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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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Related in: MedlinePlus

cAMP suppresses TNF-α-induced cell death in L929 cells via CREB-mediated transcription. A, L929 cells were treated with forskolin (10 μM), PGE2 (10 μM), epinephrine (100 μM), or 6-MB-cAMP (100 μM) for 30 min. Phosphorylation of CREB and expression of CREB and actin were analyzed by immunoblotting (IB). B, L929 cells were treated with or without actinomycin D (ActD, 1 μg/ml, 30 min) prior to forskolin treatment (10 μM, 30 min), followed by stimulation with or without 2 ng/ml TNF-α for 12 h. Cell death was measured by PI staining. The percentages of cell death are shown in the right panel as mean ± SD; n = 3. Left panel is representative of three independent experiments. C, L929 cells were transfected with CREB siRNAs or the negative control (NC) siRNA (200 nM each). After 72 h, cells were stimulated with or without forskolin (10 μM, 30 min). Phosphorylation of CREB and expression of CREB and actin were measured by immunoblotting. D, L929 cells were transfected with CREB siRNAs or the negative control siRNA (200 nM each). After 48 h, cells were pretreated with or without forskolin (10 μM, 30 min), followed by stimulation with or without 10 ng/ml TNF-α for 24 h. Cell death was monitored by PI staining. The percentages of cell death are shown in the right panel as mean ± SD; n = 3. Left panel is representative of three independent experiments.
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Figure 3: cAMP suppresses TNF-α-induced cell death in L929 cells via CREB-mediated transcription. A, L929 cells were treated with forskolin (10 μM), PGE2 (10 μM), epinephrine (100 μM), or 6-MB-cAMP (100 μM) for 30 min. Phosphorylation of CREB and expression of CREB and actin were analyzed by immunoblotting (IB). B, L929 cells were treated with or without actinomycin D (ActD, 1 μg/ml, 30 min) prior to forskolin treatment (10 μM, 30 min), followed by stimulation with or without 2 ng/ml TNF-α for 12 h. Cell death was measured by PI staining. The percentages of cell death are shown in the right panel as mean ± SD; n = 3. Left panel is representative of three independent experiments. C, L929 cells were transfected with CREB siRNAs or the negative control (NC) siRNA (200 nM each). After 72 h, cells were stimulated with or without forskolin (10 μM, 30 min). Phosphorylation of CREB and expression of CREB and actin were measured by immunoblotting. D, L929 cells were transfected with CREB siRNAs or the negative control siRNA (200 nM each). After 48 h, cells were pretreated with or without forskolin (10 μM, 30 min), followed by stimulation with or without 10 ng/ml TNF-α for 24 h. Cell death was monitored by PI staining. The percentages of cell death are shown in the right panel as mean ± SD; n = 3. Left panel is representative of three independent experiments.

Mentions: Our previous data suggest that cAMP regulates TNF-α-induced cell death in fibroblasts via CREB-mediated transcription [14]. Consistent with this notion, forskolin-, PGE2-, epinephrine-, and 6-MB-cAMP-induced CREB phosphorylation at Ser133 (Figure 3A), which is required for CREB activation [31-33], corresponded to the extent these cAMP elevators activated intracellular cAMP (Figure 2B) and suppressed TNF-α-induced cell death in L929 cells (Figure 2A). To further analyze the mechanism by which cAMP suppresses TNF-α-induced cell death in L929 cells, the cells were pretreated with or without forskolin, followed by treatment with TNF-α in the presence or absence of the RNA synthesis inhibitor actinomycin D. Because 10 ng/ml TNF-α rapidly induced more than 70% cell death in the presence of actinomycin D (data not shown), the concentration of TNF-α was titrated down. 2 ng/ml TNF-α treatment for 12 h led to only 7% cell death in the absence of actinomycin D, which was significantly suppressed by forskolin (Figure 3B). However, in the presence of actinomycin D the same dose of TNF-α resulted in about 35% cell death, though actinomycin D itself showed no detectable effect on the survival of L929 cells (Figure 3B). These data are consistent with the previous finding in the literature that blockade of de novo protein synthesis significantly enhances TNF-α-induced cell death. [34,35] The suppression of TNF-α-induced cell death by forskolin was abolished by actinomycin D (Figure 3B). These data suggest that cAMP suppresses TNF-α-induced cell death in L929 cells in a transcription-dependent manner.


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 suppresses TNF-α-induced cell death in L929 cells via CREB-mediated transcription. A, L929 cells were treated with forskolin (10 μM), PGE2 (10 μM), epinephrine (100 μM), or 6-MB-cAMP (100 μM) for 30 min. Phosphorylation of CREB and expression of CREB and actin were analyzed by immunoblotting (IB). B, L929 cells were treated with or without actinomycin D (ActD, 1 μg/ml, 30 min) prior to forskolin treatment (10 μM, 30 min), followed by stimulation with or without 2 ng/ml TNF-α for 12 h. Cell death was measured by PI staining. The percentages of cell death are shown in the right panel as mean ± SD; n = 3. Left panel is representative of three independent experiments. C, L929 cells were transfected with CREB siRNAs or the negative control (NC) siRNA (200 nM each). After 72 h, cells were stimulated with or without forskolin (10 μM, 30 min). Phosphorylation of CREB and expression of CREB and actin were measured by immunoblotting. D, L929 cells were transfected with CREB siRNAs or the negative control siRNA (200 nM each). After 48 h, cells were pretreated with or without forskolin (10 μM, 30 min), followed by stimulation with or without 10 ng/ml TNF-α for 24 h. Cell death was monitored by PI staining. The percentages of cell death are shown in the right panel as mean ± SD; n = 3. Left panel is representative of three independent experiments.
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Figure 3: cAMP suppresses TNF-α-induced cell death in L929 cells via CREB-mediated transcription. A, L929 cells were treated with forskolin (10 μM), PGE2 (10 μM), epinephrine (100 μM), or 6-MB-cAMP (100 μM) for 30 min. Phosphorylation of CREB and expression of CREB and actin were analyzed by immunoblotting (IB). B, L929 cells were treated with or without actinomycin D (ActD, 1 μg/ml, 30 min) prior to forskolin treatment (10 μM, 30 min), followed by stimulation with or without 2 ng/ml TNF-α for 12 h. Cell death was measured by PI staining. The percentages of cell death are shown in the right panel as mean ± SD; n = 3. Left panel is representative of three independent experiments. C, L929 cells were transfected with CREB siRNAs or the negative control (NC) siRNA (200 nM each). After 72 h, cells were stimulated with or without forskolin (10 μM, 30 min). Phosphorylation of CREB and expression of CREB and actin were measured by immunoblotting. D, L929 cells were transfected with CREB siRNAs or the negative control siRNA (200 nM each). After 48 h, cells were pretreated with or without forskolin (10 μM, 30 min), followed by stimulation with or without 10 ng/ml TNF-α for 24 h. Cell death was monitored by PI staining. The percentages of cell death are shown in the right panel as mean ± SD; n = 3. Left panel is representative of three independent experiments.
Mentions: Our previous data suggest that cAMP regulates TNF-α-induced cell death in fibroblasts via CREB-mediated transcription [14]. Consistent with this notion, forskolin-, PGE2-, epinephrine-, and 6-MB-cAMP-induced CREB phosphorylation at Ser133 (Figure 3A), which is required for CREB activation [31-33], corresponded to the extent these cAMP elevators activated intracellular cAMP (Figure 2B) and suppressed TNF-α-induced cell death in L929 cells (Figure 2A). To further analyze the mechanism by which cAMP suppresses TNF-α-induced cell death in L929 cells, the cells were pretreated with or without forskolin, followed by treatment with TNF-α in the presence or absence of the RNA synthesis inhibitor actinomycin D. Because 10 ng/ml TNF-α rapidly induced more than 70% cell death in the presence of actinomycin D (data not shown), the concentration of TNF-α was titrated down. 2 ng/ml TNF-α treatment for 12 h led to only 7% cell death in the absence of actinomycin D, which was significantly suppressed by forskolin (Figure 3B). However, in the presence of actinomycin D the same dose of TNF-α resulted in about 35% cell death, though actinomycin D itself showed no detectable effect on the survival of L929 cells (Figure 3B). These data are consistent with the previous finding in the literature that blockade of de novo protein synthesis significantly enhances TNF-α-induced cell death. [34,35] The suppression of TNF-α-induced cell death by forskolin was abolished by actinomycin D (Figure 3B). These data suggest that cAMP suppresses TNF-α-induced cell death in L929 cells in a transcription-dependent manner.

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