Galectin-8 Contributes to Human Trophoblast Cell Invasion

Abstract

Galectins are a class of lectins that are extensively expressed in all organisms. Galectins are involved in a range of functions, including early development, tissue regeneration, cancer and inflammation. It has been shown that galectin-8 is expressed in the villous and extravillous trophoblast (EVT) cells of the human placenta; however, its physiological role in pregnancy establishment has not been elucidated. Taking these factors into account, we investigated the functional role of galectin-8 in HTR-8/SVneo cells-a human EVT cell line-and human primary cytotrophoblast cells isolated from a first-trimester placenta. We analyzed the effects of recombinant human galectin-8 (rh galectin-8) on the adhesion, migration and invasion of HTR-8/SVneo cells. We used qPCR, cell-based ELISA (cELISA) and gelatin zymography to study the effects of galectin-8 on mediators of these processes, such as integrin subunits alpha-1 and beta-1 and matrix metalloproteinases (MMPs)-2 and -9, on the mRNA and protein levels. Further, we studied the effects of galectin-8 on primary cytotrophoblast cells' invasion. Galectin-8 stimulated the adhesion, migration and invasion of HTR-8/SVneo cells, as well as the invasion of primary cytotrophoblasts. In addition, the MMP-2 and -9 levels were increased, while the expression of integrins alpha-1 and beta-1 was not affected. Galectin-8 has the ability to positively affect EVTs' invasion, so it can be considered a significant factor in the trophoblast cell invasion process.

Keywords: MMPs; extravillous trophoblast; galectin-8; integrins; invasion.

Figure 1

(a) The effects of rh galectin-8 on HTR-8/SVneo cell viability. Results are presented as percentages of untreated control values (mean + SEM). The experiment was performed four times with six replicates each. (b) The effects of rh galectin-8 (50, 100 and 200 ng/mL) on the HTR-8/SVneo cell cycle. Results are presented as the percentages of cells in the sub-G1, G0/G1, S, G2/M phases of the cell cycle. The experiments were performed three times in three replicates. (c) Representative graphs of cell cycle analysis showing percentages of various phases of the cell cycle for the control and each galectin-8 treatment group (50, 100 and 200 ng/mL). * p < 0.05 and ** p < 0.01.

Figure 2

(a) Representative images of comet assay categories: A—no DNA damage, B—low, C—medium, D—high and E—total DNA damage. (b) The effect of exogenously added rh galectin-8 on the DNA damage of HTR-8/SVneo cells, as determined using the comet assay. Results are presented as the percentage of cells with damaged DNA (mean + SEM). C = control, CP = control with hydrogen peroxide (H₂O₂). The experiments were performed three times with two replicates each. **** p < 0.0001.

Figure 3

The effect of exogenously added rh galectin-8 on HTR-8/SVneo cell adhesion. Adhesion was examined on (a) plastic, (b) Matrigel and (c) collagen type I-coated surfaces after 2 h incubation at 37 °C at 5% CO₂. The experiment was performed three times in triplicate. Results are presented as percentages of untreated control values = C (mean + SEM). L = lactose (0.1 M). * p < 0.05 and ** p < 0.01.

Figure 4

The effects of rh galectin-8 with and without lactose on (a) HTR-8/SVneo cell migration. Experiments were performed three times in duplicate. The effects of exogenously added rh galectin-8 on (b) HTR-8/SVneo and (c) primary cytotrophoblast cell invasion. C = control, L = lactose (0.1 M). Results are presented as percentages of untreated control values (mean + SEM). The experiments were repeated three times in duplicate. * p < 0.05, ** p < 0.01.

Figure 5

The effects of rh galectin-8 on integrin subunits (a) alpha-1 and (b) beta-1 at the mRNA level. The results are presented as relative mRNA expression levels from three different experiments in duplicate (mean + SEM). C = control. The effects of exogenously added rh galectin-8 on HTR-8/SVneo integrin (c) alpha-1 and (d) beta-1 subunit protein expression using a cell-based ELISA (cELISA) assay. Results are presented as percentages of untreated control values (mean + SEM). The experiment was performed 4 times with at least four replicates each. C = control.

Figure 6

The effects of rh galectin-8 on (a) MMP-2 and (b) MMP-9 relative mRNA expression. The results are presented as a fold change in mRNA expression from three different experiments in duplicate (mean + SEM). C = control. The effects of exogenously added rh galectin-8 on (c) MMP-2 and (d) MMP-9 levels. Results are presented as percentages of untreated cell control values (mean + SEM). C = control. The experiments were performed at least three times in duplicate. * p < 0.05, ** p < 0.01.