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The pivotal role of protein inhibitors of activated STATs (PIAS) in regulating trophoblastic functions
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Abstracts / Journal of Reproductive Immunology 81 (2009) 113–175
By using IMAC copper protein chips for SELDITOF, 28 high signal proteins (>10 A) were detected exclusively in serum and 16 exclusively in FF. Further 4 proteins have been detected in almost all FF, but in only 13–35% of sera samples. Approximately 80% of GC expressed IGFBP5. The other genes were expressed less frequently. After 10 days of culture GC expressed up to 5 out of the analyzed genes, while GC from short term culture (< 4 days) did not express more than 2 genes. The combination of expressed genes was very variable and no preferential pattern could be detected yet. FF specific proteins may serve as a new tool for prognostic evaluation of follicle and oocyte quality with respect to fertilization and implantation. Presence of proteins which should be absent in FF may be used for blood contamination control or may indicate pathologic processes. Gene expression in individual GC is very inhomogenous. The level of variabilty or the dominance of specific patterns may become an indicator for follicle and oocyte quality. doi:10.1016/j.jri.2009.06.254 P46 The pivotal role of protein inhibitors of activated STATs (PIAS) in regulating trophoblastic functions L. Khachaturyan, T.G. Poehlmann, M. Weber, A.L.L. Forti, D.M. Morales, J.S. Fitzgerald, E. Schleussner, U.R. Markert Placenta-Labor, Department of Obstetrics, FriedrichSchiller-University, Jena, Germany Signal Transducer and Activator of Transcription 3 (STAT3) is an intracellular signaling molecule which, in trophoblastic cells, can be phosphorylated at its tyr705 (p-STAT3 tyr705) residue by Leukemia Inhibitory Factor (LIF) receptor binding. This phosphorylation correlates with invasiveness. Protein inhibitors of activated STATs (PIAS) antagonize STAT function by interfering with STAT DNA-binding and are, therefore, expected to regulate trophoblast invasion. We have analyzed PIAS1, PIAS3, STAT1, STAT3, Janus Kinase 1 (JAK1), nuclear Progesterone Induced Blocking Factor (PIBF) and SOCS3 spontaneous expression in the following 6 qualitatively different trophoblastic cell lines by gel electrophoresis and Western blotting: JEG-3 (choriocarcinoma), ACH-3P (hybrid of
JEG-3 and 1st trimester trophoblast), AC1-M59 (hybrid of JEG-3 and 3rd trimester trophoblast), HTR8/SVneo (extravillous trophoblast), JAR (choriocarcinoma), JAR SOCS3 (Suppressor of Cytokine Signaling 3)-silenced (JAR transfected with vector encoding small hairpin RNA for SOCS3 silencing). Freshly isolated 1st and 3rd trimester trophoblast cells have been analyzed for PIAS1 and PIAS3 expression. For intracellular localization immunoperoxidase staining of p-STAT3 tyr705 has been performed on JAR and JEG-3 cells. All analyzed cell lines express PIAS1, but JEG-3, ACH-3P, AC1-M59 as well as 1st and 3rd trimester trophoblast do not express detectable PIAS3. The same PIAS3 negative cell lines express significantly less STAT3-alpha/–beta, nPIBF and SOCS3, but significantly more STAT1 and JAK1. 1st and 3rd trimester trophoblast do not express PIAS3. Nuclear appearance of pSTAT3 is more pronounced in PIAS3 negative JEG-3 cells than in PIAS3 positive JAR cells. The presence or absence of PIAS3 correlates positively or negatively with a variety of intracellular molecules, which underlines its leading role in controling cellular functions. doi:10.1016/j.jri.2009.06.255 P47 Inhibition of ERK1/2 does not affect LIF-induced STAT3 ser727 phosphorylation in trophoblastic cells D.M. Morales, U.R. Markert Placenta-Labor, Department of Obstetrics, FriedrichSchiller-University, Jena, Germany RAS/MAPK is an intracellular signalling pathway, which is involved in the regulation of various cellular processes including proliferation and invasion in numerous cell types. ERK1/2 which is a member of this pathway, has been described controversially to interfere with signal transducer and activator of transcription 3 (STAT3) activation by phosphorylation of its serine residue. STAT3 is major inducer of trophoblast invasiveness. It has been demonstrated that Leukemia Inhibitory Factor (LIF) induces RAS and STAT3 activation. Here we have tested effects of LIF on ERK1/2 and STAT3 ser727 phosphorylation in trophoblastic cells. JEG-3 choriocarcinoma cells were stimulated with LIF and STAT3 ser727 and ERK1/2 phosphorylation were studied by Western blot analysis. The specific inhibitor U0126 that blocks ERK1/2 activation was used to ana-
Abstracts / Journal of Reproductive Immunology 81 (2009) 113–175
By using IMAC copper protein chips for SELDITOF, 28 high signal proteins (>10 A) were detected exclusively in serum and 16 exclusively in FF. Further 4 proteins have been detected in almost all FF, but in only 13–35% of sera samples. Approximately 80% of GC expressed IGFBP5. The other genes were expressed less frequently. After 10 days of culture GC expressed up to 5 out of the analyzed genes, while GC from short term culture (< 4 days) did not express more than 2 genes. The combination of expressed genes was very variable and no preferential pattern could be detected yet. FF specific proteins may serve as a new tool for prognostic evaluation of follicle and oocyte quality with respect to fertilization and implantation. Presence of proteins which should be absent in FF may be used for blood contamination control or may indicate pathologic processes. Gene expression in individual GC is very inhomogenous. The level of variabilty or the dominance of specific patterns may become an indicator for follicle and oocyte quality. doi:10.1016/j.jri.2009.06.254 P46 The pivotal role of protein inhibitors of activated STATs (PIAS) in regulating trophoblastic functions L. Khachaturyan, T.G. Poehlmann, M. Weber, A.L.L. Forti, D.M. Morales, J.S. Fitzgerald, E. Schleussner, U.R. Markert Placenta-Labor, Department of Obstetrics, FriedrichSchiller-University, Jena, Germany Signal Transducer and Activator of Transcription 3 (STAT3) is an intracellular signaling molecule which, in trophoblastic cells, can be phosphorylated at its tyr705 (p-STAT3 tyr705) residue by Leukemia Inhibitory Factor (LIF) receptor binding. This phosphorylation correlates with invasiveness. Protein inhibitors of activated STATs (PIAS) antagonize STAT function by interfering with STAT DNA-binding and are, therefore, expected to regulate trophoblast invasion. We have analyzed PIAS1, PIAS3, STAT1, STAT3, Janus Kinase 1 (JAK1), nuclear Progesterone Induced Blocking Factor (PIBF) and SOCS3 spontaneous expression in the following 6 qualitatively different trophoblastic cell lines by gel electrophoresis and Western blotting: JEG-3 (choriocarcinoma), ACH-3P (hybrid of
JEG-3 and 1st trimester trophoblast), AC1-M59 (hybrid of JEG-3 and 3rd trimester trophoblast), HTR8/SVneo (extravillous trophoblast), JAR (choriocarcinoma), JAR SOCS3 (Suppressor of Cytokine Signaling 3)-silenced (JAR transfected with vector encoding small hairpin RNA for SOCS3 silencing). Freshly isolated 1st and 3rd trimester trophoblast cells have been analyzed for PIAS1 and PIAS3 expression. For intracellular localization immunoperoxidase staining of p-STAT3 tyr705 has been performed on JAR and JEG-3 cells. All analyzed cell lines express PIAS1, but JEG-3, ACH-3P, AC1-M59 as well as 1st and 3rd trimester trophoblast do not express detectable PIAS3. The same PIAS3 negative cell lines express significantly less STAT3-alpha/–beta, nPIBF and SOCS3, but significantly more STAT1 and JAK1. 1st and 3rd trimester trophoblast do not express PIAS3. Nuclear appearance of pSTAT3 is more pronounced in PIAS3 negative JEG-3 cells than in PIAS3 positive JAR cells. The presence or absence of PIAS3 correlates positively or negatively with a variety of intracellular molecules, which underlines its leading role in controling cellular functions. doi:10.1016/j.jri.2009.06.255 P47 Inhibition of ERK1/2 does not affect LIF-induced STAT3 ser727 phosphorylation in trophoblastic cells D.M. Morales, U.R. Markert Placenta-Labor, Department of Obstetrics, FriedrichSchiller-University, Jena, Germany RAS/MAPK is an intracellular signalling pathway, which is involved in the regulation of various cellular processes including proliferation and invasion in numerous cell types. ERK1/2 which is a member of this pathway, has been described controversially to interfere with signal transducer and activator of transcription 3 (STAT3) activation by phosphorylation of its serine residue. STAT3 is major inducer of trophoblast invasiveness. It has been demonstrated that Leukemia Inhibitory Factor (LIF) induces RAS and STAT3 activation. Here we have tested effects of LIF on ERK1/2 and STAT3 ser727 phosphorylation in trophoblastic cells. JEG-3 choriocarcinoma cells were stimulated with LIF and STAT3 ser727 and ERK1/2 phosphorylation were studied by Western blot analysis. The specific inhibitor U0126 that blocks ERK1/2 activation was used to ana-