JNK-1 and p38, which also correlates with the transient phosphorylation of c-Jun and ATF-2 (13). Overexpression of a kinase-inactive JNK or a phosphorylation-defective ATF-2 inhibits activation of the E-selectin promoter (13). Thus, at least two cytokine-induced signaling pathways, NF-{kappa}B/I{kappa}B{alpha} and JNK/p38 kinases, are important for maximal activation of the E-selectin gene.

The dual-specificity phosphatase family is exemplified by MAP kinase phosphatase (MKP)–1 (also known as CL100, 3CH134, Erp and hVH-1). Other mammalian dual-specificity phosphatases include PAC-1, B23 (also known as hVH-3), VHR, MKP-2 (also known as hVH-2 and TYP-1), MKP-3 (also known as rVH6), MKP-X, hVH-5, MKP-4, and M3/6 (18–24). MKP-1 is induced by oxidative stress, uridine 5'-triphosphate–stimulation, or growth factors in a variety of cell types, and has been suggested to play a role in the regulation of many cellular stress responses mediated by extracellular signal-regulated kinase (ERK), JNK, or p38 kinases (18, 25–29). Recently, investigators have suggested that the expression of MKP-1, induced by stress kinase activation, may feedback to downregulate JNK activity in HeLa cells or fibroblasts (27, 30). The role of MKP-1 in endothelial confluent culture and MKP-1–mediated reduction in JNK activity has been correlated with enhanced expression of cyclin D1 (31–34). Information is limited, however, regarding the possible role of dual-specificity phosphatases in regulation of cytokine-induced JNK and p38 activation in endothelial cells.

Negative regulation of the E-selectin gene is less well understood. The I{kappa}B{alpha} protein may act as a labile repressor of NF-{kappa}B–mediated signaling (6). In addition, inhibitors of the JNK and p38 kinase pathways might be expected to decrease E-selectin expression. Previous investigators have suggested that the activation of p38 and JNK kinases by dual phosphorylation on Tyr and Thr is a reversible process and that dual-specificity phosphatases may regulate these kinases (20, 21). In this report, we examine the regulation of JNK-1 and p38 in human endothelial cells treated with inflammatory cytokines with a focus on the possible role of the dual-specificity phosphatases MKP-1 and MKP-2 in limiting cytokine-induced MAPK signaling. Activation of JNK-1 and p38 was transient within both the cytoplasmic and nuclear compartments but was prolonged by cycloheximide treatment suggesting that protein synthesis is required to limit this signaling cascade. MKP-1 mRNA and nuclear protein levels were induced by TNF-{alpha} or IL-1ß and a maximum effect was at 60 min after induction, which correlated with the termination of stress kinase activity and inhibition the TNF-{alpha}–induced E-selectin expression. These results suggest that the dual-specificity phosphatases, such as MKP-1, may play an important role in limiting the inflammatory effects of TNF-{alpha} and IL-1ß.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture, Transfections, and Cytokine Treatment
Human umbilical vein endothelial cells (HUVECs) obtained from collagenase-digested umbilical veins (35) were cultured in an M199 complete medium that included 20% fetal bovine serum, 100 µg/ml porcine intestinal heparin, 50 µg/ml endothelial mitogen, 50 U/ml penicillin, 50 µg/ml streptomycin, and 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid in gelatin-coated plates. For HUVEC cell transfections, conditions were established using lipofectamine reagent (Gibco BRL, Grand Island, NY). HUVEC cells were plated on 10 cm dishes and grown up to 50% confluency in complete medium in a 5% CO2 incubator at 37°C. For each transfection, 5 µg of DNA was taken into 100 µl serum-free OptiMEM medium, and 20 µl of LipofectAmine reagent was diluted in 100 µl of OptiMEM medium. DNA solution was mixed with LipofectAmine solution and incubated at room temperature for 45 min. HUVEC cells were washed with serum-free medium after complexes were formed. For each transfection, 0.8 ml of serum-free medium was added to the tube containing the DNA–Lipofectamine complexes overlaid onto the washed cells and incubated at 37°C incubator for 2 h. Complete medium without antibiotics was then added to cover the entire plate and, after 12 h of incubation, cells were washed and complete medium with antibiotics was added.

For experimental cytokine induction, confluent monolayers of endothelial cells were exposed to 100 U/ml recombinant human TNF-{alpha} (Genentech, San Francisco, CA), 10 U/ml recombinant IL-1ß (Biogen, Cambridge, MA), or 50 ng/ml phorbol 12-myristate 13-acetate, (Sigma, St. Louis, MO) and 3.5 µg/ml ionomycin (Calbiochem, San Diego, CA) added to complete media as indicated. Cycloheximide (Sigma) was used at a concentration 10 µg/ml.

Measurements of Reporter Gene Expression
Endothelial cells were transfected, harvested, and assayed for reporter proteins as described above and previously (10). Relative transfection efficiency was determined by cotransfection with plasmid thymidine kinase (pTK)–growth hormone or pTK-luciferase promoter (1 µg/60 mm plate) obtained from Promega (Madison, WI). For experimental cytokine induction, confluent monolayers of endothelial cells were exposed to recombinant human TNF-{alpha} at a final concentration of 20 ng/ml in complete media. A fragment containing the region 578 to +35 of the E-selectin promoter was generated by polymerase chain reaction amplification as described previously (10), gel-purified, and subcloned into the SmaI site of the reporter plasmid pCAT3. For CAT assay, the protocol for liquid scintillation assay was used as described in Promega handbook. The expression vectors for MKP1 were generously provided by Dr. N. K. Tonks. Transfection efficiency was monitored using a control plasmid (pCH110; Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). Luciferase reporter and ß-galactosidase activities were measured as described previously (13).

Nuclear and Cytoplasmic Extracts
After experimental treatment of HUVECs, nuclear and cytosolic extracts were prepared as described previously (32). All buffers additionally contained the following cocktail of protease inhibitors and phosphatase inhibitors: 1 mM phenylmethanesulfonyl fluoride, 10 µg/ml each leupeptin and aprotinin, 1.5 µg/ml pepstatin A, 1 mM sodium fluoride, and 1 mM sodium orthovanadate.

Immune-Complex Kinase Assays
Nuclear and cytoplasmic extracts were prepared from control or TNF-{alpha}–treated HUVECs as described above and diluted 1:5 with Triton lysis buffer (20 mM Tris, pH 7.4, 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM {alpha}-glycerophosphate, 1 mM Na orthovanadate, 2 mM pyrophosphate, 1 mM phenylmethylsulphonyl fluoride, 10 µg/ml leupeptin). The JNK-1 protein kinase was immunoprecipitated by incubation for 1 h at 4°C with rabbit polyclonal JNK-1 antibodies (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) bound to protein-A Sepharose (Pharmacia-LKB Biotechnology, Inc.). The immunoprecipitates were washed twice with TLB and twice with kinase buffer (20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [pH 7.4], 20 mM {alpha}-glycerophosphate, 20 mM MgCl2, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate). The kinase assays were initiated by the addition of 1 µg of substrate protein (glutathione s-transferase [GST]–ATF-2) and 50 µM ({gamma}-32P–ATP) (10 Ci/mmole) in a final volume of 22 µl, as described previously (16). The reactions were terminated after 15 min at 30°C by addition of Laemmli sample buffer. Control experiments demonstrated that the phosphorylation reaction was linear with time for at least 30 min under these conditions. The phosphorylation of the substrate proteins was examined by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis followed by autoradiography.

Western Blot
Nuclear extracts from 5 x 106 TNF-{alpha}–treated HUVECs were electrophoresed on 8% SDS–polyacrylamide gels and transferred to nitrocellulose in 25 mM Tris, 192 mM glycine, and 5% methanol at 100 V for 1 h. Antiserum specific for the phosphorylated forms of ATF-2 (Thr 71) or p38 (Tyr 182) were obtained from New England Biolabs (Beverly, MA) and used at 1:1,000 dilution for 16 h at 4°C. Rabbit polyclonal antisera to MKP-1 (C19), MKP-2 (S18), p38, JNK-1, or c-Jun (Santa Cruz Biotechnology, Inc.) were used at concentration 1:100, 1;100, 1;1,000, 1:1,000 or 1:5,000 dilution, respectively, for 16 h at 4°C. Immunoreactive proteins were detected according to the enhanced chemiluminescent protocol (Amersham Corp., Arlington Heights, IL) using 1:10,000 horseradish peroxidase–linked donkey anti-rabbit secondary antiserum. Blots were exposed to film for 1–15 min.

Northern Blot Analysis
Total RNA from 107 cells was isolated by the Trizol method. RNA was separated by electrophoresis on a 1% agarose formaldehyde gel, transferred to Hybond-N membrane (Amersham Corp.) and immobilized by ultraviolet (UV) irradiation using a UV Stratalinker 2400 (Stratagene, La Jolla, CA). Blots were prehybridized for 4 h and then hybridized overnight at 42°C with {alpha} 32P–labeled probe (Megaprime kit, Amersham Corp.). The probe used for MKP-1 mRNA was a 1,950 bp EcoRI fragment from pMT-CL100 (18) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was a 780 bp Pst1-Xba1 fragment from pHcGAP (American Tissue Culture Collection, Rockville, MD).

Silencing of MKP-1 and Transient Transfection
MKP-1–silencing RNA oligos were selected from the DUSP1 sequence and synthesized from Dharmacon, Inc. (Lafayette, CO). Vectors expressing MKP-1 SiRNA and its scrambled sequence were obtained from Dr. Robert Z. Orlowski's laboratory and used in a transient transfection assay (36). MKP-1 SiRNA oligos were cloned into expression vector containing pLPCX and pSilencer 1.0-U6. As a control, a scrambled oligo containing identical nucleotide to MKP-1 SiRNA was cloned into the same expression vector. HUVEC cells were plated in gelatin-coated 12-well plates and grown in growth medium containing endothelial growth factors for HUVEC described earlier (13). Conditions for transient transfections of plasmids and silencing RNA in HUVEC and human pulmonary artery endothelial (HPAE) cells were established using lipofectamine 2000 (Invitrogen, Carlsbad, CA) and the GenePORTER 2 transfection reagent (GTS, San Diego, CA). GenePORTER-dependent transfections were done as follows: cells were plated at 60–70%. The next morning, cells were rinsed twice with phosphate-buffered saline and 900 µl of serum-free medium was added to each well and incubated at 37°C. To prepare the siRNA complexes, silencing RNA for green fluorescence protein (GFP) or MKP-1 was diluted in buffer B (Gene PORTER, composition not described, patented by GTS) at concentration of 50 µM and incubated at room temperature for 5 min. Diluted DNA was added to the diluted GenePORTER 2 reagent and then mixed and incubated for 10 min at room temperature. This mixture was directly added to the cells in the serum-free medium. At 4-h after transfection, growth media was added without removing the transfection reagent. At 12 h after transfection, cells were transferred into complete growth medium for another 36 h.

Statistical Analysis
Values are presented as means ± SE (n = 3 or more for each assay condition). Data were analyzed by one-way analysis of variance with Bonferroni correction, and significance in all cases was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transient Activation of Nuclear and Cytoplasmic JNK-1 Kinase in Response to TNF-{alpha} Stimulation of Endothelium
We assessed the time course and subcellular localization of JNK-1 activity in HUVEC cells after treatment with TNF-{alpha} (Figure 1A). Immunoprecipitation kinase assays were used to measure JNK-1 activity in either cytoplasmic or nuclear extracts as described in MATERIALS AND METHODS. The majority of the JNK-1 activity was in the cytoplasm, and peak activity of this kinase in both cellular compartments was observed at 15–30 min after cytokine addition. After 60 min, the JNK-1 kinase activity in both cytoplasm and nucleus returned to uninduced levels (Figure 1A, lanes 1–5). To test whether these differences in JNK-1 activity could be accounted for by changes in amount of JNK-1 protein, the level of JNK-1 protein in the nucleus was measured by Western blot (Figure 1B, lanes 1–5). There was no significant change in nuclear JNK-1 levels or subcellular distribution.



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  		Figure 1. Transient induction of JNK-1 activity in HUVEC treated with TNF-{alpha}. (A) Immune complex kinase assays: nuclear (top panel) and cytoplasmic extracts (lower panel) were prepared from control or TNF-{alpha} treated HUVECs. The JNK-1 protein kinase was immunoprecipitated by incubation for 1 h at 4°C with rabbit polyclonal JNK-1 antibodies. The kinase assays were performed in the presence of 1 µg of substrate protein GST–ATF-2 (1–109) and 50 µM ({gamma}-32P–ATP). The phosphorylation of the substrate proteins was examined by SDS–polyacrylamide gel electrophoresis followed by autoradiography. Arrows indicate position of {alpha}32-P–ATP–labeled ATF-2. Lane 1, untreated; Lanes 2–5, TNF-{alpha} (100 U/ml); Lanes 6–9, TNF-{alpha} (100 U/ml) and cycloheximide (10 µg/ml); Lane 10, cycloheximide (10 µg/ml) alone. Values obtained from three independent experiments are means ± SD. *Significant difference (P < 0.05) compared with TNF treatment at different time points. (B) Western blot analysis of nuclear JNK-1 levels. Nuclear extracts from cells treated as in A were electrophoresed on 8% SDS–polyacrylamide gels, transferred to nitrocellulose, and probed with rabbit anti–JNK-1 antibody. Immunoreactive proteins were detected according to the enhanced chemiluminiscence using 1:10,000 horseradish peroxidase–linked donkey anti-rabbit secondary antiserum. Lane 1, untreated; Lanes 2–5, TNF-{alpha} (100 U/ml); Lanes 6–9, TNF-{alpha} (100 U/ml) and cycloheximide (10 µg/ml).

 
Cycloheximide Prolongs the Activity of JNK-1 Kinase in TNF-{alpha}–Induced Endothelial Cells
To determine the role of protein synthesis in the regulation of JNK-1 activity, we tested the effect of cycloheximide on cytokine-induced JNK-1 signaling. Treatment of endothelial cells with TNF-{alpha} in the presence of cycloheximide led to a prolonged activation of JNK-1 activity in both the cytoplasmic and nuclear compartments (Figure 1A, lanes 6–9). In addition, there was a modest increase in JNK-1 activity observed upon treatment of HUVEC with cycloheximide alone. There were no significant changes in nuclear JNK-1 levels or subcellular distribution as examined by Western blot (Figure 1B). Thus, the increase in kinase activity was not due to alterations in the levels of JNK protein. These results suggest that the activity of JNK-1 kinase in cytoplasm and nucleus is regulated by labile protein inhibitor(s).

Induction of MKP-1 Nuclear Protein by IL-1ß and TNF-{alpha} Cytokines: Correlation with the Termination of JNK/p38 Kinase Activity
Previous investigators have suggested that p38 and JNK-1 kinases are reversibly activated by dual phosphorylation on Tyr and Thr, and that the dual-specificity phosphatases may regulate these kinases (25, 26). We have previously observed that the p38 and JNK-1 kinases are transiently activated in response to TNF-{alpha} (13). To examine the p38 kinase activation in response to a second inflammatory cytokine, IL-1ß, and to directly assay the phosphorylation of the p38 protein, we performed Western blot analysis with antisera specific for the phosphorylated form of p38 (Tyr 182). Treatment with IL-1ß resulted in transient phosphorylation of the p38 kinase. This phosphorylation of p38 peaked at 15–30 min and the majority of p38 was unphosphorylated at 60 min (Figure 2A). Total levels of p38 in cytoplasmic extracts were not altered by cytokine treatment. Our results are consistent with the notion that inflammatory cytokines act to regulate p38 at the level of phosphorylation. The transient phosphorylation of p38 (and JNK-1) correlated with the transient phosphorylation of nuclear ATF-2 as measured by Western blot analysis with antisera specific for the phosphorylated form of ATF-2 (Figure 2B). Thus, p38/JNK-1 signaling of ATF-2 phosphorylation is terminated within 1 h of cytokine treatment.



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  		Figure 2. Time-course of expression of nuclear MKP-1 and MKP-2 after induction of HUVEC with IL-1ß: correlation of MKP-1 expression with the termination of p38 and ATF-2 phosphorylation. (A) Western blot analysis of cytoplasmic extracts from HUVEC cells treated (10 U/ml) for times indicated. Proteins were electrophoresed on 8% SDS–polyacrylamide gels, transferred to nitrocellulose, and probed with antisera specific for the phosphorylated p38 (Tyr 182) (top panel) or total p38 (lower panel). Immunoreactive proteins were detected according to the enhanced using 1:10,000 horseradish peroxidase–linked donkey antirabbit secondary antiserum. Values are means ± SD of three independent experiments. Fold increases in p38 phosphorylation were normalized to total p38 in the cell lysates. Dotted bars, p-p38; black bars, p38. (B) Western blot of nuclear extracts from HUVEC cells treated with IL-1ß as in A above. Blot was probed with antisera specific for the phosphorylated form of ATF-2 (Thr 71) (top panel), MKP-1(C19) (middle panel), or MKP-2 (lower panel). Values from three independent experiments are means ± SD. Fold increases in ATF-2 phosphorylation were normalized to total ATF-2 in the nuclear extracts. *Significant increase in MKP-1 expression and ATF-2 phosphorylation compared with control (P < 0.05), respective lanes. Diagonally hatched bars, p-ATF-2; horizontally hatched bars, MKP-1; shaded bars, MKP-2.

 
To examine the potential role of dual-specificity phosphatases in limiting p38/JNK-1 kinase activity, we measured the expression of nuclear MKP-1 protein in HUVEC cells treated with IL-1ß. The MKP-1 levels showed a dramatic increase at 1 h after treatment with IL-1 (Figure 2B). The appearance of MKP-1 nuclear protein correlated with the termination of p38/JNK-1 signaling (Figure 2B, lane 4). At 1 h after cytokine induction, MKP-1 levels were elevated, whereas p38 activation and ATF-2 phosphorylation had returned to uninduced levels (Figure 2B, lane 4). The same time-course of MKP-1 induction was observed in response to TNF-{alpha} (Figure 3A). The MKP-1 protein was detectable at 60 to 90 min after induction but was undetectable thereafter, suggesting that the MKP-1 protein is short-lived. To determine the role of protein synthesis in the regulation of JNK-1 activity, we tested the effect of cycloheximide on cytokine-induced MKP-1 activation. Endothelial cells treated with cycloheximide did not induce MKP-1; however, cells treated with only TNF-{alpha} showed significant induction of MKP-1. In the presence of cycloheximide, TNF-{alpha}–induced MKP-1 expression was completely inhibited, suggesting that protein synthesis is required for induced expression of MKP-1 (Figure 3B). This time-course of MKP-1 expression is consistent with a model in which MKP-1 is induced in response to stress kinase activation and serves to feed back to dephosphorylate p38 and JNK-1 and terminate the activity of these kinases.



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  		Figure 3. Time-course of expression of nuclear MKP-1 after induction of HUVEC with TNF-{alpha}. (A) Western blot of nuclear extracts from HUVECs treated with TNF-{alpha} (100 U/ml) for times indicated. Blot was probed with antisera specific for the phosphorylated form of ATF-2 (Thr 71) (top panel), MKP-1 (middle panel), or MKP-2 (lower panel). Immunoreactive proteins were detected according to the enhanced chemiluminiscence using 1:10,000 horseradish peroxidase–linked donkey anti-rabbit secondary antiserum. Values from three independent experiments are means ± SD. Fold increase in ATF-2 phosphorylation were normalized to total ATF-2 in the nuclear extracts. *Significant increase in MKP-1 expression and ATF-2 phosphorylation compared with control (P < 0.05), respectively. (B) Effect of cycloheximide on TNF-{alpha}–induced MKP-1 expression. Western blot of nuclear and cytoplasmic extracts after TNF-{alpha} with and without cycloheximide (10 µg/ml) treatment. Blot was probed with antisera specific for MKP-1. Western blot is representative of three independent experiments. *Significant increase in MKP-1 expression compared with cycloheximide + TNF control (P < 0.05), respectively.

 
Nuclear MKP-2 Levels Are Not Regulated by Cytokines
To test whether inflammatory cytokines induce the expression of another dual-specificity phosphatase, MKP-2, we performed Western blots on nuclear extracts from HUVEC stimulated with IL-1ß and TNF-{alpha} (Figures 2B and 3A). Nuclear levels of MKP-2 phosphatase were detectable prior to cytokine treatment and were not significantly altered in the presence of IL-1ß. Thus we conclude that the expression of MKP-2 is constitutive and not induced by inflammatory cytokines.

Induction of MKP-1 mRNA in Endothelial Cells Treated with Inflammatory Cytokines
The expression of MKP-1 has been reported to be regulated at the transcriptional level after oxidative stress, growth factor stimulation, or heat shock (18, 23). We tested whether or not the MKP-1 was induced at the mRNA level after cytokine treatment of endothelial cells. Northern blot analysis showed a dramatic induction of MKP-1 mRNA after 1 h induction with IL-1ß or phorbol ester (Figure 4B) and a small induction with TNF-{alpha} (Figure 4A). Thus, this shows that the MKP-1 gene is upregulated 1 h after activation with inflammatory cytokines. The levels of MKP-1 mRNA returned to baseline after 90 min of cytokine treatment. This time-course is consistent with the time in which the MKP-1 gene is upregulated by stress-related kinase stimulation (30). The significance of the difference in MKP-1 levels with the different cytokines is unclear. There was no alteration in the levels of GAPDH mRNA, suggesting that these concentrations of cytokines do not nonspecifically alter mRNA levels. These results, therefore, suggest that the MKP-1 phosphatase is induced at the transcriptional level, and that new synthesis of this phosphatase correlates with the termination of p38 and JNK-1 activity after cytokine induction.



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  		Figure 4. Northern blot analysis of MKP-1 mRNA levels in cytokine-induced HUVEC. Cells were treated with cytokines as described in MATERIALS AND METHODS and total cellular RNA was isolated by the Trizol method, separated by electrophoresis on a 1% agarose formaldehyde gel, transferred to Hybond-N membrane, and immobilized by UV irradiation with a UV Stratalinker 2400 (Stratagene). Blots were hybridized overnight at 42°C with 32P-labeled probe for human MKP-1 (upper panel) or GAPDH (lower panel). (A) Total RNA after TNF-{alpha} treatment, lanes 2–4, 100 U/ml. (B) Total RNA after IL-1ß (10 U/ml) treatment. Fold increases in MKP-1 message were normalized with total GAPDH in the cytokine-treated cell lysates. Northern blots are representative of at least three independent experiments. *Significant increase in MKP-1 expression compared with control (untreated) cells.

 
Effect of MKP-1 Silencing on TNF-{alpha}–Induced JNK-1 and p38 Activation
To understand further the role of MKP-1 expression in regulation of JNK-1 and p38 activation, we studied the reduced MKP-1 expression in HUVEC cells obtained by the SiRNA technique. In the transfection assay, we used an oligo-based delivery of SiRNA and performed direct transfection of SiRNA oligos into cultured HUVEC. Using GFP-labeled SiRNA, we first optimized the conditions for HUVEC cell transfection and acheived > 90% transfection efficiency using the Gene-Porter and Lipofectamine 2000 reagents. Transfected cells were treated with TNF-{alpha} and tested for MKP-1 expression. We observed significant reduction in TNF-induced MKP-1 expression in MKP-1–SiRNA–transfected cells compared with the GFP-SiRNA–transfected cells (Figure 5A, upper level). Nuclear and cytoplasmic extracts were prepared and tested for JNK-1 and p38 phosphorylation. In response to TNF-{alpha}, sustained phosphorylation of JNK-1 and p38 was observed (Figures 5A and 5B). Basal level of p38 phosphorylation was also increased after SiRNA treatment, suggesting that downregulation of MKP-1 prolonged the phosphorylation of JNK-1 and p38 kinases. Endothelial cells transfected with SiRNA for MKP-1 showed sustained activation of ATF-2 phosphorylation. These results clearly suggest the role of MKP-1 in attenuating TNF-induced p38 and JNK phosphorylation.




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  		Figure 5. Effect of downregulation of MKP-1 on JNK phosphorlyation in HUVEC. (A) Cells at 60–70% confluency were transfected with MKP-1 SiRNA at 50 µM concentration using GenePORTER reagent (GTS). At 48 h after transfection, cells were treated with TNF-{alpha}, and cytoplasmic and nuclear extracts were prepared. Western blot of nuclear extracts from HUVECs treated with TNF-{alpha} (100 U/ml) are presented for times indicated. Blot was probed with antisera specific for the MKP-1 (top panel), phosphorylated form of ATF-2 (middle panel), or phosphorylated JNK and total JNK after TNF treatment (lower panel). (B) Cells transfected with MKP-1 SiRNA as shown in (A) were further analyzed for p38 activation. Cytoplasmic extracts were Western blotted for total p38 protein level and phosphorylated p38 levels were measured. (C) Data obtained from three different experiments were subjected to densitometric scanning. Values from three independent experiments are means ± S.D. *Significant differences in MKP-1 protein level, p38, and ATF2 phosphorylation level after TNF and SiRNA treatment. (P < 0.05). Upper graph: red bars, MKP-1; light blue bars, ATF-2; pink bars, P-JNK; dark blue bars, total JNK. Lower graph: black bars, total p38; red bars, p-p38.

 
Selective Inhibition of E-Selectin Expression by MKP-1 in HUVEC Cells
Because MKP-1 has previously been reported to dephosphorylate ERK, JNK, and p38 kinases (34), we next sought to determine whether TNF-{alpha}–induced MKP-1 participates in the regulation of E-selectin expression. We established the conditions for transient transfections in HUVEC cells and studied the TNF-{alpha} regulation of E-selectin–CAT expression. Coexpression of MKP-1 with E-selectin–CAT resulted in a dose-dependent inhibition of TNF-{alpha}–induced E-selectin–CAT expression (Figure 6). This effect was specific, as we did not find any change in the cytomegalovirus (CMV) promoter-driven ß galactosidase activity or the thymidine kinase promoter-driven luciferase activity (TK-luciferase). Also, the dominant negative mutant of MKP-1 was less effective in inhibition of E-selectin expression. It is known that protein tyrosine kinase phosphorylation inhibitors can block E-selectin expression. Therefore, we propose the possibility that the dual-specificity phosphatase MKP-1 may have a direct role in regulating cytokine-induced adhesion molecule expression.




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  		Figure 6. Effect of MKP-1 on E-selectin expression in transient transfection assay. (A) HUVEC cells were transfected using lipofectamine protocol and, 24 h after transfection, treated with TNF (100 U/ml, Calbiochem). Cells were harvested after 24 h and CAT assays were performed as described in MATERIALS AND METHODS. E-selectin–CAT promoter construct along with increasing amounts of CMV–MKP-1 (lanes 3–6) and mutant MKP-1 expression vector were transfected. Either CMV-ß gal or TK-luciferase plasmids were used as internal control for transfection efficiency. Relative CAT activity is presented as a mean of three different experiments. Values are means ± SD of three independent experiments in triplicate. *and **A significant difference (P < 0.05) compared with vector-control–transfected cells. E-selectin promoter expression was further examined in the presence of plasmid expressing MKP-1 SiRNA. HUVEC cells were transfected with E-selectin promoter construct along with MKP-1 U6 and scrambled SiRNA–expressing vectors using lipofectamine 2000 reagent (Invitrogen). Downregulation of MKP-1 expression and its effect on E-selectin CAT were selected for statistical analysis. ***Significant difference (P < 0.05) compared with vector-control–transfected cells. (B) Along with E-selectin promoter, TK-Luciferase promoter construct was examined to understand MKP-1–dependent inhibition of promoter activity. Values are means ± SD of three independent experiments in triplicate.

 
To complement the results obtained with the MKP-1 mutant in TNF-{alpha}–induced E-selectin transactivation, we next reduced endogenous endothelial MKP-1 expression using the silencing RNA method. With this approach, earlier reports from other laboratories have shown that MKP-1 expression can be significantly blocked by a vector expressing MKP-1 silencing RNA (36). Along with the E-selectin promoter, a vector expressing the MKP-1 SiRNA and Scr-RNA were transfected using lipofectamine 2000. At 48 h after transfection, cells were treated with TNF-{alpha} for 12 h. E-selectin promoter activity was then determined (Figure 6). As compared with Scr-RNA–expressing cells, MKP-1–SiRNA–expressing cells showed a higher expression of E-selectin CAT, strongly suggesting the role of MKP-1 in regulating E-selectin expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this article, we have examined the expression of the inducible phosphatase, MKP-1, and the constitutive phosphatase, MKP-2, in endothelial cells. We have shown that the activation of the stress kinase pathway in response to inflammatory cytokines is regulated in human endothelial cells. We found that the transient activation of JNK-1 kinase could be prolonged by inhibitors of protein synthesis, suggesting that labile protein(s) play a role in the negative regulation of cytokine-induced JNK-1 kinase activity. Importantly, MKP-1 was induced both at the mRNA level and at the protein level within 1 h of cytokine stimulation, and activation of MKP-1 was inversely correlated with JNK-1 activity.

Previous investigators have reported that JNK-1 may be regulated by MKP-1 and other dual-specificity phosphatases (26, 27, 37). MKP-1 is activated by oxidative stress, uridine 5'-triphosphate stimulation, or growth factors in a variety of cell types (18, 23, 26–29). Recently, it has been reported that stress kinase activation appears to upregulate the transcription of the MKP-1 gene, and induction of MKP-1 has been suggested to feedback and downregulate stress kinases after cellular stress responses (27, 30). Our results suggest that this phosphatase feedback mechanism may play an important role in limiting the response of endothelial cells to inflammatory cytokines.

Many known members of the dual-specificity phosphatase family exhibit rapid induction by cellular stress and growth factors (18, 23, 26–29). Our results suggest that JNK-1 activity is regulated in both the nucleus and the cytoplasm, as treatment with cycloheximide prolongs JNK-1 activity in both cellular compartments. The MKP-1 protein is nuclear and thus it is unlikely that MKP-1 could account for regulation of cytoplasmic JNK-1 or p38 kinase activity (Figure 1A). Whereas most known members of the dual-specificity phosphatase family are nuclear, cytoplasmic phosphatases, such as MKP-3, have been identified (21, 22). Although MKP-3 selectively inhibits ERK1 activity and not p38 or JNK, this suggests that cytoplasmic phosphatases other than MKP-3 may regulate p38 and JNK (22). The expression of MKP-3 or other cytoplasmic dual-specificity phosphatases has not been examined in endothelial cells. In addition, our results suggest that JNK activity may be regulated by constitutive, short-lived phosphatases, as treatment with cycloheximide alone increased the activity of JNK-1 kinase. Although we have observed constitutive expression of MKP-2 in endothelial cells, it is likely that additional constitutive phosphatases in this cell type, may play a role in regulating JNK-1 activity prior to cytokine induction.

Other investigators have reported transient activation of JNK in endothelial cells in response to TNF-{alpha}, IL-1ß, or CD40 signaling (5, 13), and have suggested that homologous desensitization to cytokine stimulation may reflect negative regulation of JNK. These studies did not identify which JNK isoforms were activated in endothelial cells. Our results confirm the transient induction of JNK with the JNK-1 isoform regulated in both nuclear and cytoplasmic compartments. In addition, we have shown that protein synthesis inhibitors prolong JNK-1 activity and the induction of the MKP-1 phosphatase (but not MKP-2) correlates with the termination of JNK-1 activity in endothelial cells. Although we examined the regulation of JNK-1, a variety of JNK isoforms may be regulated by MKP-1 (25, 26). MKP-1 was observed at 1 h after induction with either TNF-{alpha} or IL-1ß. For homologous desensitization to occur, a stimulus-specific regulation of JNK induction would have to be invoked, such as by yet-uncharacterized phosphatases, in addition to MKP-1, which may play a role in limiting responses to individual cytokines. Regulation of stress kinase stimulation after cytokine induction of endothelial cells may involve members of the dual-specificity phosphatase family, which selectively regulate p38/JNK and not ERK, such as the M3/6 phosphatase (22), as ERK levels did not appear to change upon TNF-{alpha} treatment (13).

Our results suggest that dual-specificity phosphatases may regulate JNK/p38 kinase activation and E-selectin expression (see Figure 7). After silencing MKP-1, we found a significant increase in JNK/p38 phosphorylation correlating with its activation. This is direct evidence for the role of MKP-1 in regulating kinase activity or E-selectin expression after cytokine induction. Additional known members of the of the dual-specificity phosphatase family, including M3/6, B23, VHR, MKP4, and MKP-X could potentially be involved in the regulation of p38/JNK phosphorylation; however, this has not been investigated in endothelium (18–24). There may also be novel constitutive or cytokine-inducible phosphatases, which are expressed in endothelial cells that have not been identified or characterized as yet. Mice lacking MKP-1 did not show any demonstrable alteration in ERK activity and no clear phenotype was noted, suggesting that other phosphatases may perform these redundant functions (38, 39). It would be interesting to determine whether the level of JNK-1 induction or E-selectin expression in response to cytokines was altered in these animals. Overexpression of MKP-1 has been shown to negatively regulate ATF-2–dependent and NF-{kappa}B–dependent gene transcription (13, 26). In addition, we have found that MKP-1 overexpression in HUVEC cells selectively inhibits the E-selectin transcription in transient transfection experiments. It is known that a high-level expression of MKP-1 appears to lead to the loss of substrate specificity (38) and may potentially act nonspecifically to inhibit many signaling pathways. Therefore, we have assayed the E-selectin expression at the level where MKP-1 expression does not inhibit the TK-luciferase or ß-galactosidase activities.



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  		Figure 7. Regulation of TNF-{alpha} induced E-selectin expression by MKP-1. In endothelial cells, cytokines stimulation results in transient activation of JNK/p38 protein kinases through a signaling cascade in which the upstream activator, MAPK kinase kinase1, phosphorylates and activates MAPK kinase 3–7, which in turn phosphorylate and activate the JNK and p38 MAPKs (for review see Ref. 14). JNK and p38 kinases phosphorylate c-Jun and ATF-2, increasing the transactivating properties of these transcription factors (15–17). JNK and p38 are constitutively present in the nucleus and are associated with the transcriptional activators, and their phosphorylation status is regulated by MKP-1.

 
Maximal induction of E-selectin transcription requires signaling through both the NF-{kappa}B and JNK/p38 pathways. These two pathways are rapidly activated; however, there are notable differences in the duration of activation. Nuclear translocation of NF-{kappa}B in endothelial cells treated with TNF-{alpha} occurs by 15 min, and persists over many hours. E-selectin transcription requires continuous presence of the activating cytokine, and the continuous presence of NF-{kappa}B in the nucleus (6). Activation of JNK/p38 kinases and the subsequent phosphorylation of ATF-2 and c-Jun also occur within 15 min. In contrast to NF-{kappa}B, both JNK and p38 kinase activity and phosphorylation of ATF-2 and c-Jun are terminated within 1 h of cytokine induction. The mechanism by which the E selectin transcription is negatively regulated is not completely understood; however, protein synthesis is required for transcriptional repression, suggesting that labile protein inhibitors regulate the rate of E-selectin mRNA synthesis (6–8). The I{kappa}B{alpha} protein may act as a labile repressor of NF-{kappa}B-mediated signaling (6, 40). Our results suggest that MKP-1 may act as labile repressors of the p38/JNK-signaling pathway. Thus, multiple negative regulatory mechanisms appear essential to inhibit cytokine-induced expression of E-selectin with the activation of the JNK/p38 kinases—a common theme in the regulation of many genes involved in inflammatory responses—and, via dual-specificity phosphatases, may act to limit the inflammatory effects of TNF-{alpha} and IL-1ß.


   Acknowledgments
 
The authors thank Kay Case for her excellent technical assistance and Dr. David Pearse for his help on the statistical analysis. This work was supported by research grants from the National Institutes of Health to T.C. (HL 35716, PO1 HL 36028) and J.G.N.G. (HL 58064).

Received in original form October 28, 2003

Received in final form June 25, 2004


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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