Growth Factor Control of Cultured Rat Uterine Stromal Cell Proliferation is Progesterone Dependent1

Abstract

Uterine stromal cells undergo mitosis and differentiate into the decidua just prior to the expected time of implantation in humans and rodents. We have utilized a culture system that will be suitable for study of the molecular mechanisms regulating stromal cell proliferation. Stromal cells were isolated from the uteri of ovariectomized rats and were cultured in chemically defined medium. Cultured cells express the mesenchymal markers vimentin and desmin. They do not express the epithelial marker cytokeratin. Serum-starved stromal cells were stimulated to proliferate in a time frame consistent with the cell cycle through addition of a panel of growth factors (basic fibroblast growth factor [bFGF], epidermal growth factor, platelet-derived growth factor, transforming growth factor alpha, insulin-like growth factor I) and hormones to the culture medium. None of the growth factors tested significantly stimulated proliferation in the absence of progesterone. Furthermore, progesterone was the only steroid of those tested that stimulated mitosis in the presence of growth factors. Stromal cell proliferation in response to progesterone and bFGF was dose dependent and saturable. Addition of the progesterone receptor antagonist mifepristone (RU486) and an inhibitor of tyrosine kinase receptor activation (suramin) abolished stromal cell mitosis. Progesterone receptors and fibroblast growth factor receptor 1 (FGFR1) were identified by immunoblot analysis in proliferating stromal cells. Taken together, these results show that cultured stromal cells maintain progesterone-dependent cell cycle control that is mediated via progesterone receptors. Moreover, the data indicate that bFGF control of stromal cell proliferation is modulated via a specific isoform of FGFR1 containing the three-loop immunoglobulin-like domain.

Stimulation of a rat uterine stromal cell line in culture reveals a molecular switch for endocrine-dependent differentiation

“…Proliferation was measured using the MTT assay. We showed previously (Piva et al 1996, Rider et al 1998) that there is a linear relationship between absorbance and cell number under the conditions used in these proliferation assays.…”

“…Isolation of the uterine stromal cell lines has been described in detail previously (Piva et al 1996). Cells (UIII) from the same passage (between passages 15 and 25) were used to determine treatment effects within an experiment by propagating a sufficient number of cells for each experiment in Medium 199 containing 10% FBS (Rider et al 1998).…”

“…Cells (UIII) from the same passage (between passages 15 and 25) were used to determine treatment effects within an experiment by propagating a sufficient number of cells for each experiment in Medium 199 containing 10% FBS (Rider et al 1998). Quiescence was induced by culturing stromal cells for 72 h in serum-free, phenol red-free Dulbecco's Modified Eagle medium (Gibco, Grand Island, NY, USA) and MCDB-105 (Sigma) in a 3:1 mixture containing insulin (5 µg/ml) and supplements as detailed elsewhere (Piva et al 1996). Quiescent cells were stimulated to synchronously re-enter the cell cycle by adding progesterone (1 µM), estradiol 17-(10 nM) and FGF (50 ng/ml).…”

“…Cholera toxin (10 ng/ml) and IL-11 (10 ng/ml) were added to some cultures to stimulate differentiation. Doseresponse studies showed that the maximal proliferative response of uterine stromal cells occurs at these sex steroid concentrations (Piva et al 1996). Proliferation was measured using the MTT assay.…”

“…Stromal cells do not divide without progesterone, and implantation in mice lacking the progesterone receptor fails, in part, because the uterine stromal cells cannot undergo differentiation (Lydon et al 1995). Previous research in our laboratory (Piva et al 1996, Rider et al 1998, Jones et al 2000, Rider 2002) was directed towards the isolation and characterization of rat uterine stromal cell lines that could be used to identify hormone-dependent effects on uterine stromal cells. These cell lines (UI-UIII) have been extensively characterized and their response to proliferative agents is consistent and repeatable.…”