Abstract
The aim of this study was to investigate the role of estrogens in Leydig cell tumor proliferation. We used R2C rat Leydig tumor cells and testicular samples from Fischer rats with a developed Leydig tumor. Both experimental models express high levels of aromatase and estrogen receptor alpha (ERalpha). Treatment with exogenous 17beta-estradiol (E(2)) induced proliferation of R2C cells and up-regulation of cell cycle regulators cyclin D1 and cyclin E, the expression of which was blocked by addition of antiestrogens. These observations led us to hypothesize an E(2)/ERalpha-dependent mechanism for Leydig cell tumor proliferation. In determining the molecular mechanism responsible for aromatase overexpression, we found that total and phosphorylated levels of transcription factors cyclic AMP-responsive element binding protein and steroidogenic factor 1 (SF-1) were higher in tumor samples. Moreover, we found that tumor Leydig cells produce high levels of insulin-like growth factor I (IGF-I), which increased aromatase mRNA, protein, and activity as a consequence of increased total and phosphorylated SF-1 levels. Specific inhibitors of IGF-I receptor, protein kinase C, and phosphatidylinositol 3-kinase determined a reduction in SF-1 expression and in IGF-I-dependent SF-1 recruitment to the aromatase PII promoter. The same inhibitors also inhibited aromatase expression and activity and, consequently, R2C cell proliferation. We can conclude that one of the molecular mechanisms determining Leydig cell tumorigenesis is an excessive estrogen production that stimulates a short autocrine loop determining cell proliferation. In addition, cell-produced IGF-I amplifies estrogen signaling through an SF-1-dependent up-regulation of aromatase expression. The identification of this molecular mechanism will be helpful in defining new therapeutic approaches for Leydig cell tumors.
Highlights
The etiology and pathogenesis of human testicular tumors are poorly defined
TM3 cells were cultured in DMEM/F-12 supplemented with 5% horse serum (HS), 2.5% fetal bovine serum (FBS), and antibiotics (Invitrogen S.R.L.); R2C cells were cultured in Ham/F-10 supplemented with 15% HS, 2.5% FBS, and antibiotics (Invitrogen)
Aged animals have a high incidence of spontaneous Leydig cell neoplasm [8, 28], a phenomenon not observed in younger animals, allowing us to use them as a good in vivo model to confirm results obtained in cell lines
Summary
Reproductive system tumors are uncommon in general, with the few exceptions of Leydig cell and ventral prostatic neoplasms in some rat strains [3] or non-inbred mice [4]; analogously to the human [5], chronic administration of estrogens induces testicular tumors. A useful model used to investigate whether excess estrogens might have a central role in the mechanism leading to testicular tumorigenesis are transgenic mice overexpressing aromatase and presenting enhancement of circulating 17h-estradiol (E2) levels [6]. Whereas the effects of estrogen on mammary gland tumorigenesis in human and rodents are well known, the role of aromatase overexpression and in situ estrogen production in testicular tumorigenesis has not been clearly defined. We used the R2C rat Leydig tumor cell line; to validate our in vitro data in an in vivo model, we used Leydig cell tumors from older Fisher rats characterized by an exceptionally high incidence of spontaneous neoplasm with aging [8]
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