Estradiol-17beta regulates mouse uterine epithelial cell proliferation through insulin-like growth factor 1 signaling

Abstract

Estradiol-17beta (E(2)) causes cell proliferation in the uterine epithelium of mice and humans by signaling through its transcription factor receptor alpha (ERalpha). In this work we show that this signaling is mediated by the insulin-like growth factor 1 receptor (IGF1R) expressed in the epithelium, whose activation leads to the stimulation of the phosphoinositide 3-kinase/protein kinase B pathway leading to cyclin D1 nuclear accumulation and engagement with the canonical cell cycle machinery. This cyclin D1 nuclear accumulation results from the inhibition of glycogen synthase kinase 3beta (GSK3beta) activity caused by an inhibitory phosphorylation by protein kinase B. Once the IGF1 pathway is activated, inhibition of ER signaling demonstrates that it is independent of ER. Inhibition of GSK3beta in the absence of E(2) is sufficient to induce uterine epithelial cell proliferation, and GSK3beta is epistatic to IGF1 signaling, indicating a linear pathway from E(2) to cyclin D1. Exposure to E(2) is the major risk factor for endometrial cancer, suggesting that downstream activation of this IGF1-mediated pathway by mutation could be causal in the progression to ER-independent tumors.

Fig. 1.

Inhibition of GSK3β induces uterine luminal epithelial DNA synthesis. The GSK3β inhibitor, SB415286, was introduced into the uterine lumen of ovariectomized mice, and DNA synthesis was estimated by BrdU incorporation in transverse sections of uteri harvested 15 h after administration and 2 h after an i.p. injection of BrdU. As a control, 50 ng of E₂ was administered s.c. in oil, a regimen that results in maximal DNA synthesis 15 h after treatment. In some experiments, the pure estrogen antagonist ICI 182,780 was injected i.p. at time 0. (A) Quantitative estimates of BrdU incorporation expressed as percentage of luminal epithelial cells in S phase after the treatments shown on the x axis. Statistical comparisons were performed by Student's t test. (B) Representative transverse sections of mouse uteri immunostained for BrdU incorporation (1 and 2) or cyclin D1 nuclear localization (3 and 4) using appropriate antibodies in ovariectomized control uteri either untreated (1 and 3) or treated with SB415286 (2) or LiCl (4) administered intraluminally. These immunostaining experiments show the increased DNA synthesis after treatment that is restricted to the uterine epithelium and the nuclear localization of cyclin D1 after inhibition of GSK3β.

Fig. 2.

E₂ treatment increases IGF1 expression in the uterine stroma and IGF1R signaling in the luminal epithelium. (A) Representative Western blot of uterine epithelial protein extracts isolated at the times shown after E₂ treatment of ovariectomized mice and probed with anti-phospho-IGF1Rβ, -IGF1Rβ, and -β-tubulin as a loading control as indicated. (B) Quantitative estimate by densitometry of the induction of IGF1 signaling in the epithelium after E₂ treatment relative to the β-tubulin control. Hatched bars, phospho-IGF1Rβ; open bars, IGF1Rβ subunit. (C) In situ hybridization of transverse sections of uteri of control (1 and 4) and E₂-treated (2, 3, 5, and 6) mice probed by using antisense (1, 2, 4, and 5) or sense (3 and 6) probes. Purple precipitate represents the hybridization signal and shows dramatic up-regulation after E₂ treatment principally in the stroma but also in the luminal and glandular epithelium. (Magnification: 1–3, ×10; 4–6, ×40.)

Fig. 3.

E₂ signals through IGF1 and GSK3β to induce DNA synthesis in the uterine epithelium. (1–4) Transverse sections of uteri immunostained for BrdU incorporation from mice 15 h after the following treatments: control ovariectomized (1), s.c. E₂-treated (2), s.c. E₂ plus PPP given intraluminally (3), and s.c. E₂ plus PPP and SB415286 given intraluminally (4). (5 and 6) Transverse sections immunostained for cyclin D1 and harvested 4 h after treatment of E₂ alone (5) and E₂ with PPP administered intraluminally (6). The brown precipitate indicates the specific reaction, and the experiment shows the inhibition of nuclear cyclin D1 accumulation by PPP. (7 and 8) Similar sections stained in the absence of the primary antibody to cyclin D1 (7) or BrdU (8).

Fig. 4.

Inhibition of IGF1R signaling blocks E₂-induced cell proliferation, which is reversed by inhibition of GSK3β. (A and B) Intraluminal injection of PPP blocks IGF1R phosphorylation. (A) Western blot showing that E₂-induced IGF1R phosphorylation is inhibited by PPP using the antibodies described in Fig. 1. (B) Quantitation of three independent Western blots showing the PPP inhibition. The inhibition is statistically different from the E₂-treated group. (C) Quantification of the uterine luminal epithelial DNA synthetic response to the treatments shown of the x axis. The PPP treatment significantly inhibits the E₂ response, which is significantly reversed by concurrent inhibition of GSK3β; P values are from Student's t test.

Fig. 5.

E₂-induced pathway in the luminal epithelium that leads to the canonical cell cycle machinery. The points of inhibition of PPP, SB415285, and LiCl are indicated.