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J. Biol. Chem., Vol. 277, Issue 18, 15229-15232, May 3, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/18/15229    most recent C100641200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kawaguchi, M. Articles by Huang, S.-K. Search for Related Content PubMed PubMed Citation Articles by Kawaguchi, M. Articles by Huang, S.-K. Social Bookmarking                      What's this?

ACCELERATED PUBLICATION
Activation of Extracellular Signal-regulated Kinase (ERK)1/2, but Not p38 and c-Jun N-terminal Kinase, Is Involved in Signaling of a Novel Cytokine, ML-1^*

Mio Kawaguchi, Luiz F. Onuchic^§¶, and Shau-Ku Huang

From the  Johns Hopkins Asthma and Allergy Center, ^§ Division of Nephrology, Department of Medicine and the Johns Hopkins University School of Medicine, Baltimore, Maryland 21224 and the ^¶ Division of Nephrology, University of Sao Paulo School of Medicine, Sao Paulo-SP-01246903 Brazil

Received for publication, November 7, 2001, and in revised form, February 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

A novel cytokine, ML-1, was recently discovered, which shares a similar sequence homology with, but is functionally distinct from, IL-17 (Kawaguchi, M., Onuchic, L., Li, X. D., Essayan, D. M., Schroeder, J., Xiao, H. Q., Liu, M. C., Krishnaswamy, G., Germino, G., and Huang, S. K. (2001) J. Immunol. 167, 4430-4435). To determine the signaling mechanisms of ML-1, we investigated activation of mitogen-activated protein (MAP) kinases induced by ML-1. Results show that ML-1 induces in a time-dependent fashion the expression of IL-6 and IL-8 in both primary bronchial epithelial cells (PBECs) and human umbilical vein endothelial cells (HUVECs). ML-1 activated a MAP kinase and an extracellular signal-regulated kinase (ERK)1/2 but not p38 or the c-Jun N-terminal kinase (JNK) in both cell types. Selective MAP kinase kinase (MEK)1/2 inhibitors, PD98059 and U0126, inhibited, in a dose-dependent manner, ML-1-induced expression of IL-6 and IL-8. These findings suggest that ML-1-induced IL-6 and IL-8 production is mediated through the activation of ERK1/2 in both cell types.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Epithelial and endothelial cells play an important role in the regulation of inflammatory processes via their ability to express a wide range of cytokines such as IL-6¹ and IL-8 in response to various stimuli (1-4). Recently, we and others found a novel cytokine, ML-1 (Ref. 5; or IL-17F, Refs. 6 and 7), sharing a significant degree of sequence homology with IL-17 (8-11). Although members of the IL-17 gene family are classified based on sequence similarity, the genes are selectively expressed in different types of cells and tissues (5). Furthermore, distinct function among members of the IL-17 gene family has been demonstrated (5-11). Whereas IL-17 expression is restricted to activated PBMCs and activated Th0 cells, but not activated Th2 cells, ML-1 is expressed in various cell types and tissues. ML-1 is expressed in liver, lung, ovary, and fetal liver, in contrast to IL-17. It is also expressed in activated CD4⁺ T cells, basophils, PBMCs, and mast cells (9). In contrast to ML-1 and IL-17, two other members of the IL-17 family, IL-17B and IL-17C, were not detected in activated CD4⁺ T cells (9, 10).

ML-1 is able to induce IL-6, IL-8, and intercellular adhesion molecule (ICAM)-1 in primary bronchial epithelial cells, and its gene expression is significantly up-regulated in bronchial alveolar lavage cells of asthmatic subjects following segmental allergen challenge (5). The molecular nature of the receptor for ML-1 is at present unclear, and the signaling pathways involved are as yet to be discovered. The MAP kinase family, including extracellular signal-regulated kinase (ERK)1/2, p38 MAP kinase, and c-Jun N-terminal kinase (JNK), has been shown to play significant roles in mediating signals triggered by cytokines, growth factors, and environmental stress and are involved in various cellular functions (12-15). To examine the role of MAP kinase activation in ML-1-induced signaling, we investigated whether ML-1 induces IL-6 and IL-8 expression via activation of MAP kinases in both primary bronchial epithelial cells (PBECs) and human umbilical vein endothelial cells (HUVECs). In this communication, we demonstrate for the first time that ML-1-induced expression of IL-6 and IL-8 is mediated through the activation of ERK1/2 in both cell types.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Generation of Human Recombinant ML-1-- Human recombinant ML-1 was generated as described previously (5). The coding sequence of ML-1 was amplified by PCR and subcloned into pcDNA 3.1 (Invitrogen) to generate a C-terminal His fusion gene. The vector pcDNA 3.1 was transfected into COS-7 cells by an Effectene Reagent (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. ML-1 was purified with affinity purification by Ni-NTA-agarose beads (Qiagen) for His-tagged proteins. Then the concentration of ML-1 protein was quantified by Bradford assay (Bio-Rad), and the protein was stored at 80 °C until used.

Cell Cultures-- PBECs were purchased from Clonetics (San Diego, CA) and were cultured in bronchial epithelial basal medium (Clonetics) containing 0.5 ng/ml human recombinant epidermal growth factor (EGF), 52 µg/ml bovine pituitary extract, 0.1 ng/ml retinoic acid, 0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 10 µg/ml transferrin, 0.5 µg/ml epinephrine, 6.5 ng/ml triiodothyronine, 50 µg/ml gentamicin, and 50 pg/ml amphotericin-B (Clonetics). HUVECs were obtained from Clonetics. The cells were cultured in endothelial cell growth medium (EGM; Clonetics), containing 12 µg/ml bovine brain extract, 10 ng/ml EGF, 1 µg/ml hydrocortisone, 50 µg/ml gentamicin, 50 ng/ml amphotericin B, and 5% fetal bovine serum amphotericin-B (Clonetics). Both PBECs and HUVECs were incubated at 37 °C in humidified 5% CO₂ and cultured for no more than three passages prior to the analysis.

Analysis of IL-6 and IL-8 in PBECs and HUVECs-- PBECs and HUVECs were treated with 10 or 100 ng/ml ML-1 over several time points. For analysis of gene expression, total RNA was extracted using RNeasy (Qiagen) from 1 × 10⁶ cells 4 h after stimulation or exchange of media. The protocol for cDNA synthesis was the same as described previously (5). The sequences of PCR primers for IL-6: forward, 5'-ATGAACTCCTTCTCCACAAGCGC-3' and reverse, 5'-GAAGAGCCCTCAGGCTGGACTG-3' and IL-8: forward, 5'-TCTGCAGCTCTGTGTGAAG-3' and reverse, 5'-TAATTTCTGTGTTGGCGCA-3'. The amplification reaction was performed for 23 cycles with denaturation at 94 °C for 45 s, annealing at 56 °C for 45 s, and extension at 72 °C for 45 s. PCR products were detected by ethidium bromide staining and normalized by the intensity of an amplified housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, as described previously (5). The expected size for IL-8 was 154 bp, for IL-6, 628 bp, and for glyceraldehyde-3-phosphate dehydrogenase, 450 bp. IL-6 and IL-8 protein levels in the collected supernatants were determined with a commercially available ELISA kit (BIOSOURCE, Camarillo, CA) according to the manufacturer's instructions.

Detection of MAP Kinases-- For analysis of activation of MAP kinases, PBECs were treated with ML-1 (100 ng/ml) for several time periods and in some cases with or without treatment with the MEK1/2 inhibitors, PD98059 and U0126 (1-50 and 0.1-10 µM, respectively; Calbiochem, La Jolla, CA; Refs. 16 and 17), p38 inhibitor, SB202190 (0.5-10 µM; Ref. 18), or a vehicle control (Me₂SO) for 1 h. The final concentration of Me₂SO did not exceed 0.1% (v/v). Following treatment, the cells were washed with ice-cold phosphate-buffered saline, and the cell pellets were immediately lysed in cold lysis buffer (20 mM Tris, pH 7.4, 4 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, 200 µg/ml leupeptin, 50 mM NaF, 5 mM Na₄P₂O₇, 1 mM Na₃VO₄, and 1% Nonidet P-40; all purchased from Sigma). Extracts (1 × 10⁶ cell equivalents/lane) were suspended with an equal volume of 2× loading buffer (0.1 M Tris-HCl, pH 6.8, 4% SDS, 0.005% bromphenol blue, and 20% glycerol) containing 2-mercaptoethanol (0.7 M) and were subjected to 4-20% Tris-glycine gel electrophoresis (NOVEX, San Diego, CA). Gels were then transferred to polyvinylidene difluoride membranes (Bio-Rad) with a Trans Blot apparatus (NOVEX). The membranes were immersed overnight in Tris-buffered saline/Tween 20 containing 5% nonfat dry skim milk (Carnation, Los Angeles, CA).

Immunoreactive proteins were detected using Abs against various kinases and phosphorylated kinases. The Abs used were rabbit anti-ERK1/2 Ab, anti-phospho-ERK1/2 Ab, anti-p38 Ab, anti-JNK Ab, and anti-phospho-JNK Ab (New England Biolabs, Beverly, MA), and rabbit anti-phospho-p38 Ab (Santa Cruz Biotechnology, Santa Cruz, CA). All Abs were suspended in Tris-buffered saline/Tween 20 containing 5% skim milk for 1 h. After washing, the membranes were incubated with peroxidase-linked donkey anti-rabbit Ig Ab (Amersham Biosciences) for 1 h. After washing, membrane-bound anti-rabbit Ig Ab was visualized with enhanced chemiluminescence. Western blotting detection reagents (Amersham Biosciences) and Hyper ECL luminescence detection film (Amersham Biosciences) were used. A panel of kinase protein controls was included and run in parallel. These controls included nonphosphorylated and phosphorylated ERK2 proteins, phosphorylated JNK control cellular extracts from UV-treated 293 cells, and phosphorylated p38 control cellular extracts from anisomycin-treated C-6 glioma cells. (All controls were purchased from New England Biolabs.)

Data Analysis-- The statistical significance of differences was determined by analysis of variance (ANOVA). Any difference with p values less than 0.05 was considered significant. When ANOVA indicated a significant difference, the Scheffe F-test was used to determine the difference between groups.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Stimulation of PBECs and HUVECs with ML-1 elicited a time-dependent increase in IL-6 and IL-8 production (Fig. 1). ML-1 significantly induced IL-6 and IL-8 production at two different doses (10 and 100 ng/ml) and at two different time points (24 and 48 h). A significant induction of IL-8 production from both cell types was seen at a 12-h time point at a dose of 100 ng/ml. To determine the signaling pathway involved in ML-1-induced cytokine expression, untreated or ML-1-stimulated cells were lysed at various time points, and Western blotting analyses were performed using various Abs against members of the MAP kinase family, namely ERK1/2, p38, and JNK. Whereas no activation of p38 and JNK kinases was seen at any time points (Fig. 2, A and B), even after a 4-h stimulation of the cells with ML-1, Western blotting analysis revealed the phosphorylation of ERK1/2 reached a maximum at 20 and 10 min in PBECs and HUVECs, respectively and returned to baseline levels by 60 min (Fig. 2, A and B). In addition, preincubation of the cells with a MEK inhibitor, PD98059 (10 µM), diminished the activation of ERK1/2 in both PBECs and HUVECs, and preincubation of Me₂SO did not affect the phosphorylation of ERK1/2 (Fig. 2, C and D).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Analysis of IL-6 and IL-8 expression in PBECs and HUVECs stimulated by ML-1. IL-6 (A) and IL-8 (B) protein release in the supernatant was determined by ELISA as described under "Experimental Procedures." The results were expressed as mean ± S.D. (n = 6). *, p < 0.05 was considered significant versus control.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Kinetic activation of ERK1/2 by ML-1 in PBECs (A) and HUVECs (B). The cells were incubated with or without ML-1 (100 ng/ml) for different time points as indicated. Western blotting analysis was performed with Abs against different MAP kinases as indicated, and various kinase control proteins and cellular extracts were included as described under "Experimental Procedures." Effect of PD98059 on ML-1-induced phosphorylation of ERK1/2 in PBECs (C) and HUVECs (D) is shown. The cells were preincubated with PD98059 (10 µM) or Me2SO vehicle (DMSO) control for 1 h, followed by stimulation of PBECs and HUVECs with medium or ML-1 for 20 or 10 min, respectively. The results shown are representative of three separate experiments.

We next asked whether the activation of ERK1/2 was necessary for the stimulation of IL-6 and IL-8 production in both cell types. The cells were stimulated with ML-1 in the presence or the absence of PD98059. As demonstrated in Fig. 3, PD98059 inhibited, in a dose-dependent manner, the production of both IL-6 and IL-8, whereas pretreatment of the cells with Me₂SO did not affect cytokine protein release in both PBECs and HUVECs (Fig. 3, A and B). Decreased levels of gene expression for both IL-6 and IL-8 were also noted in PD98059-treated but not in vehicle-treated cells, suggesting that the inhibitory effect was at the level of transcription (Fig. 3, C and D). This inhibitory effect of PD98059 was further confirmed by the use of an additional inhibitor, U0126 (Fig. 3, A and B).

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Effect of ERK1/2 inhibitors on IL-6 (A) and IL-8 (B) protein production in PBECs and HUVECs is shown. The cells were preincubated with varying concentrations of PD98059, U0126, SB202190, or Me2SO vehicle control for 1 h, followed by stimulation with ML-1 (100 ng/ml) for 24 h. The results are expressed as the mean ± S.D. (n = 6). *, p < 0.05 when compared with cultures without the addition of inhibitors. C, semiquantitative analysis of gene expression for IL-6 (C) and IL-8 (D) by reverse transcription (RT)-PCR. The cells were preincubated as described above and treated with ML-1 for 4 h. RT-PCR was performed as described under "Experimental Procedures." The results shown are representative of three separate experiments.

ML-1 is a new cytokine produced by activated CD4⁺ T cells, basophils, PBMCs, and mast cells, and increased expression of the ML-1 gene was seen at sites of allergen challenge in four asthmatic subjects, suggesting its role in allergic inflammatory responses (5). However, the biological function of ML-1 in homeostasis and in disease, such as airway inflammation, is not completely understood. Also, the sequence of ML-1 is shorter than that of IL-17F (5-7), suggesting a differential splicing mechanism. We demonstrate, in this report, that ML-1 is able to induce both IL-6 and IL-8 production through the activation of the ERK1/2 kinase in PBECs and HUVECs. MAP kinases are known to play a central role in the airway epithelial activation in response to various stimuli such as TNF-, IL-1, diesel exhaust particles, and influenza virus infection (12, 19-22). Also, MAP kinases, including ERK1/2, are involved in cytokine signaling in HUVECs (15, 23, 24). In our study, the involvement of ERK kinase, but not p38 or JNK, in ML-1-induced IL-6 and IL-8 production was demonstrated using primary bronchial epithelial cells and endothelial cells. Interestingly, previous data showed that ERK1/2, but not p38 or JNK, may play an important role in cytokine release in primary epithelial cells (12), although all three members of the MAP kinase family are involved in cytokine expression. Indeed IL-17, which shows high homology with ML-1, also activates only ERK1/2 kinase in PBECs (18). It is noted, however, that induction of both IL-6 and IL-8 is not completely inhibited by ERK1/2 kinase inhibitors, PD98059 and U0126, suggesting that additional signaling pathways are involved in these cytokines induced by ML-1 in these cell types. Further work is needed to uncover the signaling pathways of ML-1 and its function in healthy and diseased states.

	ACKNOWLEDGEMENTS

We thank Beverly Plunkett, Eva Ehrlich, Katsushi Miura, and Kiichi Hirota for excellent technical assistance.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants AI-40274 and AI-34002 (to S. K. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6801. Tel.: 1-410-550-2006; Fax: 1-410-550-2527; E-mail: skhuang@mail.jhmi.edu.

Published, JBC Papers in Press, March 12, 2002, DOI 10.1074/jbc.C100641200

	ABBREVIATIONS

The abbreviations used are: IL, interleukin; ERK1/2, extracellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; JNK, c-Jun N-terminal kinase; PBMC, peripheral blood mononuclear cells; PBEC, primary bronchial epithelial cells; HUVEC, human umbilical vein endothelial cells; Ab, antibody; NTA, nitrilotriacetic acid, ELISA, enyzyme-linked immunosorbent assay.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/18/15229    most recent C100641200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kawaguchi, M. Articles by Huang, S.-K. Search for Related Content PubMed PubMed Citation Articles by Kawaguchi, M. Articles by Huang, S.-K. Social Bookmarking                      What's this?