Concanavalin A—induced liver cell damage: Activation of intracellular pathways triggered by tumor necrosis factor in mice☆☆☆

AbstractBackground & Aims: Concanavalin A (con A) induces tumor necrosis factor (TNF)-dependent hepatocyte apoptosis resembling immune-mediated fulminant hepatic failure in humans. Intracellular pathways originating at the TNF receptor are either linked to apoptosis, nuclear factor (NF)-κB translocation, or Jun kinase (JNK) activation. The aim of this study was to study TNF-dependent pathways after con A injection in vivo. Methods: Con A, con A plus anti-TNF, and control buffer were injected into BALB/c mice. Immunofluorescence, Western blot, Northern blot, gel shift, Erk, and JNK activity and DNA fragmentation experiments were performed at different time points after injection. Results: DNA fragmentation in hepatocytes was increased 4–24 hours after con A injection. JNK was activated maximally (>20-fold) directly after con A injection, whereas binding and nuclear translocation of NF-κB was maximal after 4 hours. All pathways were blocked by anti-TNF. JNK activation was specific because related ERK 1 + 2 were not activated after con A. High nuclear expression of c-Jun was already evident 1 hour after con A injection; however, in contrast to JNK, anti-TNF treatment did not block c-Jun nuclear expression and DNA binding. Conclusions: In the con A model, activation of TNF-dependent pathways is associated with apoptosis of hepatocytes. Their modulation in vivo may have implications to develop new therapeutic strategies to prevent apoptosis.GASTROENTEROLOGY 1998;114:1035-104

In vivo activation of mitogen-activated protein kinases in rat intestinal neoplasia

AbstractBACKGROUND & AIMS: To investigate whether mitogen-activated protein kinase (MAPK) cascades might play a role in the progression of colon cancer, c-Jun N-terminal kinase (JNK) and extracellular signal regulating kinase (ERK) activity during colonic tumorigenesis were examined.METHODS: The 1,2-dimethylhydrazine (DMH)-induced colon carcinoma model was used to study the activation of these kinases during intestinal carcinogenesis. Male Sprague-Dawley rats were injected with DMH for 24 weeks. Normal-appearing intestinal mucosa from control and treated animals and DMH-induced intestinal tumors were assayed for JNK and ERK activity using solid phase in vitro kinase assays. Tumors were typed for mutations in the K-ras gene.RESULTS: There was little or no difference in JNK and ERK activity in hyperproliferative mucosa from DMH-treated animals compared with normal mucosa from control animals. However, in 16 colonic neoplasms, an average of 23-fold and 29-fold increases in JNK and ERK activities were observed, respectively, over control levels. In addition, activating protein-1 binding was strongly induced in the colonic tumors. Activation did not correlate with the presence of mutations in K-ras.CONCLUSIONS: Both the JNK and ERK MAPKs are highly activated during late progression of colorectal carcinoma. This change is dependent on the tumorigenic state rather than changes in proliferation.(Gastroenterology 1997 Nov;113(5):1589-98

Glutamine metabolism stimulates intestinal cell MAPKs by a cAMP-inhibitable, RAF-independent mechanism

AbstractBackground & Aims: Infectious diarrhea caused by viruses plus enterotoxigenic bacteria is often more severe than diarrhea induced by either pathogen alone. We postulated that the increased cell adenosine 3',5'-cyclic monophosphate (cAMP) concentration observed during infection by enterotoxigenic organisms retards the intestinal repair process by blocking activation of mitogen-activated protein kinases (MAPKs) in proliferating intestinal cells. Methods: We evaluated the effects of glutamine on MAPK activity, thymidine incorporation, and cell number in glutamine-starved and -sufficient rat intestinal crypt cells (IEC-6). Results: In glutamine-starved cells, 10 mmol/L glutamine in the absence of serum stimulated [3H]thymidine incorporation 8-fold. This effect was inhibited by 60% with 8-(4-chlorophenylthio) (8-CPT)-cAMP (100 μmol/L) + isobutyl methylxanthine (100 μmol/L). In cells not starved of glutamine, glutamine stimulated thymidine incorporation by 3-fold, and 8-CPT-cAMP completely blocked the mitogenic effect. Inhibition of proliferation by cAMP persisted for at least 68 hours after cAMP removal. In vitro kinase assays showed that glutamine signaling requires an intact ERK (extracellular signal–related kinase) pathway in unstarved cells. In starved cells, at least one other pathway (JNK) was activated by glutamine, and the mitogenic inhibition by 8-CPT-cAMP was incomplete. Other intestinal fuels (glucose and acetate) were not mitogenic. Conclusions: Increased levels of intracellular cAMP inhibit ERKs but only partially reduce glutamine-stimulated proliferation in enterocytes adapted to low glutamine.GASTROENTEROLOGY 2000;118:90-10