Interaction of Smad6 with Hox proteins and uses thereof

Abstract

The present invention describes a novel interaction between Smad6 and the Hox genes in nuclear transcriptional regulation following BMP signal transduction. The present invention further provides methods of using this novel Smad6/Hox protein interaction to regulate gene expression, regulate bone formation and control osteoporosis. Further provided are methods of screening for compounds that interfere with the novel Smad6/Hox protein interaction, thereby resulting in expression of a Hox protein-repressed gene and/or stimulating bone formation.

1 . A method of regulating bone formation in an individual, comprising the step of: 
 (a) administering a composition that alters the binding activity of Smad6 protein in said individual, wherein an increase in Smad6 protein results in an increase in Smad6/Hoxc-8 complexes which maintains transcriptional repression of genes involved in bone formation, wherein a decrease in Smad6 protein binding activity results in a decrease in Smad6/Hoxc-8 complexes, which relieves transcriptional repression of genes involved in bone formation, thereby regulating bone formation in said individual.    
   
   
       2 . The method of  claim 1 , wherein said composition is selected from the group consisting of a transgene encoding Smad6, an antisense molecule directed towards Smad6, an antibody directed towards Smad6 and Hox proteins.  
   
   
       3 . The method of  claim 1 , wherein said genes involved in bone formation are selected from the group consisting of osteopontin, osteoprotegrin, OPGL and RANK.  
   
   
       4 . A method of regulating nuclear bone morphogenetic protein signaling in an animal, comprising the step of: 
 (a) administering a composition that alters the binding activity of available Smad6 protein in a cell in said individual, wherein an increase in available Smad6 protein results in an increase in Smad6/Hoxc-8 complexes which maintains transcriptional repression of genes involved in bone formation, wherein a decrease in said available Smad6 protein binding activity results in a decrease in Smad6/Hoxc-8 complexes which relieves transcriptional repression of genes involved in bone formation, thereby regulating nuclear BMP signaling.    
   
   
       5 . The method of  claim 4 , wherein said composition is selected from the group consisting of a gene encoding Smad6, an antisense molecule directed towards Smad6, an antibody directed towards Smad6 and Hox proteins.  
   
   
       6 . The method of  claim 4 , wherein said genes involved in bone formation are selected from the group consisting of osteopontin, osteoprotegrin, OPGL and RANK.  
   
   
       7 . A method of screening for a compound that disrupts transcriptional repression of a gene, comprising the steps of: 
 (a) combining Smad6 proteins and Hoxc-8 proteins in the presence and absence of a compound; and    (b) detecting complex formation between said Smad6 proteins and said Hoxc-8 proteins, wherein a lack of complex formation between said Smad6 proteins and said Hoxc-8 proteins in the presence of said compound is indicative of a compound that disrupts transcriptional repression of a gene.    
   
   
       8 . The method of  claim 7 , wherein said detection of Smad6/Hoxc-8 protein complex formation is by means selected from the group consisting of a gel shift assay and a reporter transfection assay.  
   
   
       9 . A method of screening for a compound that disrupts transcriptional repression of a gene, comprising the steps of: 
 (a) combining a Smad6/Hoxc-8 complex and a DNA molecule in the presence and absence of a compound, wherein said DNA molecule comprises a Hox DNA binding element; and    (b) determining the amount of binding by said Smad6/Hoxc-8 protein complex to said DNA molecule, wherein less binding in the presence of said compound than in the absence of said compound is indicative of a compound that disrupts transcriptional repression of said gene.    
   
   
       10 . The method of  claim 9 , wherein said DNA binding by said Smad6/Hoxc-8 protein complex is determined by means selected from the group consisting of a gel-shift assay, a competitive binding assay, pull-down assay and immunoprecipitation assay.  
   
   
       11 - 14 . (canceled)  
   
   
       15 . A method of regulating expression of gene that binds Hoxc-8, wherein binding by Hoxc-8 results in transcriptional repression of said gene, comprising the step of: 
 altering the amount of Smad6 protein, wherein an increase in said Smad6 protein binding activity results in an increase in Smad6/Hoxc-8 protein complexes, wherein an increase in said Smad6/Hoxc-8 protein complexes maintains said transcriptional repression of said gene, wherein a decrease in said Smad6 protein binding activity results in a decrease in Smad6/Hoxc-8 protein complexes, wherein a decrease in Smad6/Hoxc-8 protein complexes relieves said transcriptional repression of said gene, thereby regulating expression of said gene.    
   
   
       16 . The method of  claim 15 , wherein said gene is selected from the group consisting of osteopontin, osteoprotegrin, OPGL and RANK.  
   
   
       17 . The method of  claim 15 , wherein said Smad6 protein is increased by means selected from the group consisting of overexpression of a Smad6 gene and upregulation of a Smad6 gene.  
   
   
       18 . The method of  claim 15 , wherein said Smad6 protein is decreased by means selected from the group consisting of antisense hybridization to Smad6 RNA, antibody binding to a Smad6 protein and mutagenesis of a gene encoding Smad6.  
   
   
       19 . The method of  claim 15 , further comprising the step of: 
 increasing the amount of Smad1 protein, wherein said Smad1 protein binds said Hoxc-8, thereby relieving said transcriptional repression of said gene.    
   
   
       20 . A method of inducing transcription of a gene encoding osteopontin, osteoprotegrin, OPGL or RANK comprising the steps of: 
 inhibiting Smad6, wherein in the presence of Smad1, said inhibition of Smad6 removes transcriptional repression of a gene encoding osteopontin, thereby inducing transcription of said gene.