FLRG, an activin-binding protein, is a new target of TGFβ transcription activation through Smad proteins

The FLRG gene encodes a secreted glycoprotein that binds to activin and is highly homologous to follistatin, an activin ligand. We cloned the promoter region of the human FLRG gene, and defined the minimal region necessary for transcription activation in a reporter-system assay. We showed that the fragment between positions -130 and +6, which consists of multiple consensus Sp1-binding sites, is required for the constitutive expression of the FLRG gene. We demonstrate here that FLRG mRNA expression is rapidly induced by TGF beta or by transfection with Smad protein expression vectors in human HepG2 cells. We investigated the transcription-regulation mechanism of FLRG expression in HepG2 cells following treatment with TGF beta. By deletion and point-mutation analysis of the FLRG promoter, we identified a Smad-binding element involved in the TGF beta -inducible expression of the FLRG gene. Moreover, transactivation of the FLRG promoter by TGF beta was compromised by dominant-negative mutants of Smad3 and Smad4 proteins. In addition, get electrophoresis mobility-shift assays demonstrated the specific interaction of Smad3 and Smad4 proteins with the Smad-binding element consensus motif found in the FLRG promoter. Taken together, our data imply that Smad proteins participate in the regulation of expression of FLRG, a new target of TGF beta transcription activation.