REGULATION OF EXPRESSION OF Timp1 AND Timp3 mRNA IN RAT GRANULOSA CELLS DURING THE PERIOVULATORY PERIOD

In the ovary, the matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) have been postulated to play a critical role in extracellular matrix remodeling associated with ovulation as well as luteal formation and regression. We have observed by in situ hybridization that an LH stimulus results in an increase in granulosa cell expression of Timp1 and Timp3 mRNA. In the present study, we investigated the regulatory mechanisms controlling mRNA expression of Timp1 and Timp3 in vitro using preovulatory granulosa cells. Granulosa cells were isolated from immature 23 day old rat ovaries at 48h after eCGpriming (10 IU). The cells were cultured in the absence or presence of LH (1 IU/ml) for 0, 2, 4, 8, 12, 16 or 24h (N=3 except where noted). Timp1 expression was highest at 4h after LH addition and then decreased gradually over the remaining 24h of culture. LH induced a biphasic increase of Timp3 expression at 2h (early response) and 16h (late response). A drop of Timp3 mRNA levels occurred at 8h after LH treatment in luteinizing granulosa cells. The stimulatory effect of LH on Timp1 and Timp3 expression was blocked by inhibitors for PKA (H89) and PKC (GF109203X), demonstrating that Timp1 and Timp3 expression was mediated by the LH-induced activation of these signaling mediators. In addition, cyclohexamide blocked the LH-induced Timp3 mRNA expression, indicating the requirement of new protein synthesis for Timp3 expression, whereas cyclohexamide had no effect on Timp1 expression. To further explore Timp1 and Timp3 regulation, cells were cultured with the progesterone receptor antagonist RU486, the prostaglandin-endoperoxide synthase 2 inhibitor NS-398, or the EGF receptor tyrosine kinase selective inhibitor (AG1478). Timp1 and Timp3 expression was not affected by NS- 398. RU486 had no effect on Timp1 expression but reduced Timp3 late mRNA expression which suggested progesterone involvement in the regulation of Timp3 expression induced at 16h. AG1478 could block Timp1 and Timp3 early response but had no effect on Timp3 late response(N=2), indicating EGF receptor activation was involved in Timp1 and early Timp3 mRNA expression at 2h. Timp1 and Timp3 have been reported to be multifunctional other than their classically described roles as MMP inhibitors. They may act as autocrine/paracrine factors in ovarian processes involving celluar proliferation, differentiation, or steroidogenesis. The differential regulation between Timp1 and Timp3 may correlate with their unique roles in the processes of ovulation and luteinization. (Supported by NIH RR15592 and AG17164) (poster)