Hoxa-9 represses transforming growth factor-beta-induced osteopontin gene transcription

Smad2 and Smad3 are downstream transforming growth factor-beta (TGF-beta) s ignaling molecules. Upon phosphorylation by its type I receptor, Smad2 or S mad3 forms a complex with Smad4 and translocates to the nucleus where the c omplex activates target gene transcription. In the present study, we report that Smad3 binds directly to the osteopontin (OPN) promoter and that Smad4 interacts with the Hox protein and displaces it from its cognate DNA bindi ng site in response to TGF-beta stimulation. In gel shift assays, the gluta thione S-transferase-Smad3 fusion protein was found to bind to a 50-base pa ir DNA element (-179 to -229) from the OPN promoter. Also, we found that bo th Hoxc-8 and Hoxa-9 bound to a Hox binding site adjacent to Smad3 binding sequence. Interestingly, Smad4, the common partner for both bone morphogeni c protein and TGF-beta signaling pathways, inhibited the binding of Hox pro tein to DNA. FLAG-tagged Smad4 coimmunoprecipitated with HA-tagged Hoxa-9 f rom cotransfected COS-1 cells, demonstrating an interaction between Smad4 a nd Hoxa-9. Transfection studies showed that Hoxa-9 is a strong transcriptio nal repressor; it suppresses the transcription of the luciferase reporter g ene driven by a 124-base pair OPN promoter fragment containing both Smad3 a nd Hox binding sites. Taken together, these data demonstrate a unique TGF-b eta -induced transcription mechanism. Smad3 and Smad4 exhibit different fun ctions in activation of OPN transcription. Smad4 binds directly to the OPN promoter as a sequence-specific activator, and Smad4 displaces the transcri ption repressor, Hoxa-9, by formation of Smad4/Hox complex as part of the t ranscription mechanism in response to TGF-beta stimulation.