The expression and cellular distribution of transforming growth factor-1 (TGF beta 1) through TGF beta 3 and TGF beta type I-III receptor messenger ribonucleic acid (mRNA) and protein were analyzed in leiomyomata from patients receiving GnRH agonist (GnRHa; leuprolide acetate) compared to those in untreated controls. Standard reverse transcription-PCR revealed that the unaffected myometrium and leiomyomata from leuprolide-treated and untreated patients express TGF beta 1-3 and TGF beta type I-III receptor mRNA. The myometrial and leiomyomata smooth muscle cells were the primary site of TGF beta 1-3 and TGF beta type I and II receptor mRNA and protein expression, as determined by in situ hybridization and immunohistochemical localization. These observations indicate that leiomyomata express a higher of level of TGF beta and TGF beta receptor mRNA and protein than unaffected myometrium during the secretory phase of the menstrual cycle, and women who received leuprolide acetate therapy had a substantially lower level of expression than untreated controls. Furthermore, competition-based quantitative reverse transcription-PCR using synthetic internal standards revealed that leiomyomata express a significantly higher number (copies per cell) of TGF beta type II receptor mRNA, followed by TGF beta 1, TGF beta type I receptor, TGF beta 2, and TGF beta 3 (P < 0.05). However, there was a significant decrease in the levels (copies per cell) of TGF beta 1, TGF beta 3, and TGF beta type I and type II receptor mRNA expression in leiomyomata from leuprolide-treated compared to untreated patients (P < 0.05). The data provide further evidence that leiomyomata express mRNA and protein for all components of the TGF beta system, and GnRHa therapy results in down-regulation of their expression. More specifically, these data suggest that TGF beta 1 and TGF beta 3 may play a more important role in leiomyomata growth than TGF beta 2, which leads us to propose that lowering TGF beta and receptor expression may have a direct effect on leiomyomata regression.

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.

Tissue remodelling involving extracellular matrix (ECM) turnover plays a major role in leiomyoma growth and regression, regulated by the combined action of matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs). We postulated that leiomyomata express MMP and TIMP mRNA and protein, and their expression is inversely regulated during tumour growth and gonadotrophin releasing hormone agonist (GnRHa)-induced regression. We therefore examined the expression of mRNA and protein for MMPs (interstitial collagenase, MMP-1; gelatinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and TIMPs (TIMP-1 and TIMP-2) in leiomyoma and matched unaffected myometrium from GnRHa (lupron)-treated and untreated patients. Reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme analysis revealed that leiomyomata and myometrium expressed MMP-1, -2, -3 and -9, as well as TIMP-1 and -2 mRNA. Quantitative RT-PCR indicated that leiomyomata and myometrium during the secretory phase of the menstrual cycle expressed higher levels of MMP and TIMP mRNA compared to the proliferative phase (P < 0.05), with low to undetectable levels of MMP-1, -2 and -3 mRNA in the tumours. GnRHa therapy induced an overall reduction in MMP and TIMP mRNA expression in both leiomyomata and myometrium, but a significant decrease in TIMP-1, and an increase in MMP mRNA expression compared with untreated tumours (P < 0.05). Immunohistochemically, MMP-1, -2, -3 and -9 and TIMP-1 and -2 proteins were localized in leiomyomata and myometrial smooth muscle cells, arteriole wall and connective tissue fibroblasts, with an overall increase in MMP and a decrease in TIMP staining intensity in GnRHa-treated groups. The results suggest that MMP and TIMP expression in leiomyoma and myometrium are hormonally regulated, and that GnRHa-induced tumour regression is accompanied by an increase in MMP expression with a concomitant decrease in TIMP-1 expression, which may potentially provide an environment favouring ECM degradation.

The expression of granulocyte macrophage-stimulating factor (GM-CSF) and GM-CSF receptors in the human endometrium suggests an autocrine/paracrine role for GM-CSF in this tissue. Using primary cultures of isolated endometrial glandular epithelial and stromal cells, the present study examined: (i) the cell specific expression of GM-CSF and GM-CSF receptor mRNA and protein; (ii) direct action of GM-CSF on the rate of DNA synthesis and cell proliferation; and (iii) regulation of GM-CSF expression through its interaction with transforming growth factor (TGF)-beta1 in these cells. Quantitative reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay and immunocytochemistry indicates that glandular epithelial and stromal cells express GM-CSF, GM-CSF alpha and GM-CSF beta receptor mRNA and protein. The epithelial cells express a significantly higher level of GM-CSF mRNA than stromal cells while both types produce low concentrations of protein. At 0.01-100 ng/ml GM-CSF did not have a significant effect on the rate of [3H]-thymidine incorporation or proliferation of epithelial and stromal cells. However, GM-CSF (1 ng/ ml) up-regulates its own protein expression, but does not effect TGF-beta1 mRNA protein expression in epithelial and stromal cells, and actually inhibits the cell-associated TGF-beta1 protein in stromal cells (P<0.05). At 1 ng/ ml TGF-beta1 up-regulates its own mRNA and protein expression in epithelial and stromal cells (P<0.05), with no significant effect on GM-CSF expression. Co-treatment of the cells with GM-CSF + TGF-beta1 resulted in an increased production of GM-CSF protein as well as TGF-beta1 mRNA and protein expression by epithelial and stromal cells, compared with untreated controls (P<0,001). In conclusion, the results suggest that GM-CSF is not a mitogenic factor for endometrial glandular epithelial and stromal cells, however, in an interactive manner with TGF-beta1 it regulates its own and the expression of TGF-beta1.