Proposed model: genistein (Gen) initiates cytosolic signaling from IGF-IR and ERα interaction to enhance ER transcription in uterine LM cells.
Gen binding with ERα in cytoplasm induces interactions between ERα and IGF-IR, leading to early phosphorylation of Shc (P-Shc) and mitogen activated protein kinase MAPK, which may then in turn lead to activation of ER at Ser sites resulting in enhanced ER transcriptional activity and up-regulation of IGF-I expression, thus resulting in a positive feedback regulatory mechanism and/or autocrine mechanism that stimulates LM cell proliferation.

Effects of genistein (Gen) on human uterine LM cell proliferation. Cell proliferation assays were performed as described in Materials and Methods.
LM cells were treated with vehicle (control), 1 µM ICI 182,780 (ICI), 10 µM PD 98 059 (PD) and 1 µg/ml of Gen alone, or Gen in combination with 1 µM ICI 182,780 (ICI+Gen) or 10 µM PD 98 059 (PD+Gen) for 24, 72 and 168 h, and then cell proliferation was assessed. All experiments were repeated at least four independent times with six samples. Cell proliferation ratios were calculated by dividing absorbance values on the y-axis with the vehicle control (set at 1). Results are presented as mean ± SEM (n = 4). a, b and c indicate groups which are statistically different (P < 0.05).

Real-time PCR measurement of selected estrogen-responsive genes expressed in human uterine LM cells and human uterine SMC treated with 0.3% DMSO (Control) or 1 µg/ml of genistein (Gen) in phenol red-free DMEM/F-12 containing 10% charcoal-stripped fetal calf serum for 24 h.
The relative mRNA expression levels of PR, IGF-I and A-myb in LM cells and SMC were determined by using ΔΔC_T method as described in Materials and Methods. All experiments were repeated at least three independent times with duplicate samples. Fold changes were calculated by dividing values on the y-axis with the vehicle control (set at 1). Results are presented as mean ± SEM (n = 3). a and b, statistical differences between LM cells and SMC (P < 0.05). *P < 0.05 versus control.

Genistein (Gen)-induced interactions between ERα and IGF-IR in human uterine LM cells.
Interactions between ERα and IGF-IR were determined by using immunoprecipitation (IP) as described in Materials and Methods. LM cells were treated with 0.3% DMSO (Control) at 0 min, or 1 µg/ml of Gen at 5, 10 and 15 min, or 1 µM ICI 182,780 (ICI) for 10 min, or Gen in combination with 1 µM ICI 182,780 (ICI+Gen) for 10 min. The blots presented are representative examples of experiments that were performed at least three times with repetitive results. Molecular weight markers are indicated on the right of the blot. The histograms indicated below are the quantitative representation after densitometry of data (mean ± SD) (n= 3) of three independent experiments. Values of ERα band densities in LM cells were divided by the vehicle control (set at 1), *P< 0.05 versus vehicle control containing 0.3% DMSO. **P< 0.05 versus Gen-treated at 10 min.

ER transactivation in human uterine LM cells and human uterine SMC.
LM cells and SMC were transfected with pRL-CMV normalization plasmid and 3×-Vit-ERE-TATA-Luc reporter plasmid. Following transfection, cells were treated with 0.3% DMSO (Control), 10 nM E₂, 1 µg/ml of genistein (Gen) and 10 µM PD 98059 (PD), or Gen in combination with 1 µM ICI 182,780 (ICI+Gen) or 10 µM PD 98059 (PD+Gen) in phenol red-free DMEM/F-12 containing 10% charcoal-stripped fetal calf serum for 48 h. Luciferase assays were performed by using the dual-luciferase reporter assay. Each value was normalized to the internal luciferase control, and fold activation was calculated by dividing values by the vehicle control (set at 1). All experiments were repeated at least three independent times with duplicate samples, and the values represent the mean ± SEM and the average coefficient of variance for each value is <10%. a, b or c, groups which are statistically different (P < 0.05).

Effects of genistein on ERα and ERβ expression levels in human uterine LM cells and human uterine SMC.
(A) Real-time RT–PCR analysis of the relative expression of ERα and ERβ mRNA in LM cells and SMC in 0.3% DMSO (Control) or treated with 1 µg/ml of genistein (Gen) in phenol red-free DMEM/F-12 containing 10% charcoal-stripped fetal calf serum for 24 h. HPRT served as a loading control. Experiments were repeated at least three independent times with duplicate samples. Fold changes were calculated by dividing values on the y-axis with the vehicle control (set at 1). Results are presented as mean ± SEM (n = 3). (B) A representative western blot of the protein expression of ERα and ERβ in LM cells and SMC cultured in phenol red-free DMEM/F-12 containing 10% charcoal-stripped FBS for 24 h. Molecular weight markers are indicated on the right of the blot. (C) Time course of the protein expression of ERα and ERβ in LM cells in 0.3% DMSO (Control) at 0 min or treated with 1 µg/ml of genistein in phenol red-free DMEM/F-12 containing 10% charcoal-stripped fetal calf serum for 5, 10, 15, 30, 60 min, 24, 72 and 168 h. HPRT was served as a loading control. (D) Time course of the protein expression of ERα and ERβ in SMC treated with 0.3% DMSO (Control) at 0 min or 1 µg/ml of genistein in phenol red-free DMEM/F-12 containing 10% charcoal-stripped fetal calf serum for 5, 10, 15, 30, 60 min, 24, 72 and 168 h. The blots presented are representative examples of experiments that were performed at least three times with repetitive results. Molecular weight markers are indicated on the right of the blot. The histograms shown below are the quantitative representation after densitometry of data (mean ± SD) of three independent experiments. Values of ERα and ERβ band densities in LM cells and SMC were divided by the vehicle control (set at 1).*P < 0.05 versus vehicle control containing 0.3% DMSO. **P < 0.05 compared to 24 h.

Effects of genistein on phosphorylation of P44/42 MAPK, ERα and Shc in human uterine LM cells and phosphorylation of P44/42 MAPK in human uterine SMC.
(A) Time course of the protein expression of phosphorylation of P44/42 MAPK (P-P44/42) and total P44/42 MAPK (total P44/42) in LM cells treated with 0.3% DMSO (Control) at 0 min or 1 µg/ml of genistein in phenol red-free DMEM/F-12 containing 10% charcoal-stripped fetal calf serum for 5, 10, 15, 30, 60 min, 24, 72 and 168 h. (B) Protein expression of phosphorylation of P44/42 MAPK (P-P44/42), phospho-ERα Serine-118 (P-ERα) and total P44/42 MAPK (Total P44/42) in LM cells treated with 0.3% DMSO (Control), 1 µg/ml of genistein (Gen), 1 µM ICI 182,780 (ICI) and 10 µM PD 98059 (PD) alone, or Gen in combination with 1 µM ICI 182,780 (ICI+Gen) or Gen in combination with 10 µM PD 98059 (PD+Gen) for 10 min. (C) Protein expression of phosphorylated Shc (P-Shc) and total Shc (Shc) in LM cells treated with 0.3% DMSO (Control) at 0 min, or 1 µg/ml of Gen at 5, 10, 15 and 30 min. (D) Time course of the protein expression of P-P44/42 and total P44/42 in SMC treated with 0.3% DMSO (Control) at 0 min or 1 µg/ml of Gen phenol red-free DMEM/F-12 medium containing 10% charcoal-stripped fetal calf serum for 5, 10, 15, 30, 60 min, 24, 72 and 168 h. The blots presented are representative examples of experiments that were performed at least three times with repetitive results. Molecular weight markers are indicated on the right of the blot. The bar graphs indicated below are the quantitative representation after densitometry of data (mean ± SD) (n = 3) of three independent experiments. Values of P-P44/42, P-ERα and P-Shc band densities in LM cells or SMC were divided by the vehicle control (set at 1), respectively. *P < 0.05 versus vehicle control containing 0.3% DMSO. a, b, c and d indicate groups which are statistically different (P < 0.05).