Smads in human trophoblast cells: expression, regulation and role in TGF-β-induced transcriptional activity

Abstract

Smad proteins have recently been shown to be downstream signaling molecules that transduce TGF-β signals from cell surface to the nucleus. To determine the mechanisms of TGF-β action in human trophoblast cells, we investigated the expression and regulation of Smad2, 3, 4, and 7 mRNAs in a normal trophoblast cell line, NPC, and a cell line derived from choriocarcinoma, JEG-3. Messenger RNAs for Smad2, 3, 4 and 7 were detected in both NPC and JEG-3 cells. TGF-β1 induced modest increases in Smad2 and Smad4 mRNA levels without affecting Smad3 mRNA expression in both cell lines. Significant increases in Smad7 mRNA levels in both NPC and JEG-3 cells following TGF-β1 treatment were observed. TGF-β1 also induced promoter activity of the Smad7 gene, indicating a direct effect at the level of gene transcription. The transcriptional activity of TGF-β was examined in JEG-3 cells using two TGF-β responsive reporter constructs, p3TP-Lux and pAR3-Lux. We found that Smad3 and to a lesser extent, Smad2 and Smad4, enhanced, while Smad7 inhibited, TGF-β1-induced transcriptional activities. The basal and TGF-β1-induced transcription can be blocked by overexpression of a dominant negative TGF-β type II receptor. Taken together, these findings demonstrate that in human trophoblast cell lines, the Smad pathway involved in TGF-β signal transduction is functional and that TGF-β plays an autocrine role in regulating gene expression.