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O. ORTMANN, H. WIESE and G. EMONS

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C Grundker, L Schlotawa, V Viereck and G Emons

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. DESIGN: 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).

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G. Emons, O. Ortmann, U. Fingscheidt, P. Ball and R. Knuppen

Abstract. Dispersed pituitary cells from adult female rats were preincubated for different time periods (0– 12 h) in the absence or presence of 10−9 moestradiol (E2) or 4-hydroxyoestradiol (4-OHE2). Then the media were changed and the cells incubated for 4 h with either vehicle, or E2, or 4-OHE2 and additionally with different concentrations (10−11– 10−7 m) of gonadotrophin-releasing hormone (GnRH). Treatment of pituitary cells with E2 for 4 h (i.e. no preincubation with E2) significantly decreased the LH-response to GnRH at concentrations ≥ 10−10 m of the decapeptide. During a transition time of approximately 10 h (i.e. in cultures preincubated with E2 or vehicle for 2, 4, 6 or 8 h and then coincubated with E2 or vehicle and GnRH for 4 h) no differences between E2-and vehicle-treated cultures were observed. After 14 and 16 h of E2-treatment (i.e. 10 or 12 h preincubation and 4 h coincubation with GnRH) the LH-responses to GnRH in these cultures were significantly higher than in the respective controls. A nearly identical reaction pattern was observed when 4-OHE2 was used instead of E2.

In a second series of experiments dispersed rat pituitary cells were suspended in a carrier gel and continuously perifused with medium, using small chromatography columns. When these cells were exposed for 4 min to 10−9 m GnRH at 60 or 48 min intervals, they reacted with reproducible pulsatile LH-discharges during at least 6 subsequent stimuli with the decapeptide. When E2 (10−9 m) was added to the perifusion medium, the LH-responses to GnRH were significantly reduced, starting 36 min after the onset of E2-treatment. These data indicate: 1) In the rat, the negative oestrogen effect is at least in part directly mediated at the pituitary level. 2) The sensitizing effect of oestrogens on rat gonadotrophs to GnRH is significant already after 14 to 16 h. 3) E2 and the catecholoestrogen 4-OHE2 have the same effects in this system. 4) The negative E2-effect on GnRH-induced LH-release is significant after only 36 min, a finding bringing up the question of a non-genomic mechanism.

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G. EMONS, U. FINGSCHEIDT, O. ORTMANN, R. STURM and R. KNUPPEN

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G. Emons, R. Knuppen, P. Ball and K.J. Catt

Abstract. The sensitizing effect of oestrogens on GnRH-stimulated LH release was evaluated in pituitary cells from adult female rats, cultured for 2 days in the presence of 10−13 to 10−6 m concentrations of oestradiol and selected A- and D-ring modified oestrogens. With almost all steroids tested, bell-shaped dose-response curves with comparable LH-maxima but different EDmax values were obtained for the LH response to a submaximal GnRH stimulus (5 × 10−10 m). Maximal LH response to 5 × 10−10 m GnRH were found at the following oestrogen concentrations: oestradiol and 4-hydroxyoestradiol = 10−11 m; 2-methyloestradiol = 10−9 m; 2-hydroxyoestradiol = 10−8 m; with 4-methyloestradiol no significant maximum was observed. When cells were pretreated with 10−13, 10−11 and 10−9 m oestradiol, or 4-hydroxyoestradiol, or 2-hydroxyoestradiol, and exposed to increasing concentrations of GnRH (10−11 to 10−7 m), an almost 10-fold decrease in the ED50 for GnRH was observed after pretreatment with 10−11 m oestradiol and 4-hydroxyoestradiol. With 2-hydroxyoestradiol at this concentration, the sensitizing effect was much less pronounced. Increasing the steroid concentration to 10−9 m slightly decreased the effect of oestradiol and 4-hydroxyoestradiol, whereas it increased the effect of 2-hydroxyoestradiol. Thus, at the target cell 4-hydroxyoestradiol has the same potency as oestradiol, while 2-hydroxyoestradiol is significantly less active. The sensitizing effect of oestradiol is only slightly decreased by the presence of a methyl group in position 2, but is markedly reduced by a methyl group in position 4. Our results also demonstrate the high sensitivity of the pituitary to oestrogen-induced enhancement of GnRH-stimulated gonadotrophin release, as well as the decrease of the positive effect at high oestrogen concentrations. The bell-shaped dose-response curves for oestrogen action should be taken into account when evaluating the effects of oestrogens and their derivatives upon gonadotrophin release from the pituitary gland.

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O. ORTMANN, G. EMONS, R. KNUPPEN and K.J. CATT

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G. EMONS, O. ORTMANN, U. FINGSCHEIDT, P. BALL and R. KNUPPEN

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G. Emons, H. Schuppe, M. Peter, C. Brack and P. Ball

Abstract. Ovariectomized ewes were infused for different times (2–24 h) during the breeding season (September to Februray) with oestradiol (E2, 2 μg/h) or the catecholoestrogen 4-hydroxyoestradiol (4-OHE2, 10 μg/h). At these infusion rates comparable plasma levels of E2 and 4-OHE2 were obtained when steady state was reached after 3 h (E2: 20 ± 4 pg/ml; 4-OHE2: 22 ± 3 pg/ml). When E2 was infused for at least 6 h, all animals had significant LH-surges, starting 14-16 h after the beginning of oestrogen treatment, even when E2 was infused for up to 24 h. 4-OHE2, however, had only to be infused for 4 h to induce significant LH-surges in all animals tested. When E2 was infused for 12 h at a rate of 100 μg/h, the LH-surges in these ewes were significantly lower than the LH-surges in the same animals treated for 12 h with E2at a rate of 2 μg/h.

These data indicate:

  1. Once E2 has been administered at a specific infusion rate for a critical time period of 6 h, LH-surges occur, no matter whether the E2-infusion is continued or stopped.
  2. For the catecholoestrogen 4-OHE2 this critical time period amounts only to 4 h, if comparable plasma levels of either oestrogen are achieved. This might hint at a prolonged intracellular action of 4-OHE2 as compared to E2.
  3. At extremely high infusion rates of E2 (100 μg/h for 12 h) the positive oestrogen effect is significantly impaired, a finding supporting the concept of a bell-shaped dose-response relationship between oestrogens and their positive effect on LH-secretion.