The effect of RAR-α on the ability of ATRA to decrease levels of phospho-Smad2/Smad3.

The ability of ATRA to decrease levels of phospho-Smad2/Smad3 is dependent on RAR-α. (A) Treatment for 4 days with ATRA together with TGF-β1 enhances the expression of CD14 by HL-60R compared with cells treated with TGF-β1 alone (means ± SEMs). (B) In contrast to wild-type HL-60, ATRA is unable to reduce levels of phospho-Smad2 induced by TGF-β1 in RAR-α–mutant HL-60 cells (HL-60R) treated simultaneously with ATRA and TGF-β1 (lane 3 compared with lane 4). Treatments were for 24 hours. (C-E) Immunohistochemical staining showed that ATRA is unable to reduce the TGF-β1–dependent Smad3 nuclear localization in RAR-α–mutant HL-60 cells treated for 18 hours with ATRA and TGF-β1 (C) as seen in wild-type HL-60 cells (D). Original magnification × 400. The arrow on the left shows a cell with both nuclear and cytoplasmic staining, and the arrow on the right shows a cell with only cytoplasmic staining. (E) Quantitation of the data in panel C was obtained by assessment of the staining patterns in 1000 cells in 5 different fields of treated HL-60R cells. For panels A, B, and E, treatments were (1) vehicle (gray); (2) ATRA, 10 nM (black and gray striped); (3) TGF-β1, 10 ng/mL (black); or (4) the combination of ATRA and TGF-β1 (black and white striped).