Gdf11/Smad signalling and Cdx proteins cooperate to activate the Hoxc8 early enhancer in HepG2 cells.

Developing anatomy along the head-tail axis of bilaterian embryos is specified, to a large extent, by the overlapping patterns of expression of the Hox genes. Hox gene enhancers respond to a variety of signals in order to regulate these discreet domains of expression. For mouse Hoxc8, the 399bp "early enhancer" plays a major role. Activation of this enhancer is now examined using luciferase expression constructs transfected into HepG2 cells. Constructs are activated by the combined actions of Gdf11/Smad and Cdx protein signalling pathways, both of which are functional in early embryos. Each of these pathways alone has little stimulatory effect. Stimulation by the two pathways together exceeds the sum of the effects of each pathway alone, indicating synergistic activity. By mutation analysis, two Smad binding motifs are identified as mediators of the Gdf11 effect and two Cdx binding motifs mediate the Cdx effect. The two Smad motifs and one of the Cdx sites are conserved from fish to mammals. Gdf11 stimulation is partially inhibited by Specific Inhibitor of Smad3, suggesting that Smad3 plays a part in signal transduction. Fgf2 increases luciferase activation by the Hoxc8 enhancer, but not, apparently, by specific interactions with either Gdf11 or Cdx effects.