Effects of geldanamycin on signaling through activator-protein 1 in hypoxic HT29 human colon adenocarcinoma cells.
One of the characteristic responses of HT29 human colon adenocarcinoma cells to hypoxic stress is the induction of c-jun expression and binding to the activator-protein 1 (AP-1) element. To study the mechanism of c-jun activation during hypoxia, inhibitors of signaling pathways leading to the activation of AP-1 transcription factor were used. One of them, the benzoquinone ansamycin geldanamycin (GA) Mr-90,000 heat-shock protein (hsp90)-binding antibiotic, is known to disrupt signaling pathways by inducing destabilization of the enzyme complexes and degradation of signaling intermediates involving the proteasome. In our experiments, GA inhibited both basal and hypoxia-induced c-jun expression (IC50 = 75 nM). GA also abolished the hypoxia-induced increase in c-Jun NH2-terminal kinase (JNK1) catalytic activity and demonstrated an inhibitory effect on stress-activated protein kinase/ERK kinase-1 (SEK1); other participants in the mitogen-activated protein kinase and p38 signal transduction pathways were not affected to the same degree. GA treatment led to a decrease in the nuclear content of c-Jun but not that of c-Fos or of activating transcription factor 2. Functional consequences of these effects were suggested by the inhibition of AP-1 binding in hypoxic HT29 cells in the presence of GA. Pretreatment with the proteasome inhibitor lactacystin before the addition of GA resulted in the elevation of overall c-jun level, but it was unable to restore the hypoxia-induced c-jun expression. Our results demonstrate that GA acts as a highly potent inhibitor of hypoxia-induced c-jun expression, affecting the activation of JNK and of the AP-1 transcription factor. However, the effect of GA cannot be attributed solely to the inhibition of signaling through JNK, and additional mechanisms remain to be identified.
Vasilevskaya
I A
IA
Department of Medicine, University of Pennsylvania, Philadelphia 19104, USA. vasilevs@mail.med.upenn.edu
O'Dwyer
P J
PJ
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D016227:Benzoquinones; D015687:Cell Hypoxia; D019073:HT29 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D047029:Lactams, Macrocyclic; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D011494:Protein Kinases / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology*; D015290:Second Messenger Systems; D015398:Signal Transduction / Q000187:drug effects*; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D000903:Antibiotics, Antineoplastic; D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D011809:Quinones; D018808:Transcription Factor AP-1; D011494:Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; C001277:geldanamycin