Endogenous EMMPRIN Expression by Human Uterine Fibroblast Cells Regulates Metalloproteinase Production, Proliferation, and Decidualization.

Abstract

The uterine endometrium undergoes extensive proliferation and remodeling in preparation for embryo implantation. This tissue remodeling occurs, in part, through upregulation of matrix metalloproteinases (MMPs) in uterine stromal fibroblasts. Extracellular matrix metalloproteinase inducer (EMMPRIN) stimulates MMP production in a number of carcinoma cell lines and also in human uterine fibroblast (HUF) cells. We have shown in previous studies that human uterine epithelial cells (HES) express the cell surface form of EMMPRIN and secrete full length EMMPRIN via microvesicle shedding. This soluble form of EMMPRIN acts in a paracrine manner on underlying stromal fibroblast cells (HUF) to stimulate MMPs. The goals of the present study were to determine 1) whether soluble EMMPRIN is the major secretory product of HES cells that stimulates MMP production by HUF cells; 2) whether expression of EMMPRIN by HUF cells is necessary for these cells to be able to respond to HES secreted EMMPRIN; and 3) whether EMMPRIN expression in HUF cells is important for cell proliferation and decidualization. We harvested conditioned medium from HES cells and confirmed that this contains the full length form of EMMPRIN via immunoblotting. Treatment of HUF cells with either unconcentrated or 50-fold concentrated HES-cell conditioned medium (p<0.05) increased MMP-1, -2, and -3 mRNA levels without altering endogenous EMMPRIN mRNA levels. Removal of the soluble form of EMMPRIN from HES-cell conditioned medium by immunodepletion using a specific anti-human EMMPRIN antibody caused a reduction in MMP stimulation (7.7 fold for MMP-1, 4.2 fold for MMP-2 and 12.4 fold for MMP-3 (p<0.05)). Our results also showed that endogenous expression of EMMPRIN by HUF cells was important for MMP stimulation by HES-cell conditioned medium. Transfection of HUF cells with EMMPRIN siRNAs resulted in a dramatic reduction in the stimulatory effect of HES-cell conditioned medium on MMP mRNA levels (57 fold MMP-1, 69 fold MMP-2) but a marked increase in MMP-3 mRNA levels (36.7 fold, p<0.05). Treatment of uterine fibroblasts with EMMPRIN siRNAs also decreased the proliferative response of these cells to serum by 40% as measured by DNA synthesis assays. Effects of EMMPRIN siRNA on decidualization of stromal cells was determined by treating cells for 10 days with estradiol, progesterone and cAMP in the presence or absence of EMMPRIN siRNAs. Immunoblotting showed that EMMPRIN protein levels were significantly reduced by day 4 of treatment and nondetectable on days 6-10. Levels of prolactin mRNA, a marker of decidualization, increased 150-200 fold in control cells by days 6-8 but were increased less than 50 fold in EMMPRIN siRNA transfected cells. MMP-7 mRNA levels were increased 20-30 fold in control cells but only 5-7 fold in EMMPRIN siRNA transfected cells. These data indicate that EMMPRIN expression by uterine fibroblast cells is important not only for regulation of MMP production but also appears to play a role in regulating proliferation and decidualization of these cells. Future studies will focus on the role of EMMPRIN in cell cycle regulation in uterine stromal cells. (Supported by SCCPRR U54HD20093). (platform)