Testosterone dependent androgen receptor stabilization and activation of cell proliferation in primary human myometrial microvascular endothelial cells  Wolf Dietrich, M.D., Aulona Gaba, M.D., Zyhdi Zhegu, M.D., Christian Bieglmayer, M.D., Mario Mairhofer, Ph.D., Mario Mikula, Ph.D., Walter Tschugguel, M.D., Iveta Yotova, Ph.D.  Fertility and Sterility  Volume 95, Issue 4, Pages 1247-1255.e2 (March 2011) DOI: 10.1016/j.fertnstert.2010.11.012 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Characterization of HMMEC primary cultures. (A) FACS analysis of CD31 expression is shown on the left. The black line represents FACS profiles of HMMECs stained with mouse isotype control antibody. The red line shows CD31 expressing HMMECs. Immunofluorescence analysis (middle panel) of von Willebrand Factor VIII (vWF) in green and DAPI (blue) in HMMECs at passage four are presented. Original magnification, ×20. Gel electrophoresis after quantitative RT-PCR analysis of ER-α expression levels in MCF7 HUVEC and two independent cell preparations of HMMECs, showing a lack of ER-α gene expression in HMMECs. MCF7 cells served as a positive control of ER-α gene expression. GAPDH represents the housekeeping gene. (B) Representative immunoblots from four independent experiments, showing protein levels of AR, ER-β, and CYP19 in HMMECs. β-Actin was used as a loading control. LNCap, HUVEC, T-HESC, and tissue samples from placenta were used as positive controls for AR, ER-β, or CYP19 expression, or all three. Erythroid progenitor cells (EPCs) were used as a negative control for AR expression. (C) Effects of T and E2 on HMMEC cell proliferation. Effects of different concentrations of T (physiologic, 10−8 M; pharmacologic, 10−6 and 10−4 M), E2 (physiologic, 10−12 M; pharmacologic, 10−6 and 10−9 M), 4 × 10−5 M PD 98059 and 15% FBS 12 hours after their administration in HMMECs that were serum starved for 12 hours. Cell proliferation was compared with ETOH-treated controls arbitrarily set to 1. Data are expressed as mean ± SD of five independent experiments; ∗P<0.05; ∗∗P<0.005, relative to baseline levels of untreated cells. Fertility and Sterility 2011 95, 1247-1255.e2DOI: (10.1016/j.fertnstert.2010.11.012) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 T-induced HMMEC proliferation is not due to autocrine E2 production. (A) Quantitative RT-PCR analysis of ER-β and CYP19 gene expressions in HMMECs 12 hours after administration of T (10−8 or 10−6 M), T (10−6 M) + letrozole (L; 10−7 M), or T (10−6 M) + PD 98059 (4 × 10−5 M). In the case of combined treatments, the cells were pretreated for 1 hour with respective inhibitors. Average data from five independent experiments, each performed in triplicate are shown. The levels of expression of every gene were normalized to the GAPDH housekeeping gene, and expression levels relative to the control arbitrarily set to 1 are shown. (B) Western blot analysis for ER-β and CYP19 protein levels in HMMECs 12 hours after treatment described in (A) are shown on the left panel, and corresponding proliferation analysis are shown on the right panel. Representative blots from five independent experiments are shown. β-Actin was used as loading control. (C) Cell proliferation was measured and compared to the ETOH-treated controls; ∗∗P<0.005, relative to baseline levels of untreated cells. (D) CYP19 activity assay using tritiated water release after conversion of the 1β- [3H]-androstenedione to E2. Average values from a scintillometric counter of four independent experiments are presented in counts per minute (cpm). In Jeg-3 cells (used as a positive control), CYP19 activity could be inhibited as shown by the decrease in counts after inhibition of the enzyme with L. The cpm levels shown for HMMECs are in the range of the background; ∗∗P<0.005. Fertility and Sterility 2011 95, 1247-1255.e2DOI: (10.1016/j.fertnstert.2010.11.012) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 AR-mediated cell proliferation control in HMMECs. (A) Effects of T (10−6 M), 4× 10−5 M PD 98059, PD + T, Flu (1 μM), and Flu + T on HMMEC proliferation, measured 12 hours after administration. In the case of combined treatments, the cells were pretreated for 1 hour with the appropriate inhibitor. Average data from five independent experiments each performed in triplicate are shown. Cell proliferation was measured and compared with the ETOH-treated controls; ∗P<0.05, relative to baseline levels of untreated cells. (B) Western blot analysis of AR levels in HMMECs 12 hours after treatment with either T or T + PD is shown on the left. Values for AR were normalized by α-tubulin. Representative blots from five independent experiments and graphical representation of averaged values of normalized AR are shown as AR levels relative to the control; ∗P<0.05. (C) Quantitative RT-PCR analysis of AR gene expressions in HMMECs 12 hours after either T (10−8 or 10−6 M) administration or T in combination with PD. In the case of combined treatments, the cells were pretreated for 1 hour with PD. Average data from five independent experiments are shown, each performed in triplicate. The levels of expression of every gene were normalized to the GAPDH housekeeping gene, and expression levels relative to the control are shown. Fertility and Sterility 2011 95, 1247-1255.e2DOI: (10.1016/j.fertnstert.2010.11.012) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 4 T induces AR-stabilization in HMMECs. (A) Effect of short-term (4 hours) and long-term (12 hours) MG-132 treatment on AR and Raf-1 stability in HMMECs. HMMECs were cultured in complete (serum-containing) M199 media to 80% confluence, incubated with either 0.1 or 1 μM proteasome inhibitor MG-132, and subjected to Western blot analysis for AR and Raf-1 after 4 or 12 hours. Representative immunoblots from five independent experiments for each time point (right and middle panel) and graphical representation of averaged values of normalized AR and Raf-1 (left panel) 12 hours after MG-132 administration are shown as AR or Raf-1 protein levels relative to the control; ∗P<0.05; ∗∗P<0.005. Values of every protein were normalized by β-actin. (B) Effects of either T (10−6 M) or MG-132 (0.1 or 1 μM) treatments on AR and Raf-1 stability in HMMECs. HMMECs were serum starved for 12 hours and further treated as indicated for an additional 12 hours. Representative immunoblots from five independent experiments (right panel) and a graphical representation of averaged values of normalized AR and Raf-1 (left panel) 12 hours after T or MG132 administration are shown as AR or Raf-1 protein levels relative to the control; ∗P<0.05; ∗∗P<0.005. Values of every protein were normalized by α-tubulin. (C) Quantitative RT-PCR analysis of PDGF-A, PDGF-B, VEGF-A, and VEGF-C in HMMECs 12 hours after either T (10−8 or 10−6M) or E2 (10−6, 10−9, or 10−12 M) administration. Average data from three independent experiments are shown, each performed in triplicate. The levels of expression of every gene were normalized to the GAPDH housekeeping gene and expression levels relative to the control are shown; ∗P<0.05. Fertility and Sterility 2011 95, 1247-1255.e2DOI: (10.1016/j.fertnstert.2010.11.012) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions