Cancer Research Cancer Epigenetics Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kurinna, S. M.
Right arrow Articles by Ruvolo, P. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kurinna, S. M.
Right arrow Articles by Ruvolo, P. P.
[Cancer Research 64, 7852-7856, November 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Ceramide Promotes Apoptosis in Lung Cancer-Derived A549 Cells by a Mechanism Involving c-Jun NH2-Terminal Kinase

Svitlana M. Kurinna1, Chun Chui Tsao1, Alina Felicia Nica1,2, Tilahun Jiffar1 and Peter P. Ruvolo1,2

1 Division of Cell Signaling, Institute of Molecular Medicine, University of Texas Health Science Center, and 2 University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ceramide regulates diverse signaling pathways involving cell senescence, the cell cycle, and apoptosis. Ceramide is known to potently activate a number of stress-regulated enzymes, including the c-Jun NH2-terminal kinase (JNK). Although ceramide promotes apoptosis in human lung cancer-derived A549 cells, a role for JNK in this process is unknown. Here, we report that ceramide promotes apoptosis in A549 cells by a mechanism involving JNK. The JNK inhibitor SP600125 proved effective at protecting cells from the lethal effects of ceramide. To understand which JNK-mediated pathway may be involved, a number of JNK target proteins were examined, including the transcription factor, c-Jun, and the apoptotic regulatory proteins Bcl-XL and Bim. A549 cells exhibited basal levels of phosphorylated c-Jun in nuclear fractions, revealing that active c-Jun is present in these cells. Ceramide was found to inhibit c-Jun phosphorylation, suggesting that JNK-mediated phosphorylation of c-Jun is not likely involved in ceramide-induced apoptosis. Ceramide did not promote Bcl-XL phosphorylation. On the other hand, ceramide promoted phosphorylation of Bim and induced translocation of active JNK from the nucleus to the cytoplasm and mitochondrial fraction. Ceramide-mediated changes in localization of JNK were consistent with the observed changes in phosphorylation status of c-Jun and Bim. Furthermore, ceramide promoted Bim translocation to the mitochondria. Mitochondrial localization of Bim has been shown recently to promote apoptosis. These results suggest that JNK may participate in ceramide-induced apoptosis in A549 cells by a mechanism involving Bim.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The c-Jun NH2-terminal kinases (JNKs) are a family of enzymes that are commonly activated in response to stress and hence are also known as stress-activated protein kinases (SAPKs; refs. 1, 2, 3 ). At present, three JNK family members have been identified (JNK1, JNK2, and JNK3). The best-studied target of JNK is c-Jun (4, 5, 6, 7) . JNK phosphorylation of c-Jun at serines 63 and 73 promotes activation of this transcription factor (2 , 3) . The c-Jun protein has been implicated in both the induction and prevention of apoptosis (8) . JNK activation of c-Jun is necessary for apoptosis in myeloid and lymphoid cells, because use of a dominant-negative c-Jun mutant blocks programmed cell death in these cells (5) . One possible mechanism to explain how activation of c-Jun may promote cell death involves the induction of the proapoptotic Fas ligand (9) . Still, c-Jun has been shown to regulate numerous cell processes, and whether c-Jun activation promotes or prevents programmed cell death is highly dependent on the given cell type (8) . Thus, it is not surprising that JNK activation alone is not sufficient to induce apoptosis in many cell types (10) .

Early studies to determine the role of JNK in apoptosis have focused on its role as a c-Jun kinase (3, 4, 5, 6, 7) . Recent studies have identified novel targets for JNK, suggesting that this SAPK likely activates other regulatory pathways in addition to those that involve c-Jun. These JNK targets include many proteins that regulate apoptosis, such as BCL2 (11, 12, 13) , Bcl-XL (14) , and Bim (15) . JNK appears to play a key role in the intrinsic (i.e., mitochondria mediated) apoptotic pathway but is not required in the extrinsic (i.e., death receptor mediated) apoptotic pathway, because cells derived from jnk 1–/– jnk 2–/– mice were resistant to killing by intrinsic pathway-mediated pathway stress challenges (e.g., UV), but these cells were still sensitive to Fas (16) . Furthermore, JNK is required for the activation of proapoptotic Bax, cytochrome c release, and caspase activation during UV-induced apoptosis in mouse embryonic fibroblasts (17) . These studies suggest a strong link between stress activation of JNK and activation of the apoptosome via the mitochondria. However, activation of JNK does not always result in cell death. Studies in human lung cancer-derived cell lines indicate that JNK plays a prosurvival role in these cells, and the enzyme contributes to oncogenic transformation (18, 19, 20) .

Cells treated with ceramide may undergo programmed cell death, become growth arrested, or, in rare cases, become stimulated to proliferate. The diversity of biological responses of cells to ceramide reflects the complexity of the role of this sphingolipid as a second signal molecule (21, 22, 23) . Ceramide treatment of A549 cells promotes apoptosis in a caspase-dependent process (24) . Chalfant et al. (25 , 26) have discovered that ceramide activates protein phosphatase 1 (PP1) dephosphorylation of serine/arginine-rich proteins, resulting in alternative splicing of at least two critical apoptotic regulators (Bcl-XL and caspase 9). Although JNK is known to be regulated by ceramide, the exact role for this enzyme in ceramide-induced cell death in A549 cells is unknown. The goal of the present study was to determine whether a role for JNK exists in ceramide-induced apoptosis in A549 cells. Results suggest that JNK participates in ceramide-mediated apoptosis by a process that may involve the apoptotic regulatory protein Bim.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
All of the reagents used were purchased from commercial sources unless otherwise stated.

Cell Lines.
A549 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in DMEM supplemented with 1% nonessential amino acids and 10% fetal bovine serum at 37°C in 5% CO2.

Analysis of Cell Viability and Apoptosis.
Cells were treated with 50 µmol/L C6-ceramide (BioMol, Plymouth Meeting, PA) or 50 µmol/L C6-dihydroceramide (BioMol) for 24 hours. Where appropriate, cells were pretreated for 1 hour with 1 µmol/L SP600125 (Calbiochem, La Jolla, CA) or 10 µmol/L SB203580 (Calbiochem). Cell viability was measured by trypan blue dye exclusion, and apoptosis was analyzed by cell morphology. To observe cell morphology changes, cells were transferred to slides using a cytocentrifuge, fixed, and stained robotically with 20% May-Grunwald-Wright-Giemsa composite stain using an Aerospray Slide Stainer as per the manufacturer’s recommendations (Wescor, Logan, UT). Cells were observed by light microscopy using x40 magnification.

Cell Fractionation Studies.
Subcellular fractionation of cells was performed as described previously (27) . Where appropriate, cells were treated with 50 µmol/L C6-ceramide for 3 hours before fractionation. Cells were swelled in ice-cold hypotonic HEPES buffer [10 mmol/L HEPES (pH 7.4), 5 mmol/L MgCl2, 40 mmol/L Kcl, 1 mmol/L phenylmethylsulfonylfluoride, 10 µg/mL aprotinin, and 10 µg/mL leupeptin] for 30 minutes, aspirated repeatedly through a 25-gauge needle (30 strokes), and centrifuged for 10 minutes at 200 x g at 4°C to pellet nuclei. The supernatant from this spin was centrifuged at 10,000 x g for 18 minutes at 4°C to pellet the heavy membrane fraction containing mitochondria. The resulting heavy membrane supernatant was centrifuged at 150,000 x g for 90 minutes at 4°C to pellet the light membranes, and the remaining supernatant represented the cytosol. Protein concentration was determined using a bicinchonic acid assay kit (Pierce, Rockford, IL). Western blotting using antibodies to JNK and phospho-JNK (Cell Signaling, Beverly, MA), c-Jun and phospho-c-Jun (Cell Signaling), BCL2 (DAKO, Carpinteria, CA), Bcl-XL (Santa Cruz Biotechnology, Santa Cruz, CA), Bim (Calbiochem), actin (Santa Cruz Biotechnology), and prohibitin (Research Diagnostics, Inc., Flanders, NJ) was performed as described previously (27) .

Metabolic Labeling, Immunoprecipitation, and Immunoblotting Analysis.
A549 cells were treated with 50 µmol/L C6-ceramide for 3 hours during metabolic labeling with 32P-Pi, and Bcl-XL was analyzed by immunoprecipitation by a method similar to that described for studies with BCL2 (27) . Samples were electrophoresed in a 12% acrylamide/0.1% SDS gel, transferred to nitrocellulose, and exposed to Kodak X-Omat film at –80°C. The same blot was used for Western blotting with anti-Bcl-XL anti-sera and developed using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunohistochemistry Studies.
A549 cells were grown and treated with 50 µmol/L C6-ceramide for 3 hours in chamber slides. For permeabilization and fixation, cells were treated with paraformaldehyde and methanol. Where appropriate, nuclei were visualized with 4',6-diamidino-2-phenylindole (Molecular Probes, Eugene, OR). Cells were washed and blocked with 10% serum PBS, and then primary antibody was added. Antibodies used were phospho-JNK mouse monoclonal sera (Cell Signaling), Bcl-XL/S rabbit polyclonal sera (Santa Cruz Biotechnology), Bim polyclonal sera (Calbiochem), and phospho-c-Jun polyclonal rabbit sera (Cell Signaling). Finally, an Alexa Fluor-conjugated secondary antibody was added, and cells were visualized using a Zeiss Axioplan 2 imaging fluorescence microscope and photographed with AxioCam MRm digital camera in black and white. Alexa Fluor 488-labeled antirabbit serum and Alexa Fluor 594-labeled antimouse serum (both from Molecular Probes) were used. Alexa Fluor 488 appears green, and Alexa Fluor 594 appears red when observed using a fluorescent microscope. To determine subcellular regions of protein colocalization, individual red- and green-stained images derived from the same field were merged using Adobe PhotoShop 7.0 (Adobe, San Jose, CA). Areas of protein colocalization appear yellow. False color, deconvolution, and enhancement were accomplished with Adobe PhotoShop 7.0.

Statistics.
Statistical analysis was performed using standard t test analysis with Sigma Stat computer software (SSPS, Chicago, IL).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
JNK but not p38 Regulates Ceramide-Induced Apoptosis in A549 Cells.
C6-ceramide promotes apoptosis in A549 cells by a caspase-dependent mechanism that can be blocked by overexpression of exogenous BCL2 (24) . Because it is not known if SAPK participates in this process in these cells, studies were performed to determine whether JNK or p38 kinase is required for ceramide-induced killing. Pharmacological inhibitors for JNK (SP600125, 1 µmol/L) and p38 kinase (SB 203580, 10 µmol/L) were used to determine whether either of these stress signaling kinases can block cell killing by ceramide in A549 cells. SP600125 is a small molecule inhibitor that exhibits high specificity for JNK (28 , 29) . Cells were pretreated with 1 µmol/L SP600125 or 10 µmol/L SB 203580 for 1 hour before addition of 50 µmol/L C6-ceramide for 24 hours as described in Materials and Methods. Although the inactive C6-dihydroceramide analogue (50 µmol/L) had no effect on cell viability, 50 µmol/L C6-ceramide killed approximately half the cells after 24 h (Fig. 1A)Citation . Cells treated with C6-ceramide exhibited morphologic features of apoptosis (e.g., nuclear condensation, membrane blebbing, and cellular debris), whereas untreated cells or cells treated with C6-dihydroceramide appeared healthy (data not shown). As shown in Fig. 1ACitation , inactivation of the JNK kinase by SP600125 significantly protects A549 cells from cell death (P < 0.001). In contrast to the effects with the JNK inhibitor, suppression of p38 with SB203580 did not significantly protect A549 cells from ceramide (P > 0.05; Fig. 1ACitation ). To rule out the possibility that our SB203580 drug stock was not active, we treated A549 cells with 10 µmol/L SB 203580 for 24 hours and examined p38 activity as indicated by phosphorylation status. As shown in Fig. 1BCitation , SB203580 was effective at blocking p38 activation in these cells. These data suggest a role for JNK but not p38 in ceramide-mediated apoptosis in A549 cells.



View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Inhibition of JNK but not p38 protects A549 cells from ceramide-induced killing. In A, A549 cells were treated with 50 µmol/L C6-Dihydroceramide (C6-dihydro), 1 µmol/L JNK-specific inhibitor SP600125, 10 µmol/L p38-specific inhibitor SB203580, and 50 µM C6-ceramide and combination of 1 µmol/L SP600125 + 50 µmol/L C6-ceramide or 10 µmol/L SB203580 + 50 µmol/L C6-ceramide for 24 hours. Cells receiving both ceramide and an inhibitor were pretreated with the inhibitor for 1 hour before ceramide addition. Cell survival was accessed by trypan blue dye exclusion assay. Bars, mean ± SE from three separate experiments. In B, protein lysate (20 µg) from untreated A549 cells or cells treated with 10 µmol/L SB203580 for 24 hours was used in Western blot analysis using phospho-p38, p38, and actin anti-sera.

 
Ceramide Promotes JNK Translocation from the Nucleus to the Cytoplasm and Non-Nuclear Membranes.
The mechanism by which JNK might regulate apoptosis is not clear. Recent studies indicate the importance of mitochondrial JNK in the apoptotic process (17) . JNK activation and translocation to mitochondria in response to stress challenges have been observed in a number of human leukemia cell lines (30) . To determine whether ceramide could affect JNK subcellular compartmentalization, subcellular localization experiments were performed. Subcellular fractions containing heavy membrane (which contain mitochondrial membranes), light membrane (which contain endoplasmic reticulum, Golgi, and light membranes), nucleus, and cytosolic supernatant were isolated from untreated A549 cells and cells treated with 50 µmol/L C6-ceramide for 3 hours as described in Materials and Methods. As shown in Fig. 2ACitation , Western analysis of JNK protein levels revealed that untreated cells had most, if not all, of the JNK protein in the nuclear fraction. Prohibitin, which is localized in mitochondrial and nuclear fractions, was used as a control (31) . After ceramide treatment, a significant amount of JNK was translocated from the nuclear fraction to the heavy membrane fraction containing mitochondrial membranes. Moreover, phosphorylated JNK is detected in all of the membrane fractions after ceramide treatment but only in the nuclear fraction in untreated cells (Fig. 2A)Citation . The level of total JNK in the light membrane and cytosolic fractions is likely below the level of detection for the antibody against the total protein. Consistent with loss of JNK from the nucleus, levels of phosphorylated c-Jun are reduced in the nucleus (Fig. 2A)Citation . To verify that ceramide suppressed nuclear c-Jun phosphorylation, phosphorylated c-Jun was examined by immunofluorescence microscopy in untreated cells and cells treated with 50 µmol/L C6-ceramide for 3 hours (Fig. 2B)Citation . Nuclei were visualized by 4',6-diamidino-2-phenylindole staining in both treated and untreated cells. Untreated A549 cells exhibited distinctive nuclear staining with anti-sera against phosphorylated c-Jun, whereas phosphorylated c-Jun was barely visible in nuclei in cells treated with C6-ceramide. The results presented in Fig. 2, A and BCitation , demonstrate that phosphorylation of c-Jun is inhibited by ceramide, and because ceramide is toxic to A549 cells, activation of c-Jun is likely not required for programmed cell death in these cells.



View larger version (46K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Ceramide promotes JNK translocation from the nucleus and suppresses c-Jun phosphorylation. In A, subcellular fractionation studies were performed as described in Materials and Methods. Cells were treated with 50 µmol/L C6-ceramide for 3 hours, where appropriate. Western blot analysis using 10 µg of protein from each subcellular fraction was performed using anti-sera to JNK, phospho-JNK, phospho-c-Jun, and prohibitin. Lanes, heavy (HM), light (LM), cytosol (CYT), nuclear (NUC) membranes. In B, A549 cells that were untreated or treated with 50 µmol/L C6-ceramide for 3 hours were fixed, and then goat phospho-c-Jun polyclonal antibody was added. Fluorescent conjugated secondary antibody was used to visualize protein (green). Cells were counterstained with 4',6-diamidino-2-phenylindole (blue) to identify nuclei. Image analysis was performed as described in Materials and Methods.

 
Ceramide Promotes JNK Phosphorylation of Bim but not Bcl-XL.
Because ceramide-activated JNK does not target c-Jun in A549 cells, the question remained what effects ceramide would have on phosphorylation of other JNK substrates. A number of newly described JNK targets (e.g., BCL2, Bcl-XL, and Bim) have been identified recently (11, 12, 13, 14, 15) . Although BclII is a JNK target, BclII was not examined, because little, if any, of the protein is detected in A549 cells (data not shown). A metabolic radiolabeling experiment was performed to determine whether Bcl-XL was phosphorylated in A549 cells in response to ceramide (Fig. 3A)Citation . A549 cells were treated with 50 µmol/L C6-ceramide for 3 hours during metabolic labeling with 32P-Pi, and the phosphorylation status of Bcl-XL was examined after immunoprecipitation. Western analysis demonstrates that roughly equivalent levels of Bcl-XL protein were immunoprecipitated from control and C6-ceramide-treated samples. As shown in Fig. 3ACitation , A549 cells actually exhibit basal levels of Bcl-XL phosphorylation. Ceramide, however, did not augment Bcl-XL phosphorylation levels as determined by metabolic labeling. This result suggests that Bcl-XL phosphorylation, which is associated with loss of antiapoptotic function (14) , is not involved in ceramide-induced apoptosis in A549 cells.



View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. JNK promotes Bim but not Bcl-X L phosphorylation. In A, 2 x 107 cells were metabolically labeled with 32P-Pi and lysed, and then protein was immunoprecipitated with Bcl-XL antibody. Where appropriate, cells were treated with 50 µmol/L C6-ceramide for 3 hours. Phosphorylation was observed by autoradiography. Western blot analysis was performed to confirm detected bands were Bcl-XL. In B, protein lysate (80 µg) from untreated A549 cells or cells treated with 50 µmol/L C6-ceramide for 3 hours was used in Western blot analysis using Bim and actin anti-sera. Phosphorylation status of Bim was determined by a retardation of electrophoretic mobility.

 
Recent studies have revealed that JNK phosphorylation of Bim can be visualized by changes in electrophoretic mobility (15 , 17) . The electrophoretic migration of Bim is retarded when the protein is phosphorylated by JNK. Western analysis of Bim was performed using protein lysates from untreated cells and cells treated with 50 µmol/L C6-ceramide for 3 hours. As shown in Fig. 3BCitation , C6-ceramide promotes Bim phosphorylation. This finding suggests that Bim may be an important JNK target in ceramide-induced apoptosis in A549 cells.

The ability of ceramide to suppress c-Jun phosphorylation while promoting Bim phosphorylation suggests that ceramide might affect substrate targeting by altering subcellular localization of the kinase. To determine how ceramide affected colocalization of JNK with Bcl-XL and Bim, immunofluorescence microscopy was performed. A mouse monoclonal antibody against phospho-JNK and rabbit polyclonal antibodies against Bim and Bcl-XL were used so that cells could be simultaneously stained, and colocalization of JNK with its targets could be examined (Fig. 4)Citation . Staining for phospho-JNK appears red, and JNK target proteins appear green when observed using a fluorescent microscope. Untreated cells display robust nuclear staining with anti-phospho-JNK antibody. There is little, if any, detection of phospho-JNK staining in the cytoplasmic and non-nuclear membranes in untreated cells (Fig. 4)Citation . However, after ceramide treatment, nuclear staining with phospho-JNK is diffuse, and the cytoplasm becomes visible with a punctate pattern of staining, indicating migration to the cytoplasm and small organelles. In untreated cells, Bcl-XL is observed around the nuclear region of the cell. The merged image of untreated cells stained with Bcl-XL and JNK antibodies indicates colocalization of the proteins as observed by bright white/yellow punctuate staining (Fig. 4)Citation . Cells treated with ceramide, however, do not exhibit significant colocalization of Bcl-XL and JNK as demonstrated by the absence of the white/yellow punctate staining pattern. Consistent with the promotion of Bim phosphorylation, ceramide promoted colocalization of Bim and JNK (Fig. 4)Citation . The ability of ceramide to promote subcellular translocation of JNK is in agreement with earlier observations by Kharbanda et al. (30) showing that stress challenges promoted mitochondrial localization of JNK in association with apoptosis.



View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Ceramide promotes colocalization of JNK with Bim. A549 cells that were untreated or treated with 50 µM C6-ceramide for 3 hours were fixed, and then mouse monoclonal JNK antibody or rabbit polyclonal Bim or Bcl-XL antibody was added. Fluorescent conjugated secondary antibody was used to visualize Bim or Bcl-XL (green) and JNK (red) localization patterns using a fluorescent microscope. Image analysis was performed as described in Materials and Methods. Areas of colocalization appear white/yellow.

 
Ceramide Promotes Translocation of Bim to the Mitochondria.
Recent studies have suggested that Bim migration to the mitochondria is an event associated with apoptosis (15 , 17) . To determine whether ceramide promoted mitochondrial translocation of Bim, Western analysis was performed using protein lysates from isolated mitochondrial fractions. Heavy membrane fractions were isolated as described in "Materials and Methods" from untreated cells and cells treated with 50 µmol/L C6-ceramide for 3 hours. Although little, if any, Bim is detected in the mitochondria of untreated cells, ceramide promotes Bim translocation to the mitochondria (Fig. 5)Citation . This migration of Bim to the mitochondria in response to ceramide suggests that this translocation event may be important in ceramide-induced apoptosis in A549 cells.



View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Ceramide promotes Bim translocation to the mitochondria. In A, cells received 50 µmol/L ceramide for 3 hours, where appropriate. Heavy membrane fractions containing mitochondrial membranes were isolated as described in Materials and Methods. Western blot analysis using 10 µg of mitochondrial protein was performed using anti-sera to Bim and prohibitin.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanism of how ceramide activates JNK is not clear. Ceramide may activate SAPK via Rac-1 (32) , PKC {zeta} (33) , or TAK1 (34) . It is also not clear how SAPK-activating pathways are triggered in response to short chain ceramides like C6-ceramide. One possibility involves the conversion of exogenous short chain ceramide to endogenous long chain ceramide (35) . C16-ceramide failed to effectively kill A549 cells, although the drug was able to potently kill HL60 cells (data not shown). It is possible that C6-ceramide in A549 cells promotes the generation of another metabolite that is responsible for JNK activation (e.g., a sphingosine derivative) or that C16-ceramide fails to penetrate A549 cells to reach critical target regions (i.e., the nucleus). The latter possibility is supported by studies in human kidney proximal tubule cells and HCT116 human colon cancer cells, where it was found that short chain ceramides were toxic and long chain ceramide was not and that exogenous C16-ceramide did not reach intracellular locales, because the molecule mainly accumulated in the light membrane (36 , 37) . Although the exact mechanism is not clear, a model whereby JNK is activated by C6-ceramide via the production of long chain ceramides is supported by the study demonstrating that exogenous C6-ceramide promotes the production of endogenous C16-ceramide and C24-ceramide in A549 cells (35) .

Previous studies have demonstrated that C6-ceramide-induced apoptosis involves caspase activation, although little else is known of the signaling pathways involved in this process (24) . Ceramide has been shown to activate the PP1 (25 , 26) and affect telomerase activity (35 , 38) in A549 cells; however, the effect of C6-ceramide on well-characterized ceramide targets, such as the SAPKs or the PP2A, in these cells is unknown. Ceramide-induced killing of A549 cells appears to require JNK but does not require the p38 kinase (Fig. 1A)Citation . JNK appears activated in untreated cells, although active enzyme appears to be limited to the nucleus (Fig. 2ACitation and Fig. 4Citation ). The cells display basal levels of phosphorylated c-Jun, suggesting that this transcription factor may play a prosurvival role in these cells (Fig. 2B)Citation . JNK has been shown to activate either prosurvival or prodeath signaling cascades depending on the mode of how the enzyme itself is regulated (39) . The finding that nuclear JNK is normally active in A549 cells whereas ceramide promotes translocation of the enzyme from the nucleus in association with death suggests that there are both prosurvival and prodeath JNK pathways in A549 cells.

Interestingly, A549 cells displayed basal levels of phosphorylated Bcl-XL (Fig. 3A)Citation . This finding suggests that phosphorylated Bcl-XL is not detrimental to these cells. Another BCL2 family member, Bim, is one of the more recently identified targets of JNK (15 , 17) . The observed ceramide-induced phosphorylation of Bim by JNK in A549 cells suggests a mechanism by which JNK might participate in ceramide-mediated apoptosis in these cells. Lei and Davis (15) have demonstrated recently that JNK is a Bim kinase and that phosphorylation of Bim may be sufficient to cause Bax-induced apoptosis. JNK phosphorylation of Bim promotes its release from a cytosolic Dynein/Myosin V complex (15) . The significant increase in cytosolic phosphorylated JNK in response to ceramide in the A549 cells is consistent with the detection of Bim phosphorylation. Furthermore, the ability of ceramide to potently promote Bim translocation to the mitochondria (Fig. 5)Citation suggests this may be an important event in ceramide-induced apoptosis in these cells. Still, the role of JNK in apoptosis is controversial. In some cell types (e.g., fibroblasts, cells derived from various regions of the brain) suppression of JNK promotes chemoresistance (16 , 40) . Conversely, JNK may play an antiapoptotic role in some cell types, such as in thymocytes and mature T cells (41) . The findings reported here suggest that JNK has the capacity to be either prosurvival or proapoptotic depending on conditions. Still, it is tempting to speculate that the subcellular translocation of JNK in response to growth agonist or stress challenge will determine which potential signaling pathways are activated resulting in growth or death, respectively.


   FOOTNOTES
 
Grant support: NIH Grant CA30715-01.

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.

Requests for reprints: Peter Ruvolo, University of Texas Health Science Center, Institute of Molecular Medicine, 6770 Bertner Boulevard, DAC 950D, Houston, TX 77030. Phone: 713-500-2310; Fax: 713-500-2325; E-mail: peter.p.ruvolo{at}uth.tmc.edu

Received 5/ 5/04. Revised 7/21/04. Accepted 8/23/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pulverer BJ, Kyriakis JM, Avruch J, Nikolakaki E, Woodgett JR Phosphorylation of c-jun mediated by MAP kinases. Nature 1991;353:670-4.[CrossRef][Medline]
  2. Kyriakis JM, Avruch J Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001;81:807-69.[Abstract/Free Full Text]
  3. Derijard B, Hibi M, Wu IH, et al JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 1994;76:1025-37.[CrossRef][Medline]
  4. Jarvis WD, Grant S, Kolesnick RN Ceramide and the induction of apoptosis. Clin Cancer Res 1996;2:1-6.[Free Full Text]
  5. Verheij M, Bose R, Lin XH, et al Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 1996;380:75-9.[CrossRef][Medline]
  6. Basu S, Kolesnick R Stress signals for apoptosis: ceramide and c-Jun kinase. Oncogene 1998;17:3277-85.[CrossRef][Medline]
  7. Mechta-Grigoriou F, Gerald D, Yaniv M The mammalian Jun proteins: redundancy and specificity. Oncogene 2001;20:2378-89.[CrossRef][Medline]
  8. Shaulian E, Karin M AP-1 as a regulator of cell life and death. Nat Cell Biol 2002;4:E131-6.[CrossRef][Medline]
  9. Kasibhatla S, Brunner T, Genestier L, Echeverri F, Mahboubi A, Green DR DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-{kappa} B and AP-1. Mol Cell 1998;1:543-51.[CrossRef][Medline]
  10. Lin A Activation of the JNK signaling pathway: breaking the brake on apoptosis. Bioessays 2003;25:17-24.[CrossRef][Medline]
  11. Maundrell K, Antonsson B, Magnenat E, et al Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1. J Biol Chem 1997;272:25238-42.[Abstract/Free Full Text]
  12. Yamamoto K, Ichijo H, Korsmeyer SJ BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol Cell Biol 1999;19:8469-78.[Abstract/Free Full Text]
  13. Deng X, Xiao L, Lang W, Gao F, Ruvolo P, May WS Novel role for JNK as a stress-activated Bcl2 kinase. J Biol Chem 2001;276:23681-8.[Abstract/Free Full Text]
  14. Basu A, Haldar S Identification of a novel Bcl-xL phosphorylation site regulating the sensitivity of taxol- or 2-methoxyestradiol-induced apoptosis. FEBS Lett 2003;538:41-7.[CrossRef][Medline]
  15. Lei K, Davis RJ JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis. Proc Natl Acad Sci USA 2003;100:2432-7.[Abstract/Free Full Text]
  16. Tournier C, Hess P, Yang DD, et al Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 2000;288:870-4.[Abstract/Free Full Text]
  17. Lei K, Nimnual A, Zong WX, et al The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase. Mol Cell Biol 2002;22:4929-42.[Abstract/Free Full Text]
  18. Bost F, McKay R, Dean N, Mercola D The JUN kinase/stress-activated protein kinase pathway is required for epidermal growth factor stimulation of growth of human A549 lung carcinoma cells. J Biol Chem 1997;272:33422-9.[Abstract/Free Full Text]
  19. Bost F, McKay R, Bost M, Potapova O, Dean NM, Mercola D The Jun kinase 2 isoform is preferentially required for epidermal growth factor-induced transformation of human A549 lung carcinoma cells. Mol Cell Biol 1999;19:1938-49.[Abstract/Free Full Text]
  20. Xiao L, Lang W A dominant role for the c-Jun NH2-terminal kinase in oncogenic ras-induced morphologic transformation of human lung carcinoma cells. Cancer Res 2000;60:400-8.[Abstract/Free Full Text]
  21. Hannun YA Functions of ceramide in coordinating cellular responses in stress. Science 1996;274:1855-9.[Abstract/Free Full Text]
  22. Kolesnick RN, Kronke M Regulation of ceramide production and apoptosis. Ann Rev Physiol 1998;60:643-65.[CrossRef][Medline]
  23. Ruvolo PP Intracellular signal transduction pathways activated by ceramide and its metabolites. Pharmacol Res 2003;47:383-92.[CrossRef][Medline]
  24. Ravid T, Tsaba A, Gee P, Rasooly R, Medina EA, Goldkorn T Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cells. Am J Physiol Lung Cell Mol Physiol 2003;284:L1082-92.[Abstract/Free Full Text]
  25. Chalfant CE, Ogretmen B, Galadari S, Kroesen BJ, Pettus BJ, Hannun YA FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1. J Biol Chem 2001;276:44848-55.[Abstract/Free Full Text]
  26. Chalfant CE, Rathman K, Pinkerman RL, et al De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1. J Biol Chem 2002;277:12587-95.[Abstract/Free Full Text]
  27. Ruvolo PP, Deng X, Carr BK, May WS A functional role for mitochondrial protein kinase C {alpha} in Bcl2 phosphorylation and suppression of apoptosis. J Biol Chem 1998;273:25436-42.[Abstract/Free Full Text]
  28. Han Z, Boyle DL, Chang L, Bennett B, et al c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Investig 2001;108:73-81.[CrossRef][Medline]
  29. Weston CR, Davis RJ The JNK signal transduction pathway. Curr Opin Genet Dev 2002;12:14-21.[CrossRef][Medline]
  30. Kharbanda S, Saxena S, Yoshida K, et al Translocation of SAPK/JNK to mitochondria and interaction with Bcl-x(L) in response to DNA damage. J Biol Chem 2000;275:322-7.[Abstract/Free Full Text]
  31. Ruvolo PP, Clark W, Mumby M, Gao F, May WS A functional role for the B56 {alpha}-subunit of protein phosphatase 2A in ceramide-mediated regulation of Bcl2 phosphorylation status and function. J Biol Chem 2002;277:22847-52.[Abstract/Free Full Text]
  32. Brenner B, Koppenhoefer U, Weinstock C, Linderkamp O, Lang F, Gulbins E Fas- or ceramide-induced apoptosis is mediated by a Rac1-regulated activation of Jun N-terminal kinase/p38 kinases and GADD153. J Biol Chem 1997;272:22173-81.[Abstract/Free Full Text]
  33. Bourbon NA, Yun J, Kester M Ceramide directly activates protein kinase C {zeta} to regulate a stress-activated protein kinase signaling complex. J Biol Chem 2000;275:35617-23.[Abstract/Free Full Text]
  34. Shirakabe K, Yamaguchi K, Shibuya H, et al TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase. J Biol Chem 1997;272:8141-4.[Abstract/Free Full Text]
  35. Ogretmen B, Pettus BJ, Rossi MJ, et al Biochemical mechanisms of the generation of endogenous long chain ceramide in response to exogenous short chain ceramide in the A549 human lung adenocarcinoma cell line. Role for endogenous ceramide in mediating the action of exogenous ceramide. J Biol Chem 2002;277:12960-9.[Abstract/Free Full Text]
  36. Iwata M, Herrington J, Zager RA Sphingosine: a mediator of acute renal tubular injury and subsequent cytoresistance. Proc Natl Acad Sci USA 1995;92:8970-4.[Abstract/Free Full Text]
  37. Fillet M, Bentires-Alj M, Deregowski V, et al Mechanisms involved in exogenous C2- and C6-ceramide-induced cancer cell toxicity. Biochem Pharmacol 2003;65:1633-42.[CrossRef][Medline]
  38. Ogretmen B, Kraveka JM, Schady D, et al Molecular mechanisms of ceramide-mediated telomerase inhibition in the A549 human lung adenocarcinoma cell line. J Biol Chem 2001;276:32506-14.[Abstract/Free Full Text]
  39. Ham YM, Choi JS, Chun KH, Joo SH, Lee SK The c-Jun N-terminal kinase 1 activity is differentially regulated by specific mechanisms during apoptosis. J Biol Chem 2003;278:50330-7.[Abstract/Free Full Text]
  40. Kuan CY, Yang DD, Samanta Roy DR, et al The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development. Neuron 1999;22:667-76.[CrossRef][Medline]
  41. Nishina H, Fischer KD, Radvanyi L, et al Stress-signaling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3. Nature 1997;385:350-3.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
J. Zhang, O. Kamdar, W. Le, G. D. Rosen, and D. Upadhyay
Nicotine Induces Resistance to Chemotherapy by Modulating Mitochondrial Signaling in Lung Cancer
Am. J. Respir. Cell Mol. Biol., February 1, 2009; 40(2): 135 - 146.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Pattingre, C. Bauvy, S. Carpentier, T. Levade, B. Levine, and P. Codogno
Role of JNK1-dependent Bcl-2 Phosphorylation in Ceramide-induced Macroautophagy
J. Biol. Chem., January 30, 2009; 284(5): 2719 - 2728.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
S. Ghose, N. V. Oleinik, N. I. Krupenko, and S. A. Krupenko
10-Formyltetrahydrofolate Dehydrogenase-Induced c-Jun-NH2-Kinase Pathways Diverge at the c-Jun-NH2-Kinase Substrate Level in Cells with Different p53 Status
Mol. Cancer Res., January 1, 2009; 7(1): 99 - 107.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C.-L. Chen, C.-F. Lin, W.-T. Chang, W.-C. Huang, C.-F. Teng, and Y.-S. Lin
Ceramide induces p38 MAPK and JNK activation through a mechanism involving a thioredoxin-interacting protein-mediated pathway
Blood, April 15, 2008; 111(8): 4365 - 4374.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
C. G. Roberts, E. Gurisik, T. J. Biden, R. L. Sutherland, and A. J. Butt
Synergistic cytotoxicity between tamoxifen and the plant toxin persin in human breast cancer cells is dependent on Bim expression and mediated by modulation of ceramide metabolism
Mol. Cancer Ther., October 1, 2007; 6(10): 2777 - 2785.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
G. Kroemer, L. Galluzzi, and C. Brenner
Mitochondrial Membrane Permeabilization in Cell Death
Physiol Rev, January 1, 2007; 87(1): 99 - 163.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Kitatani, J. Idkowiak-Baldys, J. Bielawski, T. A. Taha, R. W. Jenkins, C. E. Senkal, B. Ogretmen, L. M. Obeid, and Y. A. Hannun
Protein Kinase C-induced Activation of a Ceramide/Protein Phosphatase 1 Pathway Leading to Dephosphorylation of p38 MAPK
J. Biol. Chem., December 1, 2006; 281(48): 36793 - 36802.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
V E MacRae, T Burdon, S F Ahmed, and C Farquharson
Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent.
J. Endocrinol., November 1, 2006; 191(2): 369 - 377.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
Y. Wei, D. Wang, F. Topczewski, and M. J. Pagliassotti
Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells
Am J Physiol Endocrinol Metab, August 1, 2006; 291(2): E275 - E281.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. Carlo-Stella, M. Di Nicola, M. C. Turco, L. Cleris, C. Lavazza, P. Longoni, M. Milanesi, M. Magni, M. Ammirante, A. Leone, et al.
The Anti-Human Leukocyte Antigen-DR Monoclonal Antibody 1D09C3 Activates the Mitochondrial Cell Death Pathway and Exerts a Potent Antitumor Activity in Lymphoma-Bearing Nonobese Diabetic/Severe Combined Immunodeficient Mice
Cancer Res., February 1, 2006; 66(3): 1799 - 1808.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kurinna, S. M.
Right arrow Articles by Ruvolo, P. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kurinna, S. M.
Right arrow Articles by Ruvolo, P. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online