Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2.
It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.
Bhattacharya
Sujoy
S
Department of Physiology, The Univesity of Tennessee Health Science Center, Memphis, TN 38163, USA.
Ray
Ramesh M
RM
Johnson
Leonard R
LR
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015317:Biogenic Polyamines / Q000502:physiology*; D015153:Blotting, Western; D002166:Camptothecin / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000235:genetics / Q000502:physiology; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D007150:Immunohistochemistry; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology*; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000494:pharmacology
D000972:Antineoplastic Agents, Phytogenic; D015317:Biogenic Polyamines; D018076:DNA, Complementary; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha; D045304:Cytochromes c; D003513:Cycloheximide; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D020169:Caspases; D002166:Camptothecin