MAPK regulate MMP-9 gene expression induced by IL-1β and TNF-α in cytotrophoblastic cells

Abstract

Objective: Matrix metalloproteinase (MMP) involved in trophoblast invasion. Many cytokines and growth factors can influence the invasion ability of trophoblast through regulating expression of MMP-9. Mitogen-activated protein kinase (MAPK) are a very important kinase superfamily of cellular signal trasduction. Our purpose is to study the role of MAPK including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and P38 pathway (P38) kinase in MMP-9 gene expression induced by cytokines (IL-1β and TNF-α in invasiveness of cytotrophoblastic (CTB) cells. Design: Cytotrophoblast cells were isolated from pooled first trimester placentae (7–9 week) using method established by Gacia et al (1994). Cytotrophoblast cells were grew in 10%FCS Dulbecco’s Modified Eagle Medium (DMEM). Fifth passage cells were used to do experiments. Materials/Methods: CTB cells at 2.5 × 104in 96-well plate were treated by IL-1β at 10ng /ml and TNF-α at 100ng/ml for 0min, 5min, 10min, 20min, 30min. Then ERK, JNK and P38 kinase activity assay were detected by cell ELISA in 96 well plate. The cells at 2 × 105in 6-well plate were treated by IL-1β at 10ng/ml and TNF-α at 100ng/ml for 24h. For inhibitor assay, CTB cells were treated by SB203580 (ERK inhibitor) at 10μM or/and PD098059 (P38 inhibitor) at 50μM for 30 min and then treated by IL-1β at 10ng/ml and TNF-α at 100ng/ml for 24h. All the treatment were performed in serum-free medium. General RNA was extracted from CBT cells and expression of MMP-9 gene was assayed by RT-PCR. The relative value of expression of MMP-9 was quantified from zymogrames of 3 experiments. Paired sample T-test was used for statistical analysis. Results: In CTB cells, expression of MMP-9 mRNA significantly increased after 24h treatment of IL-1β and TNF-α (p <0.05). JNK, ERK and P38 were activated on the stimulation of IL-1β and TNF-α at different time (p <0.05). Increasing expression of MMP-9 mRNA induced by IL-1β could be partly inhibited by specific inhibitor SB203580 or/and PD098059 for ERK and P38 (p <0.05). Increasing expression of MMP-9 mRNA induced by TNF-α could be down-regulated partly by specific inhibitor SB203580 or PD098059 for ERK and P38 (p <0.05). Increasing expression of MMP-9 mRNA induced by TNF-α could be down-regulated completely by combination of SB203580 and PD098059 for ERK and P38 (p <0.05). Conclusions: The expression of MMP-9 gene were significantly up-regulated by IL-1β and TNF-α in cytotrophoblast cell. JNK, ERK and P38 were activated on the stimulation of IL-1β and TNF-α, which acted on transcription factors to regulate cellular processes. Increasing expression of MMP-9 mRNA induced by IL-1β and TNF-α could be down-regulated by specific inhibitor for ERK and P38. ERK and P38 were important regulator of MMP-9 gene expression induced by IL-1β and TNF-α in cytotrophoblastic cell. Supported by: This work supported by Chinese National Key Basic Scientific Grant (Chinese 973 Project Grant).