ABSTRACT
Macrophage-colony stimulating factor (M-CSF, CSF-1) is secreted by human trophoblasts as well as endometrial cells, while its receptor c-fms is abundantly expressed by the extravillous trophoblasts anchoring the placenta to the uterus, suggesting a role for M-CSF at the maternal-fetal interface. We investigated the effect of M-CSF on the expression of fibronectin and its receptor, the alpha5 integrin, in human trophoblasts. Exposure of trophoblasts to M-CSF produced a two to three-fold increase in fibronectin and alpha5 mRNA abundance at both 24 and 72 hours of culture. A dose-dependent increase in cellular fibronectin secretion into the culture medium was detected at both time points. Immunocytochemistry showed co-localization of cellular fibronectin and alpha5 in the cells, suggesting that attachment of trophoblasts to fibronectin is mediated in part by the alpha5beta1 integrin. We conclude that M-CSF increases fibronectin expression and secretion by the human trophoblasts, and up-regulates its specific receptor, the alpha5 integrin. We hypothesize that M-CSF may partake in the autocrine/paracrine mechanisms regulating trophoblast invasion during implantation.
Subject(s)
Antigens, CD/genetics , Fibronectins/genetics , Gene Expression Regulation , Macrophage Colony-Stimulating Factor/pharmacology , Trophoblasts/metabolism , Antigens, CD/analysis , Cells, Cultured , Embryo Implantation , Female , Fibronectins/analysis , Fluorescent Antibody Technique, Indirect , Humans , Integrin alphaV , Pregnancy , RNA, Messenger/biosynthesis , Trophoblasts/chemistryABSTRACT
Osteopontin (OPN), a matrix glycosylated phosphoprotein, has been proposed to play a role(s) in basic cellular processes, such as neovascularization and tissue remodeling, which are essential to placental morphogenesis and embryo implantation. We have shown OPN to be expressed by cytotrophoblasts of the chorionic villus, and a putative progesterone regulatory element in the OPN promoter suggests hormonal regulatory control. This led us to test the hypothesis that progesterone regulates OPN expression in human cytotrophoblasts. Cytotrophoblasts isolated from human placentas were treated with combinations of progesterone, RU486, and/or aminoglutethimide, and their expression of OPN was assessed by Northern hybridization and immunocytochemistry. The expression of OPN messenger RNA (mRNA) declined as trophoblasts aggregated, but rebounded at later times when syncytia and mononuclear cytotrophoblasts coexisted in culture. Progesterone increased OPN mRNA expression by aggregating mononuclear cytotrophoblasts. Aminoglutethimide suppression of endogenous steroidogenesis by syncytiotrophoblasts inhibited OPN expression, whereas the addition of exogenous progesterone to cells treated with aminoglutethimide reversed this inhibitory effect. These observations were confirmed at the protein level by immunocytochemistry. Treatment of cytotrophoblasts with both progesterone and RU486 inhibited the up-regulatory effect on OPN mRNA associated with exposure to progesterone alone, further confirming a direct effect of progesterone. We conclude that progesterone up-regulates OPN expression in human cytotrophoblasts, and we propose that in vivo, progesterone secretion by syncytiotrophoblasts regulates the expression of OPN by the underlying cytotrophoblasts. As the receptors for OPN, alpha(v) integrins, are expressed by syncytiotrophoblasts, we postulate that these paracrine regulatory mechanisms contribute to the adhesive and/or signaling events between the two trophoblast cell types of the chorionic villus.
Subject(s)
Placenta/physiology , Progesterone/pharmacology , Sialoglycoproteins/biosynthesis , Sialoglycoproteins/genetics , Trophoblasts/metabolism , Aminoglutethimide/pharmacology , Blotting, Northern , Cell Communication/physiology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Chorionic Villi/chemistry , Female , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Mifepristone/pharmacology , Osteopontin , Pregnancy , RNA, Messenger/analysis , RNA, Messenger/genetics , Sialoglycoproteins/physiology , Trophoblasts/cytology , Trophoblasts/physiologyABSTRACT
OBJECTIVE: Decay-accelerating factor (DAF) is a complement regulatory protein that prevents complement-mediated cell lysis. Its expression in human trophoblasts depends on their anatomic location and gestational age. The mechanisms that govern the regulation of its expression in trophoblasts are not understood. The objective of the present study was to investigate the effects of trophoblast differentiation and cyclic adenosine monophosphate (cAMP) on DAF expression. METHODS: Human trophoblasts were isolated from chorionic villi, cultured, and harvested at timed intervals for total RNA extraction and Northern analysis. Expression of DAF was also assessed by immunocytochemistry. In some cultures, the trophoblasts were exposed to the cAMP agonists 8-bromo-cAMP and Sp-cAMPs or the antagonist Rp-cAMPs. RESULTS: Expression of DAF mRNA increased as the cells differentiated in culture, with the 2.2-kb transcript of membrane-bound DAF appearing first. As differentiation proceeded, the 1.5-kb transcript became the predominant mRNA form. Exposure of the cells to Rp-cAMPs delayed this process; 8-bromo-cAMP accelerated it. Sp-cAMPs selectively up-regulated the 2.2-kb mRNA transcript. Immunocytochemistry confirmed the effects of differentiation on DAF protein expression. CONCLUSION: Expression of DAF in human trophoblasts is dependent on the state of cell differentiation, and cAMP is an intracellular modulator of this process. This effect may be mediated through alternative processing of DAF mRNA in its 3'UT region, which in turn affects mRNA stability.
