Matrix metalloproteinase-26 (MMP-26) and Tissue inhibitor of metalloproteinases-4 (TIMP-4) in normal, hyperplastic, and malignant endometrium

Sammanfattning: The aim of the thesis was to study matrix metalloproteinase-26 (MMP-26) and tissue inhibitor of metalloproteinases-4 (TIMP-4) in normal, hyperplastic, and malignant endometrium. Most importantnly, both these molecules may be ivolved in the implantation process. Trophoblast tissue from human as well as mouse embryos produces pro-MMP-9. Active MMP-9 (gelatinase B) is a proteolytic enzyme with a broad substrate specificity. MMP-26 is an effective activator of pro-MMP-9, and TIMP-4 is a strong inhibitor of MMP-26. We found that endometrial expression of MMP-26 mRNA and TIMP-4 mRNA is elevated during the proliferative phase, is maximal in the early, decreases in the mid-, and is low in the late secretory phases (I, III). The expression pattern of both genes suggests their up-regulation by estrogen, and down-regulation by progesterone. This is supported by our findings of potential estrogen response elements upstream the coding sequences of both genes (II, III). In situ hybridization localised MMP-26 mRNA in epithelial and TIMP-4 mRNA in stromal cells (I, III). As expected, using immunohistochemistry we found MMP-26 protein in the epithelium (II). However, the same technique showed only occasional staining for TIMP-4 in the stroma (V). Instead, TIMP-4 protein was localised in granules of the apical part of epithelial cells. This finding suggests that TIMP-4 is produced in and secreted by the stromal cells, taken up by the epithelial cells, stored in apical granules, and finally secreted to the uterine fluid. Analysis of uterine fluid with Western blot technique demonstrated presence of TIMP-4, but not MMP-26 (V). So far, very few proteins of stromal origin have been demonstrated in the uterine fluid. Furthermore, using different antibodies, we have shown that MMP-26 is stored in epithelial cells in its active form, is not released spontaneously, and when released it is controlled by TIMP-4 in both stroma and uterine fluid. Examination of endometrial tissue obtained from IVF patients in an estrogen and progesterone substitution protocol showed that the estrogen sensitive genes MMP-26 and TIMP-4 are over-stimulated by both estrogen and progesterone. Analysis of MMP-26 and TIMP-4 expression in endometrial hyperplastia showed that both localization and amount of mRNA corresponded to that of the proliferative phase. This is in agreement with estrogen mediated regulation of these genes, as well as hyperplasia being the result of estrogen over-stimulation. The expression of MMP-26 and TIMP-4 decreases with loss of histological differentiation in endometrial cancer. This is also in agreement with the general loss of differentiated functions during progression to poorly differentiated, and more malignant tumors.