Involvement of CXCL12/CXCR4 in the motility of human first-trimester endometrial epithelial cells through an autocrine mechanism by activating PI3K/AKT signaling

Abstract

Background: CXCL12(chemokine ligand 12, CXCL12) and its receptors CXCR4 are widely expressed in maternal-fetal interface and plays an adjust role in materno-fetal dialogue and immune tolerance during early pregnancy. This study aimed to evaluate the role and mechanism of self-derived CXCL12 in modulating the functions of human first-trimester endometrial epithelial cells (EECs) and to identify the potential protein kinase signaling pathways involved in the CXCL12/CXCR4's effect on EECs.

Methods: The expression of CXCL12 and CXCR4 in EECs was measured by using immunohistochemistry, immunofluorescence, real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The effects of EEC-conditioned medium (EEC-CM) and recombinant human CXCL12 (rhCXCL12) on EEC migration and invasion in vitro were evaluated with migration and invasion assays. In-cell western blot analysis was used to examine the phosphorylation of protein kinase B (AKT), extracellular regulated protein kinases (ERKs) and phosphatidylinositol 3-kinase (PI3K) after CXCL12 treatment.

Results: CXCL12 and CXCR4 were both expressed in human first-trimester EECs at the mRNA and protein level. Both EEC-CM and rhCXCL12 significantly increased the migration and invasion of EECs (P < 0.05), which could be blocked by neutralizing antibodies against CXCR4 (P < 0.05) or CXCL12 (P < 0.05), respectively. CXCL12 activated both PI3K/AKT and ERK1/2 signaling and CXCR4 neutralizing antibody effectively reduced CXCL12-induced phosphorylation of AKT and ERK1/2. LY294002, a PI3K-AKT inhibitor, was able to reverse the promotive effect of EEC-CM or rhCXCL12 on EEC migration and invasion.

Conclusions: Human first-trimester EECs promoted their own migration and invasion through the autocrine mechanism with CXCL12/CXCR4 axis involvement by activating PI3K/AKT signaling. This study contributes to a better understanding of the epithelium function mediated by chemokine and chemokine receptor during normal pregnancy.

Keywords: CXCL12/CXCR4; Endometrial epithelial cell (EEC); Maternal-fetal interface; Motility; Phosphorylation.

Fig. 1

CXCL12 and CXCR4 were both expressed in human first-trimester EECs in vivo. Expression of CXCL12 and CXCR4 proteins in human first-trimester endometrial epithelial cells (EECs) of decidual tissues was measured with immunohistochemical analysis. CXCL12 and CXCR4-specific brown-colored staining was clearly observed in the cytoplasma of EECs (n = 5). No specific staining was observed in the negative control. Human first-trimester villi were used as positive controls

Fig. 2

CXCL12 and CXCR4 were both expressed in primary human first-trimester EEC cultures in vitro. Immunofluorescence staining of both CXCL12 and CXCR4 protein was detected in the primary cultured human first-trimester endometrial epithelial cells (EECs). a: CXCL12 expression (red fluorescence) in EECs. b: CXCR4 expression (green fluorescence) in EECs. c: DAPI staining in EECs. d: Merged picture. Magnification: × 400

Fig. 3

Expression of CXCL12 and CXCR4 mRNAs in primary human first-trimester EECs. The expression of CXCL12 and CXCR4 mRNA in human first-trimester endometrial epithelial cells (EECs) was measured by real-time polymerase chain reaction (PCR). The levels of CXCL12 and CXCR4 mRNA were equal to the ratio of the absorbance of the target gene to that of GAPDH. The results showed the relative levels of CXCR4 and CXCL12 mRNA in 6 samples and the horizontal line represents the average mRNA level of CXCL12 and CXCR4 in EECs

Fig. 4

Primary human first-trimester EEC cultures spontaneously secreted CXCL12 in vitro. Spontaneous CXCL12 secretion in the supernatants of in vitro cultured human first-trimester endometrial epithelial cells (EECs) was measured at 24, 48 and 72 h with enzyme-linked immunosorbent assay (ELISA). It was shown that human first-trimester primary cultured EECs continuously secreted CXCL12 in vitro. Data are presented as the mean ± SD (n = 3)

Fig. 5

EEC-derived CXCL12 induced EEC migration by binding to CXCR4 a Exogenous CXCL12 significantly increased EEC migration in vitro, whereas neutralizing antibodies against CXCR4 or CXCL12 effectively inhibited the CXCL12-induced migration of EECs. b EEC-CM significantly increased EEC migration in vitro, and a neutralizing antibody against CXCR4 or CXCL12 effectively inhibited the EEC-CM-induced migration of EECs. c CXCR4 or CXCL12 blocking antibody alone could markedly inhibit the migration of EECs. * P < 0.05, ** P < 0.01 vs. control; # P < 0.05, ## P < 0.01, vs. CXCL12-treated or EEC-CM treated group. Data are presented as mean ± SD (n = 3). EEC: human first-trimester endometrial epithelial cell, EEC-CM: EEC conditioned culture medium

Fig. 6

EEC-derived CXCL12 increased EEC invasion by binding to CXCR4 a Exogenous CXCL12 significantly increased EEC invasion in vitro, whereas neutralizing antibody against CXCR4 or CXCL12 effectively inhibited the CXCL12-stimulated invasion of EECs. b EEC-CM significantly increased EEC invasion in vitro, and a neutralizing antibody against CXCR4 or CXCL12 effectively inhibited EEC-CM-induced invasion of EECs. c CXCR4 or CXCL12 blocking antibody alone markedly inhibited the invasion of EECs. * P < 0.05, ** P < 0.01, compared to the control; # P < 0.05, ## P < 0.01, compared to the CXCL12-treated or EEC-CM-treated group. Data are presented as mean ± SD. (n = 3). EEC: human first-trimester endometrial epithelial cell, EEC-CM: EEC conditioned culture medium

Fig. 7

CXCL12 stimulated the activation of Akt, PI3K and ERK in EECs. Human first-trimester endometrial epithelial cells (EECs) were serum starved for 12 h and then stimulated with CXCL12 (100 ng/ml). The phosphorylation of protein kinase B (AKT), extracellular regulated protein kinases (ERK) and phosphatidylinositol 3-kinase (PI3K) were evaluated with in-cell western blot analysis. CXCL12 (at 100 ng/ml) led to time-dependent increase in phosphorylation of AKT, PI3K and ERK1/2 in EECs. Phosphorylated proteins were stained in green and total proteins stained in red. The phosphorylated to total protein ratio was normalized to 1 in the untreated control (n = 3)

Fig. 8

CXCL12 stimulated the activation of Akt, PI3K, and ERK in EECs by binding to CXCR4. The phosphorylation of protein kinase B (AKT) and extracellular regulated protein kinases (ERK) in human first-trimester endometrial epithelial cells (EECs) was inhibited by treatment with CXCR4 neutralizing antibody, LY294002 (a PI3K/AKT blocker) or U0126 (a ERK1/2 blocker). Phosphorylated proteins were stained in green and total proteins were stained in red. The phosphorylated to total protein ratio was normalized to 1 in the untreated control (n = 3)

Fig. 9

CXCL12 stimulated the migration and invasion of EECs by activating AKT/ PI3K signaling. Exogenous CXCL12 significantly increased the migration and invasion of EECs in vitro, which effect was remarkably inhibited by neutralizing antibodies to CXCR4 or PI3K/AKT blocker (LY294002). The ERK1/2 blocker (U0126) failed to block the CXCL12-induced EEC migration or invasion. * P < 0.05, ** P < 0.01 vs. control; # P < 0.05, ## P < 0.01 vs. CXCL12-treated or EEC-CM-treated group. Data are presented as mean ± SD. (n = 3). EEC: human first-trimester endometrial epithelial cell. EEC-CM: EEC conditioned medium