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.
Tissue injury and pelvic inflammation often results in peritoneal scar tissue formation. The objective of this study was to determine whether mesothelial cells which line the peritoneal cavity express matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), and if their expression is regulated by transforming growth factor-beta1, a key regulator of tissue fibrosis. For this purpose we used Met-5A cells, a cell line derived from human normal mesothelial cells, and for comparative analysis we used U-937 cells, a human monocytic/macrophage cell line. The cells were treated with transforming growth factor-beta1 (1 ng/ml) for various time periods and the levels of MMP and TIMP mRNA and protein expression were determined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The results indicate that the mesothelial cells and macrophages express MMP-1 (collagenase-1), MMP-3 (stromelysin-1), TIMP-1 and TIMP-2 mRNA and protein at various levels, with significantly higher TIMPs than MMPs, and higher MMP-1 than MMP-3 (p < 0.001). The mesothelial cells express significantly less MMP-1, higher MMP- 3 and similar levels of TIMP mRNA compared to macrophages. In a time-dependent manner, treatment of the mesothelial cells with transforming growth factor-beta1 resulted in a significant decrease in the expression of MMP-1, while increasing the expression of TIMP-1 mRNA (p = 0.05). In contrast, MMP-3 and TIMP-2 expression was unaffected in mesothelial cells and in macrophages, compared to untreated controls. There was a significant increase in secreted MMP-1 and TIMP-2 by mesothelial cells following transforming growth factor-beta1 treatments in a time-dependent manner (p = 0.05 and p = 0.01), without affecting the secretion of these proteins by macrophages. A major portion of MMP-1 in the culture conditioned media of both cell types was found in complex with TIMP-1. The ratios of MMP-1/TIMPs production were significantly higher than MMP-3/TIMPs in mesothelial cells and macrophages, and progressively decreased following transforming growth factor-beta1 treatments (p < 0.05). In conclusion, these results indicate that mesothelial cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by transforming growth factor-beta1, a mechanism that in part may influence the outcome of peritoneal tissue repair and adhesion formation.
The objective of the present study was to determine whether transforming growth factor beta (TGF-beta) regulates the expression of matrix metalloproteinases (MMP) and the tissue inhibitor of MMP (TIMP) in myometrial smooth muscle cells. Using primary cultures of human myometrial smooth muscle cells we found that these cells express MMP-1, MMP-3, TIMP-1 and TIMP-2 mRNA and protein, with significantly higher values of TIMP than MMP. We also found that TGF-beta1 (1 ng/ml) increased the expression of TIMP-1 mRNA, while it reduced the expression of MMP-1 and MMP-3 mRNA, compared with untreated controls. In addition, TGF-beta1 slightly increased the production of TIMP-1, but not TIMP-2. Production of MMP-1 and MMP-3 was reduced by treatment with TGF-beta1, compared with the untreated control. A major portion of MMP-1 released into the culture-conditioned media was in complex with TIMP-1, and the levels of this complex were reduced by treatment with TGF-beta1. In conclusion, the data indicate that myometrial smooth muscle cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by TGF-beta1. Such a differential regulation of MMP and TIMP by TGF-beta may influence the rate of extracellular matrix (ECM) turnover following tissue injury, induced during myomectomy and Caesarean section, or in leiomyomas during growth.
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