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

Abstract

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.

Abstract (Swedish)

Popular Abstract in Swedish

I avhandlingen studeras Matrix MetalloProteinase-26 (MMP-26) och Tissue Inhibitor of MetalloProteinases-4 (TIMP-4) i normalt, hyperplastiskt och malignt endometrium. Just detta protease/hämmarpar kan potentiellt vara av mycket stort intresse vid implantationsprocessen.



Trofoblastvävnad från såväl humant som murint embryo producerar proMMP-9. Aktivt MMP-9 (Gelatinase B) är ett proteolytiskt enzym med mycket bred specificitet. MMP-26 är en effektiv aktivator av proMMP-9, och TIMP-4 hämmar MMP-26 med mycket hög specificitet.



Vi har funnit att endometriets uttryck av de b?da generna för MMP-26 och TIMP-4 ökar under proliferations-fasen, är maximalt i tidig sekretionsfas, och faller därefter mycket snabbt i mid och sen sekretionsfas (I, III). Mönstret antyder att generna uppregleras av estradiol och nedregleras av progesterone. Detta antagande styrks av att vi identifierade s.k. estrogen response elements uppströms den kodande sekvensen i b?da generna (II, III). Med in situ hybridisering lokaliserade vi mRNA för MMP-26 till epitelet och TIMP-4 till stromat i normalt endometrium (I, III). MMP-26 protein återfanns som väntat i epitelet med immunhistokemi (II). Med samma teknik återfanns emellertid bara sparsamt TIMP-4 i stromat (V).



TIMP-4 lokaliserades i stället till epitelet, närmre bestämt granulaliknande strukturer apikalt i epitelcellerna. Fyndet antydde att TIMP-4 frisätts fr?n stromat, tages upp och ackumuleras i epitelcellerna, samt är där förberett för sekretion. I nästa steg undersöktes därför endometriesekretet (uterine fluid) med Western blot teknik. Vi fann TIMP-4, men inte MMP-26, i sekretet (V). Fyndet är intressant då mycket få protein i uterine fluid verkligen har visats häröra från stromat. Vidare fann vi med antikroppsteknik att MMP-26 upplagras i epitelcellerna i aktiverad form. MMP-26 frisätts inte spontant, men om så sker hämmas enzymet av TIMP-4 i s?väl stroma som uterine fluid.



Undersökning av endometrieprover fr?n patienter som genomg?r IVF behandling med substitution av b?de estradiol och progesterone, visar att östrogenkänsliga gener, som t.ex. MMP-26 och TIMP-4, överstimuleras först av östrogenet och därefter av progesterone.



Resultaten för både MMP-26 och TIMP-4 vid hyperplasi visar att både lokalisation och kvantitering är jämförbart med proliferationsfas. Detta passar med genernas östrogenreglering samt det faktum att hyperplasi är resultatet av överstimulering med östrogen.



Endometriecancer förlorar gradvis uttrycket av båda generna med tilltagande förlust av differentiering. Detta stämmer ocks? med en allmänt avtagande effekt av extern reglering då cancerceller förändras i riktning mot lägre differentierade, mer maligna tumörer.

• author: Pilka, Radovan
• supervisor: Bertil Casslén
• opponent: Professor Marbaix, Etienne, Department of Pathology, University of Louvain Medical School, Brussels, Belgium
• organization: Obstetrics and Gynaecology (Lund)
• publishing date: 2006
• type: Thesis
• publication status: published
• subject: Obstetrics, Gynecology and Reproductive Medicine
• keywords: cancer, Obstetrics, gynaecology, Cytologi, onkologi, cancerology, secretion, Biology, Biologi, Cytology, oncology, implantation, human, endometrium, mRNA, MMP-26, TIMP-4, reproduktion, andrologi, gynekologi, Obstetrik, sexuality, reproduction, sexualitet, andrology
• pages: 122 pages
• defense location: Dept. of Obstetrics and Gynecology, Lecture Hall
• defense date: 2006-04-28 13:00:00
• ISBN: 91-85481-73-4
• language: English
• LU publication?: yes
• additional info: R Pilka, C Whatling, H Domanski, S Hansson, P Eriksson and B Casslen. 2003. Epithelial expression of matrix metalloproteinase-26 (MMP-26) is elevated at midcycle in the human endometrium. Mol Human Reprod,, vol 9 pp 271-7.; R Pilka, G D Norata, H Domanski, C Andersson, S Hansson, P Eriksson and B Casslen. 2004. Matrix metalloproteinase-26 (matrilysin-2) expression is high in endometrial hyperplasia and decreases with loss of histological differentiation in endometrial cancer. Gynecol Oncol,, vol 94 pp 661-70.; R Pilka, H Domanski, S Hansson, P Eriksson and B Casslen. 2004. Endometrial TIMP-4 mRNA is high at midcycle and in hyperplasia, but downregulated in malignant tumours. Coordinated expression with MMP-26. Mol Hum Reprod,, vol 10 pp 641-50.; R Pilka, I Oborna, V Lichnovsky, P Havelka, H Fingerova, P Eriksson, S Hansson and B Casslén. . Endometrial expression of the estrogen sensitive genes MMP-26 and TIMP-4 is altered by a substitution protocol without down regulation in IVF patients. Hum Reprod, (submitted); R Pilka, V Noskova, H Domanski, C Andersson, S Hansson and B Casslén. . Endometrial TIMP-4 is derived from the stroma, accumulates in the epithelium, and is released to the uterine fluid. Mol Hum Reprod, (submitted)
• id: ce83dca8-89e4-4794-9e42-26e5cb3f22b6 (old id 546575)
• date added to LUP: 2016-04-01 17:00:58
• date last changed: 2018-11-21 20:45:57