Role of STAT signaling and autocrine action of chemokines during H2O2 induced HTR-8/SVneo trophoblastic cells invasion

During pregnancy, regulated generation of reactive oxygen species (ROS) is important for activation of signaling pathways and placentation. In the current study, the effect of H2O2 on invasion of HTR-8/SVneo cells, a human extravillous trophoblast cell line, is investigated. Treatment of HTR-8/SVneo cells for 24 hr with H2O2 (25 mu M) leads to a significant increase in invasion without affecting cell proliferation, viability, and apoptosis. Concomitantly, a significant increase in the matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinases-1 (TIMP-1) ratio is observed. Further, significant increase in phosphorylation of signal transducer and activator of transcription 1 (STAT-1) and STAT-3 (both at ser727 residue) is observed on treating HTR-8/SVneo cells with 25 mu M of H2O2 accompanied by an increase in the secretion of interleukin-8 (IL-8) and macrophage inflammatory protein-1 beta (MIP-1 beta). A significant decrease in H2O2-mediated invasion of HTR-8/SVneo cells and reduced expression of IL-8 and MIP-1 beta accompanied by decrease in MMP-9/TIMP-1 ratio are observed on inhibiting STAT-1 and STAT-3 by small interfering RNA (siRNA). Inhibition of STAT-1 activity by fludarabine and STAT-3 activity by Stattic also leads to a decrease in H2O2-mediated increase in HTR-8/SVneo cell invasion. Inhibition of IL-8 and MIP-1 beta by siRNA also leads to a significant decrease in both basal and H2O2-mediated invasion. Interestingly, inhibition of MIP-1 beta by siRNA leads to a significant reduction in H2O2-mediated increase in IL-8. However, no significant effect of IL-8 silencing on H2O2-mediated MIP-1 beta expression was observed. From the above results, it can be concluded that H2O2 activates STAT signaling, MIP-1 beta & IL-8 secretion and increases MMP-9/TIMP-1 ratio leading to an increased invasion of HTR-8/SVneo cells without affecting their viability.