Up-regulation by progesterone of proliferating cell nuclear antigen and epidermal growth factor expression in human uterine leiomyoma

Uterine leiomyoma is the most common smooth muscle cell tumor of the myometrium. Estrogen and progesterone (P-4) are believed to be physiological regulators of leiomyoma growth. We recently showed that Bel-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to its expression in the normal myometrium and that Bcl-2 protein expression in cultured leiomyoma cells was up-regulated by P-4, but down-regulated by 17 beta-estradiol (E-2). To further characterize the molecular mechanism of sex steroidal regulation of leiomyoma growth, we examined the effect of menstrual phase on proliferating cell nuclear antigen (PCNA) expression in leiomyoma and investigated whether sex steroids could influence PCNA expression in leiomyoma cells cultured under serum-free conditions by immunoblot and immunohistochemical analyses. As epidermal growth factor (EGF) has been shown to mediate estrogen action and to play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on the expression of EGF and EGF receptor (EGF-R) in cultured leiomyoma cells. The PCNA labeling index in leiomyomas was much greater in the secretory, P-4-dominated, phase than in the proliferative phase of the menstrual cycle and was significantly higher than that in the adjacent normal myometrium throughout the menstrual cycle. In monolayer cultures of leiomyoma cells, the addition of either E-2 (10 ng/mL) or P-4 (100 ng/mL) resulted in an increase in PCNA expression in the cells compared to that in control cultures, whereas in monolayer cultures of myometrial cells, the addition off, augmented PCNA expression in the cells, but P-4 did not. Immunoblot analysis of proteins extracted from cultured leiomyoma cells revealed that leiomyoma cells contained immunoreactive EGF with a molecular mass of 133 kDa and that the addition of P-4 resulted in a remarkable increase in the expression of 133- and 71-kDa immunoreactive EGF in the cells compared to that in control cultures, whereas the addition of E-2 resulted in a somewhat lower expression of immunoreactive EGF in the cells. Furthermore, immunocytochemical analysis with a monoclonal antibody to human EGF-R demonstrated that the treatment with E-2 augmented EGF-R expression in the cells compared to that in untreated cells, but P-4 did not. The concentrations of sex steroids used were within the physiological tissue concentrations found in leiomyomas and myometria. These results indicate that P-4 up-regulates the expression of PCNA and immunoreactive EGF in leiomyoma cells, whereas E-2 up-regulates the expression of PCNA and EGF-R in those cells. As it is evident that EGF plays a crucial role as a local factor in regulating leiomyoma growth, the P-4-induced increase in PCNA expression in leiomyoma cells may be mediated by P-4-induced enhanced expression of EGF-like proteins in the cells, whereas the E-2-induced increase in PCNA expression in leiomyoma cells may be mediated by E-2-induced enhanced expression of 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-like proteins and EGF-R expression in uterine leiomyoma.