Biomedical subjects

J M Fayard

Publications and source records attributed to J M Fayard.

At least 19 recordsLinked to original sources

PubMed2001

Control of cell proliferation via transduction of sPLA(2)-I activity and possible PPAR activation at the nuclear level.

Pancreatic phospholipase A2 (PLA(2)-I) stimulates U(III) cells proliferation, a rat uterine cell line, after binding to membrane receptors, internalization and translocation. Here, we demonstrate that during these steps of internalization, PLA(2)-I retains its hydrolytic activity and thus could exert its proliferative effect via nuclear phospholipids hydrolysis. Since fatty acids and eicosanoids released by such activity are known to be ligands of PPAR, we study the expression of these nuclear receptors and demonstrate that, in the experimental conditions where PLA(2)-I stimulates U(III) cells proliferation, PLA(2)-I also regulates PPAR expression indicating a possible mechanism of its proliferative effect.

Animals↗

PubMed1998

Protein kinase C inhibitors stimulate arachidonic and docosahexaenoic acids release from uterine stromal cells through a Ca2+-independent pathway.

The mechanisms underlying arachidonic acid (AA) release by uterine stromal (U(III)) cells were studied. Stimulation of AA release by calcium ionophore and PMA are inhibited by various PKC inhibitors and by calcium deprivation. These results suggest the involvement of an AA-specific cPLA2 as the release of docosahexaenoic acid (DHA) from prelabelled cells is much lower than the release of AA. The results also show a more original stimulation of AA and DHA release induced by PKC inhibitors, which is insensitive to calcium deprivation. This stimulation is not due to acyltransferase inhibition, suggesting the participation of a Ca2+-independent PLA2 (iPLA2). However, iPLA2 activity measured in U(III) cells is inhibited by the specific iPLA2 inhibitor, BEL, and is not stimulated by PKC inhibitors, in contrast with the AA and DHA release. It seems therefore that this iPLA2 cannot be involved in this mechanism. The participation of another iPLA2, BEL-insensitive, is discussed.

Acyltransferases↗

PubMed1998

Nuclear location of PLA2-I in proliferative cells.

We have previously demonstrated that pancreatic PLA2 (PLA2-I) stimulates the proliferation of UIII cells, a stromal cell line derived from normal rat uterus. In order to gain further insight into the mechanism of action of PLA2-I, we have investigated the intracellular processing of PLA2-I. Either highly proliferative or growth arrested UIII cells were analyzed. Growth arrested cells were obtained from a contact inhibited monolayer or from aristolochic acid-treated cultures. Using cellular fractionation, western blotting, immunocytochemistry and confocal microscopy, we demonstrate that endogenous PLA2-I was mainly located in the nucleus in highly proliferative cells whereas its location was cytoplasmic in non proliferative cells. When non confluent UIII cells were incubated with nanomolar amounts of exogenous PLA2-I, the enzyme was internalized and, in the majority of cells, appeared within the nucleus. Both internalization and nuclear location of exogenous PLA2-I were suppressed by the addition of aristolochic acid to the culture medium. Binding experiments performed on purified nuclear preparations showed the presence of specific cooperative binding sites for PLA2-I. Collectively our data suggest that the proliferative effect exerted by pancreatic PLA2 in UIII cells is mediated by a direct interaction of the enzyme at the nuclear level. Putative mechanisms and targets are discussed.

Animals↗

PubMed1996

Arachidonic acid up-regulates and prostaglandin E2 down-regulates the expression of pancreatic-type phospholipase A2 and prostaglandin-endoperoxide synthase 2 in uterine stromal cells.

It is well known that arachidonic acid, as a substrate of prostaglandin G/H synthase (PGHS), is converted into prostaglandins of the two-series. In this work, we attempted to determine whether arachidonic acid and prostaglandin E2 might regulate the expression of PGHS and the pancreatic-type phospholipase A2 (PLA2I), which may be involved in the liberation of arachidonic acid from membrane phospholipids. For this purpose, we used the uterine stromal cell line UIII, which produces prostaglandin E2 and expresses both the constitutive and inducible PGHS enzymes (PGHS1 and PGHS2) and PLA2 I. The results show that PGHS1, which is expressed at a high level in UIII cells, was not modified by arachidonic acid. The expression of PGHS2 and PLA2 I was up-regulated by increasing arachidonate concentrations (1-10 microM). The maximal response was obtained at 24 h, reaching a 2.3-fold and 2.6-fold increase for PGHS2 and PLA2 I expression, respectively, compared to the control level. To discriminate between the effect of arachidonic acid and that of prostaglandins, which are highly increased in the presence of exogenous arachidonic acid, we treated the cells with two inhibitors of PGHS activity, aspirin and meclofenamic acid. Both inhibitors failed to suppress the arachidonate-induced increase of PLA2 I and PGHS2 expression and even enhanced it either in the presence or absence of arachidonic acid. In contrast, the addition of prostaglandin E2 to the culture medium decreased the expression of both enzymes in a dose-dependent manner, the maximal response being reached at 1 microM. We conclude that arachidonic acid up-regulates the expression of PLA2 I and PGHS2 in the uterine stromal cells, independently of prostanoids, and that prostaglandin E2 is capable of down-regulating enzyme expression.

Animals↗

PubMed1996

Prolactin up-regulates prostaglandin E2 production through increased expression of pancreatic-type phospholipase A2 (type I) and prostaglandin G/H 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.

Animals↗

PubMed1996

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

Rat uterine stromal cells (U(III)) 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 U(III) cells as they progress from a non-confluent 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.

Animals↗

PubMed1996

Binding and internalization of extracellular type-I phospholipase A2 in uterine stromal cells.

The cellular uptake of extracellular type-I phospholipase A2 (PLA2) was investigated in rat uterine stromal cells (UIII) in culture, which were found to express the high-affinity binding site for mammalian type-I PLA2, with a measured KD of 6.4 nM, a Bmax of 0.1-1 pmol/mg of DNA at 4 degrees C, and a molecular mass of about 200 kDa. When UIII cells were treated with type-I PLA2 at 37 degrees C, the ligand specifically associated with the cells increased, reaching a plateau after 90 min of incubation, whose level was about 5-fold higher than that measured if cells were maintained at 4 degrees C. We could determine that the PLA2 was bound to plasma membrane receptors which were responsible for internalization of the ligand, and that the binding sites were still suitable for binding at the level of plasma membrane during UIII cell incubation at 37 degrees C. Proteolysis of internalized PLA2 could be clearly detected only after 90 min of UIII cell incubation with the ligand at 37 degrees C, and most of the intracellular PLA2 consisted of the apparently intact 14 kDa enzyme. By cross-linking studies, we found that most of the internalized PLA2 was not associated with the receptor, supporting the conclusion that in our experimental system a single pool of membrane receptors for mammalian type-I PLA2 undergoes cycles of ligand binding, intracellular transfer and release of PLA2, followed by restoration of binding sites on the plasma membrane. We calculated that the rate of internalization of the ligand by one receptor molecule in UIII cells at 37 degrees C is about three molecules of type-I PLA2 per h.

Animals↗

PubMed1996

Biogenesis and metabolic fate of docosahexaenoic and arachidonic acids in rat uterine stromal cells in culture.

To gain some insight into the mechanisms involved in the opposing effects of arachidonic acid and docosahexaenoic acid on the growth of rat uterine stromal cells (UIII cells), the dynamics of the uptake, conversion, and incorporation of labeled 18:2(n-6), 18:3(n-3), 20:4(n-6), 20:5(n-3), and 22:6(n-3) into lipid pools and phospholipid subclasses were examined. A very active and time-dependent conversion of [14C]18:3(n-3) to higher homologs was observed; 64.7 +/- 0.7 and 11.5 +/- 0.4% of the [14C] radioactivity incorporated in cellular lipids was recovered as 22:5(n-3) and 22:6(n-3) after 72 h incubation, respectively. The distribution of labeled fatty acids obtained after 72 h incubation with [3H]20:5(n-3) was not significantly different from that observed with 18:3(n-3). Arachidonic acid was the major fatty acid formed from [14C]18:2(n-6) and only trace amounts of 22:5(n-6) were detected. When cells were incubated for 72 h with 20:4(n-6), more than 75% of the radioactivity was recovered as arachidonate and slightly higher amounts of 22:4(n-6) and 22:5(n-6) were formed compared to those obtained after incubation with 18:2(n-6). Using both [14C]- and [3H]22:6(n-3), no significant retroconversion of labeled 22:6(n-3) occurred in the cells. More than 90% of labeled 20:4(n-6) and 22:6(n-3) taken up by the cells were esterified into phospholipids, but significant differences in their distribution among phospholipid classes and subclasses were observed. Docosahexaenoic acid was more rapidly and efficiently incorporated into phosphatidylethanolamine than 20:4(n-6) and was principally recovered in plasmalogens. Arachidonic acid was mainly incorporated in the diacyl subclasses of phosphatidylcholine and phosphatidylethanolamine and in phosphatidylinositol. The divergent profiles of these two fatty acids within the phospholipid compartments provide some information for the mechanisms of their opposite effects on UIII cell growth.

Animals↗

PubMed1995

Docosahexaenoic acid is a potent inhibitor of rat uterine stromal cell proliferation.

The effect of different families of fatty acids on the proliferation of rat uterine stromal cells (UIII) was studied. Docosahexaenoic acid (DHA) exerted a strong and dose-dependent inhibitory effect (IC50 approximately 2 microM), whereas arachidonic acid (AA) stimulated UIII cell proliferation at the optimal concentration of 10 microM. Oleic, linoleic and linolenic acids were ineffective from 0.1 to 10 microM. The inhibitory effect of DHA was independent of the eicosanoid biosynthesis and lipid peroxidation, since it was not reversed by the addition of the antioxidant BHT and no significant production of oxidized species from DHA occurred in our culture conditions.

Animals↗

PubMed1994

Oestradiol-induced changes in the composition of phospholipid classes of quail oviduct: specific replacement of arachidonic acid by docosahexaenoic acid in alkenylacyl-glycerophosphoethanolamine.

The phospholipid composition and the molecular species of the major subclasses of ethanolamine and choline glycerophospholipids were determined during the natural or oestradiol-induced development of the quail oviduct. The phospholipid concentration increased significantly during oviduct development, and the proportion of ethanolamine glycerophospholipids (EPL) remained constant while that of choline glycerophospholipids increased. The immature oviduct contained the majority of its endogenous arachidonic acid mass (71%) in EPL, mainly in alkenylacyl-glycerophosphoethanolamine (alkenylacyl-GPE) (49% of the total). Oestrogen treatment induced the depletion of 20:4,n-6 specifically from this pool, which indicates the biological importance of 20:4,n-6 molecular species in alkenylacyl-GPE as substrates for the oviduct phospholipases activated by oestradiol, and suggests that this EPL subclass is involved in the oestrogen-induced cell proliferation. Another striking result was the marked increase in 22:6,n-3 EPL molecular species following the oestradiol treatment and more particularly the strict substitution of 20:4,n-6 by 22:6,n-3 in alkenylacyl-GPE. We speculate that alkenylacyl-GPE molecular species containing 22:6,n-3 may participate in the arrest of oestrogen-induced proliferation.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

Regulation of quail oviduct phospholipase A2 activity by estradiol.

The phospholipase A2 (PLA2) activity was measured in the oviduct of immature and estradiol benzoate (EB)-treated quails. The pH profiles demonstrate the presence of two PLA2 isoforms in the avian oviduct: a neutral isoform, optimally active at pH 7-7.5 and calcium independent, responsible for most of the hydrolytic activity in the immature oviduct and poorly stimulated by estradiol; and an alkaline isoform, optimally active at pH 8-9.5 and calcium dependent, with little activity in the immature tissue but markedly stimulated by EB. After EB injection, PLA2 activation occurs at first during the prereplicative period of oviduct cells (+172% at 6 h), it is dose dependent from 0.01 to 1 mg/kg EB and can be prevented by cycloheximide together with ornithine decarboxylase activation. Moreover, estradiol was inactive on cell-free extracts of immature oviducts. These results suggest that EB increases PLA2 activity through gene activation and de novo protein synthesis. The correlation between the early stimulation of PLA2 activity and the proliferation of oviduct cells is discussed.

Animals↗

PubMed1994

Fluorimetric studies of calmodulin interactions with antiestrogens.

Recent cumulative data have shown that tamoxifen and its metabolites inhibit the activation of cAMP phosphodiesterase by calmodulin (CaM). In this study, the interaction of antiestrogens with CaM was investigated using a hydrophobic fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS). Tamoxifen (TAM) enhanced the fluorescence of TNS bound to CaM and shifted the emission maximum to lower wavelengths. These effects were concentration-dependent. No change in the apparent affinity of TNS for CaM was noted in the presence of TAM. These results suggest that TAM bound to CaM at sites distinct from those of TNS and induced a change in TNS environment. Interaction of TAM metabolites with CaM depended on the degree of alteration of the dimethylaminoethoxy side-chain. Thus, N-desmethylation or N-di-desmethylation notably reduced the interaction of the drug with the macromolecule by 24 and 77% respectively. Side-chain deamination to the primary alcohol (metabolite Y) totally suppressed the interaction. The ability of these different metabolites to interact with CaM correlates with their efficiency to inhibit CaM-dependent cAMP phosphodiesterase and their growth inhibitory potency reported previously.

Calmodulin↗

PubMed1993

Normal rat uterine stromal cells in continuous culture: characterization and progestin regulation of growth.

Stromal cells were isolated from rat uterus by sequential enzymatic digestion and density fractionation on Percoll gradient and subcultured by trypsinization. Two stable subcultures, named UII and UIII, were obtained. UII cells exhibited a spindle-shaped, elongated, fibroblast-like morphology, while UIII cells were rounded and polygonal. Both cell types expressed the intermediate filament vimentin but not cytokeratin, nor desmin, suggesting that both were of stromal origin. In UIII cells, the presence of progesterone and prolactin (PRL) receptors was demonstrated by immunocytochemical and binding studies. Cross-linking and Western blotting showed that PRL receptor in UIII cells corresponded to 3 molecular forms of 54, 42 and 32 kDa. The growth properties of these cells were studied under different conditions of culture. In fetal calf serum (FCS) supplemented medium, proliferation of UIII cells was dependent on serum concentration and was not affected by estradiol and progesterone. In 10% FCS supplemented medium, the doubling time was 41.5 +/- 0.8 h. When cultured in 10% dextran-charcoal-treated FCS, cells were maintained in a viable but quiescent state. Under these conditions, progesterone was able to induce growth of these cells in a dose-dependent manner. A 3-fold increase in DNA content was measurable in 10(-7) M progesterone-treated versus control cultures after 5 days. Reduction of serum concentration from 10% to 2% abolished the effect of progesterone suggesting that this effect requires the presence of serum factor(s). In conclusion, this study showed that uterine stromal cells, in continuous culture, retained progesterone and prolactin receptors and progesterone regulation of growth.

Animals↗

PubMed1992

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.

Animals↗

PubMed1992

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.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1992

Dietary alpha-linolenic acid deficiency and early uterine development in female rats.

Feeding rats a purified diet containing peanut oil with a low alpha-linolenic acid [18:3(n-3)] content resulted in lower amounts of (n-3) polyunsaturated fatty acids, mainly docosahexaenoic acid [22:6(n-3)], greater amounts of docosapentaenoic acid [22:5(n-6)] in uterus phospholipids, and altered postnatal uterus development when compared with rats fed a diet containing peanut and rapeseed oils. Maximal differences in uterine growth, as measured by uterine weight, protein and DNA content, occurred between d 24 and 30 postpartum and disappeared near the end of sexual development (d 40). The induction of the progesterone receptor was not affected, and serum estradiol concentrations were not significantly reduced in deficient animals. Moreover, growth response of the uterus to low doses of 17 beta-estradiol (less than 5 micrograms/kg) was significantly reduced in ovariectomized animals fed the diet containing only peanut oil. However, the maximal response of the uterus, observed with higher 17 beta-estradiol doses (5-50 micrograms/kg), was not affected. Because the two diets used differed in the content of alpha-linolenic acid, it is likely that alpha-linolenic acid deficiency in animals fed the diet containing only peanut oil was the cause of the affected uterine development.

Animals↗