FIGURE 5:
Hypoxia-induced Nodal expression persists via a SMAD-dependent positive feedback mechanism. (A) Graphical representation of SMAD-2/3 reporter activity in T47D cells cultured in 20% O2 (24 h), 0.5% O2 (24 h), or 0.5% O2 (24 h), which was followed by reoxygenation in 20% O2 (24 or 48 h). On reoxygenation, cells were cultured in the presence of a vehicle (−) or the ALK-4/5/7 inhibitor SB431542 (10 μM; +). SMAD-2/3 transcriptional activity was measured using flow cytometry in cells transfected with a SMAD-2/3–responsive GFP reporter construct. Exposure to hypoxia increased SMAD-2/3 transcriptional activity. This up-regulation in SMAD-2/3 activity was retained after 24 and 48 h of reoxygenation, and SMAD-2/3 activity was abrogated by SB431542. Bars represent the median fold change in SMAD-2/3 reporter activity ± interquartile range relative to control (20% O2) activity. Different letters are significantly different (n = 5; p < 0.05). (B) Immunofluorescence of SMAD-2/3 in MCF-7, T47D, or C81-61 cells cultured in 20% O2 (24 h), 0.5% O2 (24 h), or 0.5% O2 (24 h) followed by reoxygenation in 20% O2 (24 or 48 h). On reoxygenation, cells were cultured in the presence of a vehicle (−) or the ALK-4/5/7 inhibitor SB431542 (10 μM; +). In all cell lines, exposure to hypoxia resulted in the translocation of SMAD-2/3 to the nucleus. SMAD-2/3 was retained in the nucleus after 24 and 48 h of reoxygenation, and this nuclear localization of SMAD-2/3 was abrogated by SB431542. Scale bar: 10 μm. (C and D) Immunoblot analyses of phosphorylated SMAD-2, total SMAD-2/3, and Nodal in (C) T47D cells and (D) C81-61 cells cultured in 20% O2 (24 h), 0.5% O2 (48 h), or 0.5% O2 (24 h), which was followed by 20% O2 reoxygenation (24 or 48 h). On reoxygenation, cells were treated with either vehicle or an ALK 4/5/7 inhibitor (SB431542; 10 μM). SMAD-2 phosphorylation is elevated in hypoxia and persists during reoxygenation, and this up-regulation in phosphorylation is reduced with the addition of SB431542, with greater effects observed at 48 h than at 24 h. Relative to control (20% O2) levels, Nodal expression is elevated in hypoxia and during reoxygenation. In T47D cells (C), the elevated levels of Nodal protein observed during 24 and 48 h of reoxygenation are greatly reduced with the addition of SB431542. However, in C81-61 cells (D), SB431542 reduces Nodal expression only at 48 h of reoxygenation. The pro-Nodal (∼39 kDa) band is presented, and total SMAD-2/3 or β-actin are used as loading controls.