Selective unresponsiveness to the inhibition of p38 MAPK activation by cAMP helps L929 fibroblastoma cells escape TNF-α-induced cell death
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The cyclic AMP (cAMP) signaling pathway has been reported to either promote or suppress cell death, in a cell context-dependent manner. Our previous study has shown that the induction of dynein light chain (DLC) by cAMP response element-binding protein (CREB) is required for cAMP-mediated inhibition of mitogen-activated protein kinase (MAPK) p38 activation in fibroblasts, which leads to suppression of NF-κB activity and promotion of tumor necrosis factor-α (TNF-α)-induced cell death. However, it remains unknown whether this regulation is also applicable to fibroblastoma cells.
Intracellular cAMP was determined in L929 fibroblastoma cells after treatment of the cells with various cAMP elevation agents. Effects of cAMP in the presence or absence of the RNA synthesis inhibitor actinomycin D or small interfering RNAs (siRNAs) against CREB on TNF-α-induced cell death in L929 cells were measured by propidium iodide (PI) staining and subsequent flow cytomety. The activation of p38 and c-Jun N-terminal protein kinase (JNK), another member of MAPK superfamily, was analyzed by immunoblotting. JNK selective inhibitor D-JNKi1 and p38 selective inhibitor SB203580 were included to examine the roles of JNK and p38 in this process. The expression of DLC or other mediators of cAMP was analyzed by immunoblotting. After ectopic expression of DLC with a transfection marker GFP, effects of cAMP on TNF-α-induced cell death in GFP+ cells were measured by PI staining and subsequent flow cytomety.
Elevation of cAMP suppressed TNF-α-induced necrotic cell death in L929 fibroblastoma cells via CREB-mediated transcription. The pro-survival role of cAMP was associated with selective unresponsiveness of L929 cells to the inhibition of p38 activation by cAMP, even though cAMP significantly inhibited the activation of JNK under the same conditions. Further exploration revealed that the induction of DLC, the major mediator of p38 inhibition by cAMP, was impaired in L929 cells. Enforced inhibition of p38 activation by using p38 specific inhibitor or ectopic expression of DLC reversed the protection of L929 cells by cAMP from TNF-α-induced cell death.
These data suggest that the lack of a pro-apoptotic pathway in tumor cells leads to a net survival effect of cAMP.
KeywordsForskolin L929 Cell Intracellular cAMP Cell Death Assay Dynein Light Chain
It is known that persistent stress and depression, which leads to continuously elevated levels of stress hormones such as epinephrine, may increase tumor incidence and promote metastatic growth. Cyclic AMP (cAMP) is the first identified intracellular mediator (second messenger) of hormone action. The downstream effectors of cAMP—protein kinase A (PKA) and cAMP response element-binding protein (CREB)—have been shown to play a role in the tumorigenesis of endocrine tissues [1, 2]. Furthermore, it has been long disclosed that cAMP elevation is associated with impaired cell death of various tumor cells [3, 4, 5, 6, 7, 8, 9, 10]. Since resistance to cell death has been implicated in cancer pathogenesis, it is of great importance to elucidate the mechanisms by which cAMP plays a pro-survival role in tumor cells.
It is interesting that in non-malignant cells cAMP can either promote or suppress cell death depending on cell type and stimulus used [11, 12, 13, 14, 15]. The underlying mechanisms remain the topic of intensive studies. Our recent work has revealed that, at least in fibroblasts, the crosstalk between the cAMP signaling pathway and either JNK (c-Jun N-terminal protein kinase) or p38 pathway plays a key role in the regulation of cell death by cAMP [14, 15]. JNK and p38 are members of the mitogen-activated protein kinase (MAPK) superfamily [16, 17, 18]. The activation of JNK and p38 are typically mediated by sequential protein phosphorylation through a MAP kinase module, that is, MAPK kinase kinase (MAP3K) → MAPK kinase (MAP2K or MKK) → MAPK, in response to a variety of extracellular stimuli such as UV and tumor necrosis factor alpha (TNF-α) [19, 20, 21, 22]. In fibroblasts, the inhibition of JNK by cAMP confers resistance to UV-induced cytotoxicity . cAMP also significantly inhibits TNF-α-induced JNK activation . Even though JNK has been shown to contribute to TNF-α-induced cell death in various types of cells including fibroblasts [23, 24, 25], cAMP promotes TNF-α-induced cell death in fibroblasts because it simultaneously inhibits NF-κB activity through dynein light chain (DLC)-mediated suppression of p38 activation [14, 15]. Thus, the interplay of the pro-apoptotic pathway(s) and the pro-survival pathway(s) determines the outcome. However, it remains unknown whether the same regulation is also applicable to fibroblastoma cells.
The inhibition of either JNK or p38 by cAMP depends on CREB-mediated transcription and involves upstream MAP2K [14, 15]. However, the major effectors of cAMP-mediated inhibition of JNK or p38 activation are different. The induction of DLC is required for cAMP-mediated inhibition of p38 activation , whereas the induction of the long form of cellular FLICE-inhibitory protein (c-FLIPL) and MAPK phosphatase-1 (MKP-1) is required for cAMP-mediated inhibition of JNK activation . These observations suggest that the inhibition of JNK or p38 by cAMP could be uncoupled in certain cell context. In this work, we report that elevation of intracellular cAMP suppressed TNF-α-induced necrotic cell death in L929 fibroblastoma cells via CREB-mediated transcription. The pro-survival role of cAMP was associated with the lack of an inhibitory effect of cAMP on the pro-survival activation of p38 by TNF-α, even though cAMP significantly inhibited the activation of JNK under the same conditions. The induction of DLC, but not c-FLIPL and MKP-1, by cAMP was impaired in L929 cells. p38 selective inhibitor or enforced expression of DLC reversed the protection of L929 cells by cAMP from TNF-α-induced cell death. These data suggest that the lack of a pro-apoptotic pathway in tumor cells leads to a net survival effect of cAMP.
Materials and methods
Forskolin, prostaglandin E2 (PGE2), epinephrine, propidium iodide (PI), and actinomycin D were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against phospho-JNK, JNK, phospho-p38, phospho-CREB, CREB, and c-FLIPL were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p38, DLC, actin, and MKP-1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse TNF-α was purchased from R&D Systems (Minneapolis, MN, USA). D-JNKi1 was purchased from BioMol (Plymouth Meeting, PA, USA). SB203580 was from Calbiochem (San Diego, CA, USA). 6-MB-cAMP was from Biolog (Hayward CA, USA). ECL chemiluminescence kit was obtained from Amersham (Arlington Heights, IL, USA).
Cell culture and transfection
L929 cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Small interfering RNAs (siRNAs) that target murine CREB were designed based on nucleotides 1084 to 1102 (#1) and 749 to 767 (#2) relative to the translation start site, respectively, and purchased from Dharmacon (Lafayette, CO, USA). pcDNA3.1 Xpress-DLC has been described previously . Transfection was done with Amaxa nucleofection kit V (VCA-1003, program T-20, Gaithersburg, MD, USA), according to the manufacturer's protocol.
Intracellular cAMP was determined in L929 cells using the cAMP enzyme immunoassay kit purchased from Cayman Chemical (Ann Arbor, MI, USA). Samples were prepared exactly as described by the manufacturer.
Immunoblotting analysis was done as previously described . Briefly, adherent cells were washed with PBS and harvested with a cell scraper (Costars, Cambridge, MA, USA) in ice-cold lysis buffer (0.5% NP-40, 20 mM Tris-Cl, pH 7.6, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 10 mM PNPP, 10 μg/ml aprotinin). Cell lysates were resolved by SDS-PAGE before transferring to nitrocellulose membranes. Nitrocellulose membranes were then incubated with 5% (w/v) nonfat dry milk in washing buffer (20 mM Tris-Cl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 1 h at 37°C to block nonspecific protein binding. Primary antibodies (1:1000) were diluted in washing buffer containing 3% BSA and applied to the membranes for overnight at 4°C. After extensive washing, the membranes were incubated with peroxidase-conjugated antibodies for 1 h at room temperature and washed again. Immunoreactive bands were visualized with the ECL chemiluminescence kit.
Cell death assays
Cells were harvested by trypsin digestion. Dual staining with FITC-conjugated Annexin V and PI was carried out to detect the induction of apoptotic cell death. Cells were washed with PBS and resuspended in 200 μL of HEPES buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) containing 1 μg/ml Annexin V-FITC and 5 μg/ml PI (Annexin V/PI staining kit, BD Biosciences Pharmingen, San Diego, CA, USA). Following incubation for 15 min at room temperature, cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Annexin V-positive/PI-negative cells were apoptotic, whereas Annexin V/PI double positive cells were necrotic. A simple way to detect necrosis is PI staining. After washing with PBS, the pellet was stained with PI at a concentration of 5 μg/ml in PBS and incubated at room temperature in the dark for 5 min, which was followed by flow cytometry.
The data were shown as mean ± standard deviations (SD). The Student's t-test was used to compare the difference between the two groups. The difference was considered statistically significant when p < 0.05.
Forskolin suppresses TNF-α-induced necrotic cell death in L929 fibroblastoma cells
Various cAMP elevation agents suppress TNF-α-induced cell death in L929 cells
cAMP suppresses TNF-α-induced cell death in L929 cells via CREB-mediated transcription
The cAMP pathway activates several transcription factors, including CREB, CREM, and ATF-1 [31, 32, 33]. Among them, CREB is the major effector of the cAMP pathway [31, 32, 33]. We used CREB siRNAs to test whether CREB mediates the suppression by cAMP of TNF-α-induced cell death in L929 cells. Immunoblotting analysis revealed that CREB siRNAs specifically inhibited CREB expression and abolished the basal and forskolin-stimulated CREB phosphorylation (Figure 3C). Transfection of L929 cells with CREB siRNAs but not the negative control siRNA reversed the suppression of TNF-α-induced cell death by forskolin (Figure 3D). Moreover, CREB siRNA #2, which was more efficient than CREB siRNA #1, led to increased sensitivity of L929 cells to TNF-α-induced cell death. Consistently, the suppression by forskolin of TNF-α-induced cell death was also abolished by ACREB, a specific CREB inhibitor that utilizes its acidic amphipathic extension to prevent the basic region of CREB from binding to DNA  (data not shown). Taken together, these data suggest that CREB plays a pro-survival role in TNF-α-induced cell death in L929 cells, and cAMP suppresses TNF-α-induced cell death in L929 cells via CREB-mediated transcription.
cAMP inhibits TNF-α-induced JNK activation, but not p38 activation, in L929 cells
JNK activity is required for TNF-α-induced cell death in L929 cells
Inhibition of p38 activity reverses the protection of L929 cells by cAMP from TNF-α-induced cell death
Selective unresponsiveness to the inhibition of p38 activation by cAMP results from impaired induction of DLC
It has been long disclosed that cAMP elevation is associated with impaired cell death of various tumor cells [3, 4, 5, 6, 7, 8, 9, 10]. In this work, we show that treatment of L929 fibroblastoma cells with various cAMP elevation agents led to increased intracellular cAMP in a time-dependent manner (Figure 2B). cAMP increased following stimulation for 30 and 60 min and thereafter partially declined (Figure 2B). This increase and decline of cAMP were consistent with the so-called "biphasic" inhibition of JNK activation (Figure 4A) and the induction of MKP-1 and c-FLIPL (Figure 7A). Elevation of cAMP was associated with suppressed cell death in response to TNF-α (Figure 2A). Even though intracellular cAMP decreased partially after stimulation with forskolin for 90 min, the levels of intracellular cAMP remained much higher than no stimulation control in several hours (Figure 2B and data not shown). Recently, it has been shown that TNF-α induced a gradual, time-dependent increase in cAMP levels that reached a maximum after 8-10 h of stimulation in synovial fibroblasts . Similar increase in cAMP levels were also seen in L929 cells in response to TNF-α [see Additional file 1]. Even though the TNF-α-induced cAMP was weak and showed no statistically significant effect on total intracellular cAMP induced by forskolin [see Additional file 1], it could not be excluded the possibility that the TNF-α-induced cAMP might collaborate with cAMP elevation agents to suppress cell death. Specific blockade of the TNF-α-induced cAMP might address this issue.
Extensive studies have revealed that cAMP might promote the survival of tumor cells by various mechanisms. PKA-mediated phosphorylation of the proapoptotic Bcl-2 family protein BAD at Ser112 sequesters BAD in the cytoplasm through interaction with 14-3-3, thereby preventing BAD interaction with Bcl-2/Bcl-XL on the mitochondrial membrane . Several CREB target genes such as c-FLIPL, Bcl-2, and c-IAP-2 have been established to play an anti-apoptotic role [8, 9, 15, 41]. Elevation of cAMP in B cell precursor acute lymphoblastic leukaemia (BCP-ALL) cells is shown to profoundly inhibit DNA damage-induced cell death, which depends on the ability of elevated cAMP levels to quench DNA damage-induced p53 accumulation by increasing the p53 turnover .
In this study, our data suggest that cAMP suppresses TNF-α-induced cell death in L929 cells via CREB-mediated transcription (Figure 3 and data not shown). Blockade of transcription with actinomycin D or blockade of CREB activation with CREB siRNAs or ACREB reversed the suppression of TNF-α-induced cell death by cAMP (Figure 3 and data not shown). Therefore, the possible phosphorylation of BAD by PKA is not enough for cAMP to play a pro-survival role in TNF-α-induced cell death in L929 cells. It is not clear how CREB activation mediated the pro-survival effect of cAMP in this cell context. Since the protein levels of Bcl-2 and c-IAP2 have been implicated in the resistance of L929 cells to TNF-α-induced cell death [42, 43], the induction of certain anti-apoptotic protein(s) by CREB may play a key role in the suppression by cAMP of TNF-α-induced cell death. Besides directly inhibiting the death machinery, the CREB target gene(s) such as c-FLIPL might also suppress TNF-α-induced cell death via regulating JNK activity . The different ability of cAMP elevators to inhibit the activation of JNK (Figure 4B) was correlated with the extent these agents increased intracellular cAMP (Figure 2B), activated CREB (Figure 3A), and suppressed TNF-α-induced cell death in L929 cells (Figure 2A). Moreover, functional inhibition of JNK activity was enough to antagonize TNF-α-induced cell death in L929 cells (Figure 5). Thus, JNK inhibition should be part of the pro-survival cAMP mechanism in TNF-α-induced cell death in L929 cells.
Not only cAMP-stimulated CREB activity protected L929 cells from TNF-α-induced cell death, but also the basal CREB activity might affect the extent of cell death. L929 cells exhibited considerable basal level of phospho-CREB (Figure 3A and 3C). The basal CREB activity might render L929 cells resistant to TNF-α-induced cell death to certain extent by maintaining the protein levels of anti-apoptotic protein(s). The two siRNAs tested reduced CREB levels to different extents (Figure 3C), and the siRNA with greater CREB depletion exhibited a significant effect on TNF-α-induced cell death in the absence of an exogenous cAMP stimulus (Figure 3D). These data suggest that at a certain threshold of CREB depletion, a basal anti-apoptotic effect of CREB is lost, leading to a more significant level of cell death in the presence of TNF-α. This novel finding further suggests a protumorigenic role for CREB.
Despite that cAMP induces a similar activation of CREB in fibroblasts, cAMP promotes TNF-α-induced cell death in fibroblasts because it simultaneously inhibits NF-κB activity through DLC-mediated suppression of p38 activation [21, 22]. The inhibitory effect of cAMP on the pro-survival activation of p38 by TNF-α was lacking in L929 fibroblastoma cells, which might be due to loss of a cAMP-dependent induction of DLC. Because the enforced inhibition of p38 activation by using p38 specific inhibitor or ectopic expression of DLC reversed the protection of L929 cells by cAMP from TNF-α-induced cell death, it is the lack of a pro-apoptotic pathway that leads to a net survival effect of cAMP in L929 fibroblastoma cells. It remains unknown why the induction of DLC, but not c-FLIPL and MKP-1, by cAMP was impaired in L929 cells. Future studies are required to address this issue.
This work was supported by a grant from the National Natural Science Foundation of China (30973547) and 973 project 2010CB911904.
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