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Selective unresponsiveness to the inhibition of p38 MAPK activation by cAMP helps L929 fibroblastoma cells escape TNF-alpha-induced cell death.

Wang J, Tang R, Lv M, Zhang J, Shen B - Mol. Cancer (2010)

Bottom Line: After ectopic expression of DLC with a transfection marker GFP, effects of cAMP on TNF-alpha-induced cell death in GFP+ cells were measured by PI staining and subsequent flow cytomety.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.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-alpha-induced cell death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Immunology, Institute of Basic Medical Sciences, Beijing, PR China.

ABSTRACT

Background: 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-kappaB activity and promotion of tumor necrosis factor-alpha (TNF-alpha)-induced cell death. However, it remains unknown whether this regulation is also applicable to fibroblastoma cells.

Methods: 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-alpha-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-alpha-induced cell death in GFP+ cells were measured by PI staining and subsequent flow cytomety.

Results: Elevation of cAMP suppressed TNF-alpha-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-alpha-induced cell death.

Conclusion: These data suggest that the lack of a pro-apoptotic pathway in tumor cells leads to a net survival effect of cAMP.

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cAMP inhibits TNF-α-induced JNK activation, but not p38 activation, in L929 cells. Phosphorylation and expression of JNK and p38 in L929 cells were analyzed by immunoblotting after the following treatment. A, L929 cells were pretreated with forskolin (10 μM) for various times as indicated, followed by stimulation with or without 10 ng/ml TNF-α for 15 min. B, L929 cells were pretreated with forskolin (10 μM), PGE2 (10 μM), epinephrine (100 μM), or 6-MB-cAMP (100 μM) for 30 min, followed by stimulation with or without 10 ng/ml TNF-α for 15 min. C, L929 cells were pretreated with forskolin (10 μM) for various times as indicated, followed by stimulation with or without 20 J/m2 UV and incubation for 30 min. D, L929 cells were treated with forskolin (10 μM) for various times as indicated or treated with 10 ng/ml TNF-α for 15 min.
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Figure 4: cAMP inhibits TNF-α-induced JNK activation, but not p38 activation, in L929 cells. Phosphorylation and expression of JNK and p38 in L929 cells were analyzed by immunoblotting after the following treatment. A, L929 cells were pretreated with forskolin (10 μM) for various times as indicated, followed by stimulation with or without 10 ng/ml TNF-α for 15 min. B, L929 cells were pretreated with forskolin (10 μM), PGE2 (10 μM), epinephrine (100 μM), or 6-MB-cAMP (100 μM) for 30 min, followed by stimulation with or without 10 ng/ml TNF-α for 15 min. C, L929 cells were pretreated with forskolin (10 μM) for various times as indicated, followed by stimulation with or without 20 J/m2 UV and incubation for 30 min. D, L929 cells were treated with forskolin (10 μM) for various times as indicated or treated with 10 ng/ml TNF-α for 15 min.

Mentions: Our recent work has revealed that, at least in fibroblasts, the crosstalk between cAMP-PKA-CREB pathway and either JNK or p38 pathway plays a key role in the regulation of cell death by cAMP [14,15]. Now that cAMP suppresses TNF-α-induced cell death in L929 fibroblastoma cells via CREB-mediated transcription, it is of importance to investigate the effects of cAMP on the activation of JNK and p38 in this cell context. For this purpose, L929 cells were pretreated with or without forskolin for various periods of times and then stimulated with TNF-α for 15 min or left untreated. Immunoblotting analysis revealed that TNF-α-induced phosphorylation of JNK at Thr183 and Tyr185, which is required for JNK activation [20], was inhibited by forskolin in a biphasic manner. The inhibition occurred from 30 to 90 min and decreased at 120 min after the pretreatment with forskolin (Figure 4A). The kinetics in the inhibition of JNK activation by forskolin was correlated with its effects on intracellular cAMP (Figure 2B). However, forskolin pretreatment showed no significant effect on TNF-α-induced phosphorylation of p38 at Thr180 and Tyr182, which is required for p38 activation [21,22], under the same conditions (Figure 4A). Similar results were obtained when L929 cells were pretreated with PGE2, epinephrine, and 6-MB-cAMP (Figure 4B). The different ability of these agents to inhibit the activation of JNK was correlated with the extent these cAMP elevators increased intracellular cAMP (Figure 2B), activated CREB (Figure 3A), and suppressed TNF-α-induced cell death in L929 cells (Figure 2A). cAMP uncoupled JNK activation and p38 activation not only in response to TNF-α, but also in response to UV (Figure 4C). Furthermore, cAMP inhibited the basal level of JNK phosphorylation, but not p38 phosphorylation, in L929 cells (Figure 4D). Taken together, these data suggest that cAMP uncouples JNK activation and p38 activation in L929 cells.


Selective unresponsiveness to the inhibition of p38 MAPK activation by cAMP helps L929 fibroblastoma cells escape TNF-alpha-induced cell death.

Wang J, Tang R, Lv M, Zhang J, Shen B - Mol. Cancer (2010)

cAMP inhibits TNF-α-induced JNK activation, but not p38 activation, in L929 cells. Phosphorylation and expression of JNK and p38 in L929 cells were analyzed by immunoblotting after the following treatment. A, L929 cells were pretreated with forskolin (10 μM) for various times as indicated, followed by stimulation with or without 10 ng/ml TNF-α for 15 min. B, L929 cells were pretreated with forskolin (10 μM), PGE2 (10 μM), epinephrine (100 μM), or 6-MB-cAMP (100 μM) for 30 min, followed by stimulation with or without 10 ng/ml TNF-α for 15 min. C, L929 cells were pretreated with forskolin (10 μM) for various times as indicated, followed by stimulation with or without 20 J/m2 UV and incubation for 30 min. D, L929 cells were treated with forskolin (10 μM) for various times as indicated or treated with 10 ng/ml TNF-α for 15 min.
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Figure 4: cAMP inhibits TNF-α-induced JNK activation, but not p38 activation, in L929 cells. Phosphorylation and expression of JNK and p38 in L929 cells were analyzed by immunoblotting after the following treatment. A, L929 cells were pretreated with forskolin (10 μM) for various times as indicated, followed by stimulation with or without 10 ng/ml TNF-α for 15 min. B, L929 cells were pretreated with forskolin (10 μM), PGE2 (10 μM), epinephrine (100 μM), or 6-MB-cAMP (100 μM) for 30 min, followed by stimulation with or without 10 ng/ml TNF-α for 15 min. C, L929 cells were pretreated with forskolin (10 μM) for various times as indicated, followed by stimulation with or without 20 J/m2 UV and incubation for 30 min. D, L929 cells were treated with forskolin (10 μM) for various times as indicated or treated with 10 ng/ml TNF-α for 15 min.
Mentions: Our recent work has revealed that, at least in fibroblasts, the crosstalk between cAMP-PKA-CREB pathway and either JNK or p38 pathway plays a key role in the regulation of cell death by cAMP [14,15]. Now that cAMP suppresses TNF-α-induced cell death in L929 fibroblastoma cells via CREB-mediated transcription, it is of importance to investigate the effects of cAMP on the activation of JNK and p38 in this cell context. For this purpose, L929 cells were pretreated with or without forskolin for various periods of times and then stimulated with TNF-α for 15 min or left untreated. Immunoblotting analysis revealed that TNF-α-induced phosphorylation of JNK at Thr183 and Tyr185, which is required for JNK activation [20], was inhibited by forskolin in a biphasic manner. The inhibition occurred from 30 to 90 min and decreased at 120 min after the pretreatment with forskolin (Figure 4A). The kinetics in the inhibition of JNK activation by forskolin was correlated with its effects on intracellular cAMP (Figure 2B). However, forskolin pretreatment showed no significant effect on TNF-α-induced phosphorylation of p38 at Thr180 and Tyr182, which is required for p38 activation [21,22], under the same conditions (Figure 4A). Similar results were obtained when L929 cells were pretreated with PGE2, epinephrine, and 6-MB-cAMP (Figure 4B). The different ability of these agents to inhibit the activation of JNK was correlated with the extent these cAMP elevators increased intracellular cAMP (Figure 2B), activated CREB (Figure 3A), and suppressed TNF-α-induced cell death in L929 cells (Figure 2A). cAMP uncoupled JNK activation and p38 activation not only in response to TNF-α, but also in response to UV (Figure 4C). Furthermore, cAMP inhibited the basal level of JNK phosphorylation, but not p38 phosphorylation, in L929 cells (Figure 4D). Taken together, these data suggest that cAMP uncouples JNK activation and p38 activation in L929 cells.

Bottom Line: After ectopic expression of DLC with a transfection marker GFP, effects of cAMP on TNF-alpha-induced cell death in GFP+ cells were measured by PI staining and subsequent flow cytomety.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.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-alpha-induced cell death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Immunology, Institute of Basic Medical Sciences, Beijing, PR China.

ABSTRACT

Background: 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-kappaB activity and promotion of tumor necrosis factor-alpha (TNF-alpha)-induced cell death. However, it remains unknown whether this regulation is also applicable to fibroblastoma cells.

Methods: 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-alpha-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-alpha-induced cell death in GFP+ cells were measured by PI staining and subsequent flow cytomety.

Results: Elevation of cAMP suppressed TNF-alpha-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-alpha-induced cell death.

Conclusion: These data suggest that the lack of a pro-apoptotic pathway in tumor cells leads to a net survival effect of cAMP.

Show MeSH
Related in: MedlinePlus