GnRH I and II up-regulate MMP-26 expression through the JNK pathway in human cytotrophoblasts

Abstract

Background: Matrix metalloproteinase-26 (MMP-26), one of the main mediators of extracellular matrix (ECM) degradation, has been shown to exist in trophoblasts of human placenta and to play a role in trophoblast cell invasion. However, little is known about the regulation of MMP-26 expression in human trophoblasts. Recently, gonadotropin-releasing hormone I (GnRH I) and GnRH II have been shown to regulate the expression of MMP-2, MMP-9/tissue inhibitor of metalloproteinases 1 (TIMP-1), and urokinase plasminogen activator (uPA)/plasminogen activator inhibitor (PAI) in human trophoblasts, suggesting that these two hormones may work as paracrine and/or autocrine regulators in modulating the activities of various protease systems at the feto-maternal interface. In this study, we determined the regulatory effects of GnRH I and GnRH II on the expression of MMP-26 in human immortalized cytotrophoblast-like cell line, B6Tert-1.

Methods: Real-time PCR was used to quantify mRNA levels of MMP-26 in human trophoblast-like cell line, B6Tert-1 and primary cultured cytotrophoblasts. Western blotting was used to characterize the expression of MMP-26 and the phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) in B6Tert-1 cells after treatment with GnRH I and GnRH II.

Results: We found that GnRH I increased MMP-26 expression in B6Tert-1 cells after 12 h of treatment at both the mRNA and protein level, while GnRH II increased MMP-26 expression beginning at 3 h of treatment. Treatment of GnRH I at 1 nM resulted in maximal increase of MMP-26 mRNA and protein levels, whereas GnRH II treatment at a concentration of 100 nM was required to induce maximal increase in MMP-26 expression. In addition, we demonstrated that the activation of JNK, but not ERK1/2, was required for GnRH I and II-stimulated MMP-26 production in B6Tert-1 cells and primary cytotrophoblasts.

Conclusions: These novel findings indicated that GnRH I and II could up-regulate MMP-26 expression through the JNK signaling pathway in human trophoblast-like/trophoblast cells.

Figure 1

The characteristics of B6Tert-1 cells. A, Western blotting analyses show the expression of GnRH receptor (GnRHR) in JEG-3 and B6Tert-1 cells. Mouse pituitary gonadotrope αT3-1 cells were used as the positive control. B, The B6Tert-1 cells were cultured in the presence of 100 nM GnRH I or GnRH II for 24 h, and their invasive capacities were analyzed by invasion assay. C, The expression of MMP-26 protein in JEG-3, B6Tert-1, primary cultured cytotrophoblast (CTB) cells. The data derived from at least three independent sets of experiments (mean ± SEM; n ≥ 3), and the statistic results are presented in the column graphs (*, P < 0.05 vs. control).

Figure 2

Time-dependent effects of GnRH I and GnRH II on MMP-26 expression in B6Tert-1 cells. A and B, The expression of MMP-26 mRNA (A) and protein (B) in B6Ter-1 cells cultured with GnRH I (100 nM) for 0, 3, 6, 12, 24 or 48 h. C and D, The expression of MMP-26 mRNA (C) and protein (D) in B6Ter-1 cells cultured with GnRH II (100 nM) for 0, 3, 6, 12, 24 or 48 h. Levels of mRNA for MMP-26 in each sample were normalized to the GAPDH at the corresponding time points. Levels of protein for MMP-26 were normalized to the corresponding β-actin. The results derived from both these analyses as well as those from at least three other sets of experiments were standardized to the 0 h control and are represented (mean ± SEM; n ≥ 3) in the bar graphs (*, P < 0.05 vs. 0 h control).

Figure 3

Different dosage effects of GnRH I and GnRH II on MMP-26 expression in B6Tert-1 cells. A and B, The expression of MMP-26 mRNA (A) and protein (B) in B6Ter-1 cells cultured with increasing concentrations of GnRH I (0, 1, 10, 100 nM or 1 μM) for 24 h. C and D, The expression of MMP-26 mRNA (C) and protein (D) in B6Ter-1 cells cultured with increasing concentrations of GnRH II (0, 1, 10, 100 nM or 1 μM) for 24 h. Levels of mRNA for MMP-26 in each sample were normalized to the corresponding GAPDH. Levels of protein for MMP-26 were normalized to the corresponding β-actin. The results derived from both these analyses as well as those from at least three other sets of experiments were standardized to the untreated control and are represented (mean ± SEM; n ≥ 3) in the bar graphs (*, P < 0.05 vs. untreated control).

Figure 4

Involvement of JNK activity in GnRH I and II-induced MMP-26 production. A, B6Tert-1 cells were stimulated with GnRH I or GnRH II (100 nM) for 5, 10 and 30 min, or the cells were left untreated as a control. Phosphorylation of ERK1/2 or JNK and the total amount of ERK1/2 or JNK were determined by Western blotting analyses. B. Following 30-minute pretreatment with PD98059 (10 μM, ERK1/2 inhibitor) or SP600125 (10 μM, JNK inhibitor), B6Tert-1 cells were treated with GnRH I or GnRH II (100 nM) for 24 h and the protein levels of MMP-26 analyzed by Western blotting analyses. Levels of protein for MMP-26 were normalized to the corresponding β-actin. The results derived from both these analyses as well as those from at least three other sets of experiments were standardized to the untreated control and are represented (mean ± SEM; n ≥ 3) in the bar graphs (*, P < 0.05 vs. bar a; #, P < 0.05 vs. bar b; &, P < 0.05 vs. bar c).

Figure 5

Regulatory effects of GnRH I and GnRH II on MMP-26 expression in primary cultured CTB cells. A, The expression of MMP-26 mRNA in CTB cells cultured with GnRH I (gray) or GnRH II (black) at concentrations of 0.1, 1, 10 and 100 nM for 24 h. B, Following 30-minute pretreatment with PD98059 (10 μM, ERK1/2 inhibitor) or SP600125 (10 μM, JNK inhibitor), CTB cells were incubated with GnRH I (gray) or GnRH II (black) at concentrations of 100 nM for 24 h and the mRNA levels of MMP-26 analyzed by Real-time PCR. Levels of mRNA for MMP-26 in each sample were normalized to the corresponding GAPDH. The results derived from both these analyses as well as those from at least three other sets of experiments were standardized to the untreated control and are represented (mean ± SEM; n ≥ 3) in the bar graphs (*, P < 0.05 vs. untreated control).