Regulatory Interactions between Androgens, Hoxb5, and TGFβ Signaling in Murine Lung Development

Figure 2

Effect of DHT without and with Hoxb5 knockdown on lung histology and Hoxb5, SMAD2P, and SMAD7 spatial and cellular protein expression. (a–l) Immunohistochemistry: (a–d) Hoxb5; (e–h) SMAD2P; (i–l) SMAD7; (m) Hoxb5 and SMAD2P western blot analyses in DHT-treated lungs. Asterisk (*) represents mesenchyme regions of lung in all tissue sections. Blue staining represents specific staining for protein of interest; red staining is Texas Red counterstain. Compared to control treated lung (a), DHT led to more diffusely intense Hoxb5 mesenchymal (*) localization (b) and increased Hoxb5 protein levels (m). Hoxb5 knockdown profoundly diminished Hoxb5 staining (c) and partially prevented the ability of DHT to alter Hoxb5 mesenchymal (*) cell protein expression (d). DHT treatment diminished SMAD2P mesenchymal (*) and epithelial staining (arrows in (f) compared to control (e)) and total protein levels (m). With knockdown of Hoxb5 expression (g, h) SMAD2P staining was more diffuse than with DHT treatment alone ((h) compared to (f)). In the presence of Hoxb5 knockdown, DHT lost the inhibitory effect on SMAD2P protein (h) cellular expression. SMAD7 expression was not changed by DHT treatment (j), whereas Hoxb5 knockdown changed the mesenchymal (*) distribution of SMAD7 (k). This effect of Hoxb5 knockdown on SMAD7 persisted in the presence of DHT (l). 100x Mag; representative tissue sections from at least 3 lungs for each condition from 3 experiments. Immunostaining for different experimental conditions was done in simultaneous reactions and incubated for the same time periods, allowing direct comparison of spatial and cellular localization of the proteins studied.