NOVEL ROLE AND REGULATION OF UTERINE TIMPS

以下建议基于以下假设：TIMP-1和TIMP-3在生殖周期期间在子宫组织中显示特定的表达模式，这些表达模式由雌二醇和/或孕酮调节，并且TIMP-3的类固醇诱导的表达至少部分地通过TIMP-1介导。 为了检验这一假设，提出了两个具体目标。 具体目标1中的实验旨在检查内源性和外源性雌二醇-17 β和孕酮对子宫TIMP- 1和TIMP-3表达模式的影响，以及检查在这些激素环境下TIMP-1基因产物破坏对子宫TIMP-3表达的影响。 为了实现这一目标，将从成熟的、生殖周期的、类固醇致敏的、未成熟的雌性TIMP-1缺陷型和野生型小鼠获得子宫，并将检查TIMP-1和TIMP-3 mRNA定位和表达。 在发情周期（成熟小鼠）或暴露于外源性类固醇（未成熟小鼠）期间，将使用双重原位杂交将雌激素和孕激素受体的表达与TIMP-1和TIMP-3在特定子宫细胞类型内的表达共定位。 然后使用这些相同动物的子宫组织进行北方分析，以提供上述参数的定量分析。因此，特定目标1中概述的研究将使我们能够定性和定量确定类固醇和TIMP-1对子宫中类固醇受体与TIMP-1和TIMP-3之间时空关系的影响。 在具体目标2中，将使用细胞培养模型评估子宫TIMP-1和TIMP-3的类固醇调节机制，以及类固醇受体和TIMP- 1在该过程中的参与。从TIMP-1缺陷型和野生型小鼠获得子宫基质细胞，用雌二醇-17 β和孕酮处理细胞，通过北方分析定量雌激素和孕酮受体与TIMP-1和TIMP-3之间的时空关系。 为了进一步支持TIMP-1作为TIMP-3表达调节剂的作用，将从TIMP-1缺陷小鼠获得的基质细胞用外源性TIMP-1处理以恢复在野生型小鼠中检测到的TIMP-3表达的正常模式。 总之，这些实验将提供重要的信息，TIMP的调节和功能的机制，在小鼠子宫。 这些实验是及时和独特的，因为它们结合了一种新的模型来检查TIMP-1的功能，并提供了有关TIMP-1新描述功能的信息，这将对子宫和TIMP生物学领域产生广泛影响。

The following proposal is based upon the hypothesis that TIMP-1 and TIMP-3 display specific patterns of expression in uterine tissue during the reproductive cycle, that these patterns of expression are modulated by estradiol and/or progesterone, and that the steroid-induced expression of TIMP-3 is mediated at least in part through TIMP-1. To test this hypothesis, two specific aims are proposed. The experiments in Specific Aim 1 are designed to examine the effect of endogenous and exogenous estradiol-17beta and progesterone on the pattern of uterine TIMP- 1 and TIMP-3 expression as well as examine the effect of disruption of the TIMP-1 gene product on uterine TIMP-3 expression under these hormonal milieus. To accomplish this goal, uteri will be obtained from mature, reproductively cycling, and steroid-primed, immature female TIMP-1 deficient and wild- type mice and TIMP-1 and TIMP-3 mRNA localization and expression will be examined. Dual in-situ hybridization will be used to co- localize the expression of estrogen and progesterone receptors with that of TIMP-1 and TIMP-3 within specific uterine cell-types during the estrous cycle (mature mice) or during exposure to exogenous steroids (immature mice). Northern analysis will then be performed using uterine tissues from these same animals to provide quantitative analysis of the aforementioned parameters. As such, the studies outlined in Specific Aim 1 will allow us to qualitatively and quantitatively determine the effect of steroids and TIMP-1 on the spatiotemporal relationship among the steroid receptors and TIMP-1 and TIMP-3 in the uterus. In Specific Aim 2, the mechanisms for steroidal regulation of uterine TIMP-1 and TIMP-3, as well as the involvement of steroid receptors and TIMP- 1 in this process, will be assessed using a cell culture model. Uterine stromal cells will be obtained from TIMP-1 deficient and wild-type mice, cells will be treated with estradiol-17beta and progesterone, and the spatiotemporal relationship among estrogen and progesterone receptors and TIMP-1 and TIMP-3 will be quantitated by Northern analysis. To further support the role of TIMP-1 as a regulator of TIMP-3 expression, stromal cells obtained from TIMP-1 deficient mice will be treated with exogenous TIMP-1 to restore the normal pattern of TIMP-3 expression detected in the wild-type mice. Taken together, these experiments will provide important information on the mechanisms of TIMP regulation and function in the mouse uterus. These experiments are timely and unique as they incorporate a novel model to examine the functions of TIMP-1, as well as provide information on a newly-described function of TIMP-1 which will have broad impact on the fields of uterine and TIMP biology.