Christian Laugier

Prolactin up-regulates prostaglandin E2 production through increased expression of pancreatic-type phospholipase A2 (type I) and prostaglandin GH synthase 2 in uterine cells

Uterine stromal cells produce and release PGE2, both processes being regulated by hormonal factors. In this study, we examined the effect of PRL on the PGE2 production and release measured by radioimmunoassay. For this purpose, we used a rat uterine stromal cell line, UIII cells, which produce PGE2 and contain PRL receptors. The expression of sPLA2I and PGHS (PGHS1 and PGHS2), enzymes required for PGE2 production, was also estimated by immunocytochemistry and Western blotting in response to PRL. PRL (10 to 60 ng/ml) significantly increased the PGE2 release (up to 6-fold) and production, in a dose-dependent manner. Results show that PGHS1 and PGHS2 are both expressed constitutively in the uterine UIII cells, although PGHS2 is expressed at a low level. PRL did not increase PGHS1 expression, but stimulated the expression of sPLA2I and PGHS2 by 3.5- and 2.5-fold, respectively. These data show for the first time a regulation of sPLA2I and PGHS2 expression by PRL and may indicate that, in uterine cells, PRL enhances the PGE2 release and production by increasing the expression of both sPLA2I and PGHS2.

Prostaglandin E2 production by uterine stromal cell line UIII: Regulation by estradiol and evidence of an ethanol action

We have recently established a uterine stromal cell line (UIII). The purpose of the present study was to determine whether these cells have retained the ability to produce and release prostaglandins after several passages and whether this production was regulated. UIII cells, grown in basal conditions, released a very low amount (40.6 +/- 2.9 pg/24h/10(6) cells) of prostaglandin E2 (PGE2) though cellular content was more elevated (192 +/- 23 pg/10(6) cells). Ethanol increased the cellular content but decreased the release of PGE2, whereas estradiol 17 beta (E2) increased it in a dose-dependent manner, but had no effect on the cellular content. The PGE2 release by cells grown in medium containing 10 microM arachidonate (AA) reached 1.39 +/- 0.05 ng/24h/10(6) cells, and was further increased to 2.1 +/- 0.1 ng/24 h/10(6) cells by the addition of ethanol. Under the latter condition, E2 was ineffective. This study also showed that UIII cells expressed an immunoreactive pancreatic type 14 kD PLA2. A substantial increased 14 kD PLA2 expression was observed in ethanol-treated cells, suggesting that ethanol-effect on prostaglandin production might be partly mediated by PLA2 increase. Medium supplementation with arachidonate also resulted in a significant increase of intracellular 14 kD PLA2 expression. The present results showed that uterine stromal UIII cells have retained the enzymatic machinery to produce PGE2. Moreover these data demonstrate that ethanol and E2 affect differently uterine PGE2 production.

Phospholipase A2 inhibitors regulate the proliferation of normal uterine cells

The effects of inhibitors of phospholipase A2, cyclooxygenase, lipoxygenase and cytochrome P450 activity on the proliferation of normal rat uterine stromal cells (UIII) were studied. At non-cytotoxic doses, inhibitors of cyclooxygenase, lipoxygenase and cytochrome P450 activity had no effect; UIII cells did not lose their ability to synthesize and secrete arachidonic acid metabolites, mainly prostaglandin I2 and prostaglandin E2, after successive passages. Inhibition of prostaglandin production did not affect their proliferation. In contrast, phospholipase A2 inhibitors significantly reduced UIII cell proliferation in a reversible and dose-dependent manner. Aristolochic acid was the most potent inhibitor with an IC50 of 0.3 mumol/l on day 7 of culture. Moreover, low doses of arachidonic acid stimulated UIII cell proliferation. Thus the proliferation of normal uterine stromal cells appears to be independent of arachidonic acid oxygenated metabolites, contrary to what is observed in tumor cells, but requires an intact phospholipase A2 pathway.

Heterogeneity of progesterone receptor expression in epithelial cells of immature and differentiating quail oviduct

The localization of progesterone receptor (PR) in the quail oviduct was investigated before and after the onset of sexual maturation using an immunohistochemical technique. PR was revealed exclusively in nuclei of target cells whatever the hormonal state of the tissue (immature or not, pretreated or not with progesterone).

In vivo inhibition of basal and estrogen-induced phospholipase A2 activities by the triphenylethylene antiestrogen, tamoxifen, in immature quail oviduct

The effects of tamoxifen on oviductal phospholipase A2 activity were studied in immature quails. Injected alone, from 0.1 to 10 mg/kg tamoxifen significantly reduced basal phospholipase A2 activity 6 h after the injection, independently of the dose used. At 24 h, maximal inhibition (-50%) was observed with 0.1 mg/kg tamoxifen, while higher doses were less effective. Combined with estradiol benzoate, tamoxifen reduced even below the control value (1 mg/kg for 24 h) the increase in phospholipase A2 activity induced by estrogen.

The level of pancreatic PLA2 receptor is closely associated with the proliferative state of rat uterine stromal cells

Rat uterine stromal cells (UIII) express pancreatic type PLA2 (PLA2‐I) receptor and internalize the enzyme bound to receptors. Here, we investigate the proliferating effect and alterations in binding of PLA2‐I. There is a dramatic decline in PLA2‐I binding in UIII cells as they progress from a nonconfluent proliferating state (40,000 sites/cell) to a confluent state (1300 sites/cell). Intracellular concentration of PLA2‐I changed with the alteration in binding, suggesting that regulation in the PLA2 binding capacity may have important implications in growth control mechanisms.

Opposite regulation of cAMP concentration in the quail oviduct and the mouse uterus by tamoxifen. Correlation with estrogen-antagonist and estrogen-agonist activity.

The ability of estradiol and tamoxifen to regulate cAMP levels and cAMP phosphodiesterase activities has been determined in the quail oviduct and in the mouse uterus. In the quail, tamoxifen (1 mg/kg daily for 3 days) had no effect on oviducal growth but significantly increased cAMP concentration (+49%). Injected concurrently with estradiol, tamoxifen completely inhibited oviduct growth as well as the increase of cAMP phosphodiesterase activity induced by the hormone alone and increased cAMP concentration (+229% over estradiol treated group). In the mouse, estradiol and tamoxifen displayed uterotrophic activity and increased cAMP phosphodiesterase activity. In both groups, cAMP concentration was greatly reduced (-76% in estradiol treated group; -86% in tamoxifen treated group). The opposite regulation of cAMP levels in the quail oviduct and the mouse uterus by tamoxifen reflected large differences in the contribution of calmodulin-dependent and -independent forms of phosphodiesterase to the hydrolysis of cAMP in the two models and the fact that tamoxifen stimulated the activity of the calmodulin-independent isoenzyme, while it competitively inhibited the activation of the calmodulin-dependent isoenzyme by calmodulin. Several lines of evidence strongly suggest that the regulation of cAMP levels is involved in growth-inhibiting or growth-promoting activity of tamoxifen.

Modulation of quail oviduct adenylate cyclase activity by estradiol and progesterone

Oviduct adenylate cyclase activity of the quail was measured by radiochemical analysis following different hormonal treatments. A single injection of estradiol benzoate (EB) to immature female quails resulted in a prereplicative surge of adenylate cyclase activity. A second surge of enzyme activity was observed during the proliferative phase induced by EB. Estradiol-17 alpha, estrone, estriol and testosterone were ineffective. Tamoxifen completely inhibits the growth-promoting effect of EB and the second surge of adenylate cyclase activity but does not inhibit the prereplicative increase of enzyme activity. This prereplicative increase of adenylate cyclase activity was also observed, even in the absence of increased plasma estradiol, when estradiol-17 beta (E2) was perfused through the hepatic portal vein. Moreover, E2 had no effect on enzyme activity when added directly to the oviduct homogenate preparation, at concentrations ranging from 10(-9) to 10(-7) M. In response to progesterone injection, oviduct adenylate cyclase activity followed a different pattern, beginning its increase after 3 h and remaining elevated up to 24 h. The activation by estradiol was independent of the presence of guanylylimidodiphosphate. Moreover, the enzyme was more sensitive to forskolin at submaximal concentration in estradiol treated birds than in control. These results demonstrate that transient activation of adenylate cyclase at the early stages of the action of estradiol does not occur through the classic nuclear receptor-gene activation pathway or a membrane receptor mediated process, but involves an indirect pathway, yet to be defined.

Growth inhibition and the regulation of cyclic AMP by the triphenylethylene anti-estrogen tamoxifen in the quail oviduct

Summary— The aim of the present study was to investigate the regulation of cAMP by tamoxifen in quail oviduct. A single injection of tamoxifen to immature female quails induced a transient activation of adenylate cyclase. Enzyme activity began to increase 3 h after the injection, peaked at 6 h and then dropped to control level at 12 h. The same time‐response curves were observed following the injection of estradiol benzoate or estradiol benzoate + tamoxifen. Moreover, adenylcyclase exhibited the same sensitivity to exogenous activators (guanylylimidodiphosphate and forskolin) in the different treated groups. Phosphodiesterase activity was left unchanged during the prereplicative period and cAMP concentration was significantly increased at 6 h (+ 44.3%). Then, cAMP concentration continued to increase (+ 73.8% at 24 h) while cAMP phosphodi esterase and adenylcyclase activities remained at control levels. Injected concurrently with estradiol benzoate, tamoxifen completely inhibited the growth promoting effect of estradiol. Tamoxifen also inhibited the activation of adenylcyclase and cAMP phosphodiesterase induced by the hormone alone during the proliferative phase of the tissue. Moreover, the combined treatment led to a sustained elevation of cAMP in the oviduct, whereas estradiol benzoate alone decreased the level of cAMP. These results and those of our previous studies showing a significant correlation between the growth inhibitory potency of triphenylethylene derivatives in vivo and their efficiency to inhibit calmodulin‐dependent cAMP phosphodiesterase in vitro, strongly suggest that the differential regulation of cAMP levels by estradiol and tamoxifen is essential for the growth promoting or growth inhibiting activities of these molecules.

Dissociated effects of tamoxifen on growth and on progesterone receptor induction in quail oviduct

The estrogen agonist and antagonist activities of tamoxifen on growth and progesterone receptor induction were studied in the immature quail oviduct. Tamoxifen alone, when administered for 3 days at doses ranging from 0.01-10 mg/kg, had no effect on oviducal weight, DNA, and protein content, but significantly increased progesterone receptor concentration. When combined with estradiol benzoate (0.1 mg/kg daily for 3 days), tamoxifen completely inhibited the trophic action of estradiol while it only reduced the progesterone receptor concentration in the oviduct by 50%. This latter effect reflected more a reduction in the progesterone-responsive cell population of the tissue rather than a true estrogen antagonist effect on this specific protein induction. These results and previous data from this laboratory support the conclusions that in this model system: (1) the mechanisms involved in the induction of estrogen-sensitive cell proliferation and progesterone receptor synthesis are independent, and (2) the estrogen-antagonist activity of tamoxifen on cell proliferation is mediated through an estrogen receptor-independent pathway. Thus, the immature quail oviduct model system is particularly relevant to more detailed studies on the molecular modes of action of synthetic antiestrogens.