title: Regulation of JNK activity in the apoptotic response of intestinal epithelial cells.
abstract: We have studied apoptosis of gastrointestinal epithelial cells by examining the receptor-mediated and DNA damage-induced pathways using TNF-α and camptothecin (CPT), respectively. TNF-α requires inhibition of antiapoptotic protein synthesis by cycloheximide (CHX). CHX also results in high levels of active JNK, which are necessary for TNF-induced apoptosis. While CPT induces apoptosis, the increase in JNK activity was not proportional to the degree of apoptosis. Thus the mechanism of activation of JNK and its role in apoptosis are unclear. We examined the course of JNK activation in response to a combination of TNF-α and CPT (TNF + CPT), which resulted in a three- to fourfold increase in apoptosis compared with CPT alone, indicating an amplification of apoptotic signaling pathways. TNF + CPT caused apoptosis by activating JNK, p38, and caspases-8, -9, and -3. TNF-α stimulated a transient phosphorylation of JNK1/2 and ERK1/2 at 15 min, which returned to basal by 60 min and remained low for 4 h. CPT increased JNK1/2 activity between 3 and 4 h. TNF + CPT caused a sustained and robust JNK1/2 and ERK1/2 phosphorylation by 2 h, which remained high at 4 h, suggesting involvement of MEKK4/7 and MEK1, respectively. When administered with TNF + CPT, SP-600125, a specific inhibitor of MEKK4/7, completely inhibited JNK1/2 and decreased apoptosis. However, administration of SP-600125 at 1 h after TNF + CPT failed to prevent JNK1/2 phosphorylation, and the protective effect of SP-600125 on apoptosis was abolished. These results indicate that the persistent activation of JNK might be due to inhibition of JNK-specific MAPK phosphatase 1 (MKP1). Small interfering RNA-mediated knockdown of MKP1 enhanced TNF + CPT-induced activity of JNK1/2 and caspases-9 and -3. Taken together, these results suggest that MKP1 activity determines the duration of JNK1/2 and p38 activation and, thereby, apoptosis in response to TNF + CPT.
authors.affiliation: Dept. of Physiology, Univ. of Tennessee Health Science Center, Memphis, 38163, USA. rray3@uthsc.edu
authors.forename: Ramesh M
authors.initials: RM
authors.lastname: Ray
authors.forename: Shi
authors.initials: S
authors.lastname: Jin
authors.forename: Mitulkumar N
authors.initials: MN
authors.lastname: Bavaria
authors.forename: Leonard R
authors.initials: LR
authors.lastname: Johnson
mesh_terms: D000873:Anthracenes / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002166:Camptothecin / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000201:enzymology / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology / Q000502:physiology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D048768:MAP Kinase Kinase Kinase 4 / Q000037:antagonists & inhibitors / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D000873:Anthracenes; D000972:Antineoplastic Agents, Phytogenic; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C432165:pyrazolanthrone; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048051:p38 Mitogen-Activated Protein Kinases; D048768:MAP Kinase Kinase Kinase 4; C482779:MAP3K4 protein, human; D020169:Caspases; D002166:Camptothecin