Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention Cancer Health Disparities Conference 2009
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[Cancer Research 61, 8668-8675, December 15, 2001]
© 2001 American Association for Cancer Research

Biochemistry and Biophysics

Kinase Suppressor of Ras Determines Survival of Intestinal Epithelial Cells Exposed to Tumor Necrosis Factor1

Fang Yan, Sutha K. John and D. Brent Polk2

Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Vanderbilt University School of Medicine, Nashville, Tennessee 37232


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The single layer of epithelial cells lining the intestine that serves as an important physical and functional barrier regulating the uptake of nutrients and the exclusion of various environmental antigens is disrupted in inflammatory bowel diseases. A central cytokine in the pathogenesis of inflammatory bowel disease is tumor necrosis factor (TNF), which increases apoptosis in a number of cell types. However, details determining the fate of intestinal cells exposed to high levels of TNF are lacking. Our laboratory reported that kinase suppressor of Ras (KSR) regulates TNF activation of the Raf/mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase/ERK signaling cassette by threonine phosphorylation of Raf-1, regulating proliferation and differentiation pathways. In the present study, we expressed a dominant-negative kinase-inactive KSR and determined the survival of young adult mouse colon cells exposed to TNF. Our data show that inhibition of KSR signaling decreases survival and increases apoptosis of TNF-treated cells. Antiapoptotic pathways including nuclear factor {kappa}B activation and one of its transcriptional targets, cIAP2 (c inhibitor of apoptosis protein 2) gene expression, and ERK/MAP kinase activation are all inhibited in TNF-treated kinase-inactive KSR-expressing young adult mouse colon cells. These antiapoptotic pathways are also inhibited by antisense-mediated down-regulation of KSR. However, TNF activation of p38 or stress-activated protein kinase/c-Jun NH2-terminal kinase is not inhibited by disruption of KSR signaling. Furthermore, inhibitors of both ERK and nuclear factor {kappa}B activation synergistically enhance apoptosis of cells treated with TNF. These findings demonstrate that KSR plays a novel regulatory role in intestinal epithelial cells exposed to TNF by activating cell survival pathways.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TNF3 is a regulatory cytokine in the pathogenesis of IBD (1) . Overexpression of TNF is sufficient to induce IBD in a mouse model (2) . Furthermore, anti-TNF antibodies reverse IBD in both patients (3) and animal models (4) . TNF interacts with two distinct cell surface receptors, a Mr 55,000 receptor (TNFR1) and a Mr 75,000 receptor (TNFR2; Refs. 5, 6, 7 ), initiating a variety of cellular responses through signal transduction pathways. We reported that TNF directly regulates intestinal epithelial cell proliferation and migration in a concentration-dependent manner (6) . Lower TNF concentrations stimulate proliferation and migration through TNFR2, whereas at high concentrations, TNF inhibits both of these responses through TNFR1. Although we do not detect apoptosis in TNF-treated intestinal cells (6) , programmed cell death is seen in a number of different cell types by activation of TNFR1 (8, 9, 10) or TNFR2 (11 , 12) .

The balance between antiapoptotic and proapoptotic signals regulates the fate of cells exposed to various stimuli including TNF (13 , 14) . Antiapoptotic signals initiated by TNF, other cytokines, and growth factors include NF{kappa}B (15, 16, 17, 18, 19) , ERK1/ERK2/MAP kinase (20, 21, 22, 23) , and phosphatidylinositol 3'-kinase/Akt (23 , 24) , whereas other members of the MAP kinase family, including SAPK/JNK and p38, function as proapoptotic signals (25, 26, 27) .

The ERK/MAP kinase pathway is necessary for TNFR1-mediated differentiating effects on intestinal cells (28) . We recently reported that TNF activation of this pathway requires the kinase activity of KSR for threonine phosphorylation and activation of Raf-1 (29) . In the present study, we demonstrate that KSR is an essential kinase in TNF signal transduction regulating intestinal epithelial cell fate. To determine the effect of inhibition of upstream activation of Raf-1/ERK/MAP kinase on TNF regulation of intestinal cell proliferation, we expressed KSR as a dominant-negative, kiKSR or asKSR to decrease endogenous KSR production in YAMC cells. We show that with inhibition of KSR, TNF induces apoptosis in intestinal cells. Furthermore, the consequences of kiKSR or asKSR expression include loss of activation of the antiapoptotic NF{kappa}B and ERK/MAP kinase pathways. In contrast, there was no reduction in activation of potentially proapoptotic SAPK/JNK or p38 MAP kinases in TNF-treated colon cells expressing kiKSR or asKSR. These findings support our conclusion that KSR is a key regulatory kinase determining the fate of intestinal epithelial cells exposed to TNF.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
YAMC and MSIE cells expressing a heat-labile SV40 large T antigen with an IFN-{gamma}-inducible promoter were grown on collagen-coated culture dishes as described previously (6 , 30) . Briefly, cells were maintained in RPMI 1640 with 5% FBS under permissive conditions at 33°C in a humidified atmosphere with 5% CO2. Confluent cell monolayers were serum-starved (0.5% FBS) and IFN-{gamma}-deprived under nonpermissive conditions at 37°C for 24 h before all experiments. HeLa and A431 cells were grown in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% CO2.

Recombinant Plasmid Generation and Cellular Transfection.
asKSR-cDNA3.1(-)-expressing vector was constructed by subcloning wtKSR derived from pcDNA3-wtKSR construct (provided by Richard Kolesnick; Memorial Sloan-Kettering Cancer Center, New York, NY) by KpnI/NotI digestion into the pcDNA3.1(-) vector at NotI/KpnI sites. The pcDNA3.1(-) vector containing asKSR was confirmed by DNA sequencing using T7 promoter as 5' primer.

Transient transfection of pcDNA3.1(-)-asKSR vector was performed on cells with pcDNA3.1(-) vector used as control. For each well containing 3 x 105 cells, 10 µl of LipofectAMINE 2000 reagent (Life Technologies, Inc., Grand Island, NY) were diluted with 250 µl of OPTI-MEM I medium (Life Technologies, Inc.), incubated for 5 min at room temperature, and then combined with 2 µg of DNA in 250 µl of OPTI-MEM I medium. The DNA-LipofectAMINE 2000 reagent complex was incubated for 20 min at room temperature and then added to cells in 2.5 ml of OPTI-MEM I medium. After a 6-h incubation, YAMC and MSIE cells were changed to RPMI 1640 and incubated for 18 h. HeLa or A431 cells were changed to DMEM and incubated for 18 h. Then YAMC and MSIE cells were incubated in RPMI 1640 with 0.5% FBS at 37°C, and HeLa and A431 cells were incubated in DMEM with 0.5% FBS at 37°C for 6 h prior to all experiments. The level of KSR expression was detected using Western blot analysis with anti-KSR antibody (provided by Deborah Morrison; National Cancer Institute, Bethesda, MD).

Clonal cell lines stably expressing pFlag-cDNA3-wtKSR, pFlag-cDNA3-kiKSR, or pcDNA3 vector (KSR vectors were provided by Richard Kolesnick) were generated as described previously (29) . The kiKSR plasmid expresses a dominant-negative kiKSR as described previously (29 , 31) . Clonal cells were cultured in the presence of G418 (100 µg/ml) until 24 h prior to experiments. Expression was verified by Western blot analysis with anti-Flag M2 (Sigma Chemical Co., St. Louis, MO).

Preparation of Cellular Lysates.
Lysates were prepared from cells treated with murine TNF (100 ng/ml; Pepro Tech, Inc. Rocky Hill, NJ), for the indicated times. Cell monolayers were washed twice with ice-cold PBS and then scraped into cell lysis buffer [20 mM HEPES (pH 7.5), 1 mM orthovanadate, 50 mM ß-glycerolphosphate, 10 mM Na PPi, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 18 µg/ml phenylmethylsulfonyl fluoride, and 1% Triton X-100]. The scraped suspensions were centrifuged (14,000 x g for 10 min) at 4°C, and protein content was determined using the DC protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of cellular lysate protein were mixed with Laemmli sample buffer (32) and separated by SDS-PAGE for Western blot analysis with anti-Flag M2 (Sigma Chemical Co.), anti-phospho-I{kappa}B{alpha}, anti-I{kappa}B{alpha}, anti-phospho-p38, anti-phospho-SAPK/JNK, anti-SAPK/JNK (Cell Signaling Technology, Beverly, MA), anti-cIAP2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-ERK1/ERK2 (Promega, Madison, WI), and anti-ERK1/ERK2 (Transduction Laboratory, San Diego, CA) antibodies.

Cell Proliferation Analysis.
Cells were prepared as described above, trypsinized, and counted; 400,000 cells/well were plated and permitted to attach for 24 h. Cells were then cultured under nonpermissive conditions for 24 h before treatment with TNF or EGF (10 ng/ml; a gift from Stanley Cohen; Vanderbilt University, Nashville, TN) for 48 h. At the conclusion, the cell number was determined by counting trypsinized cell suspensions as reported previously (6) . The change in the number of untreated cells from the start of an experiment to the end of an experiment was standardized as 100% proliferation. The change in cell number in treated cell samples over the course of an experiment was then reported as a percentage relative to the untreated controls.

Immunofluorescence.
Cells were cultured on collagen-coated sterile glass cover slides and prepared as described above. In the indicated experiments, cells were preincubated for 30 min with NF{kappa}B nuclear localization inhibitory peptide SN50 (100 µg/ml; BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA), cell-permeable control peptide SN50M (100 µg/ml; Ref. 33 ), or MEK1 inhibitor PD98059 (10 µM; Cell Signaling Technology); cells were treated with TNF, washed twice with ice-cold PBS, fixed in 1% paraformaldehyde in PBS for 10 min at 4°C, and then permeabilized with methanol for 5 min at -20°C. Once slides were air-dried and washed with PBS, the cells were incubated in 10% normal donkey serum in PBS (Zymed Laboratories Inc., San Francisco, CA) for 1 h. The slides were then incubated with rabbit anti-NF{kappa}B p65 antibody (1:300; Santa Cruz Biotechnology) in PBS with 1% donkey serum overnight at 4°C. The slides were washed three times in PBS for 5 min and incubated with donkey antirabbit IgG-FITC (1:2000; Zymed Laboratories Inc.) in 10% donkey serum in PBS at room temperature. The slides were washed again and then dehydrated and mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Immunofluorescence was observed by a LSM410 Confocal Laser Scanning Microscope from Carl Zeiss, Inc. (Oberkochen, Germany).

Apoptosis Assay.
Cells were cultured on collagen-coated chamber glass slides and prepared as described above. After TNF or cell-permeable C8-ceramide (BIOMOL Research Laboratories) treatment, apoptotic cells were labeled by ApopTag in Situ Apoptosis Detection Kits (Intergen Company, Purchase, NY) using terminal deoxynucleotidyl transferase for detection of positive cells, following the manufacturer’s guidelines. Apoptotic cells were labeled by anti-digoxigenin peroxidase conjugate and 3,3'-diaminobenzidine as substrate or FITC-conjugated anti-digoxigenin and dehydrated and mounted using Vectashield mounting medium. Slides were counterstained with DAPI by using 1 µg/ml DAPI in mounting medium. The cells were observed by DIC or fluorescence microscopy. Apoptotic TUNEL-positive cells were determined by counting at least 150 cells in randomly chosen fields and expressed as a percentage of the total number of cells counted.

IKK{alpha} in Vitro Kinase Assays.
Cells were treated with TNF, and then lysates were prepared as described above, and IKK{alpha} was recovered as detailed previously (34) using polyclonal anti-IKK{alpha} antibody (Santa Cruz Biotechnology). The immunoprecipitated IKK{alpha} was recovered by centrifugation, washed with ice-cold cell lysis buffer plus 1 M NaCl, and then washed with kinase buffer [20 mM Tris-HCl (pH 7.4), 20 mM MgCl2, 20 mM ß-glycerolphosphate, 20 mM p-nitrophenolphosphate, 1 mM sodium orthovanadate, 1 mM EDTA, and 200 nM ATP with leupeptin (10 µg/ml), aprotinin (10 µg/ml), and phenylmethylsulfonyl fluoride (18 µg/ml)]. In some experiments, IKK{alpha} was solubilized in Laemmli sample buffer for Western blot analysis with anti-IKK{alpha} or anti-IKKß (Santa Cruz Biotechnology) antibodies. The in vitro kinase assays were performed by incubating immunoisolated IKK{alpha} with GST-I{kappa}B{alpha} fusion protein (Santa Cruz Biotechnology) in 30 µl of kinase buffer at 37°C for 30 min as described previously (34) . GST-I{kappa}B{alpha} fusion protein was then separated from immunoprecipitated IKK{alpha} by centrifugation at 4°C. Recovered GST-I{kappa}B{alpha} in the supernatant was precipitated by incubation with glutathione-Sepharose 4B beads (Pharmacia Biotech, Piscataway, NJ) for 30 min at room temperature. GST-I{kappa}B{alpha} conjugated to beads was washed with PBS and prepared for SDS-PAGE and Western blot analysis with anti-phospho-I{kappa}B{alpha} or anti-I{kappa}B{alpha}.

Each individual experiment was repeated on at least three separate occasions with similar results. Proliferation and apoptosis data are presented as the means and SDs of triplicate samples. The statistical significance of the differences was determined using Student’s t test analysis. The level of statistical significance was set at P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dominant-negative kiKSR Expression Converts TNF from an Antiproliferative to an Apoptotic Ligand in Intestinal Epithelial Cells.
We reported that TNF at 100 ng/ml inhibits intestinal cell proliferation through TNFR1 by a mechanism that requires ERK/MAP kinase activity (6 , 28) in a signal transduction pathway that requires KSR kinase activity (29) . To investigate KSR in TNF regulation of intestinal cell proliferation, we stably expressed a Flag-tagged dominant-negative kiKSR in a mouse colon cell line as reported previously (29) . For comparison, we treated cells with EGF and studied nontransfected cells, cells stably expressing the Flag-tagged wtKSR or vector. As we have shown previously, YAMC cells express KSR, and the clonal lines containing wtKSR and kiKSR as Flag-tagged molecules were selected based on equivalent expression of these proteins (29) . The absolute increase in cell number over the 48-h time period in unstimulated cells was (mean ± SD) 326,000 ± 14,000, 320,000 ± 11,400, 319000 ± 13,300, and 311,000 ± 29,500 for YAMC., kiKSR, wtKSR, and vector lines, respectively. For each cell line, the mean increase in unstimulated cell proliferation was set at 100%. As expected, TNF inhibits proliferation of YAMC cells; however, expression of kiKSR causes >70% loss of cells treated with TNF (Fig. 1A)Citation . In contrast, neither wtKSR nor vector expression alters the antiproliferative effects of TNF. EGF stimulates proliferation in all four clonal cell lines. To determine the relationship between the level of dominant-negative KSR expression and cell loss, clonal cell lines expressing various levels of kiKSR were selected based on anti-Flag immunoblotting (Fig. 1C)Citation . Dominant-negative KSR expression induces cell loss in TNF-stimulated cells in a manner directly proportional to the level of kiKSR expression (Fig. 1B)Citation . TUNEL staining shows that TNF enhances apoptosis only in kiKSR-expressing cells (Fig. 2, A and B)Citation . Because we have shown cell-permeable ceramide to mimic the effect of TNF (100 ng/ml) on proliferation and signal transduction (35) , we determined the effect of C8-ceramide on apoptosis. Similar to TNF, ceramide induces apoptosis only in kiKSR cells (Fig. 2A)Citation . Fig. 2CCitation shows that expression of an asKSR construct also induces apoptosis detected by TUNEL staining. The degree of apoptotic cell loss also correlates with the amount of dominant-negative KSR expression (data shown in Fig. 1, B and CCitation ). Therefore, KSR appears to determine whether cells exposed to TNF undergo differentiation or apoptosis.



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  		Fig. 1. Expression of a dominant-negative KSR reduces cell viability in TNF-treated YAMC cells. Proliferation assays were performed using nontransfected YAMC cells and stable clonal cell lines expressing kiKSR, wtKSR, or vector only (as indicated in A) or clonal cell lines with different kiKSR expression levels (B). Cells were serum-starved (0.5% FBS) for 24 h at 37°C before use and then treated with TNF (100 ng/ml) or EGF (10 ng/ml) for 48 h. Cell numbers were determined by counting trypsinized cell suspensions. Bars represent the mean change in cell number over the course of an experiment compared with nontreated cells set at 100%. Cellular lysates were prepared for Western blot analysis with anti-Flag to determine kiKSR expression levels (C). This and all other experiments shown were performed on at least three separate occasions with similar results. *, P < 0.005 compared with control.

 


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  		Fig. 2. TNF and cell permeable C8-ceramide induce apoptosis in kiKSR- and asKSR-expressing YAMC cells. Cells were treated with TNF (100 ng/ml) or C8-ceramide (100 nM) for the indicated times and fixed for TUNEL and DAPI staining. Apoptotic nuclei were labeled with FITC (B, C) or peroxidase for visualization by 3,3'-diaminobenzidine (A). Images were obtained using fluorescence or DIC microscopy. FITC- and DAPI-labeled images were taken from the same field. Arrows represent apoptotic nuclei.

 
KSR Mediates TNF-stimulated Cell Survival Pathways.
Because the cellular decision to undergo apoptosis appears to be regulated by the balance between pro- and antiapoptotic signals, we determined the effect of kiKSR expression on NF{kappa}B activation, an antiapoptotic signaling pathway in many cell lines (15, 16, 17, 18, 19) . We studied nuclear translocation of the p65 subunit of NF{kappa}B by immunostaining and confocal laser microscopy and verified function by determining cIAP2 expression. kiKSR expression, but not wtKSR or vector controls (Fig. 3A)Citation , inhibits translocation of the p65 subunit to the nucleus. cIAP2 has been identified as a target of NF{kappa}B transcriptional activity, which protects against TNF-induced apoptosis (36 , 37) . Therefore, we further studied the role of KSR on cIAP2 production. Lysates prepared from cells treated with TNF for Western blot analysis with anti-cIAP2 show that kiKSR expression inhibits TNF-stimulated cIAP2 production (Fig. 3B)Citation .



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  		Fig. 3. Expression of kiKSR inhibits TNF-stimulated NF{kappa}B nuclear translocation and c-IAP2 production. YAMC cells were treated with TNF for the indicated times. To test NF{kappa}B nuclear translocation, cells were fixed for immunostaining with anti-NF{kappa}B p65 antibody and visualized by confocal laser microscopy (A). Cellular lysates were prepared for Western blot analysis with anti-c-IAP2 or anti-ERK1/ERK2 to determine equal protein loading (B).

 
In response to TNF signaling, activated IKK{alpha} and IKKß directly phosphorylate I{kappa}B on serine sites (38) , leading to I{kappa}B degradation and NF{kappa}B transcriptional activation (39 , 40) . Expression of kiKSR inhibits TNF-stimulated I{kappa}B{alpha} phosphorylation and degradation in YAMC cells (Fig. 4A)Citation . In addition, TNF-stimulated I{kappa}B{alpha} phosphorylation and degradation are also inhibited by antisense-mediated down-regulation of endogenous KSR (Fig. 4B)Citation . Because we have shown that IKK{alpha} is the predominant kinase responsible for I{kappa}B{alpha} phosphorylation in the mouse intestinal epithelial cell (34) , we assessed IKK{alpha} kinase activity toward I{kappa}B{alpha}. Immunoisolated IKK{alpha} was prepared for in vitro kinase assays using GST-I{kappa}B{alpha} as substrate. TNF-stimulated IKK{alpha} kinase activity toward I{kappa}B{alpha} is inhibited in cells expressing kiKSR (Fig. 5)Citation . Analysis of immunoisolated IKK{alpha} shows no detectable recovery of IKKß, as reported previously (34) .



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  		Fig. 4. Expression of kiKSR (A) or asKSR (B) inhibits TNF-stimulated I{kappa}B{alpha} phosphorylation and degradation in YAMC cells. Cells were treated with TNF for the indicated times, and cellular lysates were prepared for Western blot analysis with anti-phospho-I{kappa}B{alpha} (Anti-P-I{kappa}B{alpha}), anti-I{kappa}B{alpha}, anti-ERK1/ERK2, or anti-KSR.

 


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  		Fig. 5. Expression of kiKSR inhibits TNF-stimulated IKK{alpha} kinase activity in YAMC cells. IKK{alpha} was immunoprecipitated from untreated or TNF-treated cells as indicated. IKK{alpha} kinase activity was detected by in vitro kinase assays as described in "Materials and Methods." GST-I{kappa}B{alpha} (0.5 µg) was incubated with immunoprecipitated IKK{alpha} and 200 nM ATP for 30 min at 30°C. GST-I{kappa}B{alpha} was recovered by centrifugation and separated by SDS-PAGE for detection of phosphorylated GST-I{kappa}B{alpha} by Western blot analysis with anti-phospho-I{kappa}B{alpha} (Anti-P-I{kappa}B{alpha}) or anti-I{kappa}B{alpha} antibodies. Immunoprecipitated IKK{alpha} was recovered for immunodetection with anti-IKK{alpha} or anti-IKKß. Recombinant IKK{alpha} and IKKß were used in Western blot analysis as controls. Lane 1 in the top panel contained only GST-I{kappa}B{alpha} in the kinase assay.

 
KSR Kinase Activity Is Not Required for TNF Activation of SAPK/JNK or p38 MAP Kinases.
Among the protein kinases identified to mediate apoptosis in a variety of cell types are MAP kinase family members SAPK/JNK and p38 (25, 26, 27) . Expression of kiKSR or asKSR has no effect on TNF activation of SAPK/JNK or p38 MAP kinase (Fig. 6, A and B)Citation . In contrast, ERK1/ERK2 MAP kinase activation requires KSR, as we have reported previously (29) . These data indicate that activation of SAPK/JNK and p38 MAP kinases appears to be independent of KSR kinase activity in TNF signal transduction pathways.



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  		Fig. 6. KSR kinase activity is not required for TNF-stimulated p38 and SAPK/JNK activation. kiKSR (A)- or asKSR (B)- expressing YAMC cells were treated with TNF, and cellular lysates were prepared for Western blot analysis with anti-phospho-p38 (Anti-P-p38), anti-phospho-SAPK/JNK (Anti-P-SAPK/JNK), anti-phospho-ERK1/ERK2 (Anti-P-ERK1/ERK2), or anti-SAPK/JNK antibodies.

 
NF{kappa}B and ERK/MAP Kinase Activation Synergistically Prevents Apoptosis in YAMC Cells Exposed to TNF.
To determine the relative importance of NF{kappa}B and ERK1/ERK2 in TNF antiapoptotic signal transduction, we used inhibitors of their activation. A chimeric peptide (SN50) containing the signal sequence of Kaposi’s fibroblast growth factor fused to the p50 nuclear localization sequence was shown to inhibit NF{kappa}B nuclear translocation (33) . Preincubation of YAMC cells with SN50 blocks both TNF-stimulated p65 NF{kappa}B nuclear translocation (Fig. 7A)Citation and enhanced cIAP2 expression (Fig. 7B)Citation in YAMC cells. Neither the MEK1 inhibitor (PD98059) nor the inactive mutant peptide (SN50M) alters NF{kappa}B activation or cIAP2 expression. As expected, PD98059 inhibits ERK1/ERK2 activation (Fig. 7B)Citation . Blocking ERK1/ERK2 activation induces no detectable increase of apoptosis in TNF-treated cells (Fig. 8)Citation , whereas blockade of NF{kappa}B activation in cells treated with TNF increases apoptosis to 25%. However, inhibition of both ERK1/ERK2 and NF{kappa}B synergistically increases apoptosis to >80% of cells treated with TNF. These results suggest that KSR regulates activation of two important signal transduction pathways determining whether or not cells exposed to TNF undergo apoptosis.



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  		Fig. 7. A NF{kappa}B nuclear localization-blocking peptide (SN50) inhibits TNF-stimulated NF{kappa}B nuclear translocation and c-IAP2 production. YAMC cells were treated with TNF in the absence or presence of a 30-min pretreatment with SN50 (100 µg/ml), control peptide SN50M (100 µg/ml), or PD98059 (10 µM). Cells were then fixed for immunostaining with anti-NF{kappa}B p65 antibody and visualized by confocal laser microscopy (A). Cellular lysates were prepared for Western blot analysis with anti-cIAP2, anti-phospho-ERK1/ERK2 (Anti-P-ERK1/ERK2), or anti-ERK1/ERK2 antibodies (B).

 


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  		Fig. 8. Inhibition of NF{kappa}B and ERK/MAP kinase activation synergistically induces apoptosis in cells exposed to TNF. YAMC cells were treated with TNF for 6 h in the absence or presence of a 30-min pretreatment with SN50M, PD98059, SN50, or both SN50 and PD98059 at the concentrations indicated in Fig. 8Citation . The cells were fixed for TUNEL. Apoptotic nuclei were labeled with peroxidase, and visualized by DIC microscopy as described in the Fig. 2Citation legend. Apoptotic cells are shown with TUNEL-labeled nuclei in A. The percentage of cells undergoing apoptosis is shown in B. *, P < 0.005 compared with control.

 
KSR Regulation of ERK/MAP Kinase Activation and I{kappa}B{alpha} Degradation by TNF May Depend on Cell Type.
To determine whether the role of KSR in TNF activation of NF{kappa}B and ERK/MAP kinase is cell type dependent, MSIE, HeLa, and A431 cells were transfected with asKSR. Loss of endogenous KSR production inhibits TNF-stimulated ERK1/ERK2 phosphorylation and I{kappa}B{alpha} degradation in MSIE cells, similar to YAMC cells (Fig. 9)Citation . However, asKSR expression shows no effect on ERK/MAP kinase activation and I{kappa}B{alpha} degradation in either HeLa or A431 cells. These data suggest that KSR regulation of TNF-activated antiapoptotic signaling may be cell type specific.



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  		Fig. 9. Expression of asKSR inhibits TNF-stimulated ERK/MAP kinase activation and I{kappa}B{alpha} degradation in MSIE cells, but not in HeLa or A431 cells. Cells were treated with TNF for 15 min, and cellular lysates were prepared for Western blot analysis with anti-phospho-ERK1/ERK2 (Anti-P-ERK1/ERK2), anti-I{kappa}B{alpha}, anti-ERK1/ERK2, or anti-KSR antibodies. NT, nontransfected.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is clear that TNF is a regulatory cytokine in the pathogenesis of a number of chronic inflammatory conditions. Furthermore, TNFR1 and related Fas induce apoptosis in a number of cells and tissues. Our previous work identified KSR phosphorylation and activation of Raf-1 in TNF activation of ERK1/ERK2 (29) , a MAP kinase pathway we demonstrated to be essential for the antiproliferative/differentiating effects of TNFR1 in intestinal epithelial cells (28) . Therefore, the present study was designed to examine the effect of a dominant-negative kiKSR on the TNF-regulated antiproliferative response. Expression of this protein in intestinal epithelial cells converted TNF from an antiproliferative to an apoptotic ligand, which can no longer activate the antiapoptotic signaling of NF{kappa}B, ERK1/ERK2 MAP kinases, or the serine/threonine protein kinase Akt. Furthermore, antisense-mediated down-regulation of KSR inhibits these antiapoptotic signals. Direct inhibition of NF{kappa}B and ERK1/ERK2 activation synergistically recapitulates the apoptosis seen in kiKSR-expressing TNF-treated cells. In contrast, inhibiting KSR kinase activity does not alter TNF activation of the proapoptotic MAP kinases SAPK/JNK and p38. These findings place KSR kinase activity in a key regulatory role determining the fate of cells exposed to high levels of TNF.

The results of these studies suggest a role for KSR kinase activity in regulating critical physiological responses to TNF in cells. Zhang et al. (31) have shown that TNF-regulated KSR kinase activity phosphorylates Raf-1 on threonine 268/269 in fibroblasts. A role for KSR in regulation of normal growth, development, and differentiation is implied by its isolation in genetic screening analysis as a loss of function mutation in the Ras signaling pathways of Drosophila and Caenorhabditis elegans (41, 42, 43) . Whereas our previous work on TNF inhibition of intestinal cell proliferation predicts that a dominant-negative KSR should alter the differentiation response (28 , 29) , the conversion to apoptosis was not anticipated. Furthermore, the regulation of NF{kappa}B activation by kiKSR was an unexpected and novel finding.

NF{kappa}B is held in an inactive state by I{kappa}B binding in the cytoplasm. Following a variety of stimuli, the IKKs phosphorylate I{kappa}B, leading to I{kappa}B ubiquitination and degradation, thereby releasing NF{kappa}B for nuclear translocation (recently reviewed in Refs. 44 and 45 ). Our findings indicate that blockade of KSR kinase activity results in attenuation of IKK{alpha} kinase activity isolated from TNF-treated cells (Fig. 5)Citation . Although the role of the KSR substrate Raf-1 (29 , 46) in NF{kappa}B activation is not fully understood, expression of a transforming Raf-1 induces NF{kappa}B (47) , and dominant-negative Raf-1 inhibits its activation (48) . Experiments using mutant Raf-1 constructs recently demonstrated loss of ERK1/ERK2 MAP kinase activation, but not NF{kappa}B activation or neuronal differentiation, indicating the divergence of multiple downstream pathways at Raf-1 (49) . Although we have not proven in this report that Raf-1 is an intermediate in this pathway, dominant-negative KSR inhibits TNF activation of Raf-1 (29 , 31) . Furthermore, KSR directly phosphorylates and activates Raf-1 in TNF-treated cells (29) . Using an inhibitor of ERK1/ERK2 activation, we show that NF{kappa}B activation is not downstream of MEK1 in the Raf-1 pathway (Fig. 7)Citation . Given the recently demonstrated bifurcation in Raf-1 signaling, we suggest that TNF regulation of NF{kappa}B through KSR kinase activity in intestinal cells occurs via an alternative Raf-1 pathway. One consequence of this regulation is decreased expression of cIAP2 (Fig. 3B)Citation , which is reported to mediate NF{kappa}B antiapoptotic effects (36 , 37 , 50) . Importantly, the biological consequence of this alternative pathway appears to be the balance between proapoptotic and antiapoptotic signals. We have previously reported that either cell-permeable ceramide or sphingomyelinase can activate intestinal cell Raf-1/MEK/ERK and NF{kappa}B pathways (29 , 35) . Ceramide is generated by TNF via acid and neutral sphingomyelinase hydrolytic activities toward sphingomyelin (51) and has been shown to activate KSR (31) . Importantly, sphingomyelinase activation of ERK1/ERK2 in intestinal cells requires KSR kinase activity (29) and has been shown to initiate I{kappa}B{alpha} degradation (52) . Findings by other groups that ceramide induces apoptosis (53, 54, 55) may partially be explained by cell context because our studies were performed in intestinal epithelial cells. The present study clearly shows (Figs. 4Citation and 9Citation ) that KSR mediates ERK/MAP kinase activation and I{kappa}B{alpha} degradation by TNF in two intestinal cell lines; such effects were not observed in HeLa or A431 cells. Alternatively, the responses to ceramide may be differentially mediated based on the mechanism of generation. Knockout of the acid sphingomyelinase gene asmase-/- produced fibroblasts that were completely resistant to radiation-induced apoptosis but not TNF-induced apoptosis (56) . Given that a number of TNF-stimulated effects in intestinal cells can be mimicked by ceramide, the induction of apoptosis by ceramide in KSR-inhibited mouse colon cells is not surprising.

TNF was originally isolated based on its biological function of tumor regression following bacterial infection (6) . However, in many cells, TNF fails to induce apoptosis by initiating antiapoptotic signal transduction pathways (15, 16, 17, 18, 19) . Our findings indicate that KSR kinase activity is an antiapoptotic effector of TNF signal transduction via regulation of both the Raf-1/MEK1/ERK signaling cassette and NF{kappa}B. The antiapoptotic effects of these two pathways appear to be functionally redundant, in that inhibition of either pathway alone is insufficient to fully recapitulate the effect of inhibiting both pathways (Fig. 8)Citation , whereas inhibition of both NF{kappa}B and ERK1/ERK2 MAP kinase synergistically recapitulates the effects of KSR signal blockade in TNF-treated cells. Whereas the mechanisms determining KSR kinase activity are not known, it is presumed that a number of the identified serine and threonine phosphorylation sites regulate both protein-protein interactions and kinase activity (57, 58, 59) . Regulation of KSR kinase activity is therefore a potential therapeutic target in inflammatory conditions where reducing the apoptotic effects of high-level TNF appears to be involved in the pathogenesis of disease (60, 61, 62, 63) . Alternatively, in tumor cells resistant to TNF-induced apoptosis, inhibition of KSR activity could shift the response to apoptosis (15 , 27 , 64 , 65) . Our results show that KSR kinase activity is necessary to maintain the balance between proapoptotic and antiapoptotic signals in TNF- or ceramide-treated intestinal epithelial cells. From these studies, we conclude that KSR serves a regulatory role in determining the fate of intestinal epithelial cells exposed to TNF and may therefore prove a novel therapeutic target for modulating inflammatory or tumoricidal activities.


   ACKNOWLEDGMENTS
 
We thank Sam Wells for technical advice and the Vanderbilt University Medical Center Imaging Core Research Laboratory, which is supported by NIH Grants CA68485 and DK20593.


   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 Supported by NIH Grants T32 DK07673, DK56008, and F32 DK10105 and by a Research Fellowship Award from the Crohn’s and Colitis Foundation of America and a Turner Scholar Award. Back

2 To whom requests for reprints should be addressed, at Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, S4322 MCN, 21st and Garland Avenue, Nashville, TN 37232-2576. Phone: (615) 322-7449; Fax: (615) 343-8915; E-mail: d-brent.polk{at}mcmail.vanderbilt.edu Back

3 The abbreviations used are: TNF, tumor necrosis factor; cIAP, c inhibitor of apoptosis protein; DAPI, 4',6-diamidino-2-phenylindole; DIC, differential interference contrast; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; IBD, inflammatory bowel disease; IKK, I{kappa}B kinase; KSR, kinase suppressor of Ras; MAP, mitogen-activated protein; MEK, MAP kinase/ERK kinase; MSIE, mouse small intestinal epithelial; NF{kappa}B, nuclear factor {kappa}B; SAPK/JNK, stress-activated protein kinase/c-Jun NH2-terminal kinase; TNFR, TNF receptor; YAMC, young adult mouse colon; asKSR, antisense KSR; kiKSR, kinase-inactive KSR; wtKSR, wild-type KSR; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; GST, glutathione S-transferase; MEK, MAP/ERK kinase. Back

Received 5/25/01. Accepted 10/17/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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