Role of the proteasome in the regulation of fetal fibronectin secretion in human placenta.

The goal of the current study was to examine the role of the ubiquitin-proteasome system (UPS), a pathway of intracellular degradation, in the regulation of fetal fibronectin (FFN) expression in human placenta. Primary cultures of cytotrophoblasts (CTs) and placental mesenchymal cells (PMCs) were isolated from human term placentas and were maintained in serum-free medium (SFM) in the presence of inhibitors of proteasome-mediated degradation (e.g., MG132) as well as inhibitors of other proteases. Levels of secreted FFN and interleukin (IL)-8 in culture media were quantitated by enzyme-linked immunosorbent assay (ELISA), and cell viability was assessed by trypan blue exclusion. Intracellular levels of FFN and ubiquinated proteins were measured by Western blotting, and levels of fibronectin mRNA were determined following Northern blotting. We found that proteasome inhibitors (MG132, MG262, and PSI) potently suppressed levels of secreted FFN in cultures of CTs, but they not did affect levels of IL-8. Lysosomal, calpain, and serine protease inhibitors as well as the anti-inflammatory compound sulfasalazine did not markedly affect levels of secreted FFN in CT cultures. Proteasome inhibitors did not compromise cell viability during the initial 16-18 hours of treatment and did not affect intracellular levels of FFN protein or fibronectin mRNA. The efficacy of suppression of FFN in CT culture media by proteasome inhibitors reflected their effects on intracellular accumulation of ubiquinated proteins. By contrast, the presence of proteasome inhibitors did not alter levels of secreted FFN in cultures of PMCs. We conclude that inhibitors of proteasome-mediated degradation potently and specifically suppressed extracellular expression of FFN in CTs through a cell type-specific pathway that did not involve alterations in FFN synthesis. This suggests that accumulation of ubiquinated proteins in the presence of proteasome inhibitors blocks FFN secretion or promotes the extracellular degradation of FFN. This experimental paradigm will be useful for dissecting the role of the UPS in regulating CT function.

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.

The role of placental Fas ligand in maintaining immune privilege at maternal-fetal interfaces.

It is now recognized that immunosuppressive factors synthesized by placenta may play a critical role in the maintenance of pregnancy. Over the last several years our group and others have formulated a hypothesis that trophoblast Fas ligand (FasL) plays an important role in maintaining fetal immune privilege in human pregnancy by actively promoting apoptosis (programmed cell death) of activated maternal lymphocytes bearing Fas (i.e., the FasL receptor). This review initially provides background information and updates aspects of the Fas/FasL signaling system, including the role of caspases and molecules recruited to the Fasl/Fas signaling complex and the revised functions ascribed to membrane and soluble forms of FasL. Information is then presented concerning the role of FasL at immune-privileged sites including the eye and testis. Pathways through which the placenta and tumors avoid may avoid immune clearance vis-à-vis the FasL/Fas signaling cascade are described. A model is then presented through which FasL production by human syncytiotrophoblasts and extravillous trophoblasts may protect the fetus against the cytolytic actions of activated Fas-bearing maternal lymphocytes in the intervillous space and in the placental bed, respectively. We conclude with a review of studies in support this model that specifically demonstrate trophoblast expression of FasL and identify potential lymphocyte targets (i.e., Fas-expressing maternal immune cells) of trophoblast FasL.

Glucocorticoid effects in the human placenta: evidence that dexamethasone-mediated inhibition of fibronectin expression in cytotrophoblasts involves a protein intermediate.

Oncofetal fibronectin is an extracellular matrix protein that is suggested to play an important role in regulating adherence at uterine-placental interfaces. The purpose of the present study was to elucidate a mechanism through which glucocorticoids (GCs) inhibit the synthesis of FN in human placenta as part of their matrix-suppressive action near parturition. We observed that treatment of cytotrophoblasts isolated from human term placentas for 48 h with 10(-7) mol/L dexamethasone (DEX) down-regulated levels of FN expression to 13-19% of control levels in immunoprecipitation, Northern blotting, and enzyme-linked immunosorbent assay experiments. Conversely, GC treatment increased FN expression in placental fibroblasts to 164-310% of control levels in Northern blotting and enzyme-linked immunosorbent assay procedures, suggesting that GC-mediated suppression of FN expression is specific to cytotrophoblasts. Results indicated that the DEX-mediated suppression of FN expression in cytotrophoblasts was not mediated through changes in the stability of FN messenger ribonucleic acid (mRNA). Run-on transcription assays using isolated nuclei suggested that GC treatment did not markedly affect transcription of the FN gene in cytotrophoblasts. To test whether the GC-mediated suppression of FN expression was mediated through a protein intermediate, levels of FN mRNA were examined by Northern blotting in cells treated for 48 h with and without 10(-7) mol/L DEX and cycloheximide (CHX; 125 ng/mL). We observed that CHX treatment increased FN expression in DEX-treated cells to 91% of control values. We noted that whereas the presence of 100-300 ng/mL CHX reversed the DEX-mediated inhibition of FN mRNA expression in cytotrophoblasts, it did not alter the overall rates of protein synthesis in DEX-treated and control cells. These data suggest that suppression of FN mRNA expression by GC in cytotrophoblasts requires de novo protein synthesis and is mediated through a short lived intermediate, the synthesis of which is inhibited at low concentrations of CHX. Thus, GC-induced protein intermediates may influence uterine-placental adherence by modulating levels of oncofetal FN at sites of uterine-placental contact.

Apoptosis and Fas expression in human fetal membranes.

Apoptosis (i.e. programmed cell death) plays a key role in maintaining reproductive function in the ovary, mammary and prostate glands, uterus, and testis. The purpose of the present report was to determine, based on biochemical and morphological parameters, whether cells in human fetal membranes undergo apoptosis and express Fas (CD95), a cell surface receptor that mediates apoptosis. Using the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling immunohistochemical technique, apoptotic nuclei were identified in amnion epithelial, chorionic trophoblast, and decidua parietalis cell layers of human fetal membranes at term. Electron microscopy of fetal membranes revealed ultrastructural characteristics in amnion epithelium and chorion trophoblast cell layers consistent with apoptosis, including condensation of chromatin along the periphery of the nucleus and nuclear shrinkage. The apoptotic index (percentage of terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling-positive nuclei of the total nuclei) ranged from 8-29% in amnion epithelial, chorionic trophoblast, and decidual cell layers from women at 23-30, 31-36, and 37-42 weeks gestation. The apoptotic index was statistically greater in the 37-42 week group than in the 23-30 week group in chorionic trophoblast (P < 0.05) and decidual cell (P < 0.01) layers. In contrast, the apoptotic index in the amnion epithelial cell layer was statistically greater (P < 0.05) in the 23-30 week group than in the 31-36 week group, suggesting that apoptosis may be independently regulated in amnion epithelial, chorionic trophoblast, and decidual cell types. Based on the importance of Fas in mediating apoptosis, we investigated whether Fas was expressed by human fetal membrane cells. Immunohistochemical staining of fetal membranes with anti-Fas antibody localized Fas in amnion epithelial, chorionic trophoblast, and decidua parietalis cell layers. A 266-bp band corresponding to the cytoplasmic domain of Fas was detected in samples of amnion, chorion, decidua, and placenta by RT-PCR. Northern blotting revealed a molecular weight of approximately 1.9 kilobases for Fas messenger ribonucleic acid in amniotic tissue. These data suggest that apoptosis and Fas signaling may play a role in remodeling of fetal membrane architecture across gestation.

[Oral and parenteral immunization with HIV immunosome induce the secretion of IgA specific of HIV-1 in the salivary and the production of circulatory IgA in mice and rabbits]. / Une immunisation orale et parentérale avec l'immunosome-VIH induit une sécrétion d'IgA spécifique du VIH-1 dans la salive et la production d'IgA circulants chez la souris et le lapin.

Given the sexual transmission of HIV, the establishment of a genital mucosal immunity through secretory IgA may be necessary to achieve protection. We have investigated if repeated stimulations of oral mucosa with HIV-Immunosomes would lead to the production of secretory IgA in saliva and also, if such an oral immunization could prime the immune system to an early systemic immune response following a parenteral immunisation with a low dose of the antigen. HIV-1 gp 160-specific secretory IgA were detected in the saliva of all rabbits orally immunized with HIV-Immunosomes. As early as one week after the parenteral immunization, high titers of serum IgA, IgM and IgG were detected both in mice and rabbits that had been orally stimulated with the antigen. These antibodies could neutralize HIV infectivity in vitro. Animals that were immunized only parenterally showed a very weak humoral immune response.

[Role of liposomes in the presentation of HIV envelope glycoprotein and the immune response in mice]. / Rôle des liposomes dans la présentation de la glycoprotéine de l'enveloppe du VIH et la réponse immunitaire chez la souris.

The role of antigen presentation in the induction of humoral as well as cell-mediated immune responses has been investigated by anchoring HIV-1 envelope glycoprotein (gp 160/120) into the phospholipidic bilayer of preformed liposomes to produce HIV-Immunosomes. HIV-Immunosomes induced high titres of HIV-specific antibodies when tested by ELISA, IFA and neutralization, whereas equal amounts of purified glycoprotein alone produced lower antibody response. Similarly, HIV-Immunosomes induced antigen-specific Interleukin-2 production and blastogenic response upon restimulation with the same antigen, in animals vaccinated with HIV-Immunosomes, whereas no secondary response was observed in animals vaccinated with equal amounts of purified gp 160/120. Taken together, these results underline the importance of antigen presentation in the establishment of an adequate immune status and show the potential of HIV-Immunosomes as vaccine against AIDS.