Figure 4. Loss of Apical TGF-β Responsiveness in Polarized Epithelial Cells Reflects Exclusive Basolateral TGF-β Receptor Expression.

(A) IF detection of transiently transfected Myc-TβRI and HA-TβRII in low density (LD, upper) and high density (HD, lower) monolayer cultures of AKR-2B, MDCK, and EpH4 cells. Images are represented as XZ cross-sections of transfected cells.

(B) IF detection of transiently transfected Myc-TβRI and HA-TβRII in polarized Transwell cultures of EpH4 cells. Two representative XZ cross-sections of transfected cells are shown. Nuclei (blue) were stained with DAPI.

(C) Surface biotinylation assays of endogenous TβRI and TβRII in AKR-2B, EpH4, and MDCK cells grown in low- or high-density monolayer cultures.

(D) Apical (AP) and basolateral (BL) surface biotinylation assays of endogenous TβRI and TβRII in MDCK and EpH4 cells in monolayer (low-density) or Transwell cultures (high density, polarized). Cell proteins were biotinylated either from the medium (monolayer on plastic, low density) or from the apical or basolateral medium in the case of high density polarized transwell cultures. After surface biotinylation, proteins were immunoprecipitated with streptavidin-agarose and then detected by blotting with the appropriate TGF-β receptor or E cadherin antibody. Lanes C and T reflect immunoprecipitated protein in the absence of biotinylation (C) or in monolayer for total labeling (T), respectively. E-cadherin was detected to serve as a marker of basolateral cell surfaces.

(E) Quantitative reverse transcriptase–PCR analysis of JUNB, CTGF, and SMAD7 expression in polarized EpH4 cells incubated without (−) or with TGF-β (2 hr) added either in the apical (AP) or basolateral (BL) compartment of Transwells. Results are expressed as -fold induction by TGF-β and are the mean ± SD from two independent experiments, each measured in triplicate.