Smad1 Domains Interacting with Hoxc-8 Induce Osteoblast Differentiation*
- From the Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and ‡Procter & Gamble Pharmaceuticals, Mason, Ohio 45040
Abstract
Bone morphogenetic proteins are potent osteotropic agents that induce osteoblast differentiation and bone formation. The signal transduction of bone morphogenetic proteins has recently been discovered to involve Smad proteins. Smad1 is an essential intracellular component that is specifically phosphorylated by bone morphogenetic protein receptors and translocated into the nucleus upon ligand stimulation. Previously, we have reported that Smad1 activates osteopontin gene expression in response to bone morphogenetic protein simulation through an interaction with a homeodomain transcription factor, Hoxc-8. In the present study, the interaction domains between the two proteins were characterized by deletional analysis in both yeast two-hybrid and gel shift assays. Two regions within the amino-terminal 87 amino acid residues of Smad1 were mapped to interact with Hoxc-8, one of which binds to the homeodomain. Overexpression of recombinant cDNAs encoding the Hoxc-8 interaction domains of Smad1 effectively activated osteopontin gene transcription in transient transfection assays. Furthermore, stable expression of these 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 with Hoxc-8 mimics bone morphogenetic protein signaling and is sufficient to induce osteoblast differentiation and bone cell formation.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant DK53757 (to X. C).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: 1670 University Blvd., VH G002, Birmingham, AL 35294. Tel.: 205-934-0162; Fax: 205-934-1775; E-mail cao@path.uab.edu.
- Abbreviations:
- BMP(s)
-
bone morphogenetic protein(s)
- TGF-β
-
transforming growth factor β
- MH
-
Mad homology
- FAST
-
forkhead activin signal transducer
- Hox
-
homeobox
- GST
-
glutathione S-transferase
- CMV
-
cytomegalovirus
- aa
-
amino acid(s)
- Tet
-
tetracycline
- rh
-
recombinant human
- ALP
-
alkaline phosphatase
- HD
-
homeodomain
- HID
-
Hoxc-8 interaction domain
- CR
-
conserved region
- m prefix
-
mutant
- -L
-
-NL, -M, linker region, NH2 domain and linker
- minimal regions of HID
-
respectively
-
- Received June 14, 1999.
- Revision received October 25, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
