The 3beta-hydroxysteroid
dehydrogenase/delta5-delta4
isomerase (3beta-HSD)
isoenzymes catalyze an essential step in the formation of all classes of active
steroid hormones. We have recently shown that 3beta-HSD type 1 gene expression is specifically induced by
interleukin (IL)-4 and
IL-13 in breast human
cancer cell lines and in normal human mammary epithelial cells in primary culture. There is evidence that
IL-4 stimulates bifurcating signaling pathways in which the signal transducer and activator of transcription-6 (Stat6)-signal pathway is involved in differentiation and gene regulation, whereas
insulin receptor substrate (IRS)
proteins mediate the mitogenic action of
IL-4. In fact, we have shown that Stat6 was activated by
IL-4 in all cell lines studied where
IL-4 induced 3beta-HSD expression, but not in those that failed to respond to
IL-4. The present study was designed to investigate the potential contribution of IRS
proteins and their downstream targets to IL-4-induced 3beta-HSD type 1 gene expression.
IL-4 rapidly induced IRS-1 and IRS-2 phosphorylation in ZR-75-1 human
breast cancer cell lines. Moreover,
insulin-like growth factor (
IGF)-I and
insulin, which are well known to cause IRS-1 and IRS-2 phosphorylation, increased the stimulatory effect of
IL-4 on 3beta-HSD activity. IRS-1 and IRS-2 are adapter molecules that provide docking sites for different SH2-domain-containing
proteins such as the
phosphatidylinositol (
PI) 3-kinase. In this light, the inhibition of IL-4-induced 3beta-HSD expression by
wortmannin and
LY294002, two potent
PI 3-kinase inhibitors, indicates the probable involvement of the
PI 3-kinase signaling molecules in this response to
IL-4. Furthermore, it has been suggested that the IRS
proteins are part of the signaling complexes that lead to activation of the
mitogen-activated
protein (MAP)
kinase by
insulin; thus we investigated the potential role of the MAP
kinase (MAPK) cascade in the
IL-4 action. In ZR-75-1 cells, both the activation of MAPK by
IL-4 and the IL-4-induced 3beta-HSD activity were completely blocked by
PD98059, an inhibitor of MAPK activation.
Wortmannin also blocked MAPK activation by
IL-4,
IGF-I, and
insulin, suggesting that the MAPK cascade acts as a downstream effector of PI 3-kinases. To further understand the cross-talk between signaling pathways involved in
IL-4 action, we investigated the possible involvement of
protein kinase C (PKC). The potential role of PKC was suggested by the observation that the well known PKC activator phorbol-12-myristate-13-acetate (PMA) potentiated the IL-4-induced 3beta-HSD activity. Taken together, these findings suggest the existence of a novel mechanism of gene regulation by
IL-4. This mechanism would involved the phosphorylation of IRS-1 and IRS-2, which transduce the
IL-4 signal through a PI 3-kinase- and MAPK-dependent signaling pathway. The inability of
IGF-I,
insulin, and PMA to stimulate 3beta-HSD expression by themselves in the absence of
IL-4 makes obvious the absolute requirement of an IL-4-specific signaling molecule. Our findings thus suggest that the multiple pathways downstream of IRS-1 and IRS-2 must act in cooperation with the IL-4-specific
transcription factor Stat6 to mediate the induction of 31beta-HSD type 1 gene expression in ZR-75-1 human
breast cancer cells.