Effects of an antiprogesterone agent, RU-486, on the menstrual cycle of the rhesus monkey

RU-486 (RU), a synthetic steroid with antiprogesterone receptor activity, was used to study the role of progesterone in the normal menstrual cycle of rhesus monkeys. The drug, at dosages of 2, 4, or 12 mg/kg, was administered as a single im injection during the luteal phase (days 5-8) in 16 experiments in 8 monkeys. RU had acute effects on the endometrium, as it induced menstruation within 3 days in spite of persistant progesterone elevations, thereby shortening the cycle length by about 5 days. Long term effects on menstrual cyclicity were also found with the higher RU doses. The intermenstrual interval after RU treatment increased from 31.8 +/- 2.4 (+/- SE) days after a 2 mg/kg dose of RU to 82.4 +/- 6.8 days after a 12 mg/kg dose of RU, with a control cycle length of 29.4 +/- 0.7 days. This prolonged interval was related to a delay in the completion of the follicular maturation process and, therefore, a delay in ovulation, as judged by estrogen and progesterone concentrations. Subsequent menstrual cycles were regular and ovulatory. Our results suggest that RU binds to the endometrial progesterone receptor, thereby inducing premature menstruation in the presence of elevated progesterone levels. RU also exerts long term effects on the hypothalamo-hypophyseal axis, since it significantly alters menstrual cyclicity.     

Identification of a 71 kDa protein as a putative non-genomic membrane progesterone receptor in boar spermatozoa

A putative non-genomic progesterone receptor was identified by Western blot analysis from the membrane fraction but not the cytosolic fraction of boar spermatozoa using monoclonal antibody (mAb) C-262. When the membrane and the cytosolic fractions of boar liver, kidney, uterus and spermatozoa were analyzed with mAb C-262, protein bands with molecular masses of 86 and 120 kDa were detected from the cytosolic fraction of the uterus, whereas a 71 kDa protein was detected from the membrane fraction of spermatozoa. Apparently, while the 86 and 120 kDa proteins from the uterus correspond to the genomic progesterone receptor isoforms A and B in boar, the 71 kDa protein of the sperm membrane fraction seems to be a novel membrane-associated progesterone receptor. Ligand blot assay of the membrane and the cytosolic fractions of boar spermatozoa performed with peroxidase-conjugated progesterone revealed that only the 71 kDa membrane protein binds specifically to progesterone, reinforcing the results obtained from the Western blot analysis. Also ligand blot assays performed in the presence of mAb C-262 demonstrated that mAb C-262 inhibited progesterone binding to the 71 kDa protein in a dose-dependent manner. Ligand blot assays performed in the presence of free progesterone, RU486 or estrogen revealed that binding of peroxidase-conjugated progesterone to the 71 kDa protein was inhibited by free progesterone and RU486 in a dose-dependent manner but not by estrogen, which further confirms that progesterone binds to the 71 kDa protein specifically. Furthermore, the progesterone-induced acrosome reaction was inhibited by mAb C-262 in a dose-dependent manner. These results strongly imply that spermatozoa possess a progesterone receptor in a membrane-bound form and can be influenced by progesterone via non-genomic progesterone receptor.     

Regulation of estrogen receptor alpha and progesterone receptor (isoform A and B) expression in cultured human endometrial cells.

The effects of RU 486 together with estradiol and progesterone on estrogen receptor alpha and progesterone receptor (isoforms A and B) expression were studied in human endometrial long term cultures at the mRNA and protein level. We asked whether ligand induced receptor regulation, found in mammals in vivo, is also found in human cultured endometrial cells with special regard to the progesterone isoforms A and B. Endometrial cultures were maintained for 27 days. Media were supplemented with progesterone and/or estradiol alone or in combination with RU 486. Receptor expression (estrogen receptor alpha and progesterone receptor isoform A and B) was examined at the mRNA level by RT-PCR and at the protein level by western blot analysis. All receptor types examined were expressed in our culture model. Estradiol led to a general increase of receptor expression whereas treatment with estradiol in combination with progesterone down regulated receptor expression. The receptor down regulation was not found when RU 486 was additionally supplemented into the medium. Activation or inhibition of expression due to these treatments was similar for both PR isoforms. Our results (1) show that in our culture system estradiol induced up regulation of estrogen receptor and progesterone receptor A and B and suggest that the estrogen induced up regulation is prevented by progesterone (2) a clear cut antigestagenic effect of RU 486 and (3) suggest that both progesterone isoforms are analogously regulated in our culture model. We conclude that human endometrial cell cultures are suitable for the study of the dynamics of steroid receptor expression.     

The effects of RU 486 on immune function and steroid-induced immunosuppression in vitro

The effect of RU 486 [17 beta-hydroxy-11 beta-(4-dimethylamino-phenol)17 alpha-(prop-1-ynyl)estra- 4,9diene-3-one] on [3H]thymidine incorporation into Concanavalin-A-stimulated human peripheral blood mononuclear cells and its influence on the suppressive effects of cortisol and progesterone were investigated. Cortisol suppressed lymphocyte thymidine incorporation at 10(-5), 10(-6), and 10(-7) M (17.6%, 20%, and 38% of control, respectively; P less than 0.01). Cortisol-induced suppression was reversed when low concentrations of RU 486 (10(-7) and 10(-6) M) were added. RU 486 at 10(-5) M further suppressed lymphocyte thymidine incorporation when added to cultures with cortisol. Progesterone significantly inhibited lymphocyte thymidine incorporation at 10(-5) M (8.2% of control; P less than 0.01). No reversal of progesterone-induced suppression of thymidine incorporation was seen when RU 486 was added to cultures; rather, further suppression of thymidine incorporation was seen. RU 486 alone in culture at concentrations achieved therapeutically (10(-5) M) significantly inhibited thymidine incorporation (7.2% of control; P less than 0.01). These findings suggest that RU 486 may have dose-dependent mixed agonist/antagonist effects on cortisol-induced immunosuppression. The lack of an antagonist effect of RU 486 on progesterone suggests that progesterone's immunosuppressive effects may not be receptor mediated. Finally, our findings would suggest that some immunosuppression may be seen at currently used doses of RU 486.     

Dickkopf-1, an inhibitor of Wnt signaling, is regulated by progesterone in human endometrial stromal cells

CONTEXT: Some members of the Wnt family, including ligands, receptors, inhibitors, and signaling components, are expressed in human endometrium. Dickkopf-1 (Dkk-1), a potent inhibitor of the Wnt signaling pathway, was recently found to be up-regulated in decidualizing endometrial stromal cells during the secretory phase of the menstrual cycle, suggesting regulation by progesterone. OBJECTIVES: To test the hypothesis that progesterone regulates Dkk-1 expression in human endometrial stromal cells, we investigated the following effects on stromal cell expression of Dkk-1 mRNA and protein: decidualizing stimuli (progesterone or cAMP), RU486 (an inhibitor of progesterone action), and withdrawal of progesterone. RESULTS: Short-term treatment (up to 72 h, which corresponds to the full decidualized phenotype in response to cAMP and an early response to progesterone) did not reveal regulation of Dkk-1 mRNA or protein by cAMP but did show induction of Dkk-1 expression when the cells were treated with progesterone, an effect that was blocked by RU486. In long-term cultures (from 14 to 23 d, which corresponds to the full decidualized phenotype in response to progesterone), a significant increase in Dkk-1 mRNA and protein production was observed. Addition of RU486 or withdrawal of progesterone after long-term decidualization resulted in a decrease of Dkk-1 mRNA and protein to control levels. Estradiol alone had no effect on stromal Dkk-1 expression. CONCLUSIONS: These data strongly support regulation by progesterone of Dkk-1 mRNA synthesis and protein expression in human endometrial stromal cells and that the response is specific for progesterone and independent of cAMP and estradiol.     

Divergent effects of antiprogesterone, RU 486, on progesterone, relaxin, and prolactin secretion in pregnant and hysterectomized pigs with aging corpora lutea

Porcine corpora lutea produce progesterone and relaxin during pregnancy and after hysterectomy. Peak amounts of relaxin are released into peripheral blood in both pregnant and hysterectomized animals on about day 113 (estrus = day 0 and term = 114), and this release coincides with an abrupt decrease in the progesterone concentration. RU 486, a progesterone receptor antagonist, was used to investigate the effects of interruption of progesterone binding to its receptor on luteal function and gonadotropin secretion of pigs with aging corpora lutea. RU 486 was administered orally to hysterectomized gilts (surgery on day 8) once a day (0800 h) on days 111-115 at two dosages (group 1, 2 mg/kg BW; group 2, 4 mg/kg BW). During 5 days of RU 486 treatment, plasma progesterone concentrations in both treated groups were markedly elevated (32 and 37 ng/ml for groups 1 and 2) compared with 22 ng/ml in the controls (group 3; P less than 0.01). PRL concentrations increased in both groups (9 and 13 ng/ml) and differed significantly from those of the controls (3 ng/ml) (P less than 0.04). RU 486 treatment delayed the time of relaxin peak to days 116.1 and 117.0 in groups 1 and 2 compared with day 114.1 in the controls (P less than 0.01). Pregnant gilts received RU 486 orally once a day (0800 h) at 4 mg/kg BW beginning on day 111 until parturition occurred. Parturition was induced on day 112.7 after only two RU 486 treatments compared with day 114.7 in the control group (P less than 0.01). Progesterone decreased abruptly from a pretreatment mean of 11 to less than 0.6 ng/ml during the 2 days that RU 486 was given compared with a shift from 12 to 6 ng/ml during the same period in the controls (P less than 0.01). The time of the relaxin peak was advanced to day 112.1 in RU 486-treated gilts compared with day 113.9 in the controls (P less than 0.01). Results from this study provide strong evidence that the antagonistic effect of RU 486 on progesterone receptor results in an abrupt increase in PRL and progesterone secretion in hysterectomized gilts with aging corpora lutea. In marked contrast with hysterectomized animals, the acute luteolytic effects of RU 486 depend on the presence of the uterus and/or conceptuses in the pig. Disruption of the regulatory loop of progesterone secretion by RU 486 alters the ability of corpora lutea to produce and release peak quantities of relaxin.     

Tibolone and its metabolites induce antimitogenesis in human coronary artery smooth muscle cells: role of estrogen, progesterone, and androgen receptors

Tibolone, a hormone replacement drug, protects postmenopausal women against osteoporosis and climacteric symptoms without inducing adverse effects on the endometrium and breast. Compared with other estrogens, little is known about the cardiovascular effects of tibolone. Because abnormal growth of smooth muscle cells (SMCs) is a prerequisite for coronary artery disease, here we investigated the effects of tibolone on SMC growth. We examined the effects of tibolone and its metabolites on human arterial SMC growth (DNA synthesis, cellular proliferation, cell migration, collagen synthesis) and MAPK expression. Fetal calf serum-induced SMC growth, phosphorylated MAPK expression, and platelet-derived growth factor-induced SMC-migration were concentration-dependently inhibited by tibolone and its endogenous estrogenic and progestogenic/androgenic metabolites in the following order of potency: Delta 4-tibolone>3 beta-OH-tibolone congruent with 3 alpha-OH-tibolone. The antimitogenic effects of tibolone were partially blocked by ER antagonist (ICI182780), progesterone receptor antagonist (RU486) but not by the androgen receptor antagonist (flutamide); moreover, RU486 was more potent than ICI182780. The antimitogenic effects of tibolone were completely blocked by RU486 plus ICI182780. In addition, the inhibitory effects of equimolar concentrations of the three tibolone metabolites summed up to the inhibitory effects of tibolone. In conclusion, tibolone inhibits SMC growth and MAPK phosphorylation via both its estrogenic and progestogenic metabolites, and these inhibitory effects involve both progesterone and ERs. Hence, tibolone may induce antivasoocclusive actions and protect women against coronary artery disease.     

Inhibition of proliferation and differentiation by RU 486 in human endometrial stromal and epithelial cells in vitro.

The effects of progesterone and RU 486 on cellular proliferation and differentiation in long term cultures of mixed human endometrial cells were studied. The endometrial tissue was obtained from women with normal menstrual cycles who were undergoing hysterectomy for benign growths. Estradiol supplemented cultures were treated with progesterone and/or RU 486 for 27 days. Cell number was measured by crystal violet assay, and prolactin secretion was used as a marker of differentiation. Progesterone doubled the rate of proliferation, but the addition of RU 486 reduced it to baseline again. The gestagen increased prolactin secretion up to 30 times, while the addition of RU 486 suppressed it to baseline levels. When administered to cells that were pretreated with progesterone for 15 days RU 486 abolished the progesterone effects. RU 486 alone was without any effect. Our results indicate that (1) in vitro progesterone is essential for the initiation and maintenance of proliferation and differentiation of endometrial cells and (2) RU 486 acts as a pure progesterone antagonist in our culture model.     

Progesterone and antiprogesterone (RU 38486) modulation of estrogen-inducible glycoprotein (USP-1) synthesis and secretion in rat uterine epithelial cells

Previous study in this laboratory revealed that estrogen stimulates synthesis and secretion of 97K glycoprotein [uterine secretory protein-1 (USP-1)] in rat uterine epithelial cells. In the present communication, the effects of progesterone and antiprogesterone (RU 38486) on estrogen-stimulated USP-1 biosynthesis were investigated. Ovariectomized rats were treated with estrogen for 4 days to induce USP-1 synthesis and secretion. Then, progesterone and RU 38486 were injected sc. After 6 and 12 h of treatment, uterine luminal fluid proteins were labeled for 6 h by direct administration of [35S]methionine into uterine lumen. By radioimmune precipitation assay using specific antibody against USP-1, it was shown that progesterone can reduce the USP-1 synthesis and secretion, which were prestimulated by estrogen. This antagonistic effect of progesterone on estrogen-induced USP-1 biosynthesis was also evident by the decreased staining intensity of USP-1 in uterine epithelial cells, as revealed by the immunohistochemical method. Although RU 38486 itself did not manifest any effect on estrogen-stimulated USP-1 biosynthesis, it completely inhibited the progesterone-induced decrease in synthesis and secretion. This system is believed to provide a useful model to analyze the progesterone and antiprogesterone actions on rat uterine epithelial cells.     

Endometrial and pituitary responses to the steroidal antiprogestin RU 486 in postmenopausal women

The effects of the antiprogestin RU 486 on the human endometrium were investigated. Seventeen postmenopausal women were injected with estradiol (E2) benzoate (0.625 mg/day) for 15 days. Progesterone (P) (25 mg/day) and/or RU 486 (100 or 200 mg/day) were given to groups of 2-3 women during the last 6 days of E2 benzoate treatment. Serial blood samples were drawn for the measurement of plasma E2, P, and LH and FSH. An endometrial biopsy was performed on the last day of treatment, and processed for histology or for assays of DNA polymerase alpha, E2-dehydrogenase (E2DH), and P receptor (PR). Treatment with E2 benzoate alone resulted in a marked decrease of plasma gonadotropins; in those patients who received either P, RU 486, or both, in addition to E2 benzoate, the concentrations of plasma LH and FSH were further decreased to premenopausal levels. In absence of glycerol, the affinity of RU 486 for the endometrial PR (Kd = 0.8 nM) was higher than that of P (Kd = 1.2 nM). Glycerol decreased markedly the affinity of RU 486, whereas the affinity of P for the PR was unchanged. RU 486 had negligible affinity for plasma transcortin. Either P or RU 486, but not both together, induced secretory changes in the endometrium as determined from histologic sections of tissue biopsies. Either P or RU 486 decreased DNA polymerase alpha and increased E2-DH activities in the endometrium. Unexpectedly, when P and RU 486 were given together. E2-DH activity remained at the level found in E2-treated women. In vitro cultures of proliferative endometrium treated with the synthetic progestagen R 5020 or with RU 486 also had increased E2-DH activity; RU 486 counteracted R 5020 effects. We conclude that, contrary to previous results with experimental animals, the anti-P RU 486 has some progestomimetic activity in humans under specific conditions. Paradoxically, when given together with P, RU 486 lost most of its progestomimetic activity in the endometrium and behaved as a pure antagonist.     

Progesterone antagonists increase androgen receptor expression in the rhesus macaque and human endometrium

Antiprogestins (APs) inhibit estradiol (E(2))-stimulated endometrial growth in women and nonhuman primates, but the mechanism of this "antiestrogenic" action is unknown. Here, we report that APs up-regulate endometrial androgen receptor (AR) in both women and macaques, an effect that might play a role in the antiproliferative effects of APs on the primate endometrium. In addition, because there are discrepancies in the literature on the regulation and localization of AR in the primate endometrium, we used both in situ hybridization and immunocytochemistry to evaluate hormonal influences on endometrial AR in women and macaques. In ovariectomized macaques, the following treatments were given for 4 weeks each: E(2) alone, E(2) + progesterone (P), E(2) + mifepristone (RU 486), and E(2) + P + RU 486. In women, samples were obtained during the normal menstrual cycle and after treatment with either RU 486 for 30 days at 2 mg/day, or after a single oral administration of 200 mg RU 486 on cycle day LH + 2. In macaques, E(2) significantly increased AR expression above vehicle controls; E(2) + RU 486 increased binding further; E(2) + P decreased AR binding; and E(2) + P + RU 486 treatment caused an intermediate elevation in AR binding. In macaques treated with E(2) alone, stromal AR staining was predominant, and P treatment suppressed that staining. E(2) + RU 486 or E(2) + P + RU 486 treatment produced a striking up-regulation of glandular epithelial AR staining and enhanced the stromal AR signal. In situ hybridization analyses confirmed the immunocytochemistry data. Similar induction of glandular AR staining and enhanced stromal AR staining were obtained in macaques treated with ZK 137 316 and ZK 230 211. During the natural cycle in women, stromal AR staining predominated and was greater in the proliferative than the late secretory phase. RU 486 treatment of women up-regulated glandular epithelial AR staining after either daily treatment for 30 days with 2 mg/day or after a single oral dose of 200 mg. In summary, endometrial AR was highest in the stroma during the human proliferative phase (or during E(2) treatment in macaques) and lowest during the late secretory phase in women (or after E(2) + P treatment in macaques). In both species, RU 486 induced AR expression in the glands and enhanced AR expression in stromal cells. Because androgens can antagonize E(2) action, enhanced endometrial AR expression induced by APs could play a role in the antiproliferative, "antiestrogenic" effects of APs in primates.     

Dosage-dependent requirement of BMP type II receptor for maintenance of vascular integrity

Germ-line mutations in bone morphogenic protein type II receptor (Bmpr2) confer susceptibility to pulmonary arterial hypertension (PAH), which is characterized by obstructive vascular lesions in small arteries. The molecular and cellular mechanisms that account for the etiology of this disorder remain elusive, as does the role of Bmpr2 in postnatal tissue homeostasis. Here we show that in adult mice, stably silencing Bmpr2 expression by RNA interference does not increase pulmonary arterial resistance but results in severe mucosal hemorrhage, incomplete mural cell coverage on vessel walls, and gastrointestinal hyperplasia. We present evidence that BMP receptor signaling regulates vascular remodeling during angiogenesis by maintaining the expression of endothelial guidance molecules that promote vessel patterning and maturation and by counteracting growth factor–induced AKT activation. Attenuation of this function may cause vascular dysmorphogenesis and predisposition to angioproliferative diseases. Our findings provide a mechanistic link between PAH and other diseases associated with the BMP/TGF-ß pathways, such as hereditary hemorrhagic telangiectasia and juvenile polyposis syndrome.     

RU486, a progestin and glucocorticoid antagonist, inhibits the growth of breast cancer cells via the progesterone receptor

The progestin and glucocorticoid antagonist RU486 was tested on the growth of several cell lines in culture. RU486 inhibited the growth of two progesterone receptor (RP) positive human breast cancer cell lines (MCF7 and T47D). The antiproliferative effect was dose dependent and its magnitude correlated with the RP content of the tested cells (T47D greater than estradiol-primed MCF7 greater than withdrawn MCF7). Cell growth inhibition was not prevented by the addition of dexamethasone, dihydrotestosterone, or estradiol, but the cells were rescued by low concentrations of the progestin R5020. RU486 had no effect on the growth of two RP negative human breast cancer cell lines and a rat fibroblast cell line. Moreover, RU486 had no progestin agonist activity in T47D cells when evaluated by measuring the 35S-labeling of two progestin-regulated proteins with mol wts of 48,000 and 250,000, but it totally prevented the induction of these two proteins by R5020. In conclusion, RU486 selectively inhibited the growth of human breast cancer cell lines with unoccupied RP sites and its effect was correlated with the RP concentration of these cells. We propose that RU486 is a RP-targeted drug of potential utility in breast cancer treatment.     

Progesterone effects on cell growth of U373 and D54 human astrocytoma cell lines

Astrocytomas are the most frequent primary brain tumors and constitute a leading cause of cancer-related deaths. We studied the effects of progesterone and its antagonist, RU486, on cell growth of two human astrocytoma cell lines with different evolution grade (U373, grade III; and D54, grade IV). Progesterone receptor expression was determined by Western blot. The effects of different doses of progesterone and RU486 on cell number, cell cycle, and apoptosis were analyzed for five consecutive days. Progesterone (10 nM) significantly increased the number of D54 cells from the second day of culture, and the number of U373 cells on days 3–5. RU486 (10 μM) blocked progesterone effects in both astrocytoma cell lines. Interestingly, RU486 administered without progesterone significantly reduced the number of cells from the second day of culture in both cell lines. Progesterone increased S phase of cell cycle in U373 cells (61%, on day 5). RU486 blocked the effects of progesterone on cell cycle but administered alone did not significantly change cell cycle profile. DNA fragmentation (TUNEL) assay showed that the diminution in the number of astrocytoma cells produced by RU486 was not by apoptosis. Progesterone receptor isoforms were detected in both cell lines. Our data suggest that progesterone induces cell growth of human astrocytoma cell lines through the interaction with its nuclear receptor.     

Role of proestrous progesterone secretion in suppressing basal pulsatile LH secretion during estrus of the estrous cycle

These studies examined the mechanisms responsible for the paucity of basal LH pulses during estrus. We confirmed our earlier observations that constant infusion of naloxone during estrus results in the immediate appearance of pulsatile LH secretion during estrus, consisting of LH peak heights and LH interpulse intervals that are similar to those observed during other days of the estrous cycle. We then tested whether the proestrous surge of progesterone was responsible for the suppression of pulsatile LH secretion during estrus. Three treatment regimens were used on proestrus to either block progesterone secretion (pentobarbital) or block its action (progesterone antiserum or the progesterone antagonist, RU 486). After treatment at 12.00 h on proestrus, blood samples were collected during estrus every 10 min for 4 h, and the plasma samples were analyzed for the pattern of LH secretion. Treatment with pentobarbital (35 mg/kg at 12:00 h) blocked the proestrous surges of LH and progesterone and resulted in pulsatile LH secretion during estrus. The LH interpulse interval (72 +/- 7 min) was somewhat slower than that observed in the naloxone-infused animals (54 +/- 8 min). Simultaneous treatment with pentobarbital and progesterone at 12:00 h on proestrus completely prevented the appearance of LH pulses during estrus. Treatment with either progesterone antiserum (0.5 ml, i.v.) or RU 486 (1 mg s.c.) resulted in the initiation of pulsatile LH secretion during estrus. In the RU 486-treated animals, LH peak heights and LH interpulse intervals were similar to those observed in naloxone-infused animals and during other days of the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of synergy between estrogen and progesterone on local IGF-I, IGFBP-3 and IGFBP-2 secretion by both hormone-dependent and hormone-independent breast cancer explants in vitro. Effect of tamoxifen and mifepristone (RU 486).

The aim of the present study was to investigate direct effects of estrogen (E2) or progesterone (P4) given separately vs. estrogen+progesterone on local IGF-I, IGFBP-3 and IGFBP-2 secretion. Explants obtained from estrogen receptor positive plus progesterone receptor positive (ER+/PR+) and hormone receptors negative (ER-/PR-) tumors were incubated with E2, P4 or both. Tamoxifen was added to E2-exposed cultures; mifepristone (RU 486) was added to P4, and both were given to E2+P4-supplemented cultures. In hormone-dependent and hormone-independent tissues, treatment with estrogen+progesterone increased IGF-I and IGFBP-2 secretion with concomitant decrease in IGFBP-3, in the same manner as E2 or P4 given alone. Tamoxifen decreased the E2- and E2+P4-stimulated IGF-I secretion by hormone-dependent breast cancer explants. RU 486 decreased the P4- and E2+P4-stimulated IGF-I secretion with parallel stimulation of IGFBP-3 secretion by ER+/PR+ explants. Estradiol and progesterone had a synergistic action on IGFBP-2 secretion by hormone-dependent breast cancer explants. In conclusion, the presented data suggest that there is no synergistic action of E2 and P4 in influencing IGF/IGFBPs ratio and, additionally, suggest a protective action of antiestrogen and antiprogestagen against excessive IGF-I secretion.     

Integrin beta1 subunit controls mural cell adhesion, spreading, and blood vessel wall stability

Growth, maturation, and integrity of the blood vessel network require extensive communication between the endothelial cells, which line the vascular lumen, and associated mural cells, namely vascular smooth muscle cells and pericytes. Pericytes extend long processes, make direct contact with the capillary endothelium, and promote vascular quiescence by suppressing angiogenic sprouting. Vascular smooth muscle cells are highly contractile, extracellular matrix-secreting cells that cover arteries and veins and provide them with mechanical stability and elasticity. In the damaged blood vessel wall, for example in atherosclerotic lesions, vascular smooth muscle cells lose their differentiated state and acquire a highly mitotic, so-called "synthetic" phenotype, which is thought to promote pathogenesis. Among other factors, extracellular matrix molecules and integrin family cell-matrix receptors may regulate this phenotypic transition. Here we show that the inactivation of the gene encoding the integrin beta1 subunit (Itgb1) with a Cre-loxP approach in mice leads to mural cell defects and postnatal lethality. Integrin beta1-deficient vascular smooth muscle cells display several hallmarks of the synthetic phenotype: Cell proliferation is enhanced, whereas differentiation and their ability to support blood vessels are compromised. Similarly, mutant pericytes are poorly spread but present in larger numbers. Our analysis of this mutant model shows that integrin beta1-mediated cell-matrix adhesion is a major determinant of the mural cell phenotype.     

Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum

Mifepristone (RU486), which binds with high affinity to both progesterone and glucocorticosteroid receptors (PR and GR), is well known for its use in the termination of unwanted pregnancy, but other activities including neuroprotection have been suggested. Cerebellar organotypic cultures from 3 to 7 postnatal day rat (P3-P7) were studied to examine the neuroprotective potential of RU486. In such cultures, Purkinje cells enter a process of apoptosis with a maximum at P3. This study shows that RU486 (20 microM) can protect Purkinje cells from this apoptotic process. The neuroprotective effect did involve neither PR nor GR, because it could not be mimicked or inhibited by other ligands of these receptors, and because it still took place in PR mutant (PR-KO) mice and in brain-specific GR mutant mice (GRNes/Cre). Potent antioxidant agents did not prevent Purkinje cells from this developmental cell death. The neuroprotective effect of RU486 could also be observed in pathological Purkinje cell death. Indeed, this steroid is able to prevent Purkinje cells from death in organotypic cultures of cerebellar slices from Purkinje cell degeneration (pcd) mutant mice, a murine model of hereditary neurodegenerative ataxia. In P0 cerebellar slices treated with RU486 for 6 days and further kept in culture up to 21 days, the synthetic steroid increased by 16.2-fold the survival of pcd/pcd Purkinje cells. Our results show that RU486 may act through a new mechanism, not yet elucidated, to protect Purkinje cells from death.