Steroidal Regulation and Localization of the Matrix Metalloproteinase Inhibitor Reversion-Inducing, Cysteine-Rich Protein with Kazal Motifs (RECK) Within the Mouse Uterus.

Matrix metalloproteinase-9 (MMP-9) has been shown to play a vital role in regulating the biological events which occur within the uterus from the time of post-natal development up to and including the reproductive competence of this organ. Because this MMP imparts its biological function through tissue remodeling as well as modulation of angiogenic and proliferative pathways, it is not surprising that aberrant expression and activity of MMP-9 is associated with uterine diseases such as endometriosis, dysfunctional uterine bleeding and endometrial cancer. As such, developing a thorough understanding of the factors which regulate MMP-9 expression and activity within the uterus and how these factors impact normal and abnormal uterine biology is of paramount importance. Regulation of MMP-9 occurs at the tissue level via binding with specific MMP inhibitors which include TIMP-1 and TIMP-3. Recently, RECK (reversion-inducing cysteine-rich protein with Kazal motifs) has been identified as a major regulator of MMP-9 (and MMP-2) activity. Further, RECK expression has been inversely correlated with MMP activity in a variety of carcinomas. Surprisingly, there is no information on the expression, regulation and role of this MMP inhibitor within the uterus under either normal or pathological conditions. As such, the objective of the current study was to examine uterine expression and regulation of RECK. To accomplish this objective, mature female mice (N=4/treatment group) were ovariectomized and treated with estrogen, progesterone or a combination of both steroids. Mice were sacrificed at 0, 4, 8 and 24 h after steroid administration as well as after three consecutive doses of steroids, 24 hours apart (72 h group). RECK mRNA and protein were localized by in-situ hybridization and immunohistochemistry, respectively, while whole tissue levels were respectively quantitated by qRT-PCR and Western analysis. MMP-9 activity was assessed by gelatin zymography and a gelatinase activity assay. Uterine RECK mRNA and protein expression were highest in untreated mice and significantly (P<0.05) decreased in response to estrogen administration at all time points. Progesterone alone had no effect on RECK expression while the estrogen induced down-regulation could be partially blocked by progesterone at 24 h and 72 h. Pre-treatment with ICI 182,780 and RU-486 confirmed mediation of these effects through cognate estrogen and progesterone receptor pathways, respectively. Cellular localization experiments revealed that RECK mRNA and protein were localized primarily to luminal and glandular epithelial cells and their level of expression was regulated in a similar fashion as in whole tissue by the steroids. As expected, estrogen induced MMP-9 activity in a time-dependent manner and RECK mRNA and protein expression were inversely correlated with protease activity and this estrogenic effect could be inhibited by progesterone co-administration. In summary, these studies demonstrate for the first time that RECK is expressed within the mouse uterus, its expression is regulated by steroids and its pattern of expression is inversely correlated with MMP-9 activity. We conclude from these studies that RECK may be an important steroid-regulated inhibitor of MMP-9 within the uterus.