Novel function of pregnancy-associated plasma protein A: promotes endometrium receptivity by up-regulating N-fucosylation

Abstract

Glycosylation of uterine endometrial cells plays important roles to determine their receptive function to blastocysts. Trophoblast-derived pregnancy-associated plasma protein A (PAPPA) is specifically elevated in pregnant women serum, and is known to promote trophoblast cell proliferation and adhesion. However, the relationship between PAPPA and endometrium receptivity, as well as the regulation of N-fucosylation remains unclear. We found that rhPAPPA and PAPPA in the serum samples from pregnant women or conditioned medium of trophoblast cells promoted endometrium receptivity in vitro. Moreover, rhPAPPA increased α1,2-, α1,3- and α1,6-fucosylation levels by up-regulating N-fucosyltransferases FUT1, FUT4 and FUT8 expression, respectively, through IGF-1R/PI3K/Akt signaling pathway in human endometrial cells. Additionally, α1,2-, α1,3- and α1,6-fucosylation of integrin αVβ3, a critical endometrium receptivity biomarker, was up-regulated by PAPPA, thereby enhanced its adhesive functions. Furthermore, PAPPA blockage with antibody inhibited embryo implantation in vivo, mouse embryo adhesion and spreading in vitro, as well as N-fucosylation level of the endometrium in pregnant mice. In summary, this study suggests that PAPPA is essential to maintain a receptive endometrium by up-regulating N-fucosylation, which is a potential useful biomarker to evaluate the receptive functions of the endometrium.

Figure 1

PAPPA promotes human endometrial cell receptivity. HEC-1A, Ishikawa and RL95-2 cell monolayers were differently pre-treated as indicated before CMFDA-stained JAR cells (green) were plated. (A,C,E) Attached JAR cells were photographed after 1 h under a fluorescenct microscope, (B,D,F), the respective adhesion rate was calculated as the percentage of attached JAR cells. (A) HEC-1A, Ishikawa and RL95-2 cells were pre-treated with different doses of rhPAPPA (1 ng/ml and 10 ng/ml) for 48 h. (C) HEC-1A cells were pre-incubated with non-pregnancy serum (NS) (a–c), pregnancy serum (PS) (d–f), and threatened abortion serum (TS) (g–i) in the absence (a,d,g), or presence of anti-PAPPA (b,e, h) and anti-PAPPA plus rhPAPPA (c,f,i). (E) HEC-1A cells were pre-incubated with conditional medium (CM) from JAR cells (a–c), CM after JAR cells were treated with progesterone (100 μM) (P4 CM) (d–f), CM after JAR cells were treated with progesterone (100 μM) plus RU486 (10 μM) (P4 + R CM) (g–i) in the absence (a,d,g), or presence of anti-PAPPA (b,e,h) and anti-PAPPA plus rhPAPPA (c,f,i) before JAR cells were added. *p < 0.05, **p < 0.01, ***p < 0.001. The bar represents 100 μm. The data were presented as the mean ± SEM of three independent experiments.

Figure 2

rhPAPPA promotes endometrial cell receptivity by increasing N-fucosylation. (A,B) UEA-1, LTL or LCA (5 μg/ml) was incubated in control or rhPAPPA pre-treated HEC-1A and Ishikawa cells, and the adhesion rate was analyzed. (C) Lectin flourescent staining and (D) Lectin blotting detected the levels of α1,2-, α1,3- and α1,6-fucosylation in HEC-1A and Ishikawa cells after rhPAPPA treatment. The bar represents 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. The data were presented as the means ± SEM of three independent experiments.

Figure 3

rhPAPPA up-regulates the expression of FUT1, FUT4 and FUT8 in HEC-1A cells. HEC-1A cells were treated with rhPAPPA (1 ng/ml and 10 ng/ml) for 48 h or rhPAPPA (10 ng/ml) for 24 h, 48 h and 72 h, respectively. (A,B) q-PCR detected the mRNA levels of FUT1, FUT4 and FUT8. (C,D) Immunofluorescent staining detected the cellular localization and (E,F) Western blotting analyzed the protein levels of FUT1, FUT4 and FUT8. GAPDH was used as an internal control. The bar represents 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. The data were presented as the means ± SEM of three independent experiments.

Figure 4

rhPAPPA rescues the impaired receptivity through up-regulating specific N-fucosylation. Ishikawa cells were differently transiently transfected with siRNA specific to FUT1, FUT4, FUT8 or scrambled control in the absence or presence of rhPAPPA. (A) Western blotting was performed to detect the expression of FUT1/4/8. (B) Lectin blotting and (C) Lectin fluorescent staining showed the specific N-fucosylation level. (D) Adhesion rate of JAR cells to FUT1/4/8 siRNA-transfected Ishikawa cells in the absence or presence of rhPAPPA, UEA-1, LTL or LCA, respectively, followed by analysis. GAPDH was used as an internal control. The bar represents 50 μm. **p < 0.01,***p < 0.001. The data were presented as the means ± SEM of three independent experiments.

Figure 5

rhPAPPA stimulates the expression of FUT1/4/8 through the IGF-1R/PI3K/Akt signaling pathway. Ishikawa cells were differently treated with rhPAPPA, AG1024, LY294002. (A) Westerrn blotting showed the expression of IGF-1R, p-IGF-1R (Tyr¹¹³¹), Akt, p-Akt (Tyr³⁰⁸) and p-Akt (Ser⁴⁷³). (B,C) Western blotting analyzed the molecules of the IGF-1R/PI3K/Akt signaling pathway and FUT1/4/8 expression. (D) Immunofluorescent staining of FUT1/4/8. DAPI (blue) was used for nuclear staining. GAPDH was used as an internal control. The bar represents 50 μm. The data were presented as the means ± SEM of three independent experiments.

Figure 6

rhPAPPA promotes endometrium receptivity by up-regulating the specific fucosylation of integrin αVβ3. (A) Adhesion of JAR cells to Ishikawa cells pre-treated without (a) untreated control, or with (b) IgG, (c) anti-αV, (d) anti-β3, (e) anti-αVβ3, (f) rhPAPPA + IgG, (g) rhPAPPA + anti-αVβ3, (h) rhPAPPA + anti-αVβ3 + UEA-1, (i) rhPAPPA + anti-αVβ3 + LTL, (j) rhPAPPA + anti-αVβ3 + LCA. (B) Adhesion rate of JAR cells to pre-treated Ishikawa as indicated is shown in the histogram. (C) Integrin αVβ3 was immunoprecipitated from the whole-protein lysate of untreated control and rhPAPPA-treated Ishikawa cells. The specific N-fucosylation of αVβ3 was detected by Western blotting. αV and β3 were detected to show the loading protein amount. Input showed the efficiency of immunoprecipitation. (D) Immunofluorescence and Lectin staining detected the expression and cellular localization of the specific fucosylation and αVβ3 after rhPAPPA treatment. Green, α1,2-, α1,3-, or α1,6-fucosylation; red, αVβ3; yellow (overlay), co-staining of α1,2-, α1,3-, or α1,6-fucosylation with αVβ3. DAPI (blue) was used for nuclear staining. The bar represents 50 μm. **p < 0.01, ***p < 0.001. The data were presented as the means ± SEM of three independent experiments.

Figure 7

PAPPA blockade inhibits embryo implantation in vivo, mouse embryo adhesion and spreading in vitro, as well as the specific N-fucosylation of pregnant mouse endometrium. Mouse uterine horns were injected with IgG or anti-PAPPA at pregnant day 3 (PD3). Mice were ethically scarified at PD8, and (A) the implanted embryos were photographed (a, IgG; b, no treatment; c, IgG; d, anti-PAPPA). (B) The number of implanted embryos was analyzed and is shown in the histogram. Mouse embryos were collected at PD4. Embryos were co-cultured with mouse primary endometrial cells. (C) The same embryo was photographed at 0 h, 24 h and 48 h, representing the floating, adhering and spreading status, respectively. IgG or anti-PAPPA was added to the co-culturing medium. (D) The adhesion rate was analyzed and shown in the histogram after co-culturing for 24 h. (E) 48 h later, endometrial cells and embryos were stained with CMFDA and were photographed. (F) The histogram showed the relative spreading area. Total proteins or tissues of the endometrium were harvested at PD4. (G) Lectin flourescent staining and (H) Lectin blotting detected the specific N-fucosylation of endometrium (I) Western blotting showed the expression of FUT1, FUT4 and FUT8. GAPDH was used as an internal control. The bars represent 100 μm. *p < 0.05, **p < 0.01. The data were presented as the means ± SEM of three independent experiments.