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J. Biol. Chem., Vol. 277, Issue 49, 47167-47174, December 6, 2002

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Differential Gene Regulation by Specific Gain-of-function JNK1 Proteins Expressed in Swiss 3T3 Fibroblasts^*

Sun-Young Han, Soo-Hyun Kim^§, and Lynn E. Heasley^§¶

From the Departments of  Pharmacology and ^§ Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received for publication, May 1, 2002, and in revised form, August 30, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The c-Jun N-terminal kinases (JNKs) are encoded by three genes that yield 10 isoforms through alternative mRNA splicing. The roles of each JNK isoform in the many putative biological responses where the JNK pathway is activated are still unclear. To examine the cellular responses mediated by different JNK isoforms, gain-of-function JNK1 polypeptides were generated by fusing the upstream mitogen-activated protein kinase kinase, MKK7, with p46JNK1 or p46JNK1. The MKK7-JNK fusion proteins, which exhibited constitutive activity in 293T cells, were stably expressed in Swiss 3T3 fibroblasts using retrovirus-mediated gene transfer. Swiss 3T3 cells expressing either of the MKK7-JNK polypeptides were equally sensitized to induction of cell death following serum withdrawal. To search for other cellular responses that may be selectively regulated by the JNK1 isoforms, the gene expression profiles of Swiss 3T3 cells expressing MKK7-JNK1 or MKK7-JNK1 were compared with empty vector-transfected control cells. Affymetrix Genechips identified 46 genes for which expression was increased in MKK7-JNK-expressing cells relative to vector control cells. Twenty genes including those for c-Jun, MKP-7, interluekin-1 receptor family member ST2L/ST2, and c-Jun-binding protein were induced similarly by MKK7-JNK1 and MKK7-JNK1 proteins, whereas 13 genes were selectively increased by MKK7-JNK1 and 13 genes were selectively increased by MKK7-JNK1. The set of genes selectively induced by MKK7-JNK1 included a number of known interferon-stimulated genes (ISG12, ISG15, IGTP, and GTPI). Consistent with these gene expression changes, Swiss 3T3 cells expressing MKK7-JNK1 exhibited increased resistance to vesicular stomatitis virus-induced cell death. These findings reveal evidence for JNK isoform-selective gene regulation and support a role for distinct JNK isoforms in specific cellular responses.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Members of the c-Jun N-terminal kinase (JNK)¹/stress-activated protein kinase family of mitogen-activated protein (MAP) kinases are strongly stimulated by numerous environmental stress, but also more modestly by mitogens, oncogenes, and inducers of cell differentiation and morphogenesis (1-3). The stimulation of JNK activity by diverse inducers of cell death, growth, and differentiation coupled with emerging genetic findings in invertebrates and mammals support broad roles for the JNKs in cell and developmental biology. In a manner parallel to the regulation of the related extracellular signal-regulated kinases, the JNKs are activated following phosphorylation on threonine and tyrosine by the dual-specificity MAP kinase kinases (MKKs), MKK4 and MKK7 (1, 3). The defined substrates of JNK are still somewhat ill defined, but include transcription factors such as c-Jun, ATF2, and Elk-1 (1) and non-transcription factor targets such as keratin 8 and neurofilament heavy chain proteins (4, 5). Despite extensive progress in the understanding of the JNK MAP kinase pathway, the mechanisms by which the pathway contributes to the many cellular programs where JNKs are activated are poorly defined.

The diversity of JNK functions within cells will likely be accounted for, in part, by the complexity of this family of MAP kinases. Ten mammalian JNK isoforms have been defined and are encoded by three distinct genes, jnk1, jnk2, and jnk3, the transcripts of which are alternatively spliced to yield four JNK1 isoforms, four JNK2 isoforms, and two JNK3 isoforms (1, 6). An alternative splice near the 3' end of the coding region dictates the p46 and p54 forms of the three distinct jnk gene products. In addition, alternative exon usage within the protein kinase domain IX and X of jnk1 and jnk2 yields the corresponding JNK or JNK forms of JNK1 and JNK2, which has been shown to affect the strength of association with substrates (6, 7). Thus, because of the many JNK polypeptides generated through alternative splicing, the JNK family of enzymes is sufficiently complex to encompass the many scenarios in which JNKs are known to be activated.

The precise biological functions of the JNK signal transduction pathway remain a subject of intense research. One approach to deciphering JNK functions is to manipulate the JNK expression or activity genetically. Recently described MKK7-JNK fusion proteins that exhibit a gain-of-function phenotype (8) offer an approach to increase JNK activity in cells without activating other signal pathways that are co-stimulated by cell stresses and growth factors. Moreover, the approach permits the development and analysis of different gain-of-function JNK isoforms. In the present study, we generated fibroblast cell lines stably expressing two different gain-of-function JNK1 isoforms and employed oligonucleotide arrays to identify genes for which expression is regulated by these JNK signals. The results reveal selective regulation of gene expression in fibroblasts by gain-of-function JNK1 and JNK1. Notably, the expression of a panel of interferon-stimulated genes is selectively increased by gain-of-function JNK1 and is associated with increased resistance to killing by vesicular stomatitis virus (VSV). The findings are consistent with an emerging role for the JNK pathway in the innate immune response.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- 293T cells and Swiss 3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidified 5% CO₂ incubator. For serum withdrawal experiments, cells were seeded in replicate wells of a 24-well plate in full growth medium at a density of 50,000 cells/well. The next day, the medium was aspirated and replaced with serum-free media for 3 days. Before and after serum withdrawal, the cells were trypsinized from wells and counted with an hemacytometer. For viral infection, cells were plated in 96-well plates at a density of 5,000 cells/well, and exposed for 24 h to VSV at the indicated dilution. The survival of the cells following VSV infection was measured by a colorimetric method using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI). The virus stock was prepared by a cytopathic effect assay in human WISH cells with 1 unit of human IFN- from Peprotech (Rocky Hill, NJ).

Generation of MKK7-JNK Fusion Constructs-- Zheng et al. (8) recently reported that a fusion protein consisting of MKK7 and JNK1 exhibited a gain-of-function phenotype. We created a cDNA encoding a similar polypeptide by fusing the murine MKK71 cDNA to the human p46 JNK1 or p46 JNK1 cDNA. To introduce an in-frame (Glu-Gly)₅ linker between MKK71 and the JNK proteins, the MKK71 cDNA was submitted to PCR with a forward primer, 5'-GAG ATA CTC GAG GTG GAT GTC GC-3', which contains the single MKK71 XhoI site (in bold) and the reverse primer 5'-CCA CCG GTC CCT CGC CCT CGC CCT CGC CCT CGC CCT CCC TGA AGA AGG GCA GAT G-3', which encodes the (Glu-Gly)₅ linker as a replacement for the MKK71 stop codon and an AgeI site (in bold) to facilitate ligation to a N-terminal portion of JNK PCR product. A PCR product containing an AgeI site and the sequences encoding the N-terminal portion of each JNK protein was generated. The N-terminal portion of JNK1 and JNK1 up to BlpI site was generated using the forward primer, 5'-CCA CCG GTG AGC AGA AGC AAG CGT GAC AAC AAT-3' and the reverse primer 5'-AAA TGG TCG GCT TAG CTT CTT GAT-3'. The MKK7-(Glu-Gly)₅ and the N-terminal portion of each JNK fragment were cloned into the pGEMT Easy vector (Promega) for DNA sequencing. The N-terminal fragment of JNK1 protein was excised from the pGEMT vector with AgeI and BlpI and ligated into pGEMT-MKK71-(Glu-Gly)₅ previously digested with AgeI and BlpI. The resulting MKK71-(Glu-Gly)₅/N-terminal JNK1 fragment was excised with XhoI-BlpI and ligated along with the BlpI-ClaI fragment of p46JNK1a (JNK1 C terminus) from LNCX-HA-p46JNK1 into XhoI-ClaI-digested LNCX-Flag-MKK71, thereby generating Flag-MKK71-(Glu-Gly)₅-p46JNK1 in the pLNCX retroviral expression vector (9). MKK7-JNK1 was generated in the same way, and the ligation junctions were confirmed by DNA dideoxynucleotide sequencing.

Transient Transfections-- A calcium phosphate precipitation procedure was used for transient transfection of 293T cells. Briefly, 25 µl of a DNA mixture containing 0.25 M CaCl₂ and UAS-luciferase reporter plasmid (100 ng), c-Jun-GAL4 expression vector (5 ng), pCMV-gal expression vector (50 ng), expression vectors for MKK7-JNK1 proteins (10 ng), and carrier DNA (pcDNA3) to a total of 1 µg of DNA was mixed with 25 µl of 2× HEPES-buffered saline and incubated for 15 min to allow for DNA precipitation. The DNA precipitates were then added to 293T cells previously plated in 24-well plates at 100,000 cells/well. The next day, cells were washed with phosphate-buffered saline and kept in full media for 2 days after transfection. The transfected 293T cells were then washed once with ice-cold phosphate-buffered saline and lysed in 250 µl of Luciferase Reporter Lysis Buffer (Promega). The cell lysates were centrifuged in a microcentrifuge and aliquots (80 µl) of the supernatants assayed for luciferase activity with a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI) and luciferase assay substrate from Promega. Aliquots (15 µl) of the extracts were also assayed for -galactosidase to correct for transfection efficiency. The data are presented as relative light units normalized for transfection efficiency by the corresponding -galactosidase activity.

Generation of MKK7-JNK Stable Transfectants-- Initial attempts with standard retroviral expression vectors (i.e. LNCX) failed to consistently yield Swiss 3T3 cells stably expressing the different MKK7-JNK fusion proteins. Therefore, the RevTet-On system (Clontech, Palo Alto, CA) was used, as it permits control over the expression of MKK7-JNKs in Swiss 3T3 fibroblasts. Transfectants containing pRevTet-On vector and each isoform of pRevTRE MKK7-JNK were generated by modified protocol according to the directions from the manufacturer. Briefly, pRevTet-On vector was packaged into replication-defective retrovirus using 293T cells and the retrovirus-component expression plasmids SV--A-MLV and SV--env-MLV as described (10-12). Conditioned growth medium containing secreted retroviruses was collected, supplemented with 8 µg/ml Polybrene, filtered through a 0.45-µm filter, and incubated with Swiss 3T3 cell lines for 24 h. The transduced cells were selected in growth medium containing G418 (500 µg/ml). Individual clones of Swiss 3T3 transfectants, determined by transcriptional efficiency of TRE promoter in reporter gene pBI-L vector (Clontech), were identified and used in subsequent transduction. The HindIII-ClaI fragments of the different MKK7-JNKs were excised from pLNCX-MKK7-JNK and ligated into pRevTRE vector digested with HindIII and ClaI. These cDNAs encoding MKK7-JNK1 and MKK7-JNK1 in pRevTRE vector were transduced into Swiss 3T3 cells containing pRevTet-On by retrovirus-mediated gene transfer as described above. The transduced cells were selected in growth medium containing both hygromycin (100 µg/ml) and G418 (500 µg/ml). Replicate clones of MKK7-JNK1 and MKK7-JNK1 were identified by immunoblotting for phospho-JNK and used in the studies.

Oligonucleotide Array Analysis-- Total RNA was isolated with TRIzol reagent (Invitrogen) from Swiss3T3 cell transfectants and further purified with RNeasy Mini Kits (Qiagen, Valencia, CA). Double-stranded DNA was synthesized from total RNA with the Superscript Choice System (Invitrogen). The double-stranded cDNA was isolated by phenol-chloroform extraction using Phase Lock Gels (Eppendorf, Westbury, NY), followed by ethanol precipitation with Pellet Paint (Novagen, Madison, WI) as a carrier. The cDNA was resuspended in RNase-free water and used as a template for in vitro transcription in the presence of biotinylated UTP and CTP to generate labeled cRNA. The in vitro transcription reaction was performed by using the BioArray high yield RNA transcript labeling kit (Enzo Diagnostics, Farmingdale, NY). Purification of the labeled cRNA was carried out with the Qiagen RNeasy Mini Kit. The labeled cRNA was fragmented in fragmentation buffer (40 mM Tris acetate (pH 8.1), 10 mM KOAc, 30 mM MgOAc) and hybridized to the microarrays according to the protocol from the manufacturer. The arrays used in this study were the Genechip^® Murine Genome U74 Set version 2 (Affymetrix, Santa Clara, CA). The microarrays were then washed and stained using the Genechip fluidics station according to the instructions from the manufacturer. DNA chips were scanned at a resolution of 6 µm with a HP Gene Array scanner and data were analyzed with Genechip Suite 4.0 Analysis software (Affymetrix).

RT-PCR and Real-time Quantitative PCR Analyses-- Reverse transcription of total RNA (4 µg) was performed in a volume of 20 µl using random hexamers and MMLV reverse transcriptase according to the protocol of the manufacturer (Invitrogen). Aliquots (2 µl) of the reverse transcription reactions were then submitted to PCR (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s) using primers for MKP-7, JBP, ST2L/ST2, RANTES, MIP1, IP-10, and -actin shown in Table I. Aliquots (10 µl) of the PCR reactions were electrophoresed on 1.2% agarose gels, stained with ethidium bromide, and photographed. Real-time quantitative PCR analyses were performed with SYBR^® green and a Cepheid Smart Cycler (Sunnyvale, CA) to measure mRNA levels of ISG12, an EST with accession no. AI158810 that blasts similar to ISG12, ISG15, and GTPI. Aliquots (1 µl) of reverse transcription reactions were subjected to PCR in 50 µl reactions with SYBR^® green Jumpstart Taq Readymix (Sigma) using primers shown in Table I. Initial real-time PCR amplifications were examined by agarose gel electrophoresis to verify that the primer pairs amplified a single product of the predicted (70-80 base pairs) size. 18 S ribosomal RNA levels were measured with Taqman^® ribosomal RNA control reagents from Applied Biosystems (Foster City, CA) as an endogenous control and to match the RNA samples. The real-time PCR data were analyzed with the Smart Cycler^® software (version 1.2b) to calculate the threshold cycle values for the different samples and are presented as mRNA levels in arbitrary units where the Swiss 3T3 TRE samples was assigned a value of 1.

Immunoblot Analysis-- Samples of cell extracts prepared in MAP kinase lysis buffer (0.5% Triton X-100, 50 mM -glycerophosphate (pH 7.2), 0.1 mM sodium vanadate, 2 mM MgCl₂, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl urea, 2 µg/ml leupeptin, and 4 µg/ml aprotinin) supplemented with 300 mM NaCl were resolved by 10% SDS-PAGE and transferred to nitrocellulose. The filters were blocked in Tris-buffered saline (10 mM Tris-Cl (pH 7.4), 140 mM NaCl) containing 0.1% Tween 20 and 3% nonfat dry milk and then incubated with blocking solution containing the indicated antibodies at 1 µg/ml for 12-16 h. The filters were extensively washed in Tris-buffered saline containing 0.1% Tween 20, and bound antibodies were visualized with alkaline phosphatase-coupled secondary antibodies and Lumi-Phos reagent (Pierce) according to the directions from the manufacturer. Antibodies to phospho-JNK, c-Jun, and phospho-Ser-73 c-Jun were purchased from Cell Signaling Technology (Beverly, MA), and the antibodies to FLAG and MKK7 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody to IGTP was kindly provided by Dr. Greg Taylor (14).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

MKK7-JNK1 Fusion Proteins Exhibit Gain-of-function in Transient Transfections-- To generate gain-of-function JNKs, we adapted the approach reported by Zheng et al. (8). As described under "Experimental Procedures," p46JNK1 and p46JNK1 were fused with the upstream MAPK kinase, MKK7, creating MKK7-JNK1 and MKK7-JNK1. An in-frame (Glu-Gly)₅ linker was introduced between the coding sequences of MKK7 and JNK to facilitate folding. The cDNAs encoding MKK7-JNK proteins and MKK7 protein were transiently transfected into 293T cells, and cell lysates were immunoblotted for the FLAG epitope incorporated at the N terminus of MKK7. FLAG-tagged polypeptides with the predicted molecular masses of 86 kDa for MKK7-JNK1 and MKK7-JNK1 were detected following immunoblotting of 293T extracts (Fig. 1A).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   MKK7-JNK fusion proteins transiently transfected in 293T cells stimulate c-Jun transcriptional activity. A, 293T cells were transiently transfected with the empty LNCX expression vector or LNCX encoding FLAG-tagged MKK7-JNK1, MKK7-JNK1, or MKK7 alone. Twenty-four hours after transfection, cell extracts were prepared with MAP kinase lysis buffer and aliquots containing 100 µg of total cellular protein were resolved by SDS-PAGE and submitted to immunoblot analysis with a FLAG antibody. B, 293T cells were cotransfected with a UAS-luciferase reporter plasmid (100 ng) and expression vectors encoding c-Jun-GAL4 (5 ng) and 10 ng of LNCX, LNCX-MKK7-JNK, or pcDNA3-MEKK1 as indicated. Cells were collected 24 h later and assayed for luciferase and -galactosidase activities. Luciferase activity expressed in cells transfected with only UAS-luciferase was assigned an arbitrary value of 1.

To test whether the MKK-JNK fusion proteins were constitutively activated, we measured the transactivation function of c-Jun in the absence of any stimuli. Plasmids encoding c-Jun-GAL4 fusion protein and a UAS-luciferase reporter gene were transiently transfected into 293T cells with expression vectors encoding MKK7-JNK1 fusion proteins or an activated form of MEKK1 for a positive control (Fig. 1B). Transfection of activated MEKK1, a defined activator of JNK pathway, or the MKK7-JNK fusion proteins strongly stimulated c-Jun-GAL4 transcriptional activity. MKK7-JNK1 and MKK7-JNK1 stimulated c-Jun-GAL4 activity by 10-11-fold compared with the empty vector control. This finding indicates that the MKK7-JNK1 fusion proteins function as gain-of-function forms of JNK within cells as previously described (8).

Generation of Swiss 3T3 Fibroblasts Stably Expressing Gain-of-function JNK Proteins-- To investigate the cellular action of the MKK7-JNK1 proteins in fibroblasts, we stably introduced pRevTRE vectors encoding MKK7-JNK1 and MKK7-JNK1 into Swiss 3T3 fibroblasts by retrovirus-mediated gene transfer (see "Experimental Procedures"). The resulting stable cell lines were then immunoblotted with antibodies specific for phospho-JNK and MKK7 (Fig. 2). Preliminary experiments indicated that the expression of the MKK7-JNK fusion proteins was markedly induced following incubation of the Swiss 3T3 fibroblasts with doxycycline (data not shown). However, doxycycline proved to have cytoprotective effects that masked the actions of the MKK7-JNK fusion proteins following serum-withdrawal experiments (see below). Fortuitously, the level of expression of the MKK7-JNK fusions achieved in the absence of doxycycline was similar to endogenous MKK7 and JNK protein levels. Thus, all subsequent experiments were performed on stably-transfected cells cultured in the absence of doxycycline. The phospho-JNK immunoblot in Fig. 2A shows that the MKK7-JNK fusion proteins are phosphorylated and activated in the absence of stress stimuli. Consistent with constitutive activity of MKK7-JNKs, the level of c-Jun and phospho-Ser-73 c-Jun were markedly increased in cells expressing MKK7-JNK fusion proteins (Fig. 2B). In subsequent experiments two independent Swiss 3T3 clones of MKK7-JNK1 and MKK7-JNK1 were used, with empty TRE vector transfected-Swiss 3T3 cells as control cells.

View larger version (54K): [in this window] [in a new window]   Fig. 2.   Stable expression of MKK7-JNK fusion proteins in Swiss 3T3 fibroblasts increases c-Jun protein expression and phosphorylation. Empty pRevTRE vector, pRevTRE-MKK7-JNK1, or pRev-TRE-MKK7-JNK1 was stably introduced into Swiss 3T3 Tet-On cells by retrovirus-mediated gene transfer as described under "Experimental Procedures." The transduced cells were selected in growth medium containing both hygromycin (100 µg/ml) and G418 (500 µg/ml). Extracts from the indicated transfectants were prepared and resolved by SDS-PAGE and submitted to immunoblot analyses with the indicated antibodies (A, phospho-JNK and MKK7; B, c-Jun and phospho-c-Jun).

We investigated the phenotypes of Swiss 3T3 cells expressing MKK7-JNK proteins to determine the potential functional outcomes of gain-of-function JNK expression in fibroblasts. The growth rates of MKK7-JNK-expressing cells and Swiss TRE vector cells in full media containing 10% FBS was not significantly different (data not shown). However, we observed that MKK7-JNK-expressing cells are highly sensitive to serum-restriction and readily undergo cell death in serum-free medium. The cell viability of Swiss TRE cells was only modestly affected following 3 days of serum withdrawal (Fig. 3). By contrast, the survival of Swiss 3T3 cells expressing MKK7-JNK1 or MKK7-JNK1 was dramatically reduced (14-42%) after 3 days of culture in serum-free medium.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Swiss 3T3 fibroblasts expressing MKK7-JNK proteins exhibit increased sensitivity to serum withdrawal-induced cell death. Swiss 3T3 cells transduced with the TRE vector, MKK7-JNK1 or MKK7-JNK1 were plated in 24-well plates (50,000 cells/well) in DMEM containing 10% FBS. The next day, the medium was changed to serum-free DMEM and the cells were further cultured for 3 days. Cell numbers were determined before and after serum withdrawal. The data are expressed as the percentage of cells surviving after 3 days in serum-free medium relative to the cell numbers measured prior to serum withdrawal. The data are the mean ± S.D. of three or four experiments where * indicates a significant difference with p < 0.005.

Gene Expression Profiles in Swiss 3T3 cells Transfected with Gain-of-function MKK7-JNK1 Proteins-- Although no difference in the Swiss 3T3 cells expressing MKK7-JNK1 or MKK7-JNK1 was noted with regard to proliferation or cell death induced by serum withdrawal, we tested the hypothesis that other signaling differences exist between the two transfected MKK7-JNK1 proteins. To search for differences in MKK7-JNK1 and MKK7-JNK1 signaling in Swiss 3T3 cells, the gene expression profiles of the TRE cells and the MKK7-JNK1-expressing cells growing in full medium with 10% FBS were determined with Affymetrix Genechips as described under "Experimental Procedures." As shown in Tables I and II, analysis of the Genechip data from the TRE vector control cells and duplicate clones expressing MKK7-JNK1 or MKK7-JNK1 revealed evidence of selective gene expression patterns in cells expressing MKK7-JNK1 and MKK7-JNK1. Overall, 46 genes were induced greater than 2-fold by MKK7-JNK1 and/or MKK7-JNK1 (Table II) and 43 genes were down-regulated greater than 2-fold by MKK7-JNK1 and/or MKK7-JNK1 (Table III). Among the induced genes, 20 genes were found to be induced equivalently in Swiss 3T3 fibroblasts expressing MKK7-JNK1 or MKK7-JNK1 relative to the TRE vector control cells. Of these, MKK7 mRNA is induced as a consequence of retroviral-mediated expression of the cDNA encoding the MKK7-JNK1 fusion proteins. This set of genes also included a number of transcription factors and homeodomain proteins (SHOX, SHOT, Sox-4, zinc finger homeobox 1a) as well as the JNK target, c-Jun, the induction of which is predicted from the immunoblot presented in Fig. 1. Two MAP kinase phosphatases (MKPs), MKP-1 and MKP-7, were induced equivalently by MKK7-JNK1 and MKK7-JNK1 as well as a murine EST (accession no. AW061307) equivalent to tumor necrosis factor intracellular domain-interacting protein (accession no. AF168675), which is the murine homolog of Homo sapiens c-Jun-binding protein (JBP; accession no. AF291105). Also among these genes were IL-1 receptor family member ST2L/ST2 and the murine interferon-inducible gene, IP-10. Thirteen genes were selectively induced by MKK7-JNK1 compared with MKK7-JNK1, including growth factors (proliferin and ELF-2) and growth factor-binding proteins (insulin-like growth factor-binding protein-10), small inducible cytokine A5/RANTES, protein kinases (Ack tyrosine kinase and SGK), and additional transcription factors (PEBP2B2, imprinted and ancient, and Id2). Thirteen genes were selectively induced by MKK7-JNK1 relative to MKK7-JNK1 and included GARG16, GARG49, IGTP, GTPI, interferon-induced 15-kDa protein (ISG15) and -interferon-inducible protein (ISG12), genes known to be interferon-responsive as well as two genes that are components of the ubiquitin pathway (Table II). Forty-three genes were down-regulated upon expression of MKK7-JNK1 proteins; 11 of these were down-regulated equally with either MKK7-JNK1 or MKK7-JNK1, whereas 32 genes were selectively down-regulated by MKK7-JNK1.

View this table: [in this window] [in a new window]   Table II Genes induced by MKK7-JNK1 proteins The gene expression profiles of two independent clones of Swiss 3T3 cells expressing MKK7-JNK1 or MKK7-JNK1 as well as the TRE vector control cells were assessed as described under "Experimental Procedures." The tabulated genes in Tables II and III were manually clustered into the indicated groups based on their patterns of induction. The numerical intensity values shown are the "average differences" calculated by the Affymetrix software and represents the averaged differences of the hybridization intensities to the specific oligonucleotides minus the hybridization intensities of the corresponding mismatched oligonucleotides. Duplicate microarray experiments were performed with the TRE vector cells, MKK7-JNK1 clone 2 and MKK7-JNK1 clone 7, whereas singlet GeneChips were probed with biotinylated cRNA prepared from MKK7-JNK1 clone 3 and MKK7-JNK1 clone 1.

Selected Genechip expression calls shown in Tables II and III were confirmed by immunoblot or RT-PCR analyses. In general, the immunoblot and RT-PCR assays confirmed the results from the array analysis. For example, the equivalent inductions of c-Jun, ST2/ST2L, MKP-7, IP-10, and tumor necrosis factor intracellular domain-interacting protein/JBP in MKK7-JNK1 and MKK7-JNK1-expressing cells on the Genechips were confirmed by immunoblotting and RT-PCR (Fig. 4). However, RANTES appeared to be similarly induced in the Genechip experiments by MKK7-JNK1 and MKK7-JNK1, but RT-PCR analysis indicated a greater induction in MKK7-JNK1-expressing cells (Fig. 4). Additionally, proliferin mRNA was induced similarly in MKK7-JNK1 and MKK7-JNK1-expressing cells as assessed by RT-PCR, although the Genechip experiment indicated a selective induction by MKK7-JNK1. The selective down-regulation of MIP1 in MKK7-JNK1-expressing cells predicted by the Genechip (Table III) was confirmed by RT-PCR (Fig. 4).

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Confirmation of selected gene expression changes predicted by the Genechips. A, total RNA from the indicated Swiss 3T3 transfectants was submitted to RT-PCR with the primer pairs (see Table I) specific for JBP, MKP-7, IP-10, ST2L/ST2, RANTES, proliferin, MIP1, and -actin. B, cell extracts prepared from the Swiss 3T3 transfectants were immunoblotted with antibodies to c-Jun and IGTP.

The selective induction of the IFN-inducible genes by MKK7-JNK1 (Table II) was confirmed. Induction of IGTP protein was confirmed by immunoblotting (Fig. 4B), and ISG12, murine EST (accession no. AI158810, which blasts to ISG12), ISG15 and GTPI were confirmed by real-time quantitative PCR (Fig. 5). The intensity of IGTP immunoblot was measured by densitometry, where a selective 6-8-fold induction by MKK7-JNK1 was observed. In MKK7-JNK1-expressing cells, the ISG12 genes and AI158810 genes were selectively induced by 3.6-4.6-fold. Furthermore, the ISG15 genes and GTPI genes consistently showed larger inductions in MKK7-JNK1-expressing cells relative to MKK7-JNK1-expressing cells (Fig. 5). These results thus confirm the results of the microarray experiment that a panel of interferon-stimulated genes are selectively induced in the MKK7-JNK1-expressing Swiss 3T3 cells.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Confirmation of selective induction of IFN-inducible genes by real-time quantitative PCR. Total RNA from the indicated Swiss 3T3 transfectants was subjected to quantitative PCR analysis of expression levels of three IFN-inducible genes. Primers specific to ISG12, murine EST (accession no. AI158810, which blasts to ISG12), ISG15, and GTPI (Table I) were used in PCR reactions along with SYBR® green as described under "Experimental Procedures." The mRNA level measured in TRE vector cells is assigned an arbitrary value of 1. The data are the mean ± S.D. of three experiments where * and ** indicate significant differences from the TRE values with p < 0.05 and with p < 0.01, respectively.

MKK7-JNK1-expressing Cells with Selective Induction of Interferon-induced Gene Expression Are Resistant to Virus-induced Cell Death-- The cellular response to infection by double-stranded RNA viruses has been extensively studied (reviewed in Refs. 15-17) and involves the transcriptional induction of type I interferons and the subsequent regulation of interferon-responsive genes involved in a host defense program. The selective induction of a number of interferon-responsive genes by MKK7-JNK1 (Table II; Figs. 4B and 5) suggests that the observed JNK activation in response to virus infection (18) may participate in the regulation of some interferon-responsive genes. Furthermore, we predicted that the Swiss 3T3 cells expressing MKK7-JNK1 may be more resistant to killing by virus infection. To test this hypothesis, Swiss 3T3 cell lines expressing MKK7-JNK proteins or empty TRE vector were incubated with VSV for 24 h, after which cell viability was measured (see "Experimental Procedures"). As shown in Fig. 6, viability of Swiss 3T3 TRE vector control cells and MKK7-JNK1-expressing cells was reduced in a concentration-dependent manner, such that ~50% of the cells died in response to a viral titer of 10³. By contrast, the Swiss 3T3 clones expressing MKK7-JNK1 exhibited significantly increased resistance to cell death even in response to higher titers of virus. Thus, the increased resistance of the MKK7-JNK1-expressing cells to virus infection is consistent with the induction of interferon-response genes identified by the Genechips (Table II).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Differential responses of Swiss MKK7-JNK cells to VSV-induced cell death. Swiss TRE cells and two independent clones of MKK7-JNK1 or MKK7-JNK1 cells were plated in 96 wells (5,000 cell/well). Cells were incubated for 24 h with VSV at the indicated dilution of the viral stock. The cell survival was measured by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (see "Experimental Procedures"). The data are the mean ± S.D. of three or four experiments where * indicates a significant difference with p < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The findings in this study are novel because they provide evidence for selective gene regulation by gain-of-function JNK1 and JNK1 polypeptides. In addition, the findings suggest that the alternatively spliced variants of JNK1 and JNK2 may perform specific cellular functions such as the host defense response to virus infection shown in Fig. 6. Admittedly, the gain-of-function MKK7-JNK fusion proteins should be considered pharmacological tools, as no evidence exists to support tight and persistent association of MKK7 and JNK within cells. However, these molecular reagents may provide valuable insight into isoform-specific signaling by JNK family members that is not presently afforded by other approaches. The gain-of-function JNK1 and JNK1 constructs used in our studies differ only in the respective sequences encoding the 24 amino acid  and  splices residing within protein kinase subdomains IX and X. Moreover, 17 of the 24 residues are identical between the JNK1  and  splices. Preliminary studies with an MKK7-JNK2 construct expressed in Swiss 3T3 cells reveals similar induction of the interferon-responsive genes highlighted in Table II as by the MKK7-JNK1 polypeptide.² Interestingly, the  splice of JNK2 is more similar to the  splice of JNK1 (19 identities of 24) than the  splice of JNK1 (14 identities of 24). Although no selective cellular functions have been assigned to JNK versus JNK, the alternative / sequences have been shown to significantly influence the affinity of JNK binding with c-Jun (6). In light of the higher affinity of JNK1 for c-Jun relative to JNK1 (6), it is notable that gain-of-function JNK1 and JNK1 equivalently induce c-Jun protein expression, phosphorylation and activation (Figs. 1, 2, and 4). Thus, it is unlikely that differential regulation of c-Jun is the mechanism by which MKK7-JNK1 and MKK7-JNK1 selectively induce or down-regulate the genes listed in Tables II and III.

Swiss 3T3 cells expressing gain-of-function JNK1 exhibit increased resistance to VSV-induced cell death, a finding that is paralleled by the selective induction of a set of interferon-stimulated genes in these cells (see Table II and Figs. 4 and 5). A well characterized cellular response to viral infection is the activation of the innate immune response mediated by induction of type I interferons (IFN-/) followed by the IFN-dependent induction of a host of genes including GARG16, GARG49, IGTP, GTPI, and ISG15, which presumably serve to impair viral gene expression and replication (16). Besides the stimulation of double-stranded RNA-dependent protein kinase and IB kinase, JNK activation is a potent response to viral infection as well as cellular delivery of double-stranded RNA (18). Additionally, jnk2-deficient mouse embryo fibroblasts exhibit reduced induction of type I IFN mRNA compared with normal mouse embryo fibroblasts in response to VSV or double-stranded RNA treatment (18), indicating that the JNK pathway is an important co-stimulatory input into the innate immune response. The requirement for integrated signaling of JNKs, IB kinase, and RNA-dependent protein kinase for full induction of type I IFNs is consistent with the collaboration of nuclear factor B, c-Jun/ATF2 heterodimers, and IFN regulatory factors (IRFs) in transcriptional induction of IFN- (19). Importantly, the Genechip data indicated that the mRNAs for IFN- family members were absent in TRE and MKK7-JNK1-expressing Swiss 3T3 cells. Although a significant basal expression of IFN- mRNA was detected in Swiss TRE control cells by both Genechip and RT-PCR analyses, IFN- mRNA was not further induced by the MKK7-JNK1 polypeptides.² Thus, expression of MKK7-JNK1 is not sufficient to initiate the complete innate immune response in Swiss 3T3 cells.

As previously mentioned, the mechanism by which MKK7-JNK1 selectively increases the expression of the interferon-stimulated genes cannot rely solely on c-Jun regulation. Generally, the promoters of IFN-inducible genes have been shown to contain IFN-stimulated response elements (ISREs), IFN response factor elements, and/or  interferon activation site elements. Regulation of ISREs occurs through IFN-stimulated gene factor 3 (ISGF3) comprising signal transducer and activator of transcription 1 (Stat1), Stat2, and IRF-9, which is a member of the IRF family of transcription factors (15, 17). The sequences of IFN response factor elements partially overlap with the consensus sequence of ISREs, but bind IRF family members, whereas  interferon activation site elements are recognized by Stat dimers. The JNK pathway is not presently considered to be a major regulatory pathway for either Stat family members or the IRF family. However, several reports do indicate that the JNKs may target specific members of these transcription factor families. The JNK and p38 MAPKs have been shown to be required for Stat3 regulation by the Src oncoprotein in a manner involving phosphorylation of Stat3 (20). In addition, both IRF-3 and IRF-7 have been invoked as targets of the JNK pathway (21, 22). Thus, precedent exists to support transcription factor targets other than c-Jun as putative mediators of MKK7-JNK1 induction of IFN-stimulated genes.

It is noteworthy that as many genes were down-regulated as induced in Swiss 3T3 cells expressing the MKK7-JNK1 proteins, a finding that is consistent with both down-regulation and induction of gene expression by wild-type and oncogenic forms of c-Jun (23-25). The finding of reduced Fas expression by MKK7-JNK polypeptides in Table III has been previously noted in a study where a gain-of-function MKK7 protein was expressed in HEK293 cells (26). Additionally, the decreased expression of phosphofructokinase mRNA noted in Table III is consistent with the JNK pathway-dependent inhibition of phosphofructokinase expression in response to insulin (27). Increased JNK activity has been reported to decrease Fas transcription through the cooperative action of Stat3 and c-Jun as repressors at the Fas promoter (28). In addition, c-Jun has been invoked as a repressor of Smad transcriptional activity, thereby inhibiting transforming growth factor -mediated gene expression (29, 30). Interestingly, MKK7-JNK1 appeared to be dominant in the down-regulation of many genes in Table III, where MIP1 serves as an example that was confirmed by RT-PCR (Fig. 4). Additional genes exhibiting a similar pattern of selective repression by MKK7-JNK1 in the array experiments were ceruloplasmin, superoxide dismutase 3, insulin-like growth factor-binding protein-4, and collagen type VI (Table III). Because c-Jun protein and phosphorylation are equally stimulated by MKK7-JNK1 and MKK7-JNK1, it is difficult to invoke a model where c-Jun mediates all the down-regulation events noted in Table III. Rather, it seems likely that JNK isoform-specific targets must exist to mediate selective gene repression by different JNK isoforms.

In contrast to selective regulation of interferon response genes and increased resistance to VSV by MKK7-JNK1, expression of either gain-of-function JNK1 or JNK1 proteins rendered Swiss 3T3 cells equally sensitive to serum withdrawal-induced cell death relative to the TRE vector control cells (Fig. 3). Inspection of Tables II and III failed to reveal genes for which induction or down-regulation by MKK7-JNK1 proteins would be predicted, based on the present understanding, to increase the sensitivity of the cells to apoptosis in response to serum withdrawal. Thus, the mechanism of enhanced sensitivity to serum withdrawal-induced cell death in Swiss 3T3 cells expressing either MKK7-JNK1 polypeptide is not satisfactorily addressed by the Genechip results. The Affymetrix Genechips assess the expression status of only a subset of the genes in the mouse genome, and it is possible that sensitization to serum withdrawal-induced cell death is accomplished by expression changes in genes not represented on the oligonucleotide array. Alternatively, the mechanism by which MKK7-JNK sensitizes Swiss 3T3 cells to serum withdrawal may be independent of gene transcription. In this regard, studies on UV-induced apoptosis in mouse embryo fibroblasts demonstrates a requirement for the JNK pathway, but not new protein or RNA synthesis (31). Thus, it is possible that MKK7-JNKs expressed in Swiss 3T3 cells will induce phosphorylation of cellular targets that enhance serum withdrawal-induced cell death independent of gene regulatory actions.

	ACKNOWLEDGEMENTS

We thank Dr. Greg Taylor (Duke University, Durham, NC) for the IGTP antibody and Drs. Cathy Tournier and Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical Center, Worcester, MA) for the FLAG-MKK71 cDNA. The University of Colorado Health Sciences Center Gene Expression Core Facility provided invaluable assistance with the Affymetrix Genechip analyses.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants GM61718, DK19928, and CA58157.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: Division of Renal Medicine, C-281, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-315-6065; Fax: 303-315-4852; E-mail: lynn.heasley@uchsc.edu.

Published, JBC Papers in Press, September 26, 2002, DOI 10.1074/jbc.M204270200

² S.-Y. Han and L. E. Heasley, unpublished results.

	ABBREVIATIONS

The abbreviations used are: JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, mitogen-activated protein kinase kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; FBS, fetal bovine serum; VSV, vesicular stomatitis virus; MKP, mitogen-activated protein kinase phosphatase; IFN, interferon; EST, expressed sequence tag; MAP, mitogen-activated protein; DMEM, Dulbecco's modified Eagle's medium; UAS, upstream activating sequence; RT, reverse transcription; IRF, interferon regulatory factor; ISRE, interferon-stimulated response element; RANTES, regulated on activation normal T cell expressed and secreted; JBP, c-Jun-binding protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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