The signaling pathway activated by TGFβ1 involves TGFβ1 receptor–mediated cell signaling. In an effort to identify the basis for ECAD’s regulation of the TGFβ1 signaling pathway, we measured the effect of forced expression of ECAD on the TGFβ1-mediated induction of its own gene. The exposure of primary HSCs to TGFβ1 (5 ng/mL for 12 hours) caused a 2.8-fold increase in TGFβ1 reporter gene activity in comparison

with a control, and this was abolished by ECAD (Fig. 3A, left). Similarly, TGFβ1-inducible TGFβ1 luciferase activity was also reduced by ECAD in LX-2 cells (Fig. 3A, right). To assess whether ECAD inhibits SBE-mediated gene induction in response to TGFβ1 treatment, we performed reporter gene assays in MEFs or LX-2 cells transfected GDC-0973 purchase with pGL3-(CAGA)9-MLP luciferase. As expected, ECAD overexpression significantly decreased TGFβ1-inducible SBE luciferase activity in these cells (Fig. 3B): the effects of transient and stable transfections were comparable. The SBE reporter activity in MEFs was less than 10% of that in LX-2 cells. TGFβ1 receptor–mediated cell signaling depends on Smad3/2 phosphorylation; this allows phosphorylated CYC202 cell line Smad3/2 to form

oligomers with Smad4. The resultant complex translocates into the nucleus and there acts as a transcriptional activator.10 To address the downstream link between ECAD and TGFβ1 repression, we assessed the inhibitory effect of ECAD on TGFβ1-dependent Smad3/2 phosphorylation. The treatment of mock-transfected MEFs or GFP-infected LX-2 cells with TGFβ1 enhanced Smad3/2 phosphorylation (Fig. 4A). Intriguingly, ECAD overexpression attenuated the phosphorylation of Smad3 and, to a minor extent, that of Smad2. A similar change was observed in LX-2 cells treated with TGFβ1 after

the adenoviral infection of ECAD. As we anticipated, ECAD inhibited the ability of Smad3 to 上海皓元 induce luciferase activity from an SBE-driven reporter or TGFβ1 reporter construct (Fig. 4B). Our results indicate that ECAD inhibits Smad3/2 phosphorylation and thus antagonizes Smad-dependent gene transcription. In addition to the Smad pathway, TGFβ1 receptor signaling activates other pathways such as small guanosine triphosphatase (GTPase), mitogen-activated protein kinases, and phosphatidylinositol 3-kinase.10 These pathways may crosstalk with Smad signaling.10, 16 In particular, RhoA regulates Smad phosphorylation and Smad-dependent gene induction in response to TGFβ1.16 To verify the regulatory role of RhoA in TGFβ1-dependent Smad activation, the phosphorylation status of Smad3/2 was monitored in cells treated with cell-permeable C3 toxin (a RhoA inhibitor) or cells transfected with a dominant negative mutant of ras homolog gene family A (DN-RhoA). The treatment of LX-2 cells with C3 toxin led to a reduction in Smad3/2 phosphorylation and, consequently, inhibited the ability of TGFβ1 to induce SBE luciferase activity (Fig. 5A).