It is well known that T(3) plays a crucial role in the maintenance of early pregnancy through the induction of endocrine function in villous trophoblasts. The effects of T(3) on extravillous trophoblast (EVT) function, however, remain to be elucidated. To investigate the possible role of T(3) in the regulation of EVT invasion to the decidua, we have examined whether T(3) affects EVT invasive potential and the expression of matrix metalloproteinase-2 (MMP-2), MMP-3, tissue inhibitor metalloproteinase-1, fetal fibronectin (FN), and integrin alpha(5)beta(1) in cultured early placental EVTs. Isolation and purification of trophoblasts differentiating into EVTs were performed by the enzymatic digestion of the anchoring chorionic villi, with the use of human FN-precoated culture dishes and FN-precoated Matrigel Transwells. The cells attached to the dishes were subcultured in DMEM supplemented with 10% fetal bovine serum for 48 h and were characterized by RT-PCR analysis after 24-h subculture and immunocytochemical analysis after 48-h subculture for specific EVT markers. Thereafter, the cultured cells were stepped down to a 4% fetal bovine serum condition and cultured in the presence or absence of T(3) (10(-8) m) for the subsequent 72 h. Matrigel invasion assay demonstrated that the treatment with T(3) significantly increased the number of cell projections of subsequent 24-, 48-, and 72-h cultured EVTs. RT-PCR analysis revealed that the treatment with T(3) increased the expression of MMP-2, MMP-3, fetal FN, and integrin alpha(5)beta(1) mRNA in subsequent 24-h cultured EVTs compared with those in control cultures. Immunocytochemical and Western immunoblot analyses revealed that treatment with T(3) increased the expression of MMP-2 and MMP-3 in subsequent 48-h cultured EVTs compared with those in control cultures. The present results suggest that T(3) (10(-8) m) may play a vital role in up-regulating the invasive potential of EVTs into the decidua.
IGF-I has been reported to play a role in regulating proliferation of human leiomyoma cells. There is, however, little evidence to suggest that IGF-I inhibits apoptosis in the leiomyoma cells. The present study was conducted to elucidate whether IGF-I affects apoptosis and Bcl-2 protein expression, an apoptosis-inhibiting gene product, in cultured leiomyoma cells. In addition, we examined the effect of IGF-I on proliferating cell nuclear antigen (PCNA) expression in cultured leiomyoma cells. Isolated human leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% FBS for 120 h and then stepped down to serum-free conditions for an additional 72 h in the absence or presence of graded concentrations of IGF-I (1.0, 10, and 100 ng/ml). The effects of IGF-I on Bcl-2 protein and PCNA expression in cultured leiomyoma cells were assessed by Western immunoblot analysis and immunocytochemical staining, whereas the effects of IGF-I on the cell viability and apoptosis of the cultured cells were determined by 3-(4,5-dimethylatriazol-2-yl)-2,5diphenyltetrasodium bromide (MTT) assay and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay, respectively. Immunocytochemical staining demonstrated that IGF-I treatment resulted in the increase in PCNA labeling index in cultured leiomyoma cells in a dose-dependent manner. Immunoblot analysis of proteins extracted from the cultured leiomyoma cells revealed that the addition of IGF-I (10 and 100 ng/ml) significantly increased the expression of 35-kDa immunoreactive PCNA and 26-kDa Bcl-2 protein, compared with those in control cultures. Cell survival and proliferation of cultured leiomyoma cells, assessed by MTT assay, was significantly augmented by IGF-I treatment, compared with those of control cultures. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay showed that the apoptosis-positive rate of leiomyoma cells treated with IGF-I was significantly decreased, compared with that in control cultures. The present results suggest that IGF-I plays crucial roles in leiomyoma cell growth, not only in promoting the proliferative potential by up-regulation of PCNA expression but also in down-regulating apoptosis by up-regulation of Bcl-2 protein expression in leiomyoma cells.
The present study provided evidence for the first time that progesterone down-regulates IGF-I mRNA and protein expression in cultured leiomyoma cells without affecting IGF-I receptor mRNA expression.
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