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.