Yersinia YopJ inhibits pro-inflammatory molecule expression in human bronchial epithelial cells

Respiratory Physiology & Neurobiology 140 (2004) 89–97

Yersinia YopJ inhibits pro-inflammatory molecule expression in human bronchial epithelial cells Limei Zhou, Alan Tan, Marc B. Hershenson∗ Department of Pediatrics, University of Chicago, Chicago, IL 60637-1470, USA Accepted 17 December 2003

Abstract Human bronchial epithelial cell pro-inflammatory molecule expression plays a role in the pathogenesis of airway diseases. We hypothesize that Yersinia outer protein-J (YopJ), a Yersinia virulence effector which inhibits mitogen activated protein (MAP) kinase kinases (MKKs), attenuates epithelial cell pro-inflammatory molecule expression. 16HBE14o-cells were co-transfected with cDNAs encoding Yersinia pseudotuberculosis YopJ or empty vector. Expression of YopJ reduced activation of extracellular signal regulated kinase (ERK)-2, Jun amino terminal kinase (JNK)-1 and I␬B kinase (IKK)-␤. YopJ also blocked transactivation of NF-␬B and AP-1 promoter sequences which has been shown to regulate chemokine expression. Finally, expression of YopJ reduced transcription from the IL-8, RANTES (regulated upon activation, normal T cell expressed and secreted) and intercellular adhesion molecule (ICAM)-1 promoters. We conclude that YopJ expression blocks the innate immune response in lung epithelial cells, the site of Yersinia pestis infection. Inhibition of bronchial epithelial cell responses by YopJ is consistent with the notion that MAP kinases regulates bronchial epithelial cell pro-inflammatory molecule expression. © 2004 Elsevier B.V. All rights reserved. Keywords: Airways; Epithelial cell; Inflammation; Disease airway; Effector; Virulence; Yersinia; Expression; Pro-inflammatory molecules; Kinase; Map; Mammals; Humans

1. Introduction Airway epithelial cells synthesize a number of chemokines including interleukin (IL)-8 (Devalia et al., 1993; Devlin et al., 1994), granulocyte-macrophage colony stimulating factor (GM-CSF) (Mullol et al., 1996; Adkins et al., 1998) and RANTES (for regulated upon activation, normal T-cell expressed ∗ Corresponding author. Present address: University of Michigan, 1500 E. Medical Center Dr., L2211 Women’s Hospital, Box 0212, Ann Arbor, MI 48109-0212, USA. Tel.: +1-734-764-9580; fax: +1-734-936-7635. E-mail address: [email protected] (M.B. Hershenson).

and secreted) (Stellato et al., 1995; Wang et al., 1996), each of which is increased in the airways of patients with asthma (Sousa et al., 1993; Ackerman et al., 1994; Hoshi et al., 1995; Berkman et al., 1996). In addition, bronchial epithelial cell expression of intercellular adhesion molecule (ICAM)-1, a receptor for the leukocyte integrin receptors LFA-1 and Mac-1 and most rhinoviruses, is increased in asthma (Vignola et al., 1993). We have shown in human bronchial epithelial cells that maximal tumor necrosis factor (TNF)-␣-induced interleukin-8 expression depends on the activation of two distinct signaling pathways, one constituted in part by activator protein (AP)-1 and

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the other by nuclear factor (NF)-␬B. Further, we have reported that extracellular signal regulated kinase (ERK) and Jun amino terminal kinase (JNK), members of the mitogen-activated protein (MAP) kinase superfamily, regulate AP-1 and NF-␬B-dependent gene expression (Li et al., 2002). Yersinia pestis is the infectious agent that causes bubonic plague, and Yersinia pseudotuberculosis and Yersinia enterocolitica are causal agents for gastrointestinal disorders. A Yersinia virulence factor, Yersinia outer protein-J (YopJ), has been shown to block MAP kinase and NF-␬B pathways by preventing the phosphorylation and activation of MAP kinase kinases (MKKs) and I␬B kinase (IKK)-␤ (Orth et al., 1999). Additional experiments have suggested that YopJ, by virtue of its cysteine protease activity, disrupts an essential posttranslational modification that is required for activation of MAP kinase and NF-␬B pathways. Based on the structural similarity between YopJ and ubiquitin-like protein protease (Ulp1), this may involve the cleavage of ubiquitin and ubiquitin-like protein conjugates (Orth et al., 2000). Given the requirement of MAP kinases and NF-␬B for IL-8 expression, we hypothesized that YopJ expression would attenuate the expression of pro-inflammatory molecules in human bronchial epithelial cells. We therefore examined the effects of YopJ on transcription from the IL-8, RANTES and ICAM-1 promoters, as well as on AP-1 and NF-␬B transactivation.

2. Methods 2.1. Cell culture A derivative of 16HBE14o-human bronchial epithelial cells, provided by S. White (University of Chicago) was studied. This cell line was originally established from bronchial epithelial tissue by transfection with pSVori-, which contains the origin-defective SV40 genome (Cozens et al., 1994). Unlike the parental line, these cells do not grow in distinct clusters and demonstrate improved transfection efficiency. Cultures show specific immuno-staining with pan-cytokeratin c11 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), bind galactose or galactosamine-specific lectins particular

to basal epithelial cells and express ␣1-, ␤2-, ␤3- and ␤6-integrin subunits on their cell surface (Li et al., 2002). The cells were grown on coated plates (fibronectin, 10 g/ml; collagen, 30 g/ml; bovine serum albumin, 100 g/ml) in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin and 200 mM of l-glutamine. HeLa cells were maintained in Dulbeco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin–streptomycin. 2.2. Plasmid vectors The −162/+44 fragment from the full-length human IL-8 promoter was cloned into a luciferase reporter plasmid (−162/+44 hIL-8/Luc). The reporter activities of this fragment have been shown to be identical to the full-length promoter in response to respiratory syncytial virus infection (Garofalo et al., 1996), and this fragment contains the NF-␬B, nuclear factor for IL-6 (NF-IL6) and AP-1 binding sites required for maximal TNF responses (Brasier et al., 1998). A cDNA encoding −884 to +64 of human RANTES promoter subcloned into luciferase was provided by R. Schleimer (Johns Hopkins Asthma and Allergy Center) (Stellato et al., 1999). A cDNA encoding the full-length ICAM-1 promoter subcloned into luciferase (Voraberger et al., 1991) was provided by J. Solway (University of Chicago). NF-␬B and AP-1 reporter plasmids (NF-␬B-TATA/Luc and AP-1-TATA/Luc, respectively) were purchased from Stratagene (LaJolla, CA). A hemagglutinin (HA)-tagged ERK2 was constructed by fusion a DNA fragment encoding the seven amino acid influenza HA epitope to the 5 -end of murine ERK2 (Hershenson et al., 1995). Plasmid DNAs encoding HA-tagged JNK1 (Minden et al., 1994) and ␤-galactosidase were provided by M. Rosner (University of Chicago). Construction of cDNAs encoding HA-tagged IKK␤ has been described elsewhere (Nemoto et al., 1998). Plasmids harboring the YopJ coding sequence or empty vector (pSFFV) were obtained from J. Dixon (University of Michigan) (Orth et al., 1999). A bacterial expression vector encoding recombinant GST-I␬B␣ was provided by M. Karin (University of California, San Diego) (DiDonato et al., 1996). GST-Jun (1–79) was obtained from

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J. Posada (University of Vermont) (Shapiro et al., 1996). 2.3. Transient transfection and reporter assays DNA transfections were performed using Lipofectamine (Invitrogen, Carlsbad, CA), as described (Ramakrishnan et al., 1998; Li et al., 2002). 16HBE14o-cells or HeLa cells were grown on six well plates at 70–80% confluence and then co-transfected with 0.5 ␮g of the relevant reporter construct and 50–300 ng of YopJ plasmid or empty vector. Transfection efficiency was assessed by co-transfection with 30 ng of ␤-galactosidase (pCMV–␤-gal). Twenty-four hours post-transfection, cells were serum-starved for 8 h and treated with TNF␣ (R&D Systems, Minneapolis, MN). Sixteen hours after treatment, cells were harvested and analyzed for luciferase and ␤-galactosidase activities, as described (Ramakrishnan et al., 1998). 2.4. RT-PCR Total RNA was isolated 48 h post-transfection using Trizol reagent (Invitrogen), treated with RNasefree DNase I and purified with RNeasy mini kit (Qiagen, Valencia, CA). cDNA was then synthesized from 1 ␮g of total RNA using superscript II RNase H-reverse transcriptase (Invitrogen, Carlsbad, CA). No reverse transcriptase was added as a negative control. PCR was performed to detect expression of YopJ gene. The 5 -primer was 5 -ATGATCGGACCAATATCACA-3 , and 3 -primer was: 5 -GTTAACAACAGTACCAACTC-3 . 2.5. Immunoblotting 16HBE14o-cells and HeLa cells were harvested 2 days post-transfection with empty vector pSFFV or pSFFV–YopJ. Cells were lysed in buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Triton, 40 mM ␤-glycerophosphate, 0.2 mM Na3 VO4 , 1 mM PMSF and 1% proteinase inhibitor cocktail (Sigma Chemical, St. Louis, MO). Lysates were centrifuged (5 min at 4 ◦ C) to remove cellular debris. Proteins were separated on 10% SDS–PAGE gel and transferred to nitrocellulose.

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After blocking for 1 h with 5% milk in TBS-T (0.05 M Tris–HCl, pH 7.5, 0.09% NaCl and 0.05% Tween 20), the membrane was sequentially incubated with anti-Flag monoclonal antibody (Sigma, St. Louis, MO) and goat anti-mouse HRP conjugate (Bio-Rad, Hercules, CA) in TBS-T. Signals were amplified using Supersignal West Chemiluminescent Substrate (Pierce, Rockford, IL). 2.6. In vitro kinase assays To examine the effect of YopJ on TNF␣-induced activation of ERK, JNK, and IKK␤, 16HBE14o-cells were co-transfected with cDNA encoding a hemagglutinin-tagged form of ERK2, JNK1 or IKK␤ and either empty vector or cDNA encoding YopJ. Forty-eight hours after transfection, cells were serum-starved in MEM. The next day, cells were treated with TNF␣ for 10 min. Cells were then lysed in 800 ␮l of lysis buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Triton, 40 mM ␤-glycerophosphate, 0.2 mM Na3 VO4 , 1 mM PMSF and 1% proteinase inhibitor cocktail (Sigma Chemical, St. Louis, MO). Lysates were centrifuged (5 min at 4 ◦ C) to remove cellular debris. After preclearance with protein A sepharose beads (1 h), the supernatant was immunoprecipitated with mouse monoclonal anti-HA antibody HA.11 (Covance, Princeton, NJ) precoupled to protein G-Sepharose (4 ◦ C for 3 h). Immunoprecipitates were washed three times with lysis buffer (4 ◦ C) and resuspended in 20 ␮l of kinase buffer containing 25 mM Tris (pH 7.5), 5 mM ␤-glycerophosphate, 2 mM DTT, 0.1 mM Na3 VO4 , 10 mM MgCl2 , 50 ␮M ATP, and 5 ␮Ci of [␥-32 P]-ATP. For measurements of ERK, JNK or IKK activation, GST-Elk1 (Cell Signaling Technology, Beverly, MA), GST-Jun or GST-I␬B was added as a substrate, respectively. After incubation (30 min at 30 ◦ C), the reactions were stopped with 4× SDS loading buffer. The reaction components were separated by 10% SDS–PAGE, transferred to nitrocellulose and exposed to film. To confirm the expression level of hemagglutinintagged kinase in bronchial epithelial cell immunoprecipitates, the membranes were probed with HA.11. Signals were amplified and visualized using peroxidase-linked rat anti-mouse light chain IgG (Zymed Laboratories, South San Francisco, CA) and enhanced chemiluminescence.

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3.2. Expression of YopJ blocks activation of ERK, JNK and IKKβ

Fig. 1. Expression of YopJ in 16HBE14o-cells. Total RNA and protein were extracted 48 h post-transfection, and RT-PCR (A) and immunoblotting (B) were performed to detect expression of YopJ. (A) A specific 724 bp fragment of YopJ DNA was obtained by RT-PCR. (B) Due to low transfection efficiency of 16HBE14o-cells, HeLa cells were also transfected with YopJ DNA as a positive control (lanes 1 and 2, 16HBE14o-cells; lane 3, HeLa cells).

2.7. Data analysis Each experiment was performed at least three times. Statistical significance was assessed by analysis of variance (ANOVA). Differences identified by ANOVA were pinpointed by Student Neuman–Keuls’ multiple range test. For reporter assays, changes in promoter activity were calculated as arbitrary light units/␤-galactosidase calorimetric units per hour.

YopJ has been shown to block both phosphorylation and activation of MKKs (Orth et al., 1999). We therefore, tested whether expression of YopJ blocks the activities of ERK, JNK and IKK, each of which we have shown to be required for maximal TNF␣-induced transcription from the IL-8 promoter (Li et al., 2002). 16HBE14o-cells were co-transfected with either YopJ or empty vector and cDNAs encoding HA-tagged ERK2, JNK1 or IKK␤. The activities of ERK, JNK and IKK␤ were assessed by immunoprecipitation of the epitope tag followed by in vitro kinase assay using recombinant Elk-1, c-Jun or I␬B␣ as a substrate. Expression of YopJ in human bronchial epithelial cells attenuated TNF␣-induced activation of ERK, JNK and IKK␤ (Fig. 2). 3.3. Expression of YopJ partially inhibits both AP-1 and NF-κB transactivation We have previously reported that MAP kinases regulate TNF␣-induced IL-8 expression via the activation of AP-1 and NF-␬B-dependent pathways (Li et al., 2002). To examine whether expression of YopJ attenuates AP-1 and NF-␬B transactivation, cells were co-transfected with YopJ and the appropriate reporter plasmid. YopJ attenuated TNF␣-induced AP-1 and NF-␬B transactivation (Fig. 3).

3. Results 3.1. Expression of YopJ in 16HBE14o-cells

3.4. Expression of YopJ blocks transcription from the IL-8, GM-CSF and RANTES promoters

To confirm that YopJ is expressed in 16HBE14ocells, we attempted to detect YopJ by both RT-PCR and Western blot. As there is no anti-YopJ antibody available, we first used RT-PCR to confirm that the YopJ gene was transcribed after transfection (Fig. 1A), and obtained a specific 724 bp fragment. Secondly, we performed immublotting using an antibody against the Flag C-terminal epitope tag of YopJ (Fig. 1B). Because the transfection efficiency of 16HBE14o-cells is low, HeLa cells were also transfected with YopJ DNA as a positive control. Both experiments confirmed that YopJ was properly expressed in the 16HBE14o-cells.

Like the IL-8 promoter, the promoter regions of RANTES and ICAM-1 also include AP-1 and NF-␬B response elements (Roebuck et al., 1995, 1999; Lakshminarayanan et al., 1998; Boehlk et al., 2000). We therefore hypothesized that expression of YopJ would attenuate transcription of all three pro-inflammatory genes. To test this, we co-transfected 16HBE14o-cells with IL-8, RANTES and ICAM-1 reporter plasmids and cDNAs encoding either empty vector or YopJ. After serum–starvation for 8 h, the cells were treated with TNF␣ overnight. The next day, the cells were harvested, and luciferase activities and ␤-gal activities measured. Expression

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Fig. 2. YopJ inhibits activation of ERK, JNK and IKK. Cells were transiently co-transfected with cDNAs encoding HA-tagged forms of ERK2, JNK1 or IKK␤ and either empty vector or YopJ. After serum–starvation, cells were treated with TNF␣ (10 ng/ml) for 10 min. ERK, JNK and IKK activities were assessed by immunoprecipitation of the epitope tag followed by in vitro kinase assay using recombinant Elk-1, c-Jun or I␬B␣ as a substrate, respectively (upper panels). To confirm that changes in substrate phosphorylation were due to changes in kinase activity, not expression, membranes were probed with anti-HA antibody (lower panels). Expression of YopJ attenuated activation of ERK (A), JNK (B) and IKK␤ (C). Kinase activity and expression were quantified by densitometry and the results expressed as the fold increase in activity normalized for expression (mean ± S.E.M. for three experiments are shown for each kinase assay; ∗ P < 0.05, repeated measures ANOVA plus Neuman–Keuls’ multiple comparison test).

of YopJ significantly blocked the transcription from the IL-8, RANTES and ICAM-1 promoters (Fig. 4).

4. Discussion We have found that: (1) expression of YopJ inhibits TNF␣-induced ERK2, JNK1 and IKK␤ activation; (2) attenuation of MAP kinase and IKK activity, in turn,

leads to a reduction in transactivation of both AP-1 and NF-␬B; and (3) YopJ expression significantly inhibits transcription from the IL-8, RANTES and ICAM-1 promoters. These data confirm the importance of MAP kinase pathways in the regulation of human bronchial epithelial inflammatory responses. YopJ has previously been shown to attenuate MKK activation in cervical adenocarcinoma-derived HeLa cells and human embryonic kidney (HEK293)

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Fig. 3. YopJ attenuates NF-␬B and AP-1 transactivation. 16HBE14o-cells were co-transfected with NF-␬B or AP-1 reporter plasmids, ␤-galactosidase and either empty vector or YopJ. After serum–starvation, cells were treated with TNF␣ (10 ng/ ml) overnight. Results are expressed as luciferase relative light units/␤-galactosidase activity per hour. TNF␣ significantly induced NF-␬B (A) and AP-1 (B) transactivation, whereas expression of YopJ significantly attenuated the TNF␣ signal (n = 3, different from control (*), different from TNF␣ (**), P < 0.05, ANOVA).

epithelial-like cells (Orth et al., 1999, 2000; CollierHyams et al., 2002). In the present study, we found that expression of YopJ attenuates ERK, JNK and IKK activities in 16HBE14o-cells, demonstrating that the effect of YopJ on MAP kinase and IKK function is conserved in human bronchial epithelial cells. While the effect on JNK activation appeared most pronounced, none of the kinases examined were fully inhibited by YopJ. Rather than suggesting MKK-independent pathways, we speculate that the incomplete activation we observed relates to the concentration of YopJ expression vector employed, and that larger DNA concentrations might have induced more complete inactivation.

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Fig. 4. YopJ inhibits pro-inflammatory molecule transcription in human bronchial epithelial cells. 16HBE14o-cells were co-transfected with DNA fragments encoding the human IL-8, ICAM-1 or RANTES promoters subcloned into a luciferase reporter plasmid, ␤-galactosidase and either empty vector or YopJ. After serum–starvation, cells were treated with TNF␣ (10 ng/ml) overnight. Results are expressed as luciferase relative light units/␤galactosidase activity per hour. TNF␣ significantly increased transcription from the IL-8 (A), RANTES (B) and ICAM-1 (C) promoters (n = 3, different from untreated (*), P < 0.05, ANOVA). Expression of YopJ significantly attenuated promoter activity (n = 3, different from TNF␣ (**), P < 0.05, ANOVA).

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Activation of MAP kinases induces the phosphorylation and increases the trans-activating activity of a number of nuclear transcription factors critical for expression of AP-1 family transcription factors, including the Ets family ternary complex factors (Gille et al., 1995a,b; Janknecht et al., 1995), c-Jun (Hibi et al., 1993; Westwick et al., 1994) and MEF2 (Han et al., 1997). Phosphorylation of Ets family transcription factors Elk-1 and SAP-1 increases their ability to form ternary complexes with serum response factor (SRF) and the c-fos serum response element (SRE), rapidly inducing c-Fos transcription. c-Jun and MEF2, on the other hand, enhance c-Jun transcription (Angel et al., 1988; Gille et al., 1995a,b; Janknecht et al., 1995; Marinissen et al., 1999). Thus, inhibition of MAP kinases due to YopJ expression would be expected to reduce AP-1 transactivation. Expression of YopJ indeed reduced both basal and TNF␣-stimulated AP-1 transcriptional activity. Similarly, inhibition of IKK activity by YopJ led to a significant reduction in NF-␬B transcriptional activity. We have previously shown in airway epithelial cells that, in the context of IL-8 promoter, the AP-1 sequence functions as a basal level enhancer, whereas the NF-␬B site confers both basal activity and TNF␣ responsiveness (Li et al., 2002). Accordingly, expression of YopJ induced dramatic reductions in both basal and TNF␣-induced transcription from the IL-8 promoter. Additional studies have demonstrated the importance of AP-1 and NF-␬B promoter binding sites for RANTES and ICAM-1 expression (Roebuck et al., 1995, 1999; Lakshminarayanan et al., 1998; Boehlk et al., 2000). Thus, expression of YopJ also inhibited RANTES and ICAM-1 transcription. Regulation of pro-inflammatory molecule expression in response to TNF␣ in bronchial epithelial cells is complex and appears to involve distinct signaling pathways culminating in the transcriptional activation of AP-1 and NF-B. However, we have recently shown in the context of IL-8 and GM-CSF expression that MAP kinase/ERK kinase (MEKK)-1 functions as a common activator of both AP-1 and NF-␬B signaling (Zhou et al., 2003). Selective activation of MEKK1 induced ERK, JNK and IKK activities, each of which would be expected to be attenuated by YopJ. Previous studies have shown YopJ capable of attenuating the innate immune response. YopJ blocks

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TNF␣ production and induces apoptosis in the infected macrophage (Monack et al., 1998; Palmer et al., 1998). Our data confirm that YopJ blocks the pro-inflammatory response, and extends this work by demonstrating this effect in lung tissue, the site of Y. pestis infection. Chemokine function and recruitment of leukocytes are essential for the protective innate host response. For example, neutralization of CXC chemokine receptor 2 significantly increases mortality in mice with experimental Pseudomonas aeruginosa pneumonia (Tsai et al., 2000). Thus, expression of YopJ may allow Y. pestis to avoid destruction by leukocytes and proliferate in the tissue, leading to increased morbidity and mortality. We must note a limitation of our study, namely that we did not measure the effects of YopJ on the total protein of IL-8, RANTES or ICAM-1, nor the activity of endogenous MAP kinases. Because of the low transfection efficiency of 16HBE14o-cells, we monitored the effects of YopJ in transfected cells by co-expressing reporter plasmids or epitope-tagged kinases. However, we have observed a tight correlation between reductions in IL-8 transcription and protein abundance previously (Li et al., 2002). Also, as far as we are aware, recombinant YopJ protein is unavailable. Finally, the co-tranfection of epitope-tagged kinases to examine the effects of upstream intermediates is a commonly employed strategy in the field of signal transduction. This allows the use of transient transfection rather than the more expensive and labor-intensive development of stable cell lines. In conclusion, we have demonstrated that YopJ attenuates human bronchial epithelial cell expression of pro-inflammatory molecules. These data confirm the importance of MAP kinases for the regulation of AP-1 and NF-␬B-dependent gene expression in these cells.

Acknowledgements These studies were supported by grants from the National Institutes of Health (HL56399) and the Cystic Fibrosis Foundation. The authors thank M. Rosner, J. Solway, J. Dixon, A. Brasier, and R. Schleimer for their gifts of plasmid vectors, and D. Gruenert and S. White for providing 16HBE140-cells.

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