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Activation of Ras, Raf-1 and protein kinase C in differentiating human neuroblastoma cells after treatment with phorbolester and NGF
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
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2001 (English)In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 13, no 2, 95-104 p.Article in journal (Refereed) Published
Abstract [en]

The human neuroblastoma cell line SH-SY5Y/TrkA differentiates in vitro and acquires a sympathetic phenotype in response to phorbolester (activator of protein kinase C, PKC) in the presence of serum or growth factors, or nerve growth factor (NGF). We have now investigated to what extent phorbolester and NGF cause activation of Ras and Raf-1 and the involvement of PKC in this response in differentiating SH-SY5Y/TrkA cells. NGF stimulated increased accumulation of Ras-GTP and a threefold activation of Raf-1. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect on the amount of Ras-GTP but led to a smaller activation of Raf-1. NGF caused a limited increase in phosphorylation of Raf-1 compared with TPA, and NGF-induced Raf activity was independent of PKC. Analysis of phosphorylation of the endogenous PKC substrate myristoylated alanine-rich C-kinase substrate (MARCKS), and of subcellular distribution of PKC-alpha, -delta, and -epsilon revealed that NGF only caused a very small activation of PKC in SH-SY5Y/TrkA cells. The results identify Raf-1 as a target for both TPA- and NGF-induced signals in differentiating SH-SY5Y/TrkA cells and demonstrate that signalling to Raf-1 was mediated via distinct mechanisms.

Place, publisher, year, edition, pages
2001. Vol. 13, no 2, 95-104 p.
National Category
Clinical Medicine
Identifiers
URN: urn:nbn:se:uu:diva-241454DOI: 10.1016/S0898-6568(00)00141-8PubMedID: 11257453OAI: oai:DiVA.org:uu-241454DiVA: diva2:779095
Available from: 2015-01-12 Created: 2015-01-12 Last updated: 2017-12-05Bibliographically approved
In thesis
1. The Effects of SSRI Treatment on Human Placenta and Embryo
Open this publication in new window or tab >>The Effects of SSRI Treatment on Human Placenta and Embryo
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During pregnancy, 4 - 7% of women suffer from major depressive disorder. When antidepressive treatment is needed, selective serotonin reuptake inhibitors (SSRIs) are the most commonly used. Although severe complications from SSRI treatment are rare, association with a number of adverse pregnancy and fetal outcomes has been found. Also, antenatal depression per se has been shown to affect pregnancy outcomes. The overall aim of this thesis was to examine the effects of SSRIs on human placenta and embryo.

In the first study, gene expression was investigated in placenta from depressed, SSRI-treated and healthy pregnant women, using microarray analysis. Antenatal depression and SSRI treatment induced alterations in gene expression, but only 20 genes in common were noted. Validation with qRT-PCR showed that six out of seven selected genes were altered in SSRI-treated women compared with controls, and two genes were altered between depressed women and controls.

In study two, the protein levels in placenta from depressed, SSRI-treated and healthy pregnant women were investigated, focusing on the NGF signaling pathway. NGF, phosphorylated Raf-1, ROCK2 and phosphorylated ROCK2, were altered in both SSRI-treated and depressed women, although the proteins were regulated differently in the two groups.

In the third study, human embryos were treated with fluoxetine. Embryo development and protein expression were studied. Fluoxetine had some effect on the timing of embryo developmental stages. Also, several proteins were uniquely found in fluoxetine-treated embryos compared with untreated embryos. Fluoxetine also altered the levels of proteins secreted from the embryo.

In the fourth study, the human neuroblastoma cell line SH-SY5Y/TrkA was treated with TPA and NGF. The activation of Raf-1 was investigated and the involvement of Ras and PKC was studied. Both NGF and TPA activated Raf-1, but to a different extent and via different pathways. The NGF-induced activation of Raf-1 was mediated via Ras, while TPA induced signaling via PKC.

In conclusion, SSRI treatment and antenatal depression influence placental gene and protein expression. These findings may affect placental development and function, which in turn could affect fetal development. Also, direct exposure of embryos to fluoxetine has some effects on embryo development and protein expression, which may affect the development of the fetus.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 90 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1095
Keyword
antenatal depression, embryo, embryo development, fluoxetine, gene expression, NGF, placenta, protein expression, Raf-1, ROCK, signaling pathways, SSRI
National Category
Obstetrics, Gynecology and Reproductive Medicine
Research subject
Medical Science; Obstetrics and Gynaecology
Identifiers
urn:nbn:se:uu:diva-248527 (URN)978-91-554-9227-4 (ISBN)
Public defence
2015-05-28, Sal IV, Universitetshuset, Uppsala, 09:15 (Swedish)
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Supervisors
Available from: 2015-05-06 Created: 2015-03-31 Last updated: 2015-07-07

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Söderholm, HelenaOlsson, Anna-Karin

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