The role of NUMB, a protein important for cellular differentiation and endocytosis in non-placental cells, was investigated in syncytiotrophoblast development and function in the human placenta. The BeWo choriocarcinoma cell line was used as a model for villous cytotrophoblast cells and syncytiotrophoblast to investigate NUMB’s involvement in differentiation and epidermal growth factor receptor (EGFR) endocytosis. NUMB isoforms 1 and 3 were found to be the predominant isoforms and were upregulated following forskolin-induced differentiation. Overexpression of NUMB isoforms 1 and 3 did not mediate differentiation or EGFR signaling. Immunofluorescence analysis revealed that NUMB colocalized with EGFR at perinuclear late endosomes and lysosomes following EGF stimulation. We have demonstrated for the first time that NUMB isoforms 1 and 3 are expressed in BeWo cells, are upregulated in forskolin-differentiated BeWo cells and are involved in ligand-dependent EGFR endocytosis in BeWo cells.

The fetal component of the human placenta is comprised of cells termed trophoblasts and the proper differentiation of these cells is pivotal for maintaining maternal and fetal health throughout pregnancy. The placental syncytiotrophoblast layer is a key secretory portion of the placenta and is produced through fusion of underlying progenitor cytotrophoblast cells with the multinucleated syncytiotrophoblast layer. The MacPhee laboratory has previously established that fusion or syncytialization of cytotrophoblasts is aided by expression of integrin-linked kinase (ILK), a cytoplasmic adapter protein. Nuclear enzyme Poly ADP-ribose Polymerase-1 (PARP-1) and the transcriptional repressor Snail-1 work with ILK to downregulate epithelial cell-cell adhesion. This process would also be necessary for proper trophoblast syncytialization. Thus, it was hypothesized that PARP-1 would be an important mediator of syncytiotrophoblast development. To test this hypothesis, immunofluorescence analysis of PARP-1 and Snail expression in human chorionic villi from first and second trimester as well as from term pregnancy was conducted. Furthermore, co-localization between PARP-1 and Snail-1 were evaluated. Lastly, human PARP-1 was overexpressed in the BeWo trophoblast derived cell line, under fusion promoting conditions, to directly test the ability of PARP-1 to regulate trophoblast fusion. Throughout the first and second trimester, PARP-1 and Snail were highly expressed and co-localized in villous cytotrophoblast nuclei. In contrast, PARP-1 was rarely detectable in syncytiotrophoblast nuclei, while Snail was highly expressed. Upon transient overexpression of PARP-1 in BeWo cells, fusion was robustly promoted. Furthermore, the mean number of nuclei per syncytium was markedly higher in PARP-overexpressing cells compared to control cells. However, PARP-1 overexpression did not regulate trophoblast hormonal differentiation. In conclusion, PARP-1 does appear to be a key enzyme in the process of trophoblast syncytialization. Lastly, a comparative analysis of PARP-1 was conducted in the mouse placenta. It was found that PARP-1 was highly detectable in nuclei of mononuclear trophoblast as well as the nuclei in the syncytiotrophoblast bilayer of the mouse labyrinth. Additionally, PARP-1 was localized to the nuclei of other trophoblast populations, including spongiotrophoblasts, trophoblast giant cell, and glycogen trophoblast giant cells, which are involved in the invasive pathway of trophoblast differentiation.

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The role of NUMB, a protein important for cellular differentiation and endocytosis in non-placental cells, was investigated in syncytiotrophoblast development and function in the human placenta. The BeWo choriocarcinoma cell line was used as a model for villous cytotrophoblast cells and syncytiotrophoblast to investigate NUMB’s involvement in differentiation and epidermal growth factor receptor (EGFR) endocytosis. NUMB isoforms 1 and 3 were found to be the predominant isoforms and were upregulated following forskolin-induced differentiation. Overexpression of NUMB isoforms 1 and 3 did not mediate differentiation or EGFR signaling. Immunofluorescence analysis revealed that NUMB colocalized with EGFR at perinuclear late endosomes and lysosomes following EGF stimulation. We have demonstrated for the first time that NUMB isoforms 1 and 3 are expressed in BeWo cells, are upregulated in forskolin-differentiated BeWo cells and are involved in ligand-dependent EGFR endocytosis in BeWo cells.

The aim of this project was to study ATP binding cassette (ABC) transporters in the human placenta, in particular their regulation and role in trophoblast differentiation and survival. The presence and localisation of four major placental drug transporters, multidrug resistance gene product 1 and 3 (MDR1 and 3)/ABC subfamily B members 1 and 4 (ABCB1 and 4), multidrug resistance associated proteins 1 and 2 (MRP1 and 2)/ABCC1 and 2 and breast cancer resistance protein (BCRP)/ABCG2 was initially studied in term human placenta, cultured primary trophoblast and BeWo and Jar trophoblast-like cell lines. Jar cells were found to be more similar to nondifferentiated cytotrophoblast with respect to their ABC protein expression profile, whereas BeWo cells more closely reflected differentiated syncytiotrophoblast. Treatment of primary term trophoblasts in vitro with cytokines (TNF- or IL-1) decreased expression and activity of apical transporters ABCB1/MDR1 and ABCG2/BCRP. Growth factors, on the other hand, increased BCRP expression and activity, while estradiol stimulated BCRP, MDR1 and MDR3 expression MDR1/3 functional activity. The ability of BCRP/ABCG2 to abrogate the apoptotic effects of TNF- and ceramides was studied in primary trophoblast and BeWo cells using pharmacological and molecular (siRNA) approaches. The results suggest that BCRP/ABCG2 contributes to the resistance of trophoblast cells to cytokine-induced (extrinsic) apoptosis, whereas its effects on apoptosis activated via the intrinsic mitochondrial pathway is minimal. This altered resistance was associated with increased intracellular accumulation of ceramides and reduced ability to maintain phosphatidylserine in the inner leaflet of the plasma membrane. A role for BCRP/ABCG2 in cell protection from differentiation-induced stressors was also demonstrated during the process of cell fusion associated with transient loss of plasma membrane lipid asymmetry. Finally, expression of BCRP/ABCG2 (and 9 other genes) was studied in 50 placentas from normal pregnancy and pregnancies complicated with fetal growth restriction (FGR). A marked reduction of BCRP/ABCG2 and MDR1/ABCB1 expression was observed in FGR placentas, while other transporter genes were unaffected. Collectively these data suggest that BCRP/ABCG2 and probably other ABC transporters may play a hitherto unrecognised survival role in the placenta, conferring a “stress resistance” to trophoblast cells.

The aim of this project was to study ATP binding cassette (ABC) transporters in the human placenta, in particular their regulation and role in trophoblast differentiation and survival. The presence and localisation of four major placental drug transporters, multidrug resistance gene product 1 and 3 (MDR1 and 3)/ABC subfamily B members 1 and 4 (ABCB1 and 4), multidrug resistance associated proteins 1 and 2 (MRP1 and 2)/ABCC1 and 2 and breast cancer resistance protein (BCRP)/ABCG2 was initially studied in term human placenta, cultured primary trophoblast and BeWo and Jar trophoblast-like cell lines. Jar cells were found to be more similar to nondifferentiated cytotrophoblast with respect to their ABC protein expression profile, whereas BeWo cells more closely reflected differentiated syncytiotrophoblast. Treatment of primary term trophoblasts in vitro with cytokines (TNF- or IL-1) decreased expression and activity of apical transporters ABCB1/MDR1 and ABCG2/BCRP. Growth factors, on the other hand, increased BCRP expression and activity, while estradiol stimulated BCRP, MDR1 and MDR3 expression MDR1/3 functional activity. The ability of BCRP/ABCG2 to abrogate the apoptotic effects of TNF- and ceramides was studied in primary trophoblast and BeWo cells using pharmacological and molecular (siRNA) approaches. The results suggest that BCRP/ABCG2 contributes to the resistance of trophoblast cells to cytokine-induced (extrinsic) apoptosis, whereas its effects on apoptosis activated via the intrinsic mitochondrial pathway is minimal. This altered resistance was associated with increased intracellular accumulation of ceramides and reduced ability to maintain phosphatidylserine in the inner leaflet of the plasma membrane. A role for BCRP/ABCG2 in cell protection from differentiation-induced stressors was also demonstrated during the process of cell fusion associated with transient loss of plasma membrane lipid asymmetry. Finally, expression of BCRP/ABCG2 (and 9 other genes) was studied in 50 placentas from normal pregnancy and pregnancies complicated with fetal growth restriction (FGR). A marked reduction of BCRP/ABCG2 and MDR1/ABCB1 expression was observed in FGR placentas, while other transporter genes were unaffected. Collectively these data suggest that BCRP/ABCG2 and probably other ABC transporters may play a hitherto unrecognised survival role in the placenta, conferring a “stress resistance” to trophoblast cells.

The aim of this project was to study ATP binding cassette (ABC) transporters in the human placenta, in particular their regulation and role in trophoblast differentiation and survival. The presence and localisation of four major placental drug transporters, multidrug resistance gene product 1 and 3 (MDR1 and 3)/ABC subfamily B members 1 and 4 (ABCB1 and 4), multidrug resistance associated proteins 1 and 2 (MRP1 and 2)/ABCC1 and 2 and breast cancer resistance protein (BCRP)/ABCG2 was initially studied in term human placenta, cultured primary trophoblast and BeWo and Jar trophoblast-like cell lines. Jar cells were found to be more similar to nondifferentiated cytotrophoblast with respect to their ABC protein expression profile, whereas BeWo cells more closely reflected differentiated syncytiotrophoblast. Treatment of primary term trophoblasts in vitro with cytokines (TNF- or IL-1) decreased expression and activity of apical transporters ABCB1/MDR1 and ABCG2/BCRP. Growth factors, on the other hand, increased BCRP expression and activity, while estradiol stimulated BCRP, MDR1 and MDR3 expression MDR1/3 functional activity. The ability of BCRP/ABCG2 to abrogate the apoptotic effects of TNF- and ceramides was studied in primary trophoblast and BeWo cells using pharmacological and molecular (siRNA) approaches. The results suggest that BCRP/ABCG2 contributes to the resistance of trophoblast cells to cytokine-induced (extrinsic) apoptosis, whereas its effects on apoptosis activated via the intrinsic mitochondrial pathway is minimal. This altered resistance was associated with increased intracellular accumulation of ceramides and reduced ability to maintain phosphatidylserine in the inner leaflet of the plasma membrane. A role for BCRP/ABCG2 in cell protection from differentiation-induced stressors was also demonstrated during the process of cell fusion associated with transient loss of plasma membrane lipid asymmetry. Finally, expression of BCRP/ABCG2 (and 9 other genes) was studied in 50 placentas from normal pregnancy and pregnancies complicated with fetal growth restriction (FGR). A marked reduction of BCRP/ABCG2 and MDR1/ABCB1 expression was observed in FGR placentas, while other transporter genes were unaffected. Collectively these data suggest that BCRP/ABCG2 and probably other ABC transporters may play a hitherto unrecognised survival role in the placenta, conferring a “stress resistance” to trophoblast cells.

The aim of this project was to study ATP binding cassette (ABC) transporters in the human placenta, in particular their regulation and role in trophoblast differentiation and survival. The presence and localisation of four major placental drug transporters, multidrug resistance gene product 1 and 3 (MDR1 and 3)/ABC subfamily B members 1 and 4 (ABCB1 and 4), multidrug resistance associated proteins 1 and 2 (MRP1 and 2)/ABCC1 and 2 and breast cancer resistance protein (BCRP)/ABCG2 was initially studied in term human placenta, cultured primary trophoblast and BeWo and Jar trophoblast-like cell lines. Jar cells were found to be more similar to nondifferentiated cytotrophoblast with respect to their ABC protein expression profile, whereas BeWo cells more closely reflected differentiated syncytiotrophoblast. Treatment of primary term trophoblasts in vitro with cytokines (TNF- or IL-1) decreased expression and activity of apical transporters ABCB1/MDR1 and ABCG2/BCRP. Growth factors, on the other hand, increased BCRP expression and activity, while estradiol stimulated BCRP, MDR1 and MDR3 expression MDR1/3 functional activity. The ability of BCRP/ABCG2 to abrogate the apoptotic effects of TNF- and ceramides was studied in primary trophoblast and BeWo cells using pharmacological and molecular (siRNA) approaches. The results suggest that BCRP/ABCG2 contributes to the resistance of trophoblast cells to cytokine-induced (extrinsic) apoptosis, whereas its effects on apoptosis activated via the intrinsic mitochondrial pathway is minimal. This altered resistance was associated with increased intracellular accumulation of ceramides and reduced ability to maintain phosphatidylserine in the inner leaflet of the plasma membrane. A role for BCRP/ABCG2 in cell protection from differentiation-induced stressors was also demonstrated during the process of cell fusion associated with transient loss of plasma membrane lipid asymmetry. Finally, expression of BCRP/ABCG2 (and 9 other genes) was studied in 50 placentas from normal pregnancy and pregnancies complicated with fetal growth restriction (FGR). A marked reduction of BCRP/ABCG2 and MDR1/ABCB1 expression was observed in FGR placentas, while other transporter genes were unaffected. Collectively these data suggest that BCRP/ABCG2 and probably other ABC transporters may play a hitherto unrecognised survival role in the placenta, conferring a “stress resistance” to trophoblast cells.

Proefschrift Universiteit van Amsterdam. / Met een samenvatting in het Nederlands.

Proefschrift Universiteit van Amsterdam. / Auteursnaam op omslag: Heleen Schuring-Blom. Met lit. opg. - Met samenvatting in het Nederlands.