Abstract
Purpose: :
In previous studies we have demonstrated that Rho GEF, NET1 have been shown to mediate RhoA activation and epithelial-mesenchymal transition (EMT) in TGF-β1 induced human retinal pigment epithelium cell line, ARPE-19 cells. The purpose of this study were to examine what regulate NET1 and to test whether Smad signaling cross-talks with Rho pathways during EMT induced by TGF-β1.
Methods: :
Serum-starved ARPE-19 cells were incubated with vehicle alone or 10ng/ml TGF-β1. Cells were pretreated for 1 h with vehicle (DMSO) or 10 µM of MEK, Akt, or P13K inhibitors (PD98059, Triciribine [TCN], or LY294002, respectively) and then treated with 10 ng/ml TGF-β1 for 0, 2, or 4 h. After treatment, total RNA was isolated, reverse transcribed, and amplified by PCR. Using dominant negative Smad3 and active Smad3 DNA construct, we show that these proteins are critical to TGF-β1 induced NET1 expression. We used qChIP analysis to address whether Smads directly target the NET1 promoter.
Results: :
Inhibitors of MEK, Akt, and PI3K did not inhibit the TGF-β1 induction of NET1. RT-PCR revealed that NET1 mRNA expression was highly induced by TGF-β1 treatment in ARPE-19 cells transformed with control vector, whereas TGF-β1 failed to induce NET1 mRNA expression in cells expressing either dominant-negative Smad3 or constitutively active Smad7. Cells expressing constitutively active Smad3 showed high levels of NET1 mRNA expression in the absence of TGF-β1 treatment that were similar to those observed in TGF-β1-stimulated cells. Similar results were obtained for NET1 protein expression as indicated by western blotting. ChIP analysis of ARPE-19 cells revealed an increased interaction of the NET1 promoter with Smad3 following treatment with TGF-β1.
Conclusions: :
These findings suggest that Smad3 induces NET1 production. Smad3 induces both the mRNA and protein expression of NET1 by regulating the NET1 promoter. These data define a new role for Smad as a modulator of RhoA activation while regulating TGF-β1 induced epithelial-mesenchymal transitions.
Keywords: retinal pigment epithelium • EMT (epithelial mesenchymal transition) • signal transduction