Notch-dependent RBPJκ inhibits proliferation of human cytotrophoblasts and their differentiation into extravillous trophoblasts.
Abnormal development of invasive trophoblasts has been implicated in the pathogenesis of human pregnancy diseases such as pre-eclampsia. However, critical signalling pathways controlling formation and differentiation of these cells have been poorly elucidated. Here, we provide evidence that the canonical Notch pathway, operating through Notch-dependent activation of its key regulatory transcription factor RBPJκ, controls proliferation and differentiation in villous explant cultures and primary trophoblasts of early pregnancy. Immunofluorescence of first trimester placental tissue revealed expression of RBPJκ and its co-activators, the MAML proteins, in nuclei of proliferative cell column trophoblasts (CCT) and differentiated, extravillous trophoblasts (EVTs). However, RBPJκ expression, transcript levels of the Notch target gene HES1 and activity of a Notch/RBPJκ-dependent luciferase reporter decreased during in vitro differentiation of primary cytotrophoblasts on fibronectin. Silencing of RBPJκ using silencing RNAs (siRNAs) increased proliferation of CCTs in floating villous explant cultures analysed by outgrowth and BrdU labelling. Similarly, down-regulation of the transcription factor enhanced BrdU incorporation in isolated primary cultures. However, motility of these cells was not affected. In addition, gene silencing of RBPJκ increased cyclin D1 expression in the two trophoblast model systems as well as markers of the differentiated, EVT, i.e. integrin α1, ADAM12 and T-cell factor 4. In summary, the data suggest that Notch-dependent RBPJκ activity could be required for balanced rates of trophoblast proliferation and differentiation in human placental anchoring villi preventing exaggerated trophoblast overgrowth as well as premature formation of EVTs.
Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1090, Austria.
P
P
Velicky
Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1090, Austria.
S
S
Haider
Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1090, Austria.
G R
GR
Otti
Gynmed Clinic, Vienna, Austria.
C
C
Fiala
Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1090, Austria.
J
J
Pollheimer
Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1090, Austria martin.knoefler@meduniwien.ac.at.
M
M
Knöfler
D051722:ADAM Proteins / Q000378:metabolism; D000072199:ADAM12 Protein; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation / Q000502:physiology; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000378:metabolism*; D039001:Integrin alpha Chains / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D060888:Real-Time Polymerase Chain Reaction; D051880:Receptors, Notch / Q000378:metabolism*; D058118:Transcription Factor 7-Like 2 Protein / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism
D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D039001:Integrin alpha Chains; D008565:Membrane Proteins; C497570:RBPJ protein, human; D051880:Receptors, Notch; C544894:TCF7L2 protein, human; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors; D051722:ADAM Proteins; D000072199:ADAM12 Protein; C000606480:ADAM12 protein, human
Notch signalling; RBPJκ; cytotrophoblast; placenta; proliferation