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The Journal of Immunology, 2007, 179, 1926 -1933
Copyright © 2007 by The American Association of Immunologists, Inc.
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Targeted Deletion of MKK4 Gene Potentiates TNF-Induced Apoptosis through the Down-Regulation of NF-{kappa}B Activation and NF-{kappa}B-Regulated Antiapoptotic Gene Products1

Gautam Sethi*, Kwang Seok Ahn*, Dianren Xia{dagger}, Jonathan M. Kurie{dagger} and Bharat B. Aggarwal2,*

* Cytokine Research Laboratory, Department of Experimental Therapeutics, and {dagger} Department of Thoracic, Head and Neck Medical Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
MAPK kinase 4 (MKK4) is a dual-specificity kinase that activates both JNK and p38 MAPK. However, the mechanism by which MKK4 regulates TNF-induced apoptosis is not fully understood. Therefore, we used fibroblasts derived from MKK4 gene-deleted (MKK4-KO) mice to determine the role of this kinase in TNF signaling. We found that when compared with the wild-type cells, deletion of MKK4 gene enhanced TNF-induced apoptosis, and this correlated with down-regulation of TNF-induced cell-proliferative (COX-2 and cyclin D1) and antiapoptotic (survivin, IAP1, XIAP, Bcl-2, Bcl-xL, and cFLIP) gene products, all regulated by NF-{kappa}B. Indeed we found that TNF-induced NF-{kappa}B activation was abrogated in MKK4 gene-deleted cells, as determined by DNA binding. Further investigation revealed that TNF-induced I{kappa}B{alpha} kinase activation, I{kappa}B{alpha} phosphorylation, I{kappa}B{alpha} degradation, and p65 nuclear translocation were all suppressed in MKK4-KO cells. NF-{kappa}B reporter assay revealed that NF-{kappa}B activation induced by TNF, TNFR1, TRADD, TRAF2, NIK, and I{kappa}B{alpha} kinase was modulated in gene-deleted cells. Overall, our results indicate that MKK4 plays a central role in TNF-induced apoptosis through the regulation of NF-{kappa}B-regulated gene products.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The MAPK kinase 4 (MKK4)3 (also called MAP2K4/SEK1) is a member of the MAPK family that was first isolated in 1995. MKK4 is a dual-specificity kinase that is specifically involved in the stress-activated protein kinase pathway, directly phosphorylating JNK in response to Ask1 activation (1, 2, 3). MKK4 not only activates JNK, but also phosphorylates p38 MAPK. Genetic deletion of the MKK4 gene causes embryonic death and has revealed its role in the activation of JNK and AP-1 (4), in CD95- and CD3-induced apoptosis of thymocytes (5), impaired CD28-mediated production of IL-2, proliferation of lymphocytes (6), maintenance of lymphoid homeostasis (7), hepatogenesis, (8, 9), enhancement of apoptosis, and in development of neural tubes (10, 11).

MKK4 also has a tumor suppressor role in embryonic stem cells (12). Numerous reports suggest that MKK4 is a tumor suppressor gene (13), in prostate (14, 15), ovarian (16, 17), pancreatic, biliary, and breast carcinoma (18); but other reports link it to oncogenesis (19, 20). The loss of MKK4 in pancreatic cancer has been linked to shorter patient survival (21). Although MKK4 has a role in signaling induced by CD28 and CD95 (5, 6), both members of the TNF superfamily (22), there is no information available about its role in TNF-induced apoptosis. TNF has, however, been shown to activate MKK4 in mouse bone marrow-derived macrophages (23). MKK7 has also been reported to be the major kinase that mediates TNF-induced JNK and AP-1 activation (24). Mutant MKK4 has been shown to increase the rate of TNF-induced apoptosis (25), but the mechanism by which MKK4 regulates apoptosis is not understood.

In the present report, we determined the mechanisms by which MKK4 regulates TNF-induced apoptosis using MKK4 gene-deleted (MKK4-KO) cells. Our results indicate, for the first time, that MKK4 regulates the expression of various cell-proliferative and antiapoptotic proteins whose expression is regulated by NF-{kappa}B. Down-regulation of these gene products in MKK4-KO cells was mediated through the down-regulation of TNF-mediated NF-{kappa}B activation, leading to potentiation of apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Bacteria-derived recombinant TNF, purified to homogeneity with a specific activity of 5 x 107 U/mg, was provided by Genentech. Penicillin, streptomycin, DMEM, and FBS were obtained from Invitrogen Life Technologies. Anti-beta-actin Ab, PMA, and LPS was obtained from Sigma-Aldrich. The Abs anti-p65, anti-p50, anti-I{kappa}B{alpha}, anti-TNFR-associated death domain (TRADD), anti-TNFR-associated factor (TRAF)-2, anti-NF-{kappa}B-inducing kinase (NIK), anti-cyclin D1, anti-matrix metalloproteinase-9 (MMP-9), anti-poly(ADP-ribose) polymerase (PARP), anti-inhibitor of apoptosis (IAP)1, anti-X-linked IAP (XIAP), anti-Bcl-2, and anti-Bcl-xL were obtained from Santa Cruz Biotechnology. Anti-cyclooxygenase (COX)-2 Ab was obtained from BD Biosciences. Phospho-specific anti-I{kappa}B{alpha} (Ser32/Ser36) and anti-phospho-specific p65 (Ser536) Abs were purchased from Cell Signaling Technology. Anti-I{kappa}B{alpha} kinase (IKK)-{alpha}, anti-IKK-beta, and anti-cellular FLIP (cFLIP) Abs were provided by Imgenex. Cigarette smoke condensate (CSC), prepared as previously described (26), was supplied by Dr. C. G. Gairola (University of Kentucky, Lexington, KY).

Cell lines

The mouse embryonic fibroblast line derived from MKK4-KO C57BL/6J mice and its wild-type (MKK4-WT) were provided by Dr. J. Woodgett (University of Toronto, Toronto, Ontario, Canada) and have been previously described (5). Cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Cell surface expression of TNFR1 and TNFR2

To determine the cell surface expression of TNFR1 and TNFR2, cells were harvested and suspended in Dulbecco’s PBS containing 1% FBS and 0.1% sodium azide. The cells were then preincubated with 10% goat serum for 20 min and washed, and monoclonal rabbit IgG anti-TNFR1 and anti-TNFR2 Abs were added. After a 1-h incubation at 4°C, the cells were washed and incubated for an additional 1 h in FITC-conjugated goat anti-rabbit IgG Abs and then analyzed using a FACSCalibur flow cytometer and CellQuest acquisition and analysis software (BD Biosciences).

Annexin V assay

An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phospholipid phosphatidylserine from the cytoplasmic interface to the extracellular surface. This loss of membrane asymmetry can be detected by using the binding properties of annexin V. To identify the apoptosis, we used annexin V Ab, conjugated with FITC fluorescence dye. Briefly, 1 x 105 cells/ml were pretreated with 1 µg/ml cycloheximide, treated with 1 nM TNF for 16 h at 37°C, and subjected to annexin V staining. Cells were washed in PBS, resuspended in 100 µl of binding buffer containing FITC-conjugated anti-annexin V Ab, and analyzed by flow cytometry (FACSCalibur; BD Biosciences).

PARP cleavage assay

To detect cleavage of PARP, we prepared whole cell extracts by subjecting TNF-treated cells to lysis in lysis buffer (20 mM Tris (pH 7.4), 250 mM NaCl, 2 mM EDTA (pH 8.0), 0.1% Nonidet P-40, 0.01 µg/ml aprotinin, 0.005 µg/ml leupeptin, 0.4 mM PMSF, and 4 mM sodium orthovanadate). Lysates were spun at 14,000 rpm for 10 min to remove insoluble material, resolved by 10% SDS-PAGE, and probed with PARP Ab.

EMSA analysis

To determine NF-{kappa}B activation, we performed an EMSA as previously described (27). Briefly, nuclear extracts prepared from TNF-treated cells (2 x 106/ml) were incubated with 32P end-labeled 45-mer double-stranded oligonucleotide (15 µg of protein with 16 fmol of DNA) from the HIV long terminal repeat, 5'-TTGTTACAA GGGACTTTC CGCTG GGGACTTTC CAGGGAGGCGTGG-3' (boldface indicates NF-{kappa}B binding sites), for 30 min at 37°C. The DNA protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5'-TTGTTACAA CTCACTTTC CGCTG CTCACTTTC CAGGGAGGCGTGG-3', was used to examine binding specificity of NF-{kappa}B to the DNA. The binding specificity was also examined by competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with Abs against either the p50 or the p65 subunit of NF-{kappa}B and anti-cyclin D1 for 30 min at 37°C before the complex was analyzed by EMSA. Preimmune serum was included as a negative control. The dried gels were visualized with a Storm820, and radioactive bands were quantified using ImageQuant software (Amersham Biosciences).

Western blot analysis

To determine the levels of protein expression in the cytoplasm and nucleus, we prepared extracts from TNF-treated cells and fractionated them by SDS-PAGE. To determine the expression of cyclin D1, COX-2, MMP-9, IAP1, XIAP, Bcl-2, Bcl-xL, cFLIP, and survivin, whole cell extracts from TNF-treated cells were prepared, and 30 µg of protein was resolved on SDS-PAGE and probed with specific Abs according to the manufacturer’s recommended protocol. The blots were washed, exposed to HRP-conjugated secondary Abs for 1 h, and finally detected by ECL reagent (Amersham Biosciences). The bands were quantified with a Personal Densitometer Scan v.1.30 using ImageQuant software version 3.3 (Molecular Dynamics).

IKK assay

The IKK assay was performed by a method previously described (28). Briefly, the IKK complex from whole cell extracts was precipitated with IKK-{alpha} and IKK-beta and treated with protein A/G-agarose beads (Pierce). After 2 h, the beads were washed with lysis buffer and resuspended in a kinase assay mixture containing 50 mM HEPES (pH 7.4), 20 mM MgCl2, 2 mM DTT, 20 µCi [{gamma}-32P]ATP, 10 µM unlabeled ATP, and 2 µg of substrate GST-I{kappa}B{alpha} (aa 1–54) and incubated at 30°C for 30 min. The reaction was terminated by boiling with SDS sample buffer for 5 min. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized with a Storm820. To determine the total amounts of IKK-{alpha} and IKK-beta in each sample, 30 µg of whole cell proteins was resolved on 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and then blotted with either anti-IKK-{alpha} or anti-IKK-beta Abs.

NF-{kappa}B-dependent reporter gene expression assay

To determine TNF-induced reporter gene expression, 5 x 105/ml cells were plated in 6-well plates and transiently transfected by the calcium phosphate method, with pNF-{kappa}B secretory alkaline phosphatase (SEAP, 0.5 µg) and the control plasmid pCMV-FLAG1 DNA (2 µg). After 24 h, cells were washed, exposed to 1 nM TNF for 24 h, and harvested from the cell culture medium. To determine the reporter gene expression induced by various genes, cells were transfected with 0.5 µg of pNF-{kappa}B SEAP plasmid, 1 µg of an expressing plasmid, or 0.5 µg of the control plasmid pCMV-FLAG1 for 24 h, treated with 1 nM TNF for 24 h, and then harvested from the culture medium. Cells were cotransfected with beta-galactosidase and normalized the data with beta-galactosidase assay. They were then analyzed for SEAP activity according to the protocol essentially as described by the manufacturer (Clontech Laboratories) using a 96-well fluorescence plate reader (Fluoroscan II; Labsystems), with the excitation set at 360 nm and the emission set at 460 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The purpose of study was to investigate the role of MKK4 in the regulation of TNF-induced apoptosis pathway. To investigate the role of MKK4, We used wild-type murine fibroblasts (MKK4-WT) and MKK4 gene-deleted fibroblasts (MKK4-KO). We first determined the expression of TNFR1 and TNFR2 in both MKK4-WT and MKK4-KO cells by Western blot analysis and flow cytometry analyses. TNFR1 and TNFR2 were expressed equally in both cell lines (Fig. 1, A and B).


Figure 1
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  		FIGURE 1. Effect of MKK4 wild-type and MKK4 gene deletion fibroblasts on expression levels of TNFRs. A, Whole cell extracts were prepared, resolved by SDS-PAGE, and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-TNFR1 and anti-TNFR2 Abs. B, Cell surface expression of TNFR in MKK4-WT and MKK4-KO cells. Cells were harvested, labeled with anti-TNFR1 or anti-TNFR2 Abs, and then with a FITC-conjugated goat anti-rabbit IgG Ab and analyzed by flow cytometry. Unstained cells are represented by gray-shaded histogram. C, Cells (1 x 105 cells/well) were pretreated with 1 µg/ml cycloheximide for 1 h, incubated with 1 nM TNF for 12 h, and then subjected to annexin V staining. Cells were washed, incubated with FITC-conjugated anti-annexin V Ab, and then analyzed by flow cytometry. D, Cells (1 x 106 cells/ml) were pretreated with 1 µg/ml cycloheximide for 1 h and then incubated with indicated concentrations of TNF for 16 h. Whole cell extracts were prepared, resolved by SDS-PAGE, and Western blot analysis using anti-PARP and beta-actin Abs was performed. Both results suggest that MKK4 deletion potentiates TNF-induced apoptosis. Data shown are representative of three independent experiments.

 
Deletion of MKK4 potentiates TNF-induced apoptosis

Whether deletion of MKK4 affects TNF-induced apoptosis was determined by two different methods. Because NF-{kappa}B-regulated antiapoptotic gene products counteract the apoptotic effects of TNF, TNF-induced apoptosis is normally examined in the presence of cycloheximide (29). Annexin V staining, which is used to visualize the early stages of apoptosis, showed that TNF- and cycloheximide-induced apoptosis was enhanced from 18% in MKK4-WT cells and 88% in MKK4–KO cells (Fig. 1C). A caspase-mediated PARP cleavage assay showed that MKK4 deletion potentiated TNF-induced caspase activation (Fig. 1D). These results suggest that MKK4 deletion potentiates TNF-induced apoptosis. However, treatment with TRAIL and anti-Fas Ab failed to induce apoptosis as measured by PARP cleavage in MKK4-deleted cells (data not shown).

MKK4 is required for expression of TNF-induced antiapoptotic proteins

Because TNF-induced apoptosis is regulated by the expression of various antiapoptotic proteins, including survivin, IAP1, XIAP, Bcl-xL, Bcl-2, and cFLIP (30), we determined the effect of MKK4 deletion on the TNF-induced expression of these antiapoptotic gene products. As shown in Fig. 2A, TNF induced survivin, IAP1, XIAP, Bcl-2, Bcl-xL, and cFLIP expression in a time-dependent manner in MKK4-WT fibroblasts but not in MKK4-KO cells. These results indicate that MKK4 is required for TNF-induced expression of antiapoptotic gene products.


Figure 2
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  		FIGURE 2. The effect of MKK4 deletion on the TNF-induced expression of various antiapoptotic and proliferative proteins. A, Effects of MKK4 deletion on expression of NF-{kappa}B-regulated proteins. Cells (1 x 106 cells/ml) were incubated with 1 nM TNF for the indicated times. Whole cell extracts were prepared and analyzed by Western blot analysis using Abs against survivin, IAP1, XIAP, Bcl-2, Bcl-xL, c-FLIP, and beta-actin. B, Cells (1 x 106 cells/ml) were treated with 1 nM TNF for the indicated times. Whole cell extracts were prepared and analyzed by Western blot analysis using Abs against COX-2, MMP-9, cyclin D1, and beta-actin. The results shown are representative of three independent experiments.

 
MKK4 is required for expression of TNF-induced NF-{kappa}B-dependent cyclin D1, COX-2, and MMP-9 proteins

TNF has been known to induce the expression of proliferative proteins (cyclin D1, COX-2), and MMP-9 that is involved in tumor invasion (22). To determine whether MKK4 is needed for this induction, we treated cells with TNF for the indicated intervals. We then prepared whole cell extracts, resolved them by SDS-PAGE, and performed a Western blot analysis to determine the expression of cyclin D1, COX-2, and MMP-9 (Fig. 2B). Cyclin D1, COX-2, and MMP-9 expression was induced by TNF in a time-dependent manner in MKK4-WT fibroblasts but not in MKK4-KO cells.

MKK4 is required for TNF-dependent NF-{kappa}B activation

Because the expression of all antiapoptotic and cell-proliferative gene products evaluated above is regulated by NF-{kappa}B, we determined whether MKK4 deletion affected TNF-induced NF-{kappa}B activation. Cells were treated with TNF, nuclear extracts were prepared, and the cells were analyzed for NF-{kappa}B activation by EMSA. As shown in Fig. 3A, TNF stimulated NF-{kappa}B activation in a time-dependent manner in MKK4-WT fibroblasts, and MKK4 deletion suppressed this activation.


Figure 3
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  		FIGURE 3. Time- and dose-dependent effects of TNF on NF-{kappa}B activation in MKK4-WT and MKK4-KO fibroblasts. A, One million cells were treated with 0.1 nM TNF for the indicated times; nuclear extracts were prepared, and NF-{kappa}B activation was analyzed by EMSA. B, One million cells were treated with the indicated concentrations of TNF for 30 min; nuclear extracts were prepared, and NF-{kappa}B activation was analyzed by EMSA. C, Composition of NF-{kappa}B induced by TNF in MKK4 WT cells. Nuclear extracts from untreated or TNF-treated mouse embryonic fibroblast cells were incubated with different Abs, preimmune sera, or unlabeled NF-{kappa}B oligoprobe or mutant oligoprobe, and NF-{kappa}B activity was analyzed by EMSA. The results shown are representative of three independent experiments. D, Effect of various activators on NF-{kappa}B activation in MKK4-WT and MKK4-KO fibroblasts. Cells (1 x 106 cells/ml) were pretreated with 0.1 nM TNF, 10 µg/ml LPS for 30 min, or 10 µg/ml CSC, 25 ng/ml PMA for 2 h, after which nuclear extracts were prepared; NF-{kappa}B activation was then analyzed by EMSA. E, MKK4-null MEF cells were transiently transfected with MKK4 expression vector. After 24 h, the cells were treated with indicated concentrations of TNF for 30 min. Nuclear extracts were prepared thereafter, and NF-{kappa}B activation was analyzed by EMSA.

 
Because activation of NF-{kappa}B by TNF is more robust at higher concentrations (27), we evaluated the effect of MKK4 deletion on NF-{kappa}B activation induced by higher concentrations of TNF (Fig. 3B). TNF activated NF-{kappa}B in a dose-dependent manner in MKK4-WT cells, and activation could be noted with as little as 10 pM TNF. However, NF-{kappa}B was not activated in MKK4-KO cells, even at a concentration of 10 nM TNF, activation of NF-{kappa}B was half as much as compared with MKK4-WT cells. These results indicate that MKK4 is required for TNF-induced NF-{kappa}B activation.

Because NF-{kappa}B is a complex of proteins, various combinations of RelA/NF-{kappa}B protein can constitute an active NF-{kappa}B heterodimer that binds to a specific sequence in the DNA (31). To show that the retarded band visualized by EMSA in TNF-treated cells was indeed NF-{kappa}B, we incubated nuclear extracts from TNF-stimulated cells with Abs to either the p50 (NF-{kappa}B1) or p65 (RelA) subunit of NF-{kappa}B. Both shifted the major band to a higher molecular mass (Fig. 3C), suggesting that the TNF-activated complex consists of p50 and p65 subunits. Preimmune serum had no effect, and excess unlabeled NF-{kappa}B (100-fold) caused the complete disappearance of the band, but a mutant oligonucleotide of NF-{kappa}B did not affect NF-{kappa}B binding activity.

MKK4 is required for NF-{kappa}B activation induced by LPS, PMA, or CSC

Besides TNF, NF-{kappa}B is activated by various carcinogens and inflammatory stimuli through a mechanism that may differ from that of TNF (26, 32, 33). Hence, we examined the role of MKK4 in NF-{kappa}B activation induced by LPS, PMA, or CSC. As shown in Fig. 3D, LPS, PMA, and CSC all stimulated NF-{kappa}B, and MKK4 deletion suppressed NF-{kappa}B activation in every case. These results indicated that MKK4 acts as a step in the NF-{kappa}B activation pathway that is common to all these agents.

Reconstitution of MKK4 restores NF-{kappa}B activation in MKK4-deleted cells

We next determined whether reconstitution of MKK4 can restore TNF-induced NF-{kappa}B activation in MKK4-KO cells. MKK4 was added back to MKK4-null cells by the introduction of an MKK4 expression vector and nuclear extracts prepared after TNF treatment were analyzed for NF-{kappa}B activation. As shown in Fig. 3E, MKK4 overexpression in MKK4-null cells increased TNF induced NF-{kappa}B activation in a dose-dependent manner.

MKK4 deletion inhibits TNF-induced IKK activation, I{kappa}B{alpha} phosphorylation, and I{kappa}B{alpha} degradation

Because IKK is required for TNF-induced NF-{kappa}B activation, we determined whether MKK4 deletion had an effect on TNF-induced IKK activation. An immune complex kinase assay was performed with GST-I{kappa}B{alpha} as substrate to measure the activation of IKK. As shown in Fig. 4A, TNF induced IKK activation, but MKK4 deletion significantly repressed this activation, with no effect on the expression of IKK-{alpha} or IKK-beta proteins, suggesting that MKK4 is required for TNF-induced IKK activation.


Figure 4
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  		FIGURE 4. Effects of MKK4 deletion on TNF-induced activation of IKK. A, One million cells were pretreated with 50 µg/ml proteasome inhibitor N-Ac-leu-leu-norleucinal (ALLN) for 1 h and then stimulated with 1 nM TNF for the indicated times. Whole cell extracts were incubated with anti-IKK-{alpha} Ab for 2 h, immunoprecipitated with protein A/G-agarose beads, and then analyzed by immunocomplex kinase assay using GST-I{kappa}B{alpha} as a substrate. To examine the level of expression of IKK proteins, the same whole cell extracts were resolved by SDS-PAGE and Western blotted using anti-IKK-{alpha} and anti-IKK-beta Abs. B, Effects of MKK4 deletion on TNF-induced degradation and phosphorylation of I{kappa}B{alpha}. One million cells were treated with 0.1 nM TNF for the indicated times. Cytoplasmic extract was prepared, resolved by SDS-PAGE, and electrotransferred onto a nitrocellulose membrane. Western blot analysis using anti-I{kappa}B{alpha} and anti-phospho-specific I{kappa}B{alpha} Abs was performed. C, Effect of MKK4 deletion on TNF-induced nuclear translocation and phosphorylation of the p65 subunit of NF-{kappa}B. One million cells were treated with 0.1 nM TNF for the indicated times; nuclear extract was prepared, resolved by SDS-PAGE, and electrotransferred to a nitrocellulose membrane, and then Western blot analysis using anti-p65 and anti-phospho-specific p65 Abs was performed. The results shown are representative of three independent experiments.

 
The translocation of NF-{kappa}B to the nucleus is preceded by the proteolytic degradation of I{kappa}B{alpha} (31). To determine whether the inhibition of TNF-induced NF-{kappa}B activation in MKK4-KO cells was due to inhibition of I{kappa}B{alpha} degradation, we treated them with TNF for the indicated times and analyzed the degradation of I{kappa}B{alpha} by Western blot analysis. TNF induced I{kappa}B{alpha} degradation, which preceded NF-{kappa}B translocation in control cells as early as 15 min in wild-type fibroblasts. In MKK4-KO cells, however, TNF had no effect on I{kappa}B{alpha} degradation (Fig. 4B, upper panel). Thus, MKK4 is required for the degradation of I{kappa}B{alpha}.

The proteolytic degradation of I{kappa}B{alpha} is known to require phosphorylation at Ser32 and Ser36 (31). To determine whether MKK4 deletion affects TNF-induced I{kappa}B{alpha} phosphorylation, we assayed the TNF-induced phosphorylated form of I{kappa}B{alpha} by Western blot analysis, using an Ab that recognizes the Ser32/Ser36-phosphorylated form of I{kappa}B{alpha}. TNF stimulated I{kappa}B{alpha} phosphorylation in wild-type fibroblasts, but in MKK4-KO cells, the I{kappa}B{alpha} phosphorylation induced by TNF was almost completely suppressed (Fig. 4B, middle panel).

MKK4 is required for TNF-induced phosphorylation and nuclear translocation of p65

Because the degradation of I{kappa}B{alpha} leads to nuclear translocation of the p65 subunit of NF-{kappa}B, we determined whether MKK4 deletion affects the TNF-induced nuclear translocation of p65. As shown in Fig. 4C (upper panel), TNF induced the nuclear translocation of p65 in a time-dependent manner, as early as 5 min after TNF stimulation in MKK4-WT fibroblasts. In MKK4-KO cells, TNF did not induce the nuclear translocation of p65.

Because p65 phosphorylation is required for its transcriptional activity (34), we also investigated the effect of MKK4 on p65 phosphorylation. As shown in Fig. 4C (middle panel), TNF induced the phosphorylation of nuclear p65 in a time-dependent manner, as early as 5 min after TNF stimulation in MKK4-WT fibroblasts. In MKK4-KO cells, TNF failed to induce p65 phosphorylation.

MKK4 is required for expression of TNF-induced NF-{kappa}B-dependent reporter gene expression

DNA binding of NF-{kappa}B can be by either a transcriptional activator or transcriptional repressor, as demonstrated recently (35, 36). To determine the role of MKK4 in TNF-induced NF-{kappa}B-dependent reporter gene expression, we transiently transfected cells with the NF-{kappa}B-regulated SEAP reporter construct and then stimulated them with different concentrations of TNF. NF-{kappa}B-regulated reporter gene expression was activated by TNF in a dose-dependent manner in MKK4-WT fibroblasts, but minimal activation was detected in MKK4-KO cells (Fig. 5). These results suggest that MKK4 is needed not only for the activation of IKK, nuclear translocation of p65, and binding of p65 to the DNA, but also for NF-{kappa}B-regulated reporter gene expression.


Figure 5
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  		FIGURE 5. Effect of MKK4 deletion on TNF-induced NF-{kappa}B-dependent reporter gene expression. Cells were transiently transfected with a NF-{kappa}B-binding sites-containing reporter plasmid for 24 h. After transfection, cells were treated with various concentrations of TNF for a further 24 h. The supernatants of the culture medium were assayed for SEAP activity as described in Materials and Methods. Results are fold expression of NF-{kappa}B-regulated reporter gene.

 
MKK4 is required for NF-{kappa}B-dependent reporter gene expression induced by TNFR1, TRADD, TRAF2, NIK, and IKK-beta

To delineate the site of action of MKK4 in the TNF-signaling pathway leading to NF-{kappa}B activation, cells were transfected with TNFR1, TRADD, TRAF2, NIK, IKK-beta, and p65-expressing plasmids and monitored for NF-{kappa}B-dependent SEAP expression. As shown in Fig. 6A, MKK4–WT cells transfected with plasmids expressing TNFR1, TRADD, TRAF2, NIK, and IKK-beta, and p65 induced NF-{kappa}B-dependent expression of SEAP. MKK4 deletion suppressed gene expression induced by TNFR1, TRADD, TRAF2, NIK, and IKK-beta, but not p65-induced NF-{kappa}B reporter gene expression. These results suggest that MKK4 acts at a step upstream from p65.


Figure 6
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  		FIGURE 6. Effect of MKK4 deletion on NF-{kappa}B-dependent reporter gene expression induced by TNF, TNFR1, TRADD, TRAF2, NIK, IKK-beta, and p65. A, Cells were transiently transfected with a NF-{kappa}B-binding site-containing reporter plasmid along with the indicated plasmids or TNF. After 24 h, the culture medium was harvested and assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity over that in the vector control. Results shown are representative of three independent experiments. B, Whole cell extracts from MKK4-WT and MKK4-KO cells transfected with various plasmids were prepared and analyzed by Western blotting using Abs against TNFR1, TRADD, TRAF2, NIK, IKK-beta, and p65. C, A model showing the role of MKK4 in TNF-induced activation of NF-{kappa}B. IKK-activating kinases (IKKK) may be Akt or any other kinase.

 
To verify that the expression vectors used indeed produce the predicted protein in the transfected cells, whole cell extracts were prepared and analyzed by Western blot using Abs against TNFR1, TRADD, TRAF2, NIK, IKK-beta, and p65. It was observed that these plasmids indeed enhanced the expression of the predicted proteins over the basal levels in both MKK4-WT and MKK4-KO cells (Fig. 6B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The goal of this study was to examine the role of MKK4 in regulation of apoptotic pathway activated by TNF. We found that deletion of MKK4 gene enhanced TNF-induced apoptosis, which was associated with abrogation of the expression of TNF-induced cell-proliferative (COX-2 and cyclin D1) and antiapoptotic (survivin, IAP1, Bcl-2, Bcl-xL, cFLIP, and XIAP) gene products. TNF-induced NF-{kappa}B activation, I{kappa}B{alpha} kinase activation, I{kappa}B{alpha} phosphorylation, I{kappa}B{alpha} degradation, p65 nuclear translocation, and the NF-{kappa}B reporter activity induced by TNF, TNFR1, TRADD, TRAF2, NIK, and IKK-beta were all suppressed in kinase-deleted cells (Fig. 6C).

Our results show, for the first time, that TNF-induced apoptosis is potentiated by deletion of MKK4 gene. These results are in agreement with those of a study that found that transfection of cells with the kinase-dead mutant of MKK4 increases TNF-induced apoptosis in NIH-3T3 cells (25). Similarly, apoptosis in thymocytes induced by Fas/CD95, another member of the TNF superfamily, is also enhanced by deletion of MKK4 gene (5). How MKK4 down-regulates the apoptotic effects of TNF and other cytokines is not understood. We found for the first time that several gene products that mediate suppression of apoptosis and are induced by TNF are down-modulated in MKK4-KO cells. The expression of TNF-induced antiapoptotic proteins such as, survivin, IAP1, XIAP, Bcl-2, Bcl-xL, and cFLIP was inhibited in kinase-deleted cells. All these results indicate that MKK4 has a prosurvival role. These results are consistent with our recent report that MKK4 promotes cell survival by activating PI3K through an NF-{kappa}B/PTEN-dependent pathway (37).

Although our data are similar to those of a report that indicated that inhibition of JNK and p38 MAPK both regulated by MKK4 potentiates TNF-induced apoptosis (25), it differs from other reports, which indicate that JNK is required for TNF-induced apoptosis (38, 39). It has been demonstrated that prolonged JNK1 activation promoted TNF-induced apoptosis via E3 ligase-mediated degradation of the caspase 8-inhibitor cFLIPL (40), and transient JNK activation suppressed TNF-induced apoptosis (41). Thus, it is the absence of NF-{kappa}B-mediated inhibition of JNK activation that may contribute to TNF-induced apoptosis (42). Tang et al. (43) further showed that besides inhibiting caspases, NF-{kappa}B negatively modulates TNF-induced JNK activation, partly through NF-{kappa}B-induced XIAP. This negative cross-talk, which is specific to TNF signaling and does not affect JNK activation by IL-1, contributes to the inhibition of apoptosis. Paradoxically, overexpression of Ask1 alone, an upstream kinase that can activate MKK4, can activate apoptosis and mediate TNF-induced apoptosis (44).

The expression of cell-proliferative COX-2 and cyclin D1 was also abolished in the gene-deleted cells. We found that the TNF-induced expression of MMP-9 was also abolished by deletion of MKK4. Both TNF and MMP-9 have been linked with tumor metastasis (30). Although MKK4 has been shown to be a metastasis-suppressor gene (14, 15), our results may explain the mechanism for how MKK4 acts as a suppressor of metastasis.

All the proteins down-regulated by the deletion of MKK4 have been reported to be regulated by the transcription factor NF-{kappa}B (30). Our results clearly show that deletion of the MKK4 gene abolishes TNF-induced NF-{kappa}B activation. This report is the first to systematically evaluate the role of MKK4 in TNF-induced NF-{kappa}B DNA binding, I{kappa}B{alpha} phosphorylation and degradation, IKK activation, p65 phosphorylation and nuclear translocation, and NF-{kappa}B-dependent reporter gene transcription. Our results in this study indicate that deletion of MKK4 abolishes TNF-induced I{kappa}B{alpha} phosphorylation and degradation through inhibition of IKK activation. How the deletion of MKK4 leads to suppression of IKK activation, however, is not clear, partly because the kinase that activates IKK is unknown. Several kinases, including MAPK kinase kinases MEKK1 (45) and MEKK3 (46), Akt (47), TGF-beta activated kinase 1 (48), glycogen synthase kinase 3beta (49), and various other kinases have been implicated in the IKK activation induced by TNF (50, 51). Thus, it is possible that MKK4 deletion suppresses the activation of IKK indirectly by interfering with some upstream regulatory kinases. We also found that TNF-induced p65 phosphorylation at residue 536 was also inhibited in MKK4-KO cells. Both IKK and Akt have been linked to phosphorylation of p65 at residue 536 (52). Thus, MKK4-mediated IKK activation may mediate a central role in TNF-induced NF-{kappa}B and NF-{kappa}B-regulated antiapoptotic gene products.

Overall, our results show that MKK4 can regulate TNF-induced apoptosis through the modulation of NF-{kappa}B-signaling pathway, It is possible that the tumorigenic role assigned to TNF occurs through the activation of MKK4, which could regulate apoptosis through NF-{kappa}B signaling pathway as demonstrated in this study.


   Acknowledgment
 
We thank Dawn Chalaire for carefully editing the manuscript and providing valuable comments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Clayton Foundation for Research (to B.B.A.), by Grant PO1 CA91844 on lung chemoprevention from the National Institutes of Health (to B.B.A.), and by Grant P50 CA97007 from the National Institutes of Health Head and Neck SPORE Program (to B.B.A.). Dr. Aggarwal is the Ransom Horne, Jr., Professor of Cancer Research. Back

2 Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Laboratory, Department of Experimental Therapeutics, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail address: aggarwal{at}mdanderson.org Back

3 Abbreviations used in this paper: MKK, MAPK kinase; IKK, I{kappa}B{alpha} kinase; PARP, poly(ADP-ribose) polymerase; SEAP, secretory alkaline phosphatase; COX-2, cyclooxygenase-2; MMP-9, matrix metalloproteinase-9; TRADD, TNFR-associated death domain; TRAF, TNFR-associated factor; NIK, NF-{kappa}B-inducing kinase; IAP, inhibitor of apoptosis protein; cFLIP, cellular FLIP; CSC, cigarette smoke condensate. Back

Received for publication May 7, 2007. Accepted for publication May 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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