The objective of this study was to elucidate the biological significance of GnRH and antiprogestins and antiestrogen in leiomyoma and their interactions with ovarian steroid 'add-back' therapy. Leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) were isolated and exposed to GnRH agonist (leuprolide acetate, LA), 17beta-estradiol (E2), medroxyprogesterone acetate (MPA), GnRH antagonist (Antide), estrogen antagonist, ICI182780 (Fulvestrant) and progesterone antagonists RU486 (Mifepristone) and ZK98299 (Onapristone) and combinations thereof. The rate of DNA synthesis, cell proliferation and transforming growth factor-beta (TGF-beta) expression were then determined. In both cell types, we found that in a dose-dependent manner, LA inhibited, whereas E2, MPA and the combination of E2 + MPA stimulated, the rate of DNA synthesis in these cells. Antide reversed the inhibitory effect of LA, while LA partly inhibited the stimulatory effect of the steroids. In addition, RU486, ICI182780 and ZK98299 at 0.1 micro mol/l or higher doses inhibited the rate of DNA synthesis and partly reversed the effects of E2 and/or MPA. We also found that LSMC expressed elevated levels of TGF-beta1 compared with MSMC. In both cell types, the effects of LA, E2, MPA, RU, ZK and ICI and combinations thereof on TGF-beta1 production were reflective of their effects on DNA synthesis. In line with this, TGF-beta1 was found to stimulate DNA synthesis and the E2-, TGF-beta1- or E2 + TGF-beta1-induced DNA synthesis was found to be inhibited by TGF-beta1 neutralizing antibodies and/or LA. In conclusion, the results provide further evidence that GnRH agonist- and RU486-induced leiomyoma regression is mediated in part through an interactive mechanism that results in altered cell growth and suppression of TGF-beta production.

The objective of the present study was to elucidate the presence and cellular distribution of insulin-like growth factor I (IGF-I), IGF-I receptor (IGF-IR), and IGF binding proteins (IGFBPs) in human uterine tissue at various reproductive stages, and to determine the effect of IGF-I and its interaction with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in endometrial stromal and myometrial smooth muscle cells in primary culture. Using specific antibodies, immunohistochemical observations indicated that luminal and glandular epithelial cells were the major sites of immunoreactive IGF-I, IGF-IR, and IGFBPs 1-4, followed by myometrial smooth muscle and endometrial stromal cells. The immunostaining intensity of IGF-I, IGF-IR, and IGFBPs in endometrial but not myometrial tissue was cycle-dependent and higher in the late proliferative and early/mid-secretory periods than in the late secretory and postmenopausal periods, with little immunostaining at the early proliferative phase of the menstrual cycle. Stromal and smooth cells in primary cell culture also contained immunoreactive IGF-I, IGF-IR, and IGFBPs. IGF-I at 10-100 ng/ml stimulated 3H-thymidine incorporation in quiescent stromal and smooth muscle cells with maximal effect at 100 ng/ml (p < 0.05). However, in the presence of 2% serum, which induces half-maximal stimulation, IGF-I (100 ng/ml) further increased the rate of 3H-thymidine incorporation in stromal but not smooth muscle cells (p < 0.05). The effect of IGF-I was significantly lower than that induced by EGF (10 ng/ml), PDGF-BB (10 ng/ml) and their combination (p < 0.005), and higher in stromal cells from proliferative, than secretory phase of the cycle in the presence of 2% fetal bovine serum, but not serum-free condition (p < 0.005). The effect of IGF-I on myometrial smooth muscle cells was significantly higher than that induced by EGF, but lower than that induced by PDGF-BB or by EGF+PDGF-BB, without the cycle specificity seen with stromal cells. EGF, PDGF-BB, and their combination with IGF-I, but not IGF-I alone, stimulated stromal and smooth muscle cell growth as determined by a cell proliferation assay. The results indicate that human uterine tissue at various reproductive stages contains immunoreactive IGF-I, IGF-IR, and IGFBPs 1-4. Although IGF-I alone was found to be a weak mitogenic factor for stromal and smooth muscle cells, by interacting with EGF and PDGF-BB in a cycle-dependent manner it may regulate the growth and differentiation of these and other uterine cell types.

In this study we investigated the expression of transforming growth factor-beta (TGF-beta) isoform and TGF-beta receptor mRNA and protein, and the effect of TGF-beta 1-3 on the rate of DNA synthesis and proliferation of human myometrial smooth muscle cells in vitro. To determine these, we utilized primary cultures of myometrial smooth muscle cells, standard and competitive quantitative reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, enzyme-linked immunoassay, radioreceptor assay, [3H] thymidine incorporation and cell proliferation assay. Standard RT-PCR and immunocytochemistry revealed that myometrial smooth muscle cells express TGF beta 1-3 and TGF-beta type I-III receptor (TGF-beta R) mRNA and protein. Quantitative RT-PCR, using an external synthetic RNA standard, indicated that the cells express 10 copies/cell of TGF-beta 1 and TGF-beta 2, less than one copy/cell of TGF-beta 3 and TGF-beta type IR, three copies/cell of type IIIR, and > 200 copies/cell, of TGF-beta type IIR mRNA. The cells also synthesized and released TGF-beta 1 at the rate of 7.8 +/- 0.7 ng/10(6) cells, of which 1.4 +/- 0.2 ng/10(6) cells was in an active form. The rate of [3H] thymidine incorporation or proliferation of subconfluent quiescent smooth muscle cells was not altered by TGF-beta s (0.1-10 ng/ml) under serum-free conditions, nor in the presence of 10% fetal bovine serum (FBS). TGF-beta 1-3 at 0.25-0.5 ng/ml in the presence of 2% FBS, which induces half maximal stimulation of these cells, stimulated the rate (P < 0.05), whereas at higher doses it reduced the rate of [3H]-thymidine incorporation compared to the controls. The effect of TGF-beta was partially reversible using neutralizing antibodies specific to TGF-beta 1, TGF-beta 2 (10 micrograms/ml) or TGF-beta 3 (3-6 micrograms/ml). TGF-beta s had no significant effect on cell proliferation determined by cell counting. The data indicate that human myometrial smooth muscle cells express the necessary components of the TGF-beta system, suggesting an autocrine/paracrine role for TGF-beta s in myometrium.