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


CONCLUSIONS
These data suggest that the lack of a pro-apoptotic pathway in tumor cells leads to a net survival effect of cAMP.
Wang
Jing
J
Department of Molecular Immunology, Institute of Basic Medical Sciences, Beijing, PR China.
Tang
Ruihong
R
Lv
Ming
M
Zhang
Jiyan
J
Shen
Beifen
B
D000818:Animals; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D019610:Cytoprotection / Q000187:drug effects; D004398:Dyneins / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D051379:Mice; D009336:Necrosis; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D017362:Cyclic AMP Response Element-Binding Protein; D014409:Tumor Necrosis Factor-alpha; D005576:Colforsin; D000242:Cyclic AMP; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D004398:Dyneins