Inhibition of binding of hox and homeodomain-containing proteins and uses thereof

Abstract

The present invention demonstrates that BMP-2/4 activates osteopontin gene transcription by removing Hoxc-8 binding through Smad1 interaction with the Hoxc-8 DNA binding domain. Since the DNA binding domain is conserved in all Hox and homeodomain-containing proteins, Smad1 likely interacts with all Hox or homeodomain-containing proteins. Furthermore, the present invention reveals the Smad1-mediated transcriptional mechanism in the BMP-2/4 signaling pathway and also provides information about the transcriptional roles of the Hox genes during embryonic development.

What is claimed is:  
     
         1 . A method of stimulating bone formation in an individual, comprising the step of: 
 inducing an interaction between Smad1 and a homeobox-containing transcription factor, wherein said interaction induces a BMP-responsive gene encoding a bone matrix protein which produces osteoblast and/or chondroblast differentiation thereby stimulating bone formation.    
     
     
         2 . The method of  claim 1 , wherein said interaction is induced by means selected from the group consisting of phosphorylation of Smad1, overexpression of Smad1, and mutation of said homeobox-containing transcription factor.  
     
     
         3 . The method of  claim 1 , wherein said homeobox-containing transcription factor is selected from the group consisting of Hoxc-8,Hoxa-9, Msx-1 and Msx-2.  
     
     
         4 . The method of  claim 1 , wherein said BMP-responsive gene is selected from the group consisting of osteopontin, sialoprotein, osteonectin, and osteocalcin.  
     
     
         5 . The method of  claim 1 , wherein said individual is osteopenic.  
     
     
         6 . A method of inducing gene(s) encoding bone matrix proteins, comprising the step of: 
 inducing an interaction between Smad1 and a homeobox-containing transcription factor, wherein said interaction results in a n induction of gene(s) encoding bone matrix proteins.    
     
     
         7 . A method of inducing a gene encoding osteopontin, comprising the steps of: 
 inducing an interaction between Smad1 and Hoxc-8, wherein said interaction results in removing transcriptional repression of a gene encoding osteopontin, thereby inducing said gene encoding osteopontin.    
     
     
         8 . The method of  claim 10 , wherein said interaction is induced by means selected from the group consisting of phosphorylation of Smad1, overexpression of Smad1, and mutation of said homeobox-containing transcription factor.  
     
     
         9 . A method of screening for a compound that stimulates bone formation, comprising the steps of: 
 contacting a cell with a compound; and    determining the ability of said compound to inhibit binding of Hoxc-8 to a gene, wherein inhibition of binding results in induction of said gene, thus indicating that the compound stimulates bone formation.    
     
     
         10 . The method of  claim 9 , wherein said compound is selected from the group consisting of an antibody or fragment thereof, synthetic drugs, synthetic proteins and a phosphorylated form of Smad1 or fragments thereof.  
     
     
         11 . The method of  claim 9 , wherein determination of inhibition of binding of Hoxc-8 to a gene is by a method selected from the group consisting of a gel-shift assay, transcription, Northern blotting, and Western blotting.  
     
     
         12 . The method of  claim 9 , wherein said gene is selected from the group consisting of osteopontin, sialoprotein, osteonectin, and osteocalcin.  
     
     
         13 . A method of regulating disease in an individual, comprising the step of: 
 inhibiting the binding of a homeobox-containing transcription factor to a gene involved in regulating disease in cells of said individual, wherein inhibition of binding removes transcriptional repression by the homeobox-containing protein of said gene, thereby resulting in the induction of said genes involved in regulating disease.    
     
     
         14 . The method of  claim 13 , wherein said inhibition is due to the presence of a compound that binds to the homeobox-containing transcription factor, thereby inhibiting the DNA binding ability of said homeobox-containing transcription factor.  
     
     
         15 . The method of  claim 13 , wherein said compound is selected from the group consisting of an antibody or fragment thereof, synthetic drugs, synthetic proteins and a phosphorylated form of Smad1 or fragments thereof.  
     
     
         16 . The method of  claim 13 , wherein said homeobox-containing transcription factor is selected from the group consisting of Hoxc-8,Hoxa-9, Msx1, and Msx2.  
     
     
         17 . The method of  claim 13 , wherein said individual has a disease selected from the group consisting of osteoporosis, cancer, cardiovascular disease and neurological disease.