The effect of LIF on production of TIMP -1 and 2

1. The effect of LIFon production of TIMP-1 and 2 M. H. Özörnek, U. Koldovsky EUROFERTIL Reproductive Health Center, Istanbul, Turkey Dept. of OB/GYN, Heinrich Heine University, Düsseldorf, Germany

2. Introduction • Matrix metalloproteinases (MMP) play important roles in the invasion of the trophoblast cell into the maternal endometrium during placentation. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases (TIMP).

3. Introduction • Cytokines are understood to play an important role in embryo-maternal signalling • LIF is a paracrine factor that may locally regulate implantation • LIF receptor mRNA has been identified in oocyte, embryo, blastocyst as well as trophoblast

4. Introduction • LIF is beneficial for preimplantation embryos. • In the human endometrium expression of LIF reaches maximal levels around the time of expected implantation

5. Introduction • LIF could be detected in human follicular Özörnek MH, J Reprod Med 1999 • LIF produced locally in decidual tissue Nakajima S, Int J Mol Med, 2003

6. Introduction • LIF null female mice were infertile because of failure of blastocyst implantation Robb L, J Reprod Immunol 2002

7. Endometrium Trophoblast L I F + MMP INVASION • LIF induces uPA and MMP-9 • Harvey MB, et al., Hum Reprod 1995

8. Introduction • The choriocarcinoma cell lines JEG-3, Jar and BeWo have been widely used as models for the study of trophoblasts. • It has been demonstrated that the invasiveness of the human trophoblasts depends on the production of matrix metalloproteinases (MMP).

9. Introduction • The invasive behaviour of trophoblasts can be blocked by the inhibitors of metalloproteases. They are, secreted locally into the extracellular space, where they control the activity of MMP’s. • Tissue inhibitor of metalloprotease-1 and 2 (TIMP-1, TIMP-2) secreted by the endometrium and also by the human decidua.

10. Aim • The aim of this study is the evaluation of the effect of LIF on TIMP-1 and TIMP-2 production by the choricarcinoma cell lines.

11. Materials & methods • Choriocarcinoma cell lines JEG-3, Jar and BeWo were cultured with various LIF concentrations (5, 10, 20, 100, 200 ng/ml). • Cells cultured without cytokine served as control. • After a 24h culture period in 37°C + 5% CO2, all supernatants were stored at – 80°C.

12. Materials & methods • TIMP-1 and TIMP-2 levels were measured by commercial ELISA kits (Amersham pharmacia biotech, Germany). Minimal detectable value was 1.25ng/ml for TIMP-1 and 3.0 ng/ml for TIMP-2. • Differences were evaluated by one way ANOVA tests, with P < 0.05 as the level of statistical significance.

13. TIMP-1

14. TIMP-1

15. TIMP-2

16. TIMP-2

17. Results • LIF reduce the TIMP-2 levels significantly in all three cell lines, but reduce the TIMP-1 levels significantly only in cell line JEG-3.

18. Conclusion LIF reduces the TIMP-1 and TIMP-2 levels inchoriocarcinoma cell lines. It can be speculated that these cytokine supports the trophoblast implantation by blocking the tissue inhibitors of metalloproteases.

19. Conclusion Endometrium Trophoblast L I F + MMP TIMP - INVASION