Smad1 domains interacting with hoxc-8 induce osteoblast differentiation

Bone morphogenetic proteins are potent osteotropic agents that induce osteo blast differentiation and bone formation. The signal transduction of bone m orphogenetic proteins has recently been discovered to involve Smad proteins . Smad1 is an essential intracellular component that is specifically phosph orylated by bone morphogenetic protein receptors and translocated into the nucleus upon ligand stimulation. Previously, we have reported that Smad1 ac tivates osteopontin gene expression in response to bone morphogenetic prote in simulation through an interaction with a homeodomain transcription facto r, Hoxc-8. In the present study, the interaction domains between the two pr oteins were characterized by deletional analysis in both yeast two-hybrid a nd gel shift assays. Two regions within the aminoterminal 87 amino acid res idues of Smad1 were mapped to interact with Hoxc-8, one of which binds to t he homeodomain. Overexpression of recombinant cDNAs encoding the Hoxc-8 int eraction domains of Smad1 effectively activated osteopontin gene transcript ion in transient transfection assays. Furthermore, stable expression of the se Smad1 fragments in 2T3 osteoblast precursor cells stimulated osteoblast differentiation-related gene expression and led to mineralized bone matrix formation. Our data suggest that the interaction of amino-terminal Smad1 wi th Hoxc-8 mimics bone morphogenetic protein signaling and is sufficient to induce osteoblast differentiation and bone cell formation.