This makes it unlikely that BMP ligand-trap proteins, modification or degradation of BMP-Rs,

or Everolimus R-Smad deactivation play an important role in the modulation of the BMP effect by HGF or EGF. Further, total nuclear Smad1/5/8 is not decreased by HGF treatment (Fig. 5A). Additional modulation at the ligand-receptor level is provided by BMP pathway inhibitors including BAMBI,21 Smad 6,22, 23 and Smad7, the latter already known to play a role in hepcidin regulation.7, 24 The concentrations of BAMBI and inhibitory Smads determine their effect on signaling.23 We used whole-cell lysates of primary mouse hepatocyte cultures treated with BMP and HGF or EGF to examine the protein levels of the three inhibitors. After overnight incubation, neither Smads 6 or 7 (Supporting Fig. S6A,B) nor BAMBI (data not shown) were induced by growth factor treatments. Growth factors have been reported to decrease the total Smad1 pool by proteasomal degradation.25 GSK1120212 molecular weight Treatment with HGF had no effect on total Smad1 or Smad5 (Supporting Fig. S6C,D) in the 2 hours following HGF treatment or overnight

(data not shown). Treatment with EGF also did not cause a change in total Smad 1/5/8 in whole cell lysates (Fig. 5B). The common mediator Smad4 is also a target for regulatory input and its ubiquitination leads to its degradation in the proteasome.26 Decreased Smad4 in the context of hepcidin reporter suppression by hypoxia was recently described.27 From hepatocytes treated with BMP6 with and without HGF, we blotted nuclear lysates for Smad4. Smad4 levels in

the nucleus were unaffected by HGF, indicating that the BMP signal had adequate access to co-Smad for formation of transcription complexes (Supporting Fig. S6E). Thus, the mechanism for growth factor suppression of hepcidin does not include overall degradation of the receptor-activated Smad pool or Smad4. The linker region between the two globular domains of Smad1 can be phosphorylated by several kinases, including the mitogen-activated protein kinase (MAPK) ERK2, cyclin-dependent kinases (CDK), and glycogen Tolmetin synthase kinase-3β (GSK3β)25 and the modification inhibits nuclear translocation of Smads. Growth factors including HGF and EGF induce linker phosphorylation28 acting to limit BMP signaling during development. After growth factor treatment of BMP6-stimulated hepatocytes, immunoblots for phospho-Smad1/5/8 showed moderately decreased nuclear localization of phospho-Smad/1/5/8 (Fig. 6). The difference between the growth-factor treated nuclear lysates and the control lysates was statistically significant for both growth factors by pairwise t test when four repeated experiments were analyzed together for each growth factor. We next considered modes of BMP pathway suppression that target the Smad transcriptional complex. The Smad transcriptional complex is nucleated by R-Smad/Smad4, but the binding affinity is regulated by DNA-binding coactivators or corepressors such as TGIF.