Protein kinase C-independent stimulation of activator protein-1 and c-Jun N-terminal kinase activity in human endometrial cancer cells by the LHRH agonist triptorelin.
OBJECTIVE
The expression of luteinizing hormone-releasing hormone (LHRH) and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumours, including cancers of the endometrium. The signalling pathway through which LHRH acts in endometrial cancer is distinct from that in pituitary gonadotrophs. The LHRH receptor interacts with the mitogenic signal transduction of growth factor receptors via activation of a phosphotyrosine phosphatase, resulting in down-regulation of cancer cell proliferation. In addition, LHRH activates nucleus factor kappaB (NFkappaB) and protects the cancer cells from apoptosis. This study was conducted to investigate additional signalling mechanisms of the LHRH receptor cooperating with NFkappaB in endometrial cancer cells.


METHODS
The LHRH agonist triptorelin-induced activator protein-1 (AP-1) activation was analysed using a pAP-1-SEAP reporter gene assay. Expression of c-jun mRNA was quantified using quantitative reverse transcription (RT)-PCR. c-Jun N-terminal kinase (JNK) activity was measured by quantification of phosphorylated c-Jun protein.


RESULTS
Treatment of Ishikawa and Hec-1A human endometrial cancer cells with 100 nM triptorelin resulted in a 3.1-fold and 3.5-fold activation of AP-1 respectively (P<0.05). If the cells had been made quiescent, treatment with triptorelin (100 nM) resulted in a 41.7-fold and 48.6-fold increase of AP-1 activation respectively (P<0.001). This effect was completely blocked by simultaneous treatment with pertussis toxin (PTX). A 17.6-fold and 17.3-fold increase of c-jun mRNA expression respectively (P<0.001) was obtained after 20 min of stimulation with triptorelin (100 nM). Treatment with 1 nM triptorelin resulted in a 12.5-fold or an 11.9-fold increase, and treatment with 10 pM triptorelin resulted in a 6.5-fold or a 5.2-fold increase of maximal c-jun mRNA expression respectively (P<0.001). Maximal c-Jun phosphorylation (68.5-fold and 60.2-fold, respectively, P<0.001) was obtained after 90 min incubation with triptorelin (100 nM).


CONCLUSIONS
These results suggest that the LHRH agonist triptorelin stimulates the activity of AP-1 in human endometrial cancer cells mediated through PTX-sensitive G-protein alphai. In addition, triptorelin activates JNK, known to activate AP-1. In earlier investigations we have shown that triptorelin does not activate phospholipase and protein kinase C (PKC) in endometrial cancer cells. In addition, it has been demonstrated that triptorelin inhibits growth factor-induced mitogen activated protein kinase (MAPK, ERK) activity. Thus triptorelin-induced activation of the JNK/AP-1 pathway in endometrial cancer cells is independent of the known AP-1 activators, PKC or MAPK (ERK).
Gründker
C
C
Department of Gynaecology and Obstetrics, Georg-August-University, D-37070 Gottingen, Germany.
Schlotawa
L
L
Viereck
V
V
Emons
G
G
D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D015153:Blotting, Western; D016889:Endometrial Neoplasms / Q000201:enzymology*; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000819:agonists*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D000073718:Pancreatitis-Associated Proteins; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013268:Stimulation, Chemical; D018808:Transcription Factor AP-1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017329:Triptorelin Pamoate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D018931:Antineoplastic Agents, Hormonal; D016328:NF-kappa B; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D017329:Triptorelin Pamoate; D007987:Gonadotropin-Releasing Hormone; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases