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.