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Cytokine-modulation of platelet-activating factor metabolism in human decidua during parturition
Placenta(1996),Vol. 17
A.36 Quantitation of Activin Receptor Type-I mRNA by competitive PCR in Human Trophoblast Cells. Victor h, Jessica Dy, Chun Peng, Peter C.K. Leung, Department of Obstetrics and Gynaecology, University of British Columbia, Canada. Activin has been shown to regulate production of severalhormones, such as gonadotropin-releasinghormone, human chorionic gonadotropin and progesterone, from the placenta. In an attempt to study the regulation of activin receptor gene expression in the placenta, we have establisheda competitive PCR assayto quantify the type I activin receptor (actR-I) mRNA levels. A DNA fragment of 698 bp was obtained from amplifying cDNA from term placenta using primers specific for ActR-I. An internal standard was created by deletion of this DNA fragment after it was cloned into a pDirect vector. To validate the competitive PCR assay, serial dilutions of the internal standard were co-amplified with cDNA reverse-transcribed from a constant amount of total RNA isolated from trophoblast cells and vice versa. A linear relationship was obtained when the internal standard/endogenous ActR-1 expressionratio was plotted against the amount of internal standard use. Competitive PCR assaysare currently being established and validated for the other ActR subtypes. (Supported by MRC.)
Cytokine-modulation of Platelet-activating Factor MetahoIism in Human Decidua during Parturition. H. Narahara.
Y. Tanaka, Y. Kawano, c Miyakawa, and .I. M. Johnston. Deuartmentof Obstetricsand Gvnecologv. Oita Medical ‘Unibersity, Oita, Japan, and -Depart%nt of Biochemistry, University of Texas Southwestern Medical Center, Dallas,Texas, USA.
To clarify the role of platelet-activating factor (PAF) in parturition and preterm labor, we have investigated PAF metabolism in the decidua and its modulation by endotoxin or cytokines. The decidual macrophage(M$) populations were obtained from human deciduaby enzymicdigestion, Ficoll-Paquecentrifugation, or flow cytometric sorting. The activity of a PAFinactivating enzyme, PAF-acetylhydrolase(PAF-AH) in the cultured media was assayedby the method of Miwa et al. Lipopolysaccaride (LPS), tumor necrosis factor-a (TNF-a), interleukin:lR (IL-l@, and IL-8 inhibited the PAF-AH secretionby decidual Ma, whereas macrophage colony stimulating factor (M-CSF) stimulated the enzyme secretion. The LPS-inhibition was partially reversed by IL-l receptor antagonist (IL-lra) or by neutralizing antibodiesagainstTNF-a and IL-113.The effects of TNFa, IL-lR, IL-8, and M-CSF were specificallyneutralized by the correspondingantibodies.The IL-l&inhibition was abolishedby IL-lra. It is suggestedthat M@in the decidua may play an important role -in PAF metabolism during parturition and that PAF is involved in the uathogenesisof preterm labor causedby chorioamnionitis. I -
Regulation of Trophoblast Differentiation by TGF-J3, and TGF& via Endoglin. I. Can&&, M. Letarte, M. Post, S.J.
Lye. SamuelLunenfeld ResInst, Dept. Ob/Gyn, Mount Sinai Hosp & Divs. Immunol. and Neonatol., HSC, Univ. Toronto. Transforming growth factor beta (TGF-B), a multifunctional growth factor, has been implicated in the regulation of trophoblast invasion. We used lSftrimester human placental villous explantsto investigatethe role of the different TGF-I3 isoforms and their receptors in the regulation of trophoblast differentiation. Antisense (A/S) TGF-Rl and TGF-I33 oligonucleotides (but not TGF-D2) stimulated extravillous trophoblast outgrowth and migration. A/S TGFBl and to a greater extent A/S TGF-03 stimulated FN production in villous explants.In contrastaddition of A/S TGFl32 decreased FN synthesis.Both exogenousTGF-RI and 133overcame the A/S TGF-Bl and I33stimulatoryeffect on FN production. We then investigated the role of TGF-Rreceptors on trophoblast differentiation. Immunohistochemical studies demonstrated that endoglin was highly expressedin the transition from polarized to non-polarized trophoblast during the first trimester of gestation, and weak immunoreactivity was detectedfor both receptor I (R-I) and II (R-II). A/S endoglin (but not A/S R-I and R-II) dramatically increased FN synthesis. The A/S endoglin stimulatory effect was temporally regulated as it was observed in explants of 4-7 weeks gestation and not thereafter. These data suggest that TGF-01 and I33 control the onset of trophoblast differentiation and that endoglin, in the early stages of trophoblast differentiation, is a critical component of the TGF-S receptor complex. Exyression Leukemia Troyhoblast
rind Localisation of the Receptor for Inhibitory Factor in Human and Decidua. A.M.Sharkev, A.King,
S. Verma, L. Hayes, D.E. Clark, D.S.CharnockJones, Y.W.Loke and S.K.Smith. Reproductive Department of Obstetrics and Gynaecology, University of Cambridge, Cambridge, U.K. Mice deleted for the gene encoding the LIF receptor (LIF-R) show abnormal growth and development of the placenta. This indicates that Leukemia Inhibitory Factor (LIF) plays an important role in placental development. The expression of LIF-R was examined in human trophoblast and decidua throughout pregnancy using in situ hybridisation and immunocytochemistry. LIF-R immunoreactivity and mRNA was localised in villous trophoblast throughout pregnancy, and on endothelial cells within the villi. In the decidua, extravillous trophoblast and glandular epithelium also exhibited strong LIF-R expression. However addition of recombinant human LIF did not affect 3H-thymidine incorporation by purified extravillous trophoblast. These results indicate that LIF is able to act on villous and extravillous trophoblast populations in the human placenta. The effects of LIF on troghoblast differentiation are currently under investigation.
A.36 Quantitation of Activin Receptor Type-I mRNA by competitive PCR in Human Trophoblast Cells. Victor h, Jessica Dy, Chun Peng, Peter C.K. Leung, Department of Obstetrics and Gynaecology, University of British Columbia, Canada. Activin has been shown to regulate production of severalhormones, such as gonadotropin-releasinghormone, human chorionic gonadotropin and progesterone, from the placenta. In an attempt to study the regulation of activin receptor gene expression in the placenta, we have establisheda competitive PCR assayto quantify the type I activin receptor (actR-I) mRNA levels. A DNA fragment of 698 bp was obtained from amplifying cDNA from term placenta using primers specific for ActR-I. An internal standard was created by deletion of this DNA fragment after it was cloned into a pDirect vector. To validate the competitive PCR assay, serial dilutions of the internal standard were co-amplified with cDNA reverse-transcribed from a constant amount of total RNA isolated from trophoblast cells and vice versa. A linear relationship was obtained when the internal standard/endogenous ActR-1 expressionratio was plotted against the amount of internal standard use. Competitive PCR assaysare currently being established and validated for the other ActR subtypes. (Supported by MRC.)
Cytokine-modulation of Platelet-activating Factor MetahoIism in Human Decidua during Parturition. H. Narahara.
Y. Tanaka, Y. Kawano, c Miyakawa, and .I. M. Johnston. Deuartmentof Obstetricsand Gvnecologv. Oita Medical ‘Unibersity, Oita, Japan, and -Depart%nt of Biochemistry, University of Texas Southwestern Medical Center, Dallas,Texas, USA.
To clarify the role of platelet-activating factor (PAF) in parturition and preterm labor, we have investigated PAF metabolism in the decidua and its modulation by endotoxin or cytokines. The decidual macrophage(M$) populations were obtained from human deciduaby enzymicdigestion, Ficoll-Paquecentrifugation, or flow cytometric sorting. The activity of a PAFinactivating enzyme, PAF-acetylhydrolase(PAF-AH) in the cultured media was assayedby the method of Miwa et al. Lipopolysaccaride (LPS), tumor necrosis factor-a (TNF-a), interleukin:lR (IL-l@, and IL-8 inhibited the PAF-AH secretionby decidual Ma, whereas macrophage colony stimulating factor (M-CSF) stimulated the enzyme secretion. The LPS-inhibition was partially reversed by IL-l receptor antagonist (IL-lra) or by neutralizing antibodiesagainstTNF-a and IL-113.The effects of TNFa, IL-lR, IL-8, and M-CSF were specificallyneutralized by the correspondingantibodies.The IL-l&inhibition was abolishedby IL-lra. It is suggestedthat M@in the decidua may play an important role -in PAF metabolism during parturition and that PAF is involved in the uathogenesisof preterm labor causedby chorioamnionitis. I -
Regulation of Trophoblast Differentiation by TGF-J3, and TGF& via Endoglin. I. Can&&, M. Letarte, M. Post, S.J.
Lye. SamuelLunenfeld ResInst, Dept. Ob/Gyn, Mount Sinai Hosp & Divs. Immunol. and Neonatol., HSC, Univ. Toronto. Transforming growth factor beta (TGF-B), a multifunctional growth factor, has been implicated in the regulation of trophoblast invasion. We used lSftrimester human placental villous explantsto investigatethe role of the different TGF-I3 isoforms and their receptors in the regulation of trophoblast differentiation. Antisense (A/S) TGF-Rl and TGF-I33 oligonucleotides (but not TGF-D2) stimulated extravillous trophoblast outgrowth and migration. A/S TGFBl and to a greater extent A/S TGF-03 stimulated FN production in villous explants.In contrastaddition of A/S TGFl32 decreased FN synthesis.Both exogenousTGF-RI and 133overcame the A/S TGF-Bl and I33stimulatoryeffect on FN production. We then investigated the role of TGF-Rreceptors on trophoblast differentiation. Immunohistochemical studies demonstrated that endoglin was highly expressedin the transition from polarized to non-polarized trophoblast during the first trimester of gestation, and weak immunoreactivity was detectedfor both receptor I (R-I) and II (R-II). A/S endoglin (but not A/S R-I and R-II) dramatically increased FN synthesis. The A/S endoglin stimulatory effect was temporally regulated as it was observed in explants of 4-7 weeks gestation and not thereafter. These data suggest that TGF-01 and I33 control the onset of trophoblast differentiation and that endoglin, in the early stages of trophoblast differentiation, is a critical component of the TGF-S receptor complex. Exyression Leukemia Troyhoblast
rind Localisation of the Receptor for Inhibitory Factor in Human and Decidua. A.M.Sharkev, A.King,
S. Verma, L. Hayes, D.E. Clark, D.S.CharnockJones, Y.W.Loke and S.K.Smith. Reproductive Department of Obstetrics and Gynaecology, University of Cambridge, Cambridge, U.K. Mice deleted for the gene encoding the LIF receptor (LIF-R) show abnormal growth and development of the placenta. This indicates that Leukemia Inhibitory Factor (LIF) plays an important role in placental development. The expression of LIF-R was examined in human trophoblast and decidua throughout pregnancy using in situ hybridisation and immunocytochemistry. LIF-R immunoreactivity and mRNA was localised in villous trophoblast throughout pregnancy, and on endothelial cells within the villi. In the decidua, extravillous trophoblast and glandular epithelium also exhibited strong LIF-R expression. However addition of recombinant human LIF did not affect 3H-thymidine incorporation by purified extravillous trophoblast. These results indicate that LIF is able to act on villous and extravillous trophoblast populations in the human placenta. The effects of LIF on troghoblast differentiation are currently under investigation.