Tumor necrosis factor-alpha expression in human uterine leiomyoma and its down-regulation by progesterone.

Although tumor necrosis factor-alpha (TNFalpha) has been shown mainly to inhibit proliferation and induce apoptosis in a variety of cells, no information is available regarding whether human leiomyoma cells express TNFalpha. In the present study, we examined the expression of TNFalpha in leiomyomas, in comparison with that in the adjacent normal myometrium, using immunohistochemical staining and Western immunoblot analysis with a polyclonal antibody to human TNFalpha. Furthermore, we investigated the effect of sex steroid hormones on TNFalpha expression in leiomyoma cells cultured under serum-free, phenol red-free conditions. Immunohistochemical staining showed that TNFalpha expression in leiomyoma cells was higher than that in the adjacent normal myometrial cells, being more abundant in the proliferative phase than in the secretory, progesterone (P4)-dominated, phase of the menstrual cycle. TNFalpha expression in leiomyoma cells in pregnant uterus was scarce. Western immunoblot analyses of leiomyoma and normal myometrial tissue extracts revealed that TNFalpha, with a molecular mass of 17.3 kDa, was abundantly present in leiomyoma tissue extracts, relative to normal myometrial tissue extracts, and that TNFalpha expression in leiomyoma cells was most abundant in the proliferative phase of the menstrual cycle, less abundant in the secretory phase, and least abundant in pregnant uterus; whereas no such changes in TNFalpha expression were noted in the normal myometrium. In monolayer cultures of uterine leiomyoma cells under serum-free conditions, addition of P4 (3.18 x 10(-7) mol/L) resulted in a decrease in TNFalpha expression in the cells, relative to that in control cultures, whereas treatment with 17beta-estradiol (3.67 x 10(-8) mol/L) did not affect the TNFalpha expression in the cells. The concentrations of sex steroids used were within the physiological tissue concentrations noted in leiomyoma and myometrium. The present results suggest that the abundant expression of TNFalpha may be a molecular basis characteristic of leiomyomas in the human uterus and that P4 may play a vital role in down-regulating the expression of TNFalpha in human uterine leiomyoma.

Effects of progesterone on uterine leiomyoma growth and apoptosis.

Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential. Recently we have found that the use of levonorgestrel-releasing intrauterine system (IUS) is effective in the long-term contraception and management of menorrhagic women with uterine myomas because of a striking reduction in menorrhagia. These clinical experiences prompted us to characterize the effects of progestin on the proliferation and apoptosis of leiomyoma cells cultured in vitro. As epidermal growth factor (EGF) has been shown to mediate estrogen action and play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on EGF and EGF receptor (EGF-R) expression in leiomyoma cells. In cultures of leiomyoma cells, the addition of either E(2) (10 ng/ml) or P(4) (100 ng/ml) resulted in an increase in proliferating cell nuclear antigen (PCNA) expression in the cells; whereas in cultures of normal myometrial cells, the addition of E(2) augmented PCNA expression in the cells, but P(4) did not. Immunoblot analysis revealed that leiomyoma cells contained immunoreactive EGF and that P(4) treatment resulted in an increase in EGF expression in the cells. In contrast, E(2) treatment augmented EGF-R expression in cultured leiomyoma cells, but P(4) did not. These results indicate that P(4) up-regulates the expression of PCNA and EGF in leiomyoma cells, whereas E(2) up-regulates the expression of PCNA and EGF-R in those cells. It is, therefore, conceivable that P(4) and E(2) act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF and EGF-R expression. We also found that Bcl-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to that in normal myometrium, suggesting that the abundant expression of Bcl-2 protein in leiomyoma cells may be one of the molecular bases for the enhanced growth of leiomyoma relative to that of normal myometrium in the uterus. Furthermore, Bcl-2 protein expression in leiomyoma cells was up-regulated by P(4), but down-regulated by E(2). Therefore, it seems likely that P(4) may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells.

Molecular bases for the actions of ovarian sex steroids in the regulation of proliferation and apoptosis of human uterine leiomyoma.

Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential. In order to characterize the molecular mechanism of sex steroidal regulation of leiomyoma growth, we examined whether sex steroids could influence the proliferation of leiomyoma cells. As epidermal growth factor (EGF) has been shown to mediate estrogen action and play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on EGF and EGF receptor (EGF-R) expression in leiomyoma cells. In cultures of leiomyoma cells, the addition of either estradiol (E(2); 10 ng/ml) or progesterone (P(4); 100 ng/ml) resulted in an increase in proliferating cell nuclear antigen (PCNA) expression in the cells, whereas in cultures of normal myometrial cells, the addition of E(2) augmented PCNA expression in the cells, but P(4) did not. Immunoblot analysis revealed that leiomyoma cells contained immunoreactive EGF and that P(4) treatment resulted in an increase in EGF expression in the cells, whereas E(2) treatment resulted in a lower EGF expression in the cells. By contrast, E(2) treatment augmented EGF-R expression in cultured leiomyoma cells, but P(4) did not. These results indicate that P(4) upregulates the expression of PCNA and EGF in leiomyoma cells, whereas E(2) upregulates the expression of PCNA and EGF-R in those cells. It is, therefore, conceivable that P(4) and E(2) act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF and EGF-R expression. We also found that Bcl-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to that in normal myometrium and that Bcl-2 protein expression in leiomyoma cells was upregulated by P(4), but downregulated by E(2). It seems, therefore, likely that P(4) may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells. The abundant expression of Bcl-2 protein in leiomyoma cells may be one of the molecular bases for the enhanced growth of a leiomyoma relative to that of normal myometrium in the uterus.