Effect of ATRA on dephosphorylation of Smad2.
ATRA increases dephosphorylation of Smad2. (A) ATRA reduced the levels of phospho-Smad2 induced by TGF-β1 in HL-60 cells. HL-60 cells treated sequentially showed a lower level of phospho-Smad2 when cells were incubated with 10 ng/mL TGF-β1 for 48 hours, washed, and then treated with 10 nM ATRA for 24 hours, as compared with cells similarly pretreated with 10 ng/mL TGF-β1, washed, and treated with control medium. (B-D) Cells were changed to 0.2% serum, and okadaic acid or MG-132 was added. After 3 hours, cells were washed 3 times with medium containing 5% serum and incubated for an additional 24 hours with addition of TGF-β, Vit D3, ATRA, or TGF-β plus ATRA, in the absence of the inhibitors. Lysates were analyzed by immunoblotting with an antibody specific to C–terminally phosphorylated Smad2. (B) The proteasome inhibitor MG-132 (50 μM) did not have specific effects on cells treated with ATRA. (C) Treatment with okadaic acid (100 nM) blocks the ability of ATRA (10 nM) to decrease levels of phospho-Smad2 induced by TGF-β1 (10 ng/mL). (D) Treatment with okadaic acid (100 nM) alone enhances detection of phospho-Smad2, in addition to its ability to augment levels induced by Vit D3.
