Requirement of estrogen receptor-alpha in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk

In the uterus insulin-like growth factor-1 (IGF-1) signaling can be initiated by estradiol acting through its nuclear receptor (estrogen receptor (ER)) to stimulate the local synthesis of IGF-1. Conversely, in vitro studies have demonstrated that estradiol-independent ER transcriptional activity can be induced by IGF-1 signaling, providing evidence for a cross-talk mechanism between IGF-1 and ER. To investigate whether ERalpha is required for uterine responses to IGF-1 in vivo, both wild-type (WT) and ERalpha knockout (alphaERKO) mice were administered IGF-1, and various uterine responses to IGF-1 were compared. In both WT and alphaERKO mice, IGF-1 treatment resulted in phosphorylation of uterine IGF-1 receptor (IGF-1R) and formation of an IGF-1R/insulin receptor substrate-1/ phosphatidylinositol 3-kinase signaling complex. In addition, IGF-1 stimulated phosphorylation of uterine Akt and MAPK in both WT and alphaERKO mice. However, IGF-1 treatment stimulated BrdUrd incorporation and proliferating cell nuclear antigen expression in WT uteri only. To determine whether ERalpha can be activated in vivo by IGF-1 signaling, transgenic mice carrying a luciferase gene driven by two estrogen response elements (ERE-luciferase mice) were utilized. Treatment of ovariectomized ERE-luciferase mice with IGF-1 resulted in an increase in uterine luciferase activity that was attenuated in the presence of the ER antagonist ICI 182,780. Together these data demonstrate that 1) functional signaling proximal to IGF-1R is maintained in the alphaERKO mouse uterus, 2) ERalpha is necessary for IGF-1 induction of uterine nuclear proliferative responses, and 3) cross-talk between IGF-1R and ER signaling pathways exists in vivo

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus