MUC1 is involved in trophoblast transendothelial migration

Abstract

The factors that regulate trophoblast invasion of the uterine vasculature are incompletely understood. In this paper we show that macaque trophoblasts express the mucin, MUC1, and that it is involved in trophoblast-endothelial interaction. Immunocytochemistry, Western blotting and RT-PCR analyses confirmed that MUC1 was expressed by isolated early gestation macaque trophoblasts. MUC1 was also detected in endovascular trophoblasts in sections of placental-decidual tissue during early gestation. A blocking antibody against MUC1 reduced trophoblast adhesion to uterine endothelial cells and also blocked trophoblast transendothelial migration. MUC1 is known to bind to Intercellular Adhesion Molecule-1 (ICAM-1) in other systems. Incubation in the presence of a blocking antibody against Intercellular Adhesion Molecule-1 (ICAM-1) or recombinant ICAM-1 modestly, but significantly, reduced transendothelial trophoblast migration. These results are consistent with the idea that MUC1 is involved in trophoblast adhesion to uterine endothelial cells and in trophoblast transendothelial migration.

Fig. 1. Immunofluorescence detection of MUC1 in isolated macaque trophoblasts

Trophoblast cells were cultured for 24 h then stained with CT2 (A) or B27.29 (E) anti-MUC1 antibodies and viewed by immunofluorescence microscopy (see Methods). The immunofluorescence images C and G show cells incubated with the corresponding control immunoglobulins. The images in the right-hand column are the respective matching phase contrast views. The horizontal bar represents 50 μm. The images are typical representatives from four separate experiments.

Fig. 2. Immunofluorescence detection of MUC1 in macaque placental/decidual tissue

Serial sections of paraffin-embedded placental/decidual tissues were stained with antibodies against MUC1 (CT2), PECAM-1, or cytokeratin as described in Methods. The results of incubation with control immunoglobulin for the anti-MUC1 antibody are also illustrated. Panel A-D shows a typical large uterine gland. Panel E-H shows villous tissue. Panel I-L shows an invaded artery. Panel M-O shows a different invaded vessel at higher magnification. Panel P-S shows a non-invaded vessel. The arrow indicates endothelium stained with PECAM-1. The asterisks indicate the location of the corresponding vessel lumen in adjacent sections. The bars represent 50 μm.

Fig. 3. Analysis of MUC1 expression in trophoblasts by Western blot and RT-PCR

(A) Trophoblast lysates and human Jar choriocarcinoma cell lysates were examined by Western blot using the CT2 anti-MUC1 antibody (see Methods). A representative blot is shown. (B) Total RNA was extracted from macaque trophoblast cultures and reverse transcribed. The resulting cDNA was used as a template for PCR using primers directed against MUC1 as described in Methods. PCR products were analyzed on 2% agarose gels. DNA standards (Std; left lane) were included. The middle lane shows that no band was produced in the absence of reverse transcription (RT-). The right lane (RT+) shows a prominent band of the predicted size (207 bp). The band was excised from the gel and sequenced. The sequence data confirmed identity with MUC1.

Fig. 4. Effect of anti-MUC1 antibody on trophoblast adhesion to endothelial cells

Trophoblasts were labeled with the fluorescent cell tracer CFDA-SE and added to confluent endothelial (UtMVEC) monolayers as described in Methods. Adhesion was measured after 2 h. For some experiments trophoblasts were pre-incubated with anti-MUC1 antibody or with recombinant human ICAM-1 (rICAM-1). In other experiments the endothelial cells were pre-incubated with anti-ICAM-1 or anti-E-selectin (E-sel). Adhesion is expressed as a percentage of the initial input trophoblasts and values are means ± SEMs from 3 experiments. Data were analyzed by ANOVA with Dunnett’s Multiple comparison post-test. Values marked with an asterisk were significantly different from the control (p<0.01).

Fig. 5. Effect of anti-MUC1 antibody on trophoblast transendothelial migration

Trophoblasts were labeled with the fluorescent cell tracer CFDA-SE and added to confluent endothelial monolayers cultured on TransWell FluorBlok inserts. Transendothelial migration of trophoblasts to the lower chamber was measured at different time points as described in Methods. For some experiments trophoblasts were pre-incubated with anti-MUC1 antibody or with recombinant human ICAM-1 (rICAM-1) and then added to the endothelial cells without washing. In other experiments the endothelial cells were pre-incubated with anti-ICAM-1 antibody which was maintained in the culture medium throughout the experiment. Trophoblast migration is expressed as a percentage of the initial input trophoblasts and values are means ± SEMs from 3-4 experiments. Data were analyzed by non-linear regression with an F test. The results are described in the text. Data were also evaluated by 2-way ANOVA with a Bonferroni post-test. The asterisks indicate values that are significantly different (p<0.05) from the corresponding control value.