microM Medroxyprogesterone Acetate Research Articles

Inhibition of matrix metalloproteinases prevents the synthesis of insulin-like growth factor binding protein-1 during decidualization in the baboon.

During pregnancy in the primate, uterine stromal fibroblasts are transformed into decidual cells. Decidualization is associated with extensive remodeling of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) play a pivotal role in ECM degradation. We hypothesized that MMPs also contribute to regulation of IGF binding protein-1 (IGFBP-1), a biochemical marker of primate decidual cells. We reported that IL-1beta (10 ng/ml) with steroid hormones [36 nm estradiol-17beta, 1 microm medroxyprogesterone acetate (P), and 100 ng/ml relaxin] induces in vitro IGFBP-1 synthesis. This study demonstrates that IL-1beta also induces stromelysin-1 (MMP-3) mRNA and synthesis of the latent form of MMP-3 (pro-MMP-3) protein in baboon stromal fibroblasts. In contrast, hormones (particularly P) negatively regulate MMP-3 because their addition decreases IL-1beta-induced pro-MMP-3 protein. The ERK and p38 MAPK pathways induced by IL-1beta regulate pro-MMP-3 because inhibitors PD98059 (20 microm) and SB203580 (1 microm) prevent its synthesis. The nuclear factor-kappaB inhibitory peptide, SN50 (50 microg/ml), or proteasome inhibitor, MG-132 (1 microm), did not inhibit pro-MMP-3 synthesis but appeared to enhance it. The role of MMPs in IGFBP-1 induction was investigated using a broad-spectrum MMP inhibitor, doxycycline, and specific MMP-3 inhibitor, N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH). Both inhibitors caused the dose-dependent decrease of IGFBP-1. alpha-Smooth muscle actin, which is down-regulated during decidualization, was partially up-regulated by doxycycline or N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid. This suggests that alpha-smooth muscle actin is modulated by changes in ECM caused by the action of MMPs/MMP-3. Disruption of actin filaments enhances IGFBP-1 induction. Thus, our data imply that IL-1beta-induced MMPs and particularly MMP-3 may up-regulate IGFBP-1 by disrupting the actin cytoskeleton as a result of ECM degradation.

Interleukin-1beta induces the expression of insulin-like growth factor binding protein-1 during decidualization in the primate.

Since interleukin (IL)-1 can modulate fetal/maternal interactions, we hypothesized that IL-1beta is one potential embryonic cytokine that regulates the conceptus-induced decidual response in baboon stromal fibroblasts. Treatment of stromal fibroblasts with IL-1beta (10 ng/ml, 10 min) resulted in the phosphorylation of p38 mitogen-activated protein kinase and IkappaB-alpha. This suggests that IL-1beta induces multiple signaling pathways in stromal cells that result in the activation of mitogen-activated protein kinase cascade and the transcription factor NF-kappaB. After 4 h of stimulation, IL-1beta induced gene expression of cyclooxygenase-2 (COX-2) but not cyclooxygenase-1 (COX-1). PGE2 synthesis paralleled COX-2 messenger RNA expression. The addition of hormones [36 nM estradiol-17beta, 1 microM medroxyprogesterone acetate, and 100 ng/ml relaxin] to IL-1beta-treated cells induced insulin-like growth factor binding protein-1 (IGFBP-1) messenger RNA expression after 3 days of incubation. A specific COX-2 inhibitor, NS 398 (10 nM), partially inhibited IGFBP-1 protein synthesis. In contrast, the induction of IGFBP-1 by N6, 2'-O-dibutyryladenosine 3:5'-cyclic monophosphate (dbcAMP) and hormones was not affected by NS 398 treatment. Both dbcAMP and IL-1beta, in the presence of hormones, can independently induce IGFBP-1 gene expression and decidualization. However, if IL-1beta and dbcAMP were added together, IGFBP-1 expression was inhibited. These data suggest that IL-1beta can activate multiple signaling pathways that either positively or negatively regulate IGFBP-1 gene expression and decidualization.

Interleukin-1 beta and the endometrium: an inhibitor of stromal cell differentiation and possible autoregulator of decidualization in humans.

Interleukin-1 beta (IL-1 beta), which modulates cell proliferation and differentiation in a number of cell types, is present in human endometrial stromal cells. However, both the function of IL-1 beta in endometrium and the factors that modulate its expression in endometrial stromal cells are unknown. To examine the effects of IL-1 beta on decidualization, human proliferative endometrial stromal cells were cultured for 12 days in medium (Dulbecco's Modified Eagle's medium with 2% fetal bovine serum) containing 1 microM medroxyprogesterone acetate, 10 nM estradiol, and 1 microM prostaglandin E2 (PGE2) with and without IL-1 beta (17 pg/ml). Morphologic changes as well as release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) were used as markers of decidualization. Morphologic analysis of cells exposed to IL-1 beta revealed incomplete decidualization. In addition, cells exposed to IL-1 beta released 40% less PRL and 85% less IGFBP-1 than cells cultured in the absence of IL-1 beta, and the PRL mRNA content of the IL-1 beta-exposed cells was decreased by 68%. A possible role for ovarian steroids in the modulation of IL-1 beta expression in the endometrium is suggested by the increase in IL-1 beta mRNA that occurs in late secretory endometrium and by the induction of IL-1 by estrogen and progesterone in the mouse uterus.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined effects of estradiol, leuprorelin, tamoxifen and medroxyprogesterone acetate on cell growth and steroid hormone receptors in breast cancer cells.

Both tamoxifen and medroxyprogesterone acetate have a direct antitumor effect and are widely used in breast cancer therapy. Luteinizing-hormone-releasing hormone analogs inhibit the growth of breast cancer cells and could represent an alternative treatment for patients affected by breast cancer. Our study was carried out to investigate the effect of leuprorelin (TAP-144) alone or combined with tamoxifen or medroxyprogesterone acetate in human breast cancer cells. Ineffective when used in the absence of estrogens, TAP-144 inhibited the estrogen-stimulated growth of MCF-7, CG-5 and ZR-75-1 cells cultured in medium supplemented with charcoal-treated serum. The growth of estrogen-unresponsive MDA-MB-231 cells was not affected by TAP-144. The combination of TAP-144 with tamoxifen in CG-5 cells did not determine any enhancement of inhibition of cell growth, whereas in both CG-5 and MCF-7 cells, when 1 microM TAP-144 was associated with 0.1 microM medroxyprogesterone acetate, cell growth inhibition was increased, resulting in a subadditive effect. Progesterone receptor levels of CG-5 cells were significantly increased by TAP-144 in the presence of 17 beta-estradiol with respect to those present in control and 17 beta-estradiol-treated cells.

Prostaglandin E2 enhances human endometrial stromal cell differentiation.

Endometrial stromal differentiation (decidualization) is essential for implantation of the developing blastocyst. Because prostaglandins (PGs) are synthesized in the endometrium, and PG-binding sites have been demonstrated in the proliferative endometrial stromal cell (the precursor of the decidual cell), experiments were performed to determine whether PGs are involved in the process of decidualization. Human endometrial stromal cells were cultured for 18 days in Dulbeco's Modified Eagle's Medium-2% fetal bovine serum with 1 microM medroxyprogesterone acetate (MPA) and 10 nM estradiol, with and without PGE2 or PGF2 alpha. Expression of PRL was used as a marker of decidualization. In the presence of estradiol and MPA alone (control), PRL was detected beginning on day 9 and gradually increased through day 18. In contrast, PRL was detected on day 3 in the PGE2-treated cells, and the magnitude of stimulation in these cells on days 9-12 was 1300-1400% of that in control cells. Furthermore, the PRL mRNA content of the PGE2-treated cells on day 12 was 4.6-fold greater than that in the control cells. The effect of PGE2 on PRL production was dose dependent, with a minimal effective dose of 10(-10) M. PGE2 in the absence of steroids had a minimal effect on PRL production. In contrast to PGE2, PGF2 alpha treatment had no effect on PRL expression in steroid-treated cells. These results indicate that there are synergistic effects among PGE2, estradiol, and MPA, resulting in acceleration of endometrial stromal cell differentiation and enhanced PRL expression.

Down-regulation of androgen receptor by progestins and interference with estrogenic or androgenic stimulation of mammary carcinoma cell growth

The regulatory influence of medroxyprogesterone acetate (MPA) on estrogen and androgen receptors of the human breast cancer cell lines MCF-7 and EFM-19 was explored in conjunction with the growth-promoting properties of these steroids. In the absence of steroidal stimulation, up to 1 microM MPA had no effect on the proliferation of the MCF-7 cell strain used and of EFM-19 cells. Under stimulation with 10 nM 17 beta-estradiol or 1 microM dihydrotestosterone, dose-dependent inhibition of the cell proliferation rates by 0.1-1 microM MPA was observed. Binding of MPA to the androgen receptor (Kd = 2.1 nM) but not to the estrogen receptor was demonstrable. During incubation of MCF-7 or EFM-19 cells with 1 microM MPA for 7 days, the estrogen and androgen receptor contents were down-regulated by approximately 50% and 60%, respectively. Likewise, the number of androgen-binding sites was reduced to 35% of the untreated controls after incubation of MCF-7 cells with 1 microM synthetic progestin R5020 for 7 days. The results indicate down-regulation of estrogen and androgen receptors by progestins in the absence of stimulatory effects on the proliferation of mammary carcinoma cells.

Estrone Sulfate Sulfatase Activity Is Increased duringin VitroDecidualization of Stromal Cells from Human Endometrium*

Arylsulfatase (EC 3.1.6.1) activity in human stromal cells isolated from specimens of histologically normal proliferative endometrium was increased several-fold during culture for 8-15 days in RPMI-1640 medium plus 10% charcoal-treated fetal bovine serum in the presence of a mixture of ovarian hormones (36 nM estradiol, 1 microM medroxyprogesterone acetate, and 100 micrograms/mL relaxin). The changes in sulfatase activity, determined by measuring the rate of formation of estrone from tritiated estrone sulfate, were associated with in vitro decidualization of the stromal cells, as determined by changes in secretion of PRL into daily renewed culture medium. PRL output by the cells during the last 24 h in culture and sulfatase activity in the cells collected at the end of the culture period were related to their DNA and protein contents. Sulfatase activity in the cells cultured in the presence of the ovarian hormones was comparable to the activity found in decidual cells at term pregnancy. PRL added for 1 day to cultures of stromal cells in the absence of exogenous hormones increased sulfatase activity in the cells, probably by acting in an autocrine manner, as previously demonstrated with human decidual cells during pregnancy. These experiments also revealed a hormonal regulation of stromal cell proliferation in vitro, as estimated from measurements of both DNA and protein levels per dish. Augmentation of sulfatase activity can serve as another marker of in vitro decidualization. Physiologically, an increase in this enzymatic activity may result in a preferential estrogenic stimulation of the decidualized cells by utilization of a circulating substrate, estrone sulfate. This hypothesis could explain the preferential retention of progesterone receptors in decidual cells observed immunohistochemically during the late luteal phase of the menstrual cycle, suggestive of a shift in progestogenic actions from the epithelium to the stroma.

Regulation of Prolactin Production by Progestin, Estrogen, and Relaxin in Human Endometrial Stromal Cells*

Human decidua synthesizes and secretes PRL. We identified the PRL synthesized in endometrial stromal cells and investigated the effect of medroxyprogesterone acetate (MPA), estradiol (E2), porcine relaxin (RLX), and RU486, an antiprogestin, on PRL production by stromal cells from non-pregnant endometrium in primary culture. Stromal cells were isolated from proliferative and secretory endometria and individually cultured in nutrient medium or medium supplemented with different hormone(s). The immunoreactive PRL isolated from culture medium of hormone-stimulated stromal cells was identified and compared to pituitary PRL. Bio-Gel elution pattern and mol wt analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that PRL produced by stromal cells had properties identical to those of pituitary PRL. In addition, PRL mRNA was identified in hormone-stimulated stromal cells using human pituitary PRL cDNA as a hybridization probe. Analysis of mRNA by Northern blotting showed that the size of PRL mRNA isolated from stromal cells was indistinguishable from that of PRL mRNA in human decidua and pituitary tissue. These results indicated that PRL measured in culture medium was synthesized de novo by stromal cells. The PRL content in culture medium was quantitated by RIA. The PRL production rate in stromal cells cultured without hormones ranged from 6-10 ng/day.mg cell protein. After 4-5 days of incubation with RLX or MPA alone, the PRL production rate increased about 2- to 3-fold over the control value. E2 alone had either no effect or slightly decreased the stromal cell PRL production rate. Stromal cells responded to 0.02 microM MPA, and the maximal response was at 0.1-1 microM MPA. A further increase in PRL production was found when stromal cells were treated with a combination of MPA and E2 and MPA, E2 and RLX. In the presence of MPA or MPA and E2, 0.1 ng/ml relaxin increased the PRL production rate. A potent progestin antagonist, RU486, inhibited PRL production in stromal cells treated with MPA, MPA and E2, or MPA and RLX. These results indicate that endometrial PRL production is regulated by the combined effects of steroid hormones (progestin and estrogen) and a peptide hormone (relaxin).

Direct effects of medroxyprogesterone acetate (MPA) and megestrol acetate (MGA) on rat testicular steroidogenesis.

The effects of MPA and MGA on rat testicular steroidogenesis were examined by studying: 1) serum testosterone in hCG primed animals treated with MPA or MGA, 2) testosterone synthesis in rat Leydig cells cultured with MPA or MGA, 3) MPA and MGA binding to rat testis microsomal cytochrome P-450 and 4) MPA and MGA inhibition of enzymes of rat testicular steroidogenesis. In immature rats receiving 1.0 IU of hCG per day 20 mg/kg of MPA or MGA reduced serum testosterone by 57 and 56%, respectively. In mature male rats receiving 50.0 IU of hCG per day 20 mg/kg of MPA or MGA reduced serum testosterone by 40 and 29%, respectively. In rat interstitial cells cultured with 10 ng of rat LH, 1 microM MPA or MGA inhibited testosterone production by 32 and 23%, respectively. Addition of MPA or MGA to microsomal preparations resulted in a type I cytochrome P-450 difference spectrum. MPA and MGA inhibited rat testicular 17 alpha-hydroxylase, 17,20-lyase, and the 3 beta- and 17 beta-hydroxysteroid dehydrogenases. These findings suggest that MPA and MGA inhibit rat testicular steroidogenesis in vivo and in vitro.