br Materials and methods br Results To investigate the
Materials and methods
Results To investigate the effect of 17βE on cell proliferation in the human renal tubular epithelia, BrdU uptake was measured in HRTEC primary and 3D-HRTEC cultures, allowing determination of the DNA replication rate. Representative microphotographs show BrdU uptake in HRTEC (Fig. 1A). Incubation of HRTEC with 10 nmol/L 17βE for 24 h, significantly stimulated the BrdU uptake in eight HRTEC primary cultures from different pediatric patients in comparison to their respective controls (Fig. 1B). These results demonstrated the reproducibility of 17βE effect on cell proliferation in primary cultures of HRTEC. The microphotographs in Fig. 1C and the graph in Fig. 1D, show that a significant increase in BrdU uptake was also observed in tubular structures of 3D-HRTEC cultures treated with 17βE for 24 h with respect to their controls. The number of HRTEC was also quantified to assess whether the increase in BrdU uptake in 17βE-treated cells corresponds to a subsequent increase in cell proliferation and growth. A tendency to increase cell growth was found in HRTEC at 24 h of 17βE incubation, which progressed to a significant increase in relative cell growth after 48 h of 17βE incubation (Fig. 1E) indicating that cells entering the PFI-1 mg due to the estrogen treatment end up proliferating. The increase of BrdU uptake induced by 17βE was completely abrogated by incubation with the ERs and GPER-1 inhibitors (ICI182,780 and G-15, respectively). (Fig. 2). 17βE treatment of HRTEC with ICI182,780 or G-15 alone did not significantly modify the percentage of BrdU uptake compared to control cells (Fig. 2). Of note, ICI182,780 was used at a dose (100 nmol/L) that was reported to not stimulate GPER-1 . The involvement of GPER-1 on 17βE stimulated cell proliferation was corroborated by the treatment of HRTEC with the agonist G-1. Incubation of HRTEC with G-1 (10 nmol/L for 24 h) significantly stimulated the percentage of BrdU uptake with respect to control cells, similarly to the effect produced by 17βE at the same dose (Fig. 2). Furthermore, co-incubation of HRTEC with 17βE and G-1 (1 or 10 nmol/L) did not produce potentiation or synergistic effects (data not shown). As shown in Fig. 3, ERα and ERβ are localized mainly to the cytosol of most cells in HRTEC control cultures (Fig. 3A and C), and only to the nuclei of few cells. Incubation with 17βE for 24 h significantly increased the percentage of nuclei positive labeled for ERα compared to control cells (Control: 7.8 ± 2.10%; 17βE: 15.00 ± 3.09%) (Fig. 3B). However, no significant differences were observed in the percent of nuclei labeled for ERβ between control and 17βE treated HRTEC (3.9 ± 1.39% vs 3.0 ± 0.73%) (Fig. 3D). To investigate whether β-catenin participates in the cell proliferation increase induced by estradiol, primary cultures were incubated with different concentrations of iCRT14, a specific inhibitor that antagonizes the transcriptional function of nuclear β-catenin . Treatment of HRTEC with 1 and 10 μmol/L iCRT14 for 24 h significantly inhibited the stimulation of BrdU uptake induced by 17βE (Fig. 4A). A lower dose of iCRT14 (0.1 μmol/L) partially inhibited the estradiol effect. iCRT14 alone did not modify significantly the BrdU uptake at any dose assayed. Moreover, western blot analysis (Fig. 4B) demonstrates that 17βE significantly stimulates β-catenin expression in HRTEC. Both ICI 182,780 and G-15, inhibited the increase in β-catenin expression induced by 17βE (Fig. 4B). Immunofluorescence showed that β-catenin was localized mainly to the plasma membrane of control HRTEC and to a lesser extent in the cytoplasm and nuclei (Fig. 4C). Incubation of HRTEC with 17βE, significantly increased the number of cells that translocated β-catenin into their nuclei (14 ± 2.0%), compared to control cells (8 ± 1.2%) (Fig. 4C and D), demonstrating that β-catenin is activated by 17βE in a percent of HRTEC, such as occurred with ERα whereas HRTEC incubation with G-15 or ICI182,780, in the presence or absence of 17βE, showed similar values as control cells (Fig. 4C and D).