Fig. 5.

Increased ERα expression and estrogen production in LAM tissue are responsible for LAM fibroblast proliferation and collagen formation in Arom^hum mice. (A) Cell proliferation indicated by K_i67 immunoreactivity was measured in LAM tissue. (Scale bar, 50 μm.) (B) A minimum of 1,000 nuclei were counted in sections to calculate the percent of K_i67⁺ stromal cells in UAM, LAM, and QM of WT and Arom^hum mice. Two-way ANOVA with Sidak’s multiple comparison test, *P < 0.05, n = 10. (C) The effect of letrozole (Let) treatment on stromal cell proliferation determined by calculation of the percent of K_i67⁺ nuclei in UAM and LAM of WT and Arom^hum mice treated with letrozole. Veh, vehicle. Two-way ANOVA with Tukey’s multiple comparison test, *P < 0.05, n = 8–11. (D) Cell proliferation marker proliferating cell nuclear antigen (PCNA) and protein levels of type I collagen (Col1A) were determined by immunoblotting in primary LAM fibroblasts of WT mice treated with the physiological doses of E₂ for 24 h. Tubulin protein levels served as the loading control. Data are representative of three independent experiments. mRNA levels of profibrotic genes Kiss1 (E), Ren1 (F), Emb (G), Timp1 (H), Spon2 (I), and Eln (J) were measured in UAM and LAM tissues of WT and Arom^hum mice at 3 wk of age. mRNA levels of Kiss1 and Spon2 in primary LAM fibroblasts from Arom^hum mice after MPP (10 µM) treatment (K and L) or siRNA-mediated knockdown of ERα (M and N) in the presence or absence of E₂ (10 nM). Cells were pretreated with MPP for 2 h before the addition of E₂. Veh, vehicle. GAPDH mRNA levels served as the control. Two-way ANOVA with Tukey’s multiple comparison test, *P < 0.05, **P < 0.01, n = 10 mice in each group.