27 - 30 May 2005

WNT SIGNALING AND REGULATION OF GENE EXPRESSION IN HUMAN ENDOMETRIAL STROMAL CELLS S Tulac [1], S Talbi [1], L Ailles [2], AE Hamilton [1], E Suchanek [3], IL Weissman [2], R Nusse [4], LC Giudice [1] [1] Department OB/GYN [2] Department of Pathology and Developmental Biology [4] Howard Hughes Medical Institute and Department of Developmental Biology Stanford University School of Medicine Stanford, CA USA [3] Department OB/GYN Zagreb University School of Medicine Zagreb, Croatia

Wnt family members are expressed and/or regulated in human endometrium during the menstrual cycle and in early pregnancy. Signal transduction initiated by Wnt ligand binding to its membrane receptor triggers a cascade of events, including inhibition of GSK3-beta and dephosphorylation of beta-catenin. The latter, together with two partner proteins, TCF and LEF-1, activates expression of Wnt target genes. Herein, we have investigated the signaling events and down-stream genes regulated by Wnt3a in human endometrial stromal cells (hESC) as a model of embryo-endometrial and epithelial-stromal interactions. To confirm activation of the Wnt signaling pathway, we treated hESC with Wnt3a conditioned medium (Wnt3a-CM), obtained from mouse fibroblast L cells transfected with Wnt3a cDNA and with Con-CM (L cells transfected with an empty vector). When Wnt3a-CM was added to endometrial stromal cells in vitro, beta-catenin cytoplasmic accumulation increased 2-4 fold, compared to controls, confirming that the CM contained bioactive Wnt3a. A LEF-1/TCF-GFP reporter was transduced into hESC using a lentiviral system, and cells were stimulated with Wnt3a-CM and Con-CM for 24 hrs. GFP expression monitored via FACs resulted in 2-6 fold increased reporter activity in Wnt3a treated cells, further demonstrating hESC responsiveness to Wnt3a-CM.

To determine global gene regulation by Wnt3a in hESC nondecidualized and decidualized (with progesterone after estradiol priming), ESC were treated with Wnt3a-CM and Con-CM for 24 hrs and subjected for analysis using the Affymetrix Human Genome U133A Plus 2.0 microarrays. With a 1.5 fold change taken as the cut-off, microarray analysis of non-decidualized hESC revealed up-regulation of 901 and down-regulation of 995 known genes and ESTs. Among up-regulated genes were known Wnt targets including cyclin D1, TCF-4 and a newly identified gene APCDD1 (a direct target of beta-catenin/TCF-4 signaling). New Wnt target genes were also identified and include DUSP6, FLRT2, BMP2 and others involved in cell adhesion, development, regulation of transcription and signal transduction.

Among down-regulated genes in non-decidualized hESC were carboxypeptidase M, FGF-7, TIMP-3, Dkk-1 and others involved in development, proteolysis and morphogenesis. Analysis of decidualized hESC revealed up-regulation of 1218 and down-regulation of 1056 known genes and ESTs. Known Wnt target genes including LEF-1 and keratin were up-regulated, together with newly identified Wnt targets: inhibin-alpha ADAM3a, 12, and 19, MMP3, FLRT3 and others involved in apoptosis, inflammation and motility. Some of the down-regulated genes were: TMOD, Gro1, Gro2 and LRP6. These data demonstrate different genes induced or repressed in hESC depending on their differentiation state and provide insight into processes affected by Wnt ligand in these cells.

Supported by the NIH Specialized Cooperative Centers Program in Reproduction Research (NICHD HD 31398) -08 (LCG).

List of abstracts from the 3rd International Conference on the Female Reproductive Tract