Single- and double-stranded viral RNA generate distinct cytokine and antiviral responses in human fetal membranes.

There has been growing interest in the role of viral infections and their association with adverse pregnancy outcomes. However, little is known about the impact viral infections have on the fetal membranes (FM). Toll-like receptors (TLR) are thought to play a role in infection-associated inflammation at the maternal-fetal interface. Therefore, the objective of this study was to characterize the cytokine profile and antiviral response in human FMs exposed to viral dsRNA, which activates TLR3, and viral ssRNA, which activates TLR8; and to determine the mechanisms involved. The viral dsRNA analog, Poly(I:C), induced up-regulated secretion of MIP-1α, MIP-1ß, RANTES and TNF-α, and down-regulated interleukin (IL)-2 and VEGF secretion. In contrast, viral ssRNA induced a broader panel of cytokines in the FMs by up-regulating the secretion of IL-1ß, IL-2, IL-6, G-CSF, MCP-1, MIP-1α, MIP-1ß, RANTES, TNF-α and GRO-α. Using inhibitory peptides against TLR adapter proteins, FM secretion of MIP-1ß and RANTES in response to Poly(I:C) was MyD88 dependent; MIP-1α secretion was dependent on MyD88 and TRIF; and TNF-α production was independent of MyD88 and TRIF. Viral ssRNA-induced FM secretion of IL-1ß, IL-2, IL-6, G-CSF, MIP-1α, RANTES and GRO-α was dependent on MyD88 and TRIF; MIP-1ß was dependent upon TRIF, but not MyD88; and TNF-α and MCP-1 secretion was dependent on neither. Poly(I:C), but not ssRNA, induced an FM antiviral response by up-regulating the expression of IFNß, myxovirus-resistance A, 2',5'-oligoadenylate synthetase and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G. These findings demonstrate that human FMs respond to two viral signatures by generating distinct inflammatory cytokine/chemokine profiles and antiviral responses through different mechanisms.

Local paracrine effects of estradiol are central to parturition in the rhesus monkey.

The central biochemical mechanisms involved in primate parturition are still unclear. Studies in both humans and nonhuman primates such as the baboon and rhesus monkey indicate that many factors play a part in the cascade of interactive positive feedforward loops that progressively promote parturition: changes in maternal endocrinology, a nocturnal switch in myometrial activity from low amplitude, infrequent contractures to high amplitude, high frequency contractions (see Fig. 1), dilation of the cervix and biochemical changes in the fetal membranes that lead to rupture. Here we demonstrate that infusion of the aromatase inhibitor 4-hydroxyandrostenedione (4OHA) inhibits conversion of androgen to estrogen and prevents premature delivery caused by administration of androgen to pregnant rhesus monkeys at 0.8 of pregnancy term. 4OHA also inhibited the androstenedione induced maternal endocrine and fetal membrane biochemical changes, and alteration of myometrial activity patterns. Secondly, peripheral estrogen infusions increased myometrial activity but did not produce preterm delivery or fetal membrane changes. We conclude that paracrine functions of estrogen at its site of production play critical and central roles in delivery in the non-human primate.

Production of premature delivery in pregnant rhesus monkeys by androstenedione infusion.

The endocrine mechanism involved in term and preterm delivery in primates, including pregnant women, are poorly understood. In the term monkey, fetal plasma androgen concentration rises to two hundred times the maternal concentration which remains unchanged. Placental conversion of androgen to estrogen results in increased maternal plasma estrogen concentration at term in both pregnant nonhuman primates and women. In the present study, continuous infusion of androstenedione to 0.8 gestation monkeys resulted in the premature occurrence of labor-type myometrial activity and increases in maternal plasma estrogen, oxytocin and amnion fibronectin concentrations similar to those measured at normal-term labor. Androstenedione induction of these normal-term biochemical and endocrine changes accompanied by fetal membrane rupture, cervical dilatation and live delivery provides a rich opportunity to study the molecular and physiological mechanisms of both term and preterm labor in primates.

Method to enhance transfection efficiency of cell lines and placental fibroblasts.

We report a method that allows for a 2-9-fold increase in transfection efficiency compared to the standard cationic lipid-based protocol. The method involves a brief incubation of freshly trypsinized cells with transfection complexes, followed by incubation in cell growth medium containing serum and antibiotics. The method is simple, cost-effective, and can be applied to both DNA and siRNA transfections as well as to a variety of cell types including hard-to-transfect human placental fibroblasts.

Role of hypoxia-inducible transcription factors 1alpha and 2alpha in the regulation of plasminogen activator inhibitor-1 expression in a human trophoblast cell line.

The plasminogen activator inhibitors (PAIs) play critical roles in regulating hemostatic and invasive functions of trophoblasts through suppression of plasmin-dependent fibrinolysis and extracellular matrix degradation. The expression of PAI-1 is increased under hypoxic conditions, although the mechanism remains incompletely understood. In the current study we used HTR-8/SVneo cells, a first trimester extravillous trophoblast cell line, and siRNA technology to examine the role of hypoxia-inducible transcription factors (HIFs)-1alpha and -2alpha in the regulation of PAI-1 expression. Using serum-containing and serum-free media culture media it was initially noted that levels of PAI-1, but not PAI-2 protein, were markedly induced by hypoxic (2-3% oxygen) treatment. Under hypoxic conditions, Western blotting revealed that the presence of siRNAs to HIF-1alpha and HIF-2alpha suppressed expression of their respective proteins, whereas treatment with non-targeting and cyclophilin B siRNAs did not. Importantly, incubation with siRNA to HIF-1alpha or HIF-2alpha alone reduced PAI-1 protein levels to a similar extent, with the combined treatment inducing a more profound effect. The presence of HIF siRNAs reduced levels of PAI-1 mRNA as measured by quantitative real-time PCR, indicating that HIF-1alpha and HIF-2 alpha regulate PAI-1 expression at a transcriptional level. These results indicate that both HIF-1alpha and HIF-2alpha play important and similar roles in hypoxia-mediated stimulation of PAI-1 expression in HTR-8/SVneo cells. Our findings provide insight into the physiological regulation of trophoblast PAI-1 expression in early pregnancy when placental oxygen levels are low, as well as a mechanism for over-expression of placental PAI-1 noted in pregnancies with preeclampsia.

Differential release of plasminogen activator inhibitors (PAIs) during dual perfusion of human placenta: implications in preeclampsia.

Plasminogen activator inhibitors (PAIs) play critical roles in regulating cellular invasion and fibrinolysis. An increase in the ratio of PAI-1/PAI-2 in placenta and maternal serum is suggested to result in excessive intervillous fibrin deposition and placental infarction in pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR). In the current study we used dual (maternal and fetal) perfusion of human term placentas to examine the release of PAIs to the intervillous space. ELISA revealed a significant time-dependent increase in total PAI-1 levels in maternal perfusate (MP) between 1 and 7h of perfusion. Conversely, PAI-2 levels decreased resulting in a 3-fold increase in the PAI-1/PAI-2 ratio in MP. Levels of PAI-1, but not PAI-2, in placental tissue extracts increased during perfusion. In perfusions carried out with xanthine and xanthine oxidase (X + XO), compounds used to generate reactive oxygen species (ROS), no time-dependent increase in total PAI-1 levels was observed. In addition, X + XO treatment promoted a 3-fold reduction in active PAI-1 levels in MP, indicating that ROS decrease PAI-1 release to MP. The finding of a time-dependent change in patterns of PAI expression and response to ROS indicates the utility of dual perfusion as a model to dissect mechanism(s) promoting aberrant fibrinolysis in pregnancies complicated by PE and IUGR.

Cell type-specific expression and function of toll-like receptors 2 and 4 in human placenta: implications in fetal infection.

Placental infection is associated with adverse fetal outcomes. Toll-like receptors (TLRs) are critical regulators of the innate immune response based on their ability to recognize and respond to pathogen-associated molecular patterns expressed by microbes. To date, cell-type specific expression and regulation of TLR function in human term placenta remains largely unelucidated. The goal of the current study was to examine the in vivo and in vitro patterns of TLR expression and function in major cell types of term placenta. Immunohistochemical analysis of terminal and stem villi localized TLR-2, which recognizes peptidoglycan (PG) from Gram-positive bacteria, to endothelial cells and macrophages, and to a lesser extent to syncytiotrophoblast (SCTs) and fibroblasts (FIBs). Staining for TLR-4, the receptor for Gram-negative bacterial lipopolysaccharide (LPS), was most prominent in SCTs and endothelial cells. Results from Western blotting, conventional, and quantitative PCR (qRTPCR) analyses using protein and mRNA isolated from cultures of SCTs and myofibroblasts (mFIBs) revealed that SCTs expressed TLR-2 and TLR-4, whereas mFIBs expressed only TLR-4. In addition, qRTPCR showed that LPS treatment increased TLR-2 expression in SCTs, indicating that infection with Gram-negative bacteria may enhance innate immune responses in placenta toward a broad range of microorganisms. In addition, treatment with LPS increased IL-8 levels in both SCTs and mFIBs, whereas PG treatment only stimulated IL-8 levels in SCTs. Our results indicate that there exist cell type-specific patterns of TLR function in placenta which likely regulate innate immune response at the maternal-fetal interface.

Dual in vitro perfusion of an isolated cotyledon as a model to study the implication of changes in the third trimester placenta on preeclampsia.

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-alpha, IL-1ss, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2alpha 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group. Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group. Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1beta of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1beta at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue. Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1beta only the 17kD band was detected. In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

BCR-ABL mediates arsenic trioxide-induced apoptosis independently of its aberrant kinase activity.

In the prechemotherapy era arsenic derivatives were used for treatment of chronic myelogenous leukemia, a myeloproliferative disorder characterized by the t(9;22) translocation, the Philadelphia chromosome (Ph+). In acute promyelocytic leukemia response to arsenic trioxide (As2O3) has been shown to be genetically determined by the acute promyelocytic leukemia-specific t(15;17) translocation product PML/RARalpha. Hence, we reasoned that As2O3 might have a selective inhibitory effect on proliferation of BCR-ABL-expressing cells. Here, we report that: (a) As2O3 induced apoptosis in Ph+ but not in Ph- lymphoblasts; (b) enforced expression of BCR-ABL in U937 cells dramatically increased the sensitivity to As2O3; (c) the effect of As2O3 was independent of BCR-ABL kinase activity; and (d) As2O3 reduced proliferation of chronic myelogenous leukemia blasts but not of peripheral CD34+ progenitors. In summary, these data establish As2O3 as a tumor cell-specific agent, making its clinical application in Ph+ leukemia feasible.

Regulation of tissue factor gene expression in human endometrium by transcription factors Sp1 and Sp3.

Prior studies indicate that tissue factor (TF), the primary cellular initiator of hemostasis, is persistently up-regulated in human endometrial stromal cells (HESCs) undergoing progestin-induced decidualization in vivo and in vitro. The mechanism underlying progestin enhancement of TF mRNA and protein levels in these cells involves transcriptional activation of the TF gene. Transient transfections of HESCs with the truncated TF promoters driving the luciferase reporter gene have demonstrated that the region spanning -111 to +14 bp retained differential progestin-enhancing effects. We now demonstrate that RU486 displays inhibitory effects on the progestin-induced TF promoter activity, confirming the involvement of the progesterone receptor. Since the TF minimal promoter (pTF 111 spanning -111 to +14 bp) contains three overlapping Sp1 and three Egr-1 sites, the present study determined whether Sp1 and/or Egr-1 were required for progestin-regulated TF expression. The results indicate that the three Sp1 sites are primarily responsible for both the constitutive and progestational activity of the pTF 111 promoter, whereas the Egr-1 sites have only a minor involvement in both activities. Overexpression of the Sp1 protein resulted in greater than a 6-fold induction in TF promoter activity. In contrast, no enhancement was observed when the Sp3 protein was overexpressed. The concomitant overexpression of Sp1 and Sp3 demonstrated that Sp3 completely blocked the induction of TF promoter activity by Sp1. Moreover, the addition of 10 nM mithramycin, a concentration that inhibits Sp1 binding to target DNA, blocked the progestational induction of TF mRNA expression. Immunohistochemical studies demonstrated increased Sp1 levels in perivascular stromal cells in secretory phase compared with proliferative phase endometria. In contrast, Sp3 expression was greatly decreased in stromal cells of secretory, compared with proliferative phase tissues. The levels of Egr-1 were low in both proliferative and secretory endometria. Immunocytochemistry of E2 vs. E2 + medroxyprogesterone acetate-treated HESCs demonstrated a dramatic reduction in Sp3 expression after progestin treatment, and Northern blots demonstrated progestational increases in Sp1 and reduction in Sp3 mRNA expression compared with controls. Taken together, our results demonstrate that progestin enhancement of TF gene expression in HESCs is mediated principally by Sp1. We propose that progestins regulate HESC TF gene expression in vivo by altering the ratio of Sp1 to Sp3 nuclear factors.

Cyclic adenosine 3',5'-monophosphate induces prolactin expression in stromal cells isolated from human proliferative endometrium.

In vitro decidualization of stromal cells isolated from human proliferative endometrium and cultured in RPMI-1640 containing 2% charcoal-treated fetal bovine serum and 0.1 U/ml insulin was achieved by adding to the medium cAMP derivatives [(Bu)2cAMP (db-cAMP) and 8-bromo-cAMP] or forskolin. PRL production under these conditions was demonstrated by documenting the synthesis of PRL mRNA (approximately 1.1 kilobase), the output of immunoprecipitable [35S]methionine-labeled PRL migrating as a single 23-kilodalton band during gel electrophoresis, and the time- and concentration-dependent secretion of PRL into the medium, measured by RIA (maximal on days 4-5 using 0.5 mM db-cAMP). Medroxyprogesterone acetate (1 microM) enhanced (1.7- to 2.5-fold) the effect of db-cAMP, 8-bromo-cAMP, or forskolin on PRL production, as evaluated by Western blotting analysis. Further evidence for a participation of db-cAMP in the decidualization process was provided by its ability to induce immunocytochemically detectable heat shock protein-27, insulin-like growth factor-binding protein-1, desmin, and laminin, all compounds produced by human decidual cells, but not expressed by stromal cells. The induction of PRL by cAMP may be a key step in the process of differentiation of fibroblast-like stromal cells to the decidual phenotype, as it has been previously reported by this laboratory that, under similar culture conditions, PRL itself is capable of inducing the production of heat shock protein-27, insulin-like growth factor-binding protein-1, desmin, and laminin in stromal cells isolated from proliferative endometrium.

Expression of growth hormone-independent adipogenesis by a 3T3 cell variant.

We have examined the regulation of adipogenesis of a 3T3-F442A cell variant. The variant, designated 3T3-GH-independent clone 16 (GI-16), was isolated after serum-induced adipogenic commitment. 3T3-GI-16 fibroblasts displayed a lower serum requirement for adipogenesis than the 3T3-F442A parent cell. Insulin-stimulated adipogenesis of 3T3-GI-16 cells in serum-free medium (SFM) was extensive in the absence of GH, as judged by oil red O staining or glycerol-3-phosphate-dehydrogenase activity, a property not associated with the 3T3-F442A cell. In SFM devoid of GH the concentration of insulin required to promote half-maximal adipogenesis of 3T3-GI-16 fibroblasts was 5 nM. The expression of GH-independent adipogenesis by 3T3-GI-16 cells was not due to exposure to adipogenic stimuli during routine passage, as insulin-stimulated differentiation was not a function of the inoculation density in nonadipogenic cat serum. We noted that nine proteins resolved by polyacrylamide gel electrophoresis behaved in a differentiation-dependent manner during adipogenesis of 3T3-GI-16 and 3T3-F442A fibroblasts in SFM. The concentrations of all nine proteins were regulated in a GH-independent manner during insulin-stimulated adipogenesis of 3T3-GI-16 fibroblasts. In contrast, the presence of insulin alone markedly altered the expression of only two of the proteins during differentiation of 3T3-F442A cells. The observed changes in the expression of five presently uncharacterized differentiation-dependent proteins were most likely due to employment of SFM. Our results suggest that expression of GH-independent insulin-induced adipogenesis of 3T3-GI-16 fibroblasts reflects a prior commitment by GH during our selection protocol. These results are discussed in the context of a model in which adipogenesis in vivo is postulated to proceed through the sequential action of GH and insulin on target cells.

Up-regulation of vinculin expression in 3T3 preadipose cells by growth hormone.

In an effort to define biochemical events relevant to the adipogenic action of GH, the effect of GH on expression of the cytoskeletal element vinculin was studied in 3T3-F442A preadipose cells. Results from Western blotting indicated that in serum-free medium 2 nM met-hGH induced an approximately 100% increase in vinculin expression relative to that in cells maintained in serum-free medium alone. GH-elicited alterations in vinculin expression were dose dependent. GH treatment elevated levels of tubulin to a lesser extent, whereas actin expression was unaffected by GH. Immunoprecipitation experiments revealed that GH treatment promoted a 200% increase in vinculin synthesis on day 4 relative to that in control cells. GH had no effect on phosphorylation of vinculin in 3T3-F442A cells. Based on Northern blotting, we noted that GH induced approximately a 200% increase in levels of vinculin mRNA on day 4. GH responsiveness as well as levels of vinculin were similar in 3T3-GI-16 (a highly adipogenic subclone of the 3T3-F442A cell) and 3T3-F442A cells. The GH-dependent increase in vinculin protein expression was not observed in nonadipogenic 3T3-C2 cells, suggesting that this effect of GH was related to the program of differentiation. Interestingly, levels of vinculin in nontreated 3T3-C2 cells were approximately 10-fold lower than levels in 3T3-F442A cells. GH-mediated alterations in vinculin expression in 3T3-F442A cells were abolished by treatment with fetal bovine serum, a potent mitogen. Our data indicate that increased expression of vinculin is a component of the GH-induced portion of the adipose differentiation program.

Role of insulin in growth hormone-stimulated 3T3 cell adipogenesis.

The role of insulin during GH-stimulated adipogenesis of 3T3-F442A fibroblasts was investigated. Adipogenesis in defined medium (DM), as quantified by the level of glycerol-3-phosphate dehydrogenase activity, revealed that there existed a strict requirement for both insulin and GH during adipogenesis. The concentration of insulin required to elicit half-maximal adipogenesis was approximately 20 nM. Insulin-like growth factor I was less effective than insulin in promoting adipogenesis, indicating that insulin action during differentiation was most likely mediated through the insulin receptor. Cellular viability was not compromised by the absence of insulin, as judged by colony-forming efficiency or trypan blue exclusion. Deletion of insulin from DM supplemented with 1 nM recombinant human GH reduced glycerol-3-phosphate dehydrogenase activity to uninduced levels. Removal of other individual DM constituents did not have this effect. The growth factors fibroblast growth factor, platelet-derived growth factor, and bombesin did not substitute for insulin during GH-stimulated adipogenesis. The characteristic increase in cell number observed during serum-based differentiation, reflecting clonal expansion of young adipocytes, did not occur in DM supplemented with insulin, and insulin-like growth factor I were necessary for this event. These results suggest that insulin functions in concert with GH as a coinducer of the differentiating signals.

Glucocorticoid suppression of human placental fibronectin expression: implications in uterine-placental adherence.

Increased levels of glucocorticoids are associated with human parturition whether occurring before or at term. In the present study we examined the effects of glucocorticoids on placental fibronectin (FN) expression in cytotrophoblasts, isolated from human term placentas, to provide a potential mechanism through which glucocorticoids may influence uterine-placental adherence near parturition. Based on immunoassays, relative to controls, media levels of placental FNs bearing an oncofetal epitope (onfFN) were inhibited 65-80% by treatment with 10(-7) M dexamethasone (DEX) during experiments in which cumulative levels and daily release of onfFN were measured. DEX treatment increased human CG production by cytotrophoblasts approximately 3-fold without affecting the levels of total protein, suggesting that DEX treatment did not reduce placental function. DEX and cortisol inhibited onfFN expression with an EC50 of 2 and 16 nM, respectively. Other steroids were not effective in down-regulating onfFN expression, indicating that this was a glucocorticoid-specific response. In immunoprecipitation studies, treatment of cytotrophoblasts with 10(-7) M DEX for 3 days inhibited both release of labeled FN to the media and its incorporation into cell-associated material by approximately 80%. Results from Northern blotting indicated that DEX treatment suppressed levels of FN messenger RNA approximately 90% relative to controls. Levels of labeled laminin in media were inhibited approximately 80% by a 3-day treatment with 10(-7) M DEX, suggesting that glucocorticoids may coordinately suppress the synthesis of multiple extracellular matrix proteins in cytotrophoblasts. In our model, we propose that glucocorticoids may suppress placental extracellular matrix protein synthesis, which could lead to decreased uterine-placental adherence and be associated with parturition.

Growth hormone and fibronectin expression in 3T3 preadipose cells.

In the present study we focused on the relationship between GH action and the extracellular matrix in 3T3-F442A preadipose cells. Results from Northern blotting indicated that in serum-free medium, the presence of 2 nM met-human GH down-regulated levels of fibronectin messenger RNA by approximately 40, 60, and 70% as compared with control levels on days 1, 2, and 4, respectively. GH-dependent reduction of levels of collagen alpha 1(I) mRNA expression occurred later and was less pronounced than effects on levels of fibronectin mRNA, suggesting a specificity in the matrix-altering function of GH. Western blot analyses and immunoprecipitation studies revealed that between 2 and 5 days of culture, matrix-associated fibronectin protein was reduced 70 to 90% by GH treatment. Down-regulation of fibronectin protein expression by met-human GH was dose-dependent between 2 and 0.02 nM. The presence of 2 nM insulin or insulin-like growth factor-1 promoted a 30-40% increase in fibronectin levels compared to control cells. The GH-promoted down-regulation of fibronectin expression was eliminated by concomitant addition of insulin. These data demonstrated that GH effects on matrix-associated fibronectin expression were independent of, and in opposition to, effects promoted by insulin and insulin-like growth factor-1. Treatment of culture dishes with fibronectin or collagen inhibited GH-stimulated adipogenesis 50 and 80%, respectively, compared with controls, as judged by levels of glycerol-3-phosphate dehydrogenase activity. Thus, composition of the extracellular matrix was a critical factor in GH-induced adipogenesis of 3T3-F442A fibroblasts. Our results demonstrate that GH action in 3T3 preadipose cells is intimately coupled to the biology of extracellular matrix.

Growth hormone-dependent events in the adipose differentiation of 3T3-F442A fibroblasts: modulation of macromolecular synthesis.

GH is necessary but not sufficient to induce adipose differentiation of 3T3-F442A fibroblasts in serum-free medium. Human (h) GH (2 nM) treatment of 3T3-F442A cells in serum-free medium caused a time-dependent (maximal at 48-72 h) and dose-dependent (EC50, approximately 0.2 nM) decrease (40-60%) in de nova protein synthesis. Insulin-like growth factor-I (IGF-I; 17 nM), PRL (2 nM), and glucagon (20 nM) did not decrease de novo protein synthesis, whereas bovine GH was equipotent with hGH. The half-lives of 35S-labeled proteins of 3T3-F442A cells were 21 and 57 h for cells maintained in serum-free medium for 3 days without or with hGH (2 nM), respectively. The total protein content of cells maintained in hGH (2 nM) for 1-4 days was unaffected compared to cells in serum-free medium alone. IGF-I (17 nM) treatment of cells for 4 days doubled the protein content of cells compared to control values in serum-free medium. hGH (2 nM) pretreatment of cells for 1-4 days had no effect on total RNA synthesis. hGH (2 nM) but not IGF-I (17 nM) treatment (3 days) resulted in a 7-fold decrease in cytoplasmic 18S rRNA content (as measured by DNA-RNA hybridization) of cells compared to that of control cells maintained in serum-free medium. When 3T3-F442A cells were transferred to serum-free medium there was a progressive decrease in DNA synthesis. The presence of hGH enhanced the rate at which DNA synthesis decreased for 3T3-F442A cells. IGF-I (17 nM) increased DNA synthesis by 6- and 8-fold after 2 and 3 days of IGF-I exposure. 3T3-F442A cells maintained in serum-free medium for 3 days responded to the addition of platelet-derived growth factor (2 U/ml) and insulin (1.6 microM) with a 56-fold increase in DNA synthesis, assayed 24 h later. 3T3-F442A cells treated with hGH (2 nM) for 3 days before platelet-derived growth factor and insulin addition exhibited a diminished DNA synthetic response, demonstrating that GH-exposed cells were partially refractory to mitogenic stimulation. GH had no effect on any aspect of macromolecular synthesis in 3T3-C2 cells, which have a low frequency of adipogenesis. Based upon these results a cell cycle model for the role of GH in the adipose differentiation of 3T3-F442A cells was proposed.

Steroid regulation of human placental integrins: suppression of alpha2 integrin expression in cytotrophoblasts by glucocorticoids.

Maintenance of uterine-placental attachment during human pregnancy may depend at least partly on adhesive interactions between cytotrophoblasts and their extracellular matrix (ECM). Such interactions are often mediated by integrins, signal-transducing heterodimeric transmembrane glycoproteins. We previously showed that glucocorticoid (GC) suppressed the expression of collagen and laminin in human placenta; here we show that GC also modulates the expression by human cytotrophoblasts of the integrin subunits alpha2 and beta1, components of a known receptor for these ECM ligands. Cytotrophoblasts were isolated from human term placentas, cultured up to 4 days in the presence of 0-1000 nM dexamethasone (DEX), and assayed for 1) integrin messenger RNA (mRNA) levels by Northern hybridization, 2) integrin subunit synthesis after [35S]methionine labeling, or 3) cell surface integrin levels after 125I labeling by lactoperoxidase. In four independent experiments, 100 nM DEX reduced mRNA levels for integrin alpha2 to 6+/-1% of the control value. This effect was similar between 1-4 days of treatment and was dose dependent between 1-1000 nM DEX. Cortisol treatment (100 nM) inhibited levels of integrin alpha2 mRNA, but 100 nM testosterone, estradiol, and progesterone were less effective, suggesting that this response was specific to GC. In immunoprecipitation studies, treatment of cytotrophoblasts with 100 nM DEX for 2 days reduced the rates of synthesis of the alpha2 integrin subunit as well as its expression on the cell surface to 1-10% of control levels. DEX effects on the beta1 integrin subunit were less dramatic. DEX reduced beta1 mRNA levels to only 69+/-8% of control levels, a smaller reduction compared with effects on alpha2 integrin mRNA. DEX inhibited beta1 protein synthesis and cell surface expression to 60-70% of control levels. In all experiments, DEX had no effect on total protein synthesis. Thus, our results demonstrate that GC treatment specifically and markedly down-regulates expression of alpha2 integrin subunit by human cytotrophoblasts. This finding is consistent with the concept that uterine-placental adherence across gestation may be regulated by coordinate effects on ECM ligands and cellular adhesion receptors.

A growth hormone (GH) analog can antagonize the ability of native GH to promote differentiation of 3T3-F442A preadipocytes and stimulate insulin-like and lipolytic activities in primary rat adipocytes.

The effect of amino acid substitutions introduced to the third alpha-helix in bovine GH (bGH) was investigated. A GH analog (bGH-M8), in which three amino acids were substituted to form an idealized amphiphilic alpha-helix, possessed the same specific binding affinity as wild-type bGH to cell membranes prepared from 3T3-F442A cells or rat adipocytes. However, bGH-M8 failed to stimulate preadipocyte differentiation, as measured by the level of glycerol-3-phosphate dehydrogenase activity. An equimolar concentration of bGH-M8 was inhibitory for this adipogenic effect caused by bGH at a concentration of 30 pM. bGH-M8 also failed to induce an insulin-like response and reduced lipolytic potency in rat primary adipocytes. A 10-fold excess of bGH-M8 abolished the effect of wild-type bGH in the insulin-like and lipolytic assays. Thus, bGH-M8 inhibited these actions of wild-type bGH and, therefore, appears to be a competitive antagonist. These results suggest that a major biologically active domain resides in the third alpha-helix of bGH, which is independent of amino acids important in the initial interaction of GH with its receptor.

Developmental regulation of glucocorticoid-mediated effects on extracellular matrix protein expression in the human placenta.

The extracellular matrix (ECM) protein fibronectin (FN) is a critical regulator of uterine-placental adherence. In the present report we compared the effects of glucocorticoids on FN expression in cytotrophoblast cultures isolated from human first trimester and term placentas to elucidate potential steroid-dependent cellular mechanisms associated with human parturition. Based on immunoassays, treatment of first trimester cytotrophoblasts with 10(-7) M dexamethasone (DEX) for 2 cr 4 days reduced medium levels of oncofetal FN (onfFN; i.e. FNs bearing an oncofetal epitope) to approximately 80% of control levels. Conversely, treatment of cytotrophoblasts isolated from term placentas with DEX dramatically reduced medium levels of onfFN to approximately 12% of control values. Treatment of both first trimester and term cells with 10(-6) M progestin, mineralocorticoid, or estrogen had no significant effect on onfFN expression in either cell type. Glucocorticoids specifically down-regulated medium levels of onfFN in term cells, but not in first trimester cells. In contrast, DEX treatment promoted an approximately 3- to 7-fold increase in levels of hCG in both first trimester and term cytotrophoblasts, suggesting that the effects of glucocorticoid on FN and hCG expression are elicited through independent cell-signaling pathways. In first trimester cells, DEX promoted a reduction in rates of FN and laminin synthesis to 60-70% of control levels. In term cells, DEX treatment reduced levels of FN and laminin synthesis to approximately 10% of control levels. Similarly, DEX treatment down-regulated levels of FN mRNA to approximately 60% and 10% of control values in first trimester and term cells, respectively. The first trimester of human pregnancy is associated with low levels of glucocorticoids and reduced glucocorticoid responsiveness. These conditions would favor high levels of placental ECM protein synthesis, thus stabilizing uterine-placental adherence. Conversely, elevated levels of glucocorticoids near parturition and increased glucocorticoid responsiveness would inhibit placental ECM protein synthesis, reducing uterine-placental adherence and promoting the separation of placenta from uterus.