WO2001011013A2 - The interaction of smad6 with hox proteins and uses thereof - Google Patents

The interaction of smad6 with hox proteins and uses thereof Download PDF

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WO2001011013A2
WO2001011013A2 PCT/US2000/040563 US0040563W WO0111013A2 WO 2001011013 A2 WO2001011013 A2 WO 2001011013A2 US 0040563 W US0040563 W US 0040563W WO 0111013 A2 WO0111013 A2 WO 0111013A2
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smadό
hoxc
gene
protein
compound
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PCT/US2000/040563
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WO2001011013A3 (en
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Xu Cao
Xingming Shi
Shuting Bai
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Uab Research Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity

Definitions

  • the present invention relates generally to the field o f signal transduction, transcriptional regulation and b o ne physiology. More specifically, the present invention relates to th e role by which Smad ⁇ interacts with nuclear Hox proteins during bone morphogenetic protein (BMP) signal transduction.
  • BMP bone morphogenetic protein
  • Smads Members of TGF- ⁇ superfamily transduce their signals into the cell through a family of mediator proteins called Smads .
  • Smadl Receptor-regulated Smadl , Smad5 and Smad ⁇ mediate BMP signaling, whereas Smad2 and Smad3 respond to TGF- ⁇ (9- 1 2) .
  • Smad4 Upon phosphorylation by their type I receptors, the receptor- regulated Smads interact with a common partner, Smad4, and translocate to the nucleus where the complex recruits DNA binding protein(s) to activate specific gene transcription ( 1 , 2, 1 3 - 1 5 ) .
  • Hox homeobox-containing transcription factor genes In vertebrates, there are 39 Hox homeobox-containing transcription factor genes, organized into four separate chromosome clusters, which play critical roles in the process a n d patterning of vertebrate embryonic development (28,29). These 39 genes are subdivided into 13 paralogous groups on the basis of duplication of an ancestral homeobox cluster during evolution, sequence similarity and position within the cluster (30). Each paralog group has been demonstrated to be responsible for morphogenesis of a particular embryonic domain or structure (29). There are three members in Hox paralog group VIII, Hoxb- 8, Hoxc-8 and Hoxd-8 (30).
  • Hox genes are required during vertebrate limb bud development, and particularly, Hoxb-8 was suggested to be a transcription factor involved in activating th e Sonic hedgehog gene, which is the key mediator in limb development (31 ,32). Furthermore, Northern blot analysis shows that Hoxc-8 is expressed during human embryo development a t high levels in spinal cord, backbone and limbs and at a lower level in heart (33). BMP-2/4 activates expression of Hox genes , induces osteoblast differentiation and controls patterning acro s s the anteroposterior (a-p) axis of developing limb (34).
  • a-p anteroposterior
  • Smads are mediators of the superfamily o f transforming growth factor- ⁇ (TGF- ⁇ ) signaling pathways ( 1 , 2 ) .
  • TGF- ⁇ transforming growth factor- ⁇
  • Smad ⁇ and Smad7 antagonize the TGF- ⁇ signals (3,4).
  • Smad ⁇ also interacts with phosphorylated Smadl t o prevent the formation of an active signaling complex of Smad l and Smad4 in the cytoplasm (7,8).
  • Smad ⁇ interacts with Hoxc-8 as a transcriptional corepres sor, inhibiting bone morphogenetic protein signaling in the nucleus .
  • the present invention describ that Smad ⁇ functions as a transcriptional corepressor in the nucleus of BMP signaling.
  • One object of the present invention is to describe a role for Smad ⁇ in Hox protein transcriptional regulation, a n d additionally to provide methods of using the S mad ⁇ /Hox interaction in gene regulation and methods of screening for drugs that effect the Smad ⁇ /Hox interaction.
  • a method of regulating bone formation in an individual comprising the step of: (a) administering a composition to th e individual, wherein the composition alters the activity of Smad ⁇ protein.
  • An increase in the Smad ⁇ protein results in an increase in Smad6/Hoxc-8 complexes; an increase in S mad6/Hoxc- 8 complexes maintains transcriptional repression of genes involved in bone formation.
  • a decrease in the Smad ⁇ protein activity results in a decrease in Smad ⁇ /Hoxc-8 complexes wherein a decrease in Smad6/Hoxc-8 complexes relieves transcriptional repression of genes involved in bone formation, thereby regulating bone formation in the individual.
  • a method of regulating nuclear b o n e morphogenetic protein signaling comprising the step of: ( a ) administering a composition to a cell that alters the activity o f Smad ⁇ protein.
  • An increase in the available Smad ⁇ protein results in an increase in Smad ⁇ /Hoxc-8 complexes; an increase in Smad6/Hoxc-8 complexes maintains transcriptional repression o f genes involved in bone formation.
  • a decrease in the Smad ⁇ protein binding activity results in a decrease in Smad ⁇ /Hoxc- 8 complexes, wherein a decrease in Smad ⁇ /Hoxc-8 complexes relieves transcriptional repression of genes involved in bo ne formation, thereby regulating nuclear BMP signaling.
  • a method of screening for a compound that disrupts transcriptional repression of a gene comprises the steps of: (a) combining Smad ⁇ proteins and Hoxc- 8 proteins in the presence and absence of a compound; and ( b ) detecting complex formation between the Smad ⁇ proteins and th e Hoxc-8 proteins.
  • a lack of complex formation between th e Smad ⁇ proteins and the Hoxc-8 proteins in the presence of th e compound is indicative of a compound that disrupts transcriptional repression of a gene.
  • 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 the DNA molecule comprises a Hox DNA binding element; and (b) determining the amount of binding by th e Smad6/Hoxc-8 protein complex to the DNA molecule, wherein less binding in the presence of the compound than in the ab sence of the compound is indicative of a compound that disrupts transcriptional repression of the gene.
  • a method of screening for a compound that disrupts transcriptional repression of a gene comprising the steps of: (a) combining a Smad ⁇ /Hoxc-8 protein complex with a gene in the presence and absence of a compound, wherein the gene comprises a Hox DNA binding element; and ( b ) assaying for transcription of the gene.
  • An increase in the level o f transcription in the presence of the compound relative to th e level of transcription in the absence of the compound is indicative of a compound that disrupts transcriptional repres sion of the gene.
  • a method of regulating expression o f gene that binds Hoxc-8, wherein binding by Hoxc-8 results in transcriptional repression of the gene comprising the step of: altering the binding activity of Smad ⁇ protein.
  • An increase in th e Smad ⁇ protein results in an increase in Smad ⁇ /Hoxc-8 protein complexes; an increase in the Smad6/Hoxc-8 protein complexes maintains the transcriptional repression of the gene.
  • a decrease in the Smad ⁇ protein binding activity results in a decrease in Smad ⁇ /Hoxc-8 protein complexes, wherein a decrease i n Smad ⁇ /Hoxc-8 protein complexes relieves the transcriptional repression of the gene, thereby regulating expression of the gene .
  • This method may further comprise the step of: increasing th e amount of Smadl protein, wherein the Smadl protein binds th e Hoxc-8, thereby relieving the transcriptional repression of th e gene.
  • a method of inducing transcription o f a gene encoding osteopontin comprising the steps of: inhibiting Smad ⁇ , wherein in the presence of Smadl , the inhibition o f Smad ⁇ removes transcriptional repression of a gene encoding osteopontin, thereby inducing transcription of the gene encoding osteopontin.
  • FIG. 2 shows the interaction of Smadl with Hoxc-8 in vivo .
  • Flag-tagged Smad ⁇ , Flag-tagged Smad ⁇ carboxy domain (Smad ⁇ C), Flag-tagged Smad ⁇ amino domain with linker region (Smad ⁇ N+L) and HA-tagged Hoxc-8 were co-transfected in COS-1 cells with or without ALK3 (Q233D).
  • Cell lysates were immunoprecipitated by anti-Flag antibody and the resulted complexes were analyzed by Western blotting with anti-HA antibody.
  • the expression levels of Smad ⁇ were shown by Western blot with anti-Flag antibody (middle panel), and Hoxc-8 with anti- HA antibody (bottom panel).
  • Figure 3 shows that the Smad ⁇ and Hoxc-8 form a complex on Hox DNA binding site from osteopontin promoter .
  • Figure 3A the complex of Smad ⁇ and Hoxc-8 blocks th e interaction of Smadl with Hoxc-8.
  • Gel-shift assays were performed using osteopontin Hox DNA binding element (OPN-5) as the probe (13), with 1.5 mg GST (lane 2), 1.5 mg GST-Smadl (lanes 3, 6, 8, 10), 0.2 mg GST-Hoxc-8 protein (lanes 5-10), 1 .5 mg GST-Smad ⁇ (lanes 4, 7- 10) and 0.1 mg Smad ⁇ polyclonal antibody (Smad ⁇ AB, lanes 9 and 10).
  • Figure 3B the complex of Smad ⁇ and Hoxc-8 moderately blocks the interaction of Smad4 with Hoxc-8.
  • OPN-5 was used as probe, with 1.5 mg GST (lane 2), 1.5 mg GST-Smad4 (lanes 3, 5, 7 and 9), 0.2 mg GST-Hoxc-8 protein (lanes 4-9), 1 .5 mg GST-Smad ⁇ (lanes 6-9) and 1.5 mg GST-Smadl (lanes 9 an d 1 0) .
  • FIG 4 shows that BMP-induced osteopontin gene transcription is mediated by Hoxc-8 binding site.
  • Figure 4A Smadl/Hoxc-8 interaction domain (SmadlB) induces transcription in a concentration dependent manner.
  • Hox-pGL3 construct 500 ng
  • osteopontin Hox binding site linked to SV40 promoter was co-transfected in MvlLu cells with different amounts of SmadlB expression plasmid.
  • Smads are the mediators of the superfamily o f transforming growth factor- ⁇ (TGF- ⁇ ) signaling pathways ( 1 , 2 ) .
  • TGF- ⁇ transforming growth factor- ⁇
  • Smad ⁇ and Smad7 a subgroup of Smad proteins, antagonize th e TGF- ⁇ signals (3,4).
  • These two Smads induced by TGF- ⁇ or b o n e morphogenetic protein (BMP), form stable association with activated type I receptors, which, in turn, block phosphorylation of ligand-induced Smads (5,6).
  • Smad ⁇ also interacts with phosphorylated Smadl to prevent the formation of an active signaling complex of Smadl and Smad4 in the cytoplasm (7,8).
  • Smad ⁇ interacts with Hoxc-8 as a transcriptional corepressor, inhibiting BMP signaling in th e nucleus.
  • the interaction between Smad ⁇ and Hoxc-8 was identified in a yeast two-hybrid approach, and further demonstrated by co-immunoprecipitation assays in cells.
  • Gel shift assays show that Hoxc-8 interacts with Smad ⁇ as a heterodimer when binding to DNA. More importantly, th e Smad ⁇ /Hoxc-8 complex inhibited both Smadl interaction with Hoxc-8 in gel shift assays and transcription activity mediated b y Smadl .
  • the data presented herein indicate that Smad ⁇ functions as a transcriptional corepressor in BMP signaling in the nucleus.
  • the present invention is directed towards a method o f regulating bone formation in an individual, comprising the s tep of: (a) administering a composition to the individual, wherein th e composition alters the binding activity of Smad ⁇ protein, wherein an increase in the Smad ⁇ protein results in an increase i n Smad ⁇ /Hoxc-8 complexes, wherein an increase in S mad6/Hoxc-8 complexes maintains transcriptional repression of genes involved in bone formation, wherein a decrease in the Smad ⁇ protein binding activity results in a decrease in Smad ⁇ /Hoxc-8 complexes, wherein a decrease in Smad6/Hoxc-8 complexes relieves transcriptional repression of genes involved in b one formation, thereby regulating bone formation in the individual.
  • the present invention is directed towards a method o f regulating nuclear BMP signaling, comprising the step of: ( a ) administering a composition to a cell, wherein the composition alters the binding activity of available Smad ⁇ protein, wherein a n increase in the available Smad ⁇ protein results in an increase in Smad6/Hoxc-8 complexes, wherein an increase in S mad6/Hoxc-8 complexes maintains transcriptional repression of genes involved in bone formation, wherein a decrease in the Smad ⁇ protein binding activity results in a decrease in Smad ⁇ /Hoxc- 8 complexes, wherein a decrease in Smad6/Hoxc-8 complexes relieves transcriptional repression of genes involved in b o ne formation, thereby regulating nuclear BMP signaling.
  • the present invention is directed towards a method o f screening for a compound that disrupts transcriptional repression of a gene, comprising the steps of: (a) combining a Smad ⁇ /Hoxc-8 complex and a DNA molecule in the presence an d absence of a compound, wherein the DNA molecule comprises a Hox DNA binding element; and (b) determining the amount o f binding by the Smad6/Hoxc-8 protein complex to the DNA molecule, wherein less binding in the presence of the compound than in the absence of the compound is indicative of a compound that disrupts transcriptional repression of the gene.
  • DNA binding by the Smad6/Hoxc-8 protein complex i s determined by means selected from the group consisting of a gel- shift assay, a competitive binding assay, immunoprecipitation a nd Yeast two-hybrid assay.
  • the present invention is directed towards a method o f screening for a compound that disrupts transcriptional repression of a gene, comprising the steps of: (a) combining a Smad6/Hoxc-8 protein complex with a gene in the presence an d absence of a compound, wherein the gene comprises a Hox DNA binding element; and (b) assaying for transcription of the gene, wherein an increase in the level of transcription in the pres ence of the compound relative to the level of transcription in th e absence of the compound is indicative of a compound th at disrupts transcriptional repression of the gene.
  • transcription is assayed by means selected from the group consisting of a Northern blot, a Western blot, an enzymatic assay and a chemiluminescent assay.
  • the gene is a reporter gene, and more preferably, the reporter gene is selected from th e group consisting of ⁇ -galactosidase, luciferase, secreted alkaline phosphotase and CAT assay.
  • the present invention is directed towards a method o f regulating expression of gene that binds Hoxc-8, wherein binding by Hoxc-8 results in transcriptional repression of the gene, comprising the step of: altering the amount of Smad ⁇ protein, wherein an increase in the Smad ⁇ protein results in an increase in Smad ⁇ /Hoxc-8 protein complexes, wherein an increase in th e Smad ⁇ /Hoxc-8 protein complexes maintains the transcriptional repression of the gene, wherein a decrease in the Smad ⁇ protein results in a decrease in Smad ⁇ /Hoxc-8 protein complexes , wherein a decrease in Smad ⁇ /Hoxc-8 protein complexes relieves the transcriptional repression of the gene, thereby regulating expression of the gene.
  • the present invention is directed towards a method o f inducing transcription of a gene encoding o steopontin, comprising the steps of: inhibiting Smad ⁇ , wherein in th e presence of Smadl , the inhibition of Smad ⁇ removes transcriptional repression of a gene encoding osteopontin, thereby inducing transcription of the gene encoding osteopontin.
  • the term "transcriptional repres sion by a hox protein” or "transcriptional repression by a homeodomain-containing protein shall refer to any gene whose transcription activities are repressed in the presence of the hox protein or the homeodomain-containing protein.
  • Expression vectors for Flag-tagged full length Smad ⁇ , amino-domain with linker region (Smad ⁇ NL) and carboxy-domain (Smad ⁇ C) were subcloned into a mammalian expression vector pcDNA3 (Invitrogen). HA-tagged Hoxc-8 expression vector was constructed (13). Constitutively active BMP type IA (ALK3) expression plasmid was provided by Dr. Jeffrey L. Wrana (The Hospital for Sick Children, Canada). COS-1 cells were transfected with expression constructs as indicated in Figure 2 using Tfx-50 according to manufacturer' s description (Promega). Cells were lysed 48 h post-transfection and lysates were immuno- precipitated with anti-Flag M2 antibody (Eastman Kodak) an d immuno-blotted with anti-HA antiserum (Babco).
  • Hox-pGL3 reporter bearing Hoxc-8 binding site ( - 290 to -166) was constructed into pGL3-control vector (Promega) .
  • the Hox recognition core TAAT was replaced with GCCG in Hox- pGL3 by PCR to create mutant Hox- ⁇ GL3 (mHox-pGL3).
  • MvlLu cells (5 x l 0 4 cells/22.6 mm dish) were transfected using Tfx-50 with 0.5 mg of luciferase reporter plasmid (Hox-pGL3 or mHox- pGL3 ) and different expression plasmids as indicated. Total DNA was kept constant by addition of pcDNA3 plasmid.
  • Smad ⁇ a n d Smad7 are immunolocalized in the nucleus of rat epiphyseal plate ( 17), Xenopus embryo (18) and Mink lung epithelial (Mv l Lu) cells (19).
  • the interaction of Smad ⁇ with Hoxc-8, a transcription repressor in bone morphogenetic protein signaling pathway suggests that Smad ⁇ may have a novel antagonistic function in the nucleus.
  • the initial Smad ⁇ cDNA clone (Smad ⁇ C in Figure 1 ) encodes amino acids 281 to 496 out of a 496 amino acid protein .
  • the interaction between Hoxc-8 and Smad ⁇ was further confirmed with a ⁇ -gal filter lift assay and quantified by a liquid ⁇ -gal assay ( Figure 1).
  • Figure 1 The interaction between the full length Smad ⁇ fused with th e Gal4 transcriptional activation domain was tested in the two- hybrid system, it showed a weaker interaction compared with th e carboxy-terminal domain (Smad ⁇ C). Deletion of Smad ⁇ amino- terminal domain may change the protein conformation such th at the carboxy-terminal region becomes available to interact with Hoxc-8.
  • COS-1 cells were transiently c o - transfected with expression plasmids for Flag-Smad6, HA-Hoxc-8, and/or constitutively active bone morphogenetic protein type LA receptor ALK3 (Q233D).
  • the cell lysates were immunoprecipitated with anti-Flag antibody and immuno-blotted with anti-HA antibody.
  • the results in Figure 2 demonstrate th at Smad ⁇ (lanes 7 and 8) was co-immunoprecipitated with HA- Hoxc-8.
  • Smad ⁇ C exhibits a strong interaction with Hoxc-8 (lanes 4 and 5).
  • Smad ⁇ amino-terminal with linker region (Smad ⁇ NL) failed to bind to Hoxc-8 in immuno-precipitation assay ( Figure 2, lane 6).
  • Smad proteins contain highly conserved carboxy- and amino-terminal domains (referred to as MHl and MH2 domains, respectively). The MH l domain inhibits biological activities of the MH2 domain due t o interactions between these two distal sites (23).
  • Smad ⁇ on the DNA element was confirmed by the fact that a n anti-Smad ⁇ polyclonal antibody inhibited the development of th e retarded band (lanes 9 and 10).
  • Smad4 also interacting with Hoxc-8, was examined for the same purpose in gel shift assays (Figure 3B). The complex of Smad ⁇ and Hoxc-8, however, did n o t block the interaction of Smad4 with Hoxc-8 completely ( Figure 3B, lanes 7 and 9).
  • Smad6/Hoxc-8 complex To investigate whether the Smad6/Hoxc-8 complex inhibits the interaction of Smadl with Hoxc-8 in activating gene transcription, a model was used ( 13). The interaction domains within the amino-terminal 87 amino acid residues of Smadl w ere mapped to interact with Hoxc-8. Overexpression of cDNAs encoding the Hoxc-8 interaction domains of Smadl linked to a nuclear localization signal (SmadlB) effectively activated osteopontin gene transcription. Stable expression of these S mad l fragments in 2T3 osteoblast precursor cells stimulated endogenous osteoblast differentiation-related gene expres sion and mineralized bone matrix formation.
  • SmadlB nuclear localization signal
  • Hox-pGL3 construct was co-transfected in MvlLu cells with Hoxc-8 or Smad ⁇ expression plasmid, or both.
  • overexpression of Hoxc-8 or Smad ⁇ alone inhibited SmadlB-induced transcription activity.
  • Smad ⁇ binds to BMP type I receptor to block phosphorylation of o ther regulatory Smads.
  • Smad ⁇ has also been shown to interacted with phosphorylated Smadl, inhibiting Smadl translocated into nucleus.

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Abstract

The present invention describes a novel interaction between Smad6 and the Hox genes in nuclear transcriptional regulation following BMP signal transduction. The accompanying figure shows interaction between flag-tagged, recombinant Smad6 and Hoxc-8 in the presence and absence of bone morphogenetic protein type IA receptor, ALK3. The present invention further provides methods of using this novel Smad6/Hox protein interaction, thereby resulting in expression of a Hox protein-repressed gene and/or stimulating bone formation.

Description

THE INTERACTION OF SMAD6 WITH HOX PROTEINS
AND USES THEREOF
BACKGROUND OF THE INVENTION
Cross-reference to Related Application
This non-provisional patent application claims benefit of provisional patent application U.S. Serial number 60/ 147 , 1 6 1 , filed August 14, 1999, now abandoned.
Field of the nvention
The present invention relates generally to the field o f signal transduction, transcriptional regulation and b o ne physiology. More specifically, the present invention relates to th e role by which Smadό interacts with nuclear Hox proteins during bone morphogenetic protein (BMP) signal transduction.
Description of the Related Art
Members of TGF-β superfamily transduce their signals into the cell through a family of mediator proteins called Smads . Receptor-regulated Smadl , Smad5 and Smadδ mediate BMP signaling, whereas Smad2 and Smad3 respond to TGF-β (9- 1 2) . Upon phosphorylation by their type I receptors, the receptor- regulated Smads interact with a common partner, Smad4, and translocate to the nucleus where the complex recruits DNA binding protein(s) to activate specific gene transcription ( 1 , 2, 1 3 - 1 5 ) .
Smadό and Smad7 are struturally divergent Smads a nd antagonists of TGF-β family signaling (1). Smadό and Smad7 are characterized by the stable interactions formed with b o th activated TGF-β and BMP type I receptors, thereby preventing phosphorylation of ligand-induced Smads (4,5). In addition, Smadό has also been demonstrated to interact with phosphorylated Smadl to prevent the formation of an active signaling complex of Smadl and Smad4, preferentially inhibiting the signaling pathways activated by bone morphogenetic proteins (7,16). Furthermore, it was previously demonstrated that S mad l interacts with Hoxc-8 in response to BMP stimulation (13). Hoxc- 8 functions as a transcriptional repressor. The interaction o f Smadl with Hoxc-8 dislodges Hoxc-8 binding from its element, thereby resulting in initiation of gene transcription (13).
In vertebrates, there are 39 Hox homeobox-containing transcription factor genes, organized into four separate chromosome clusters, which play critical roles in the process a n d patterning of vertebrate embryonic development (28,29). These 39 genes are subdivided into 13 paralogous groups on the basis of duplication of an ancestral homeobox cluster during evolution, sequence similarity and position within the cluster (30). Each paralog group has been demonstrated to be responsible for morphogenesis of a particular embryonic domain or structure (29). There are three members in Hox paralog group VIII, Hoxb- 8, Hoxc-8 and Hoxd-8 (30). Hox genes are required during vertebrate limb bud development, and particularly, Hoxb-8 was suggested to be a transcription factor involved in activating th e Sonic hedgehog gene, which is the key mediator in limb development (31 ,32). Furthermore, Northern blot analysis shows that Hoxc-8 is expressed during human embryo development a t high levels in spinal cord, backbone and limbs and at a lower level in heart (33). BMP-2/4 activates expression of Hox genes , induces osteoblast differentiation and controls patterning acro s s the anteroposterior (a-p) axis of developing limb (34).
The prior art is deficient in recognizing the role o f Smadό in conjunction with transcriptional regulation by Hox genes. The present invention fulfills this long-standing need a n d desire in the art and further provides methods of gene regulation and screening for drugs using the teachings of the pre sent invention.
SUMMARY OF THE INVENTION
Smads are mediators of the superfamily o f transforming growth factor- β (TGF-β) signaling pathways ( 1 , 2 ) . Smadό and Smad7, antagonize the TGF-β signals (3,4). Smadό and Smad7, induced by TGF-β or bone morphogenetic protein (BMP), form stable associations with activated type I receptors , which, in turn, block phosphorylation of ligand-induced S mads (5,6). Smadό also interacts with phosphorylated Smadl t o prevent the formation of an active signaling complex of Smad l and Smad4 in the cytoplasm (7,8). Herein, it is shown that Smadό interacts with Hoxc-8 as a transcriptional corepres sor, inhibiting bone morphogenetic protein signaling in the nucleus . The present invention describ that Smadό functions as a transcriptional corepressor in the nucleus of BMP signaling.
One object of the present invention is to describe a role for Smadό in Hox protein transcriptional regulation, a n d additionally to provide methods of using the S madό/Hox interaction in gene regulation and methods of screening for drugs that effect the Smadό/Hox interaction.
In an embodiment of the present invention, there i s provided a method of regulating bone formation in an individual, comprising the step of: (a) administering a composition to th e individual, wherein the composition alters the activity of Smadό protein. An increase in the Smadό protein results in an increase in Smad6/Hoxc-8 complexes; an increase in S mad6/Hoxc- 8 complexes maintains transcriptional repression of genes involved in bone formation. A decrease in the Smadό protein activity results in a decrease in Smadό/Hoxc-8 complexes wherein a decrease in Smad6/Hoxc-8 complexes relieves transcriptional repression of genes involved in bone formation, thereby regulating bone formation in the individual.
In another embodiment of the present invention, there is provided a method of regulating nuclear b o n e morphogenetic protein signaling, comprising the step of: ( a ) administering a composition to a cell that alters the activity o f Smadό protein. An increase in the available Smadό protein results in an increase in Smadό/Hoxc-8 complexes; an increase in Smad6/Hoxc-8 complexes maintains transcriptional repression o f genes involved in bone formation. A decrease in the Smadό protein binding activity results in a decrease in Smadό/Hoxc- 8 complexes, wherein a decrease in Smadό/Hoxc-8 complexes relieves transcriptional repression of genes involved in bo ne formation, thereby regulating nuclear BMP signaling.
In yet another embodiment of the present invention, there is provided a method of screening for a compound that disrupts transcriptional repression of a gene. This me thod comprises the steps of: (a) combining Smadό proteins and Hoxc- 8 proteins in the presence and absence of a compound; and ( b ) detecting complex formation between the Smadό proteins and th e Hoxc-8 proteins. A lack of complex formation between th e Smadό proteins and the Hoxc-8 proteins in the presence of th e compound is indicative of a compound that disrupts transcriptional repression of a gene.
In still yet another embodiment of the pre sent invention, there is provided 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 the DNA molecule comprises a Hox DNA binding element; and (b) determining the amount of binding by th e Smad6/Hoxc-8 protein complex to the DNA molecule, wherein less binding in the presence of the compound than in the ab sence of the compound is indicative of a compound that disrupts transcriptional repression of the gene.
In still yet another embodiment of the present invention, there is provided a method of screening for a compound that disrupts transcriptional repression of a gene, comprising the steps of: (a) combining a Smadό/Hoxc-8 protein complex with a gene in the presence and absence of a compound, wherein the gene comprises a Hox DNA binding element; and ( b ) assaying for transcription of the gene. An increase in the level o f transcription in the presence of the compound relative to th e level of transcription in the absence of the compound is indicative of a compound that disrupts transcriptional repres sion of the gene.
In still yet another embodiment of the pre sent invention, there is provided a method of regulating expression o f gene that binds Hoxc-8, wherein binding by Hoxc-8 results in transcriptional repression of the gene, comprising the step of: altering the binding activity of Smadό protein. An increase in th e Smadό protein results in an increase in Smadό/Hoxc-8 protein complexes; an increase in the Smad6/Hoxc-8 protein complexes maintains the transcriptional repression of the gene. A decrease in the Smadό protein binding activity results in a decrease in Smadό/Hoxc-8 protein complexes, wherein a decrease i n Smadό/Hoxc-8 protein complexes relieves the transcriptional repression of the gene, thereby regulating expression of the gene . This method may further comprise the step of: increasing th e amount of Smadl protein, wherein the Smadl protein binds th e Hoxc-8, thereby relieving the transcriptional repression of th e gene. In still yet another embodiment of the pre sent invention, there is provided a method of inducing transcription o f a gene encoding osteopontin, comprising the steps of: inhibiting Smadό, wherein in the presence of Smadl , the inhibition o f Smadό removes transcriptional repression of a gene encoding osteopontin, thereby inducing transcription of the gene encoding osteopontin.
Other and further aspects, features, and advantages o f the present invention will be apparent from the following description of the presently preferred embodiments of th e invention. These embodiments are given for the purpose o f disclosure .
BRIEF DESCRIPTION OF THE DRAWINGS
The appended drawings have been included herein s o that the above-recited features, advantages and objects of th e invention will become clear and can be understood in detail . These drawings form a part of the specification. It is to be noted , however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered t o limit the scope of the invention.
Figure 1 shows the specific interaction of Smadό with Hoxc-8 in a yeast two-hybrid system. The interaction was assayed in a liquid culture of yeast strain Y190, a strain which requires His, Leu and Trp to grow. pGBT9-Hoxc-8 and pACT2-Smadό plasmids carry Trp and Leu as their selective markers , respectively. The interaction between Smadό and Hoxc-8 enables the yeast to synthesize His and induces β-gal expression. The arbitrary units of β-gal activities for yeast bearing different plasmids were plotted as shown in the Table.
Figure 2 shows the interaction of Smadl with Hoxc-8 in vivo . Flag-tagged Smadό, Flag-tagged Smadό carboxy domain (SmadόC), Flag-tagged Smadό amino domain with linker region (Smadό N+L) and HA-tagged Hoxc-8 were co-transfected in COS-1 cells with or without ALK3 (Q233D). Cell lysates were immunoprecipitated by anti-Flag antibody and the resulted complexes were analyzed by Western blotting with anti-HA antibody. The expression levels of Smadό were shown by Western blot with anti-Flag antibody (middle panel), and Hoxc-8 with anti- HA antibody (bottom panel).
Figure 3 shows that the Smadό and Hoxc-8 form a complex on Hox DNA binding site from osteopontin promoter . Figure 3A : the complex of Smadό and Hoxc-8 blocks th e interaction of Smadl with Hoxc-8. Gel-shift assays were performed using osteopontin Hox DNA binding element (OPN-5) as the probe (13), with 1.5 mg GST (lane 2), 1.5 mg GST-Smadl (lanes 3, 6, 8, 10), 0.2 mg GST-Hoxc-8 protein (lanes 5-10), 1 .5 mg GST-Smadό (lanes 4, 7- 10) and 0.1 mg Smadό polyclonal antibody (SmadόAB, lanes 9 and 10).
Figure 3B : the complex of Smadό and Hoxc-8 moderately blocks the interaction of Smad4 with Hoxc-8. OPN-5 was used as probe, with 1.5 mg GST (lane 2), 1.5 mg GST-Smad4 (lanes 3, 5, 7 and 9), 0.2 mg GST-Hoxc-8 protein (lanes 4-9), 1 .5 mg GST-Smadό (lanes 6-9) and 1.5 mg GST-Smadl (lanes 9 an d 1 0) .
Figure 4 shows that BMP-induced osteopontin gene transcription is mediated by Hoxc-8 binding site. Figure 4A : Smadl/Hoxc-8 interaction domain (SmadlB) induces transcription in a concentration dependent manner. Hox-pGL3 construct (500 ng), containing osteopontin Hox binding site linked to SV40 promoter, was co-transfected in MvlLu cells with different amounts of SmadlB expression plasmid.
Figure 4B : The Smadό inhibits Smad I B-induced transcription in presence of Hoxc-8. Hox-pGL3 construct was co- transfected with SmadlB (300 ng), Hoxc-8 (25 ng) or Smadό ( 100 ng) expression plasmids. Figure 4C : Mutation of Hox binding site abolishes SmadlB stimulation. mHox-pGL3 (500 ng), contains mutated osteopontin Hox binding site in Hox-pGL3 construct, was co-transfected with SmadlB (300 ng), Hoxc-8 ( 25 ng) or Smadό (100 ng) plasmids. Cell lysates in A, B and C were assayed for luciferase activity normalized to Renilla luciferase levels 48 h after transfection. Experiments were repeated 3 times in triplicate.
DETAILED DESCRIPTION OF THE INVENTION
Smads are the mediators of the superfamily o f transforming growth factor- β (TGF-β) signaling pathways ( 1 , 2 ) . Smadό and Smad7, a subgroup of Smad proteins, antagonize th e TGF-β signals (3,4). These two Smads, induced by TGF-β or b o n e morphogenetic protein (BMP), form stable association with activated type I receptors, which, in turn, block phosphorylation of ligand-induced Smads (5,6). Smadό also interacts with phosphorylated Smadl to prevent the formation of an active signaling complex of Smadl and Smad4 in the cytoplasm (7,8).
Herein, it is shown that Smadό interacts with Hoxc-8 as a transcriptional corepressor, inhibiting BMP signaling in th e nucleus. The interaction between Smadό and Hoxc-8 was identified in a yeast two-hybrid approach, and further demonstrated by co-immunoprecipitation assays in cells. Gel shift assays show that Hoxc-8 interacts with Smadό as a heterodimer when binding to DNA. More importantly, th e Smadό/Hoxc-8 complex inhibited both Smadl interaction with Hoxc-8 in gel shift assays and transcription activity mediated b y Smadl . The data presented herein indicate that Smadό functions as a transcriptional corepressor in BMP signaling in the nucleus.
The present invention is directed towards methods o f using the heretofore unknown interaction between Smadό a n d the Hox proteins in transcriptional gene regulation, thereby producing a desired effect (i. e., regulating bone formation, controlling osteoporosis, etc.). A method of screening for compounds that disrupt the Smad6/Hox protein complex is further provided.
The present invention is directed towards a method o f regulating bone formation in an individual, comprising the s tep of: (a) administering a composition to the individual, wherein th e composition alters the binding activity of Smadό protein, wherein an increase in the Smadό protein results in an increase i n Smadό/Hoxc-8 complexes, wherein an increase in S mad6/Hoxc-8 complexes maintains transcriptional repression of genes involved in bone formation, wherein a decrease in the Smadό protein binding activity results in a decrease in Smadό/Hoxc-8 complexes, wherein a decrease in Smad6/Hoxc-8 complexes relieves transcriptional repression of genes involved in b one formation, thereby regulating bone formation in the individual.
The present invention is directed towards a method o f regulating nuclear BMP signaling, comprising the step of: ( a ) administering a composition to a cell, wherein the composition alters the binding activity of available Smadό protein, wherein a n increase in the available Smadό protein results in an increase in Smad6/Hoxc-8 complexes, wherein an increase in S mad6/Hoxc-8 complexes maintains transcriptional repression of genes involved in bone formation, wherein a decrease in the Smadό protein binding activity results in a decrease in Smadό/Hoxc- 8 complexes, wherein a decrease in Smad6/Hoxc-8 complexes relieves transcriptional repression of genes involved in b o ne formation, thereby regulating nuclear BMP signaling. Representative compositions are selected from the group consisting of a gene encoding Smadό, an antisense molecule directed towards Smadό, an antibody directed towards Smadό . Generally, the genes involved in bone formation are selected from the group consisting of osteopontin, osteoprotegrin, RANK an d OPGL.
The present invention is directed towards a method of screening for a compound that disrupts transcriptional repression of a gene, comprising the steps of: (a) combining Smadό proteins and Hoxc-8 proteins in the presence and absence of a compound; and (b) detecting complex formation between the Smadό proteins and the Hoxc-8 proteins, wherein a lack o f complex formation between the Smadό proteins and the Hoxc-8 proteins in the presence of the compound is indicative of a compound that disrupts transcriptional repression of a gene . Representative means of detection are a gel shift assay and a Western blot.
The present invention is directed towards a method o f screening for a compound that disrupts transcriptional repression of a gene, comprising the steps of: (a) combining a Smadό/Hoxc-8 complex and a DNA molecule in the presence an d absence of a compound, wherein the DNA molecule comprises a Hox DNA binding element; and (b) determining the amount o f binding by the Smad6/Hoxc-8 protein complex to the DNA molecule, wherein less binding in the presence of the compound than in the absence of the compound is indicative of a compound that disrupts transcriptional repression of the gene. Typically, DNA binding by the Smad6/Hoxc-8 protein complex i s determined by means selected from the group consisting of a gel- shift assay, a competitive binding assay, immunoprecipitation a nd Yeast two-hybrid assay.
The present invention is directed towards a method o f screening for a compound that disrupts transcriptional repression of a gene, comprising the steps of: (a) combining a Smad6/Hoxc-8 protein complex with a gene in the presence an d absence of a compound, wherein the gene comprises a Hox DNA binding element; and (b) assaying for transcription of the gene, wherein an increase in the level of transcription in the pres ence of the compound relative to the level of transcription in th e absence of the compound is indicative of a compound th at disrupts transcriptional repression of the gene. Generally, transcription is assayed by means selected from the group consisting of a Northern blot, a Western blot, an enzymatic assay and a chemiluminescent assay. Preferably, the gene is a reporter gene, and more preferably, the reporter gene is selected from th e group consisting of β-galactosidase, luciferase, secreted alkaline phosphotase and CAT assay. The present invention is directed towards a method o f regulating expression of gene that binds Hoxc-8, wherein binding by Hoxc-8 results in transcriptional repression of the gene, comprising the step of: altering the amount of Smadό protein, wherein an increase in the Smadό protein results in an increase in Smadό/Hoxc-8 protein complexes, wherein an increase in th e Smadό/Hoxc-8 protein complexes maintains the transcriptional repression of the gene, wherein a decrease in the Smadό protein results in a decrease in Smadό/Hoxc-8 protein complexes , wherein a decrease in Smadό/Hoxc-8 protein complexes relieves the transcriptional repression of the gene, thereby regulating expression of the gene. Representative genes are o steopontin, osteoprotegrin, OPGL and RANK. Typically, the Smadό protein is increased by means selected from the group consisting o f overexpression of a Smadό gene and upregulation of a Smadό gene, or alternatively, the Smadό protein is decreased by means selected from the group consisting of antisense hybridization t o Smadό RNA, antibody binding to a Smadό protein a n d mutagenesis of a gene encoding Smadό. This method may further comprise the step of: increasing the amount of Smadl protein, wherein the Smadl protein binds the Hoxc-8, thereby relieving the transcriptional repression of the gene.
The present invention is directed towards a method o f inducing transcription of a gene encoding o steopontin, comprising the steps of: inhibiting Smadό, wherein in th e presence of Smadl , the inhibition of Smadό removes transcriptional repression of a gene encoding osteopontin, thereby inducing transcription of the gene encoding osteopontin. As used herein, the term "transcriptional repres sion by a hox protein" or "transcriptional repression by a homeodomain-containing protein shall refer to any gene whose transcription activities are repressed in the presence of the hox protein or the homeodomain-containing protein.
The following examples are given for the purpose o f illustrating various embodiments of the invention and are n o t meant to limit the present invention in any fashion:
EXAMPLE 1
Two-hybrid library screening
A full-length Hoxc-8 coding sequence was cloned into pGBT9 (CLONTECH) to generate the pGBT9/Hoxc-8 bait plasmid. The human U2 OS osteoblast-like pACT2 cDNA library was screened with the pGBT9/Hoxc-8 bait plasmid according to th e manufacturer' s instruction (CLONTECH).
EXAMPLE 2
Immunoprecipitation and Western blot
Expression vectors for Flag-tagged full length Smadό , amino-domain with linker region (SmadόNL) and carboxy-domain (SmadόC) were subcloned into a mammalian expression vector pcDNA3 (Invitrogen). HA-tagged Hoxc-8 expression vector was constructed (13). Constitutively active BMP type IA (ALK3) expression plasmid was provided by Dr. Jeffrey L. Wrana (The Hospital for Sick Children, Canada). COS-1 cells were transfected with expression constructs as indicated in Figure 2 using Tfx-50 according to manufacturer' s description (Promega). Cells were lysed 48 h post-transfection and lysates were immuno- precipitated with anti-Flag M2 antibody (Eastman Kodak) an d immuno-blotted with anti-HA antiserum (Babco).
EXAMPLE 3
Gel shift assay
Gel-shift assays were performed (26). Smad 1 and 4 cDNAs were obtained from Dr. R. Derynck. GST-fusion constructs of Smadl and 4 and Hoxc-8 were generated ( 1 3 ) . Smadό cDNA, obtained from Dr. Ali Hemmati-Brivanlou, was cloned into pGEX-KG vector. The GST-constructs described above were transformed into BL21. The expression and purification o f the fusion proteins were performed (27). OPN5 DNA fragments were used for the gel shift assays ( 13).
EXAMPLE 4
Transfecti on
Hox-pGL3 reporter bearing Hoxc-8 binding site ( - 290 to -166) was constructed into pGL3-control vector (Promega) . The Hox recognition core TAAT was replaced with GCCG in Hox- pGL3 by PCR to create mutant Hox-ρGL3 (mHox-pGL3). MvlLu cells (5 x l 04 cells/22.6 mm dish) were transfected using Tfx-50 with 0.5 mg of luciferase reporter plasmid (Hox-pGL3 or mHox- pGL3 ) and different expression plasmids as indicated. Total DNA was kept constant by addition of pcDNA3 plasmid. Luciferase activities were assayed 48 h post-transfection using the Dual Luciferase Assay Kit (Promega) according to manufacturer' s direction. Luciferase values shown in the figures are representative of transfection experiments performed in triplicate in at least three independent experiments.
EXAMPLE S
Identification of transcription factors that, interact with Hoxc-8
To characterize the Hoxc-8-mediated transcription mechanism in bone morphogenetic protein-induced gene activation, a yeast two-hybrid system was used to identify transcription factors that interact with Hoxc-8. An intact Hoxc-8 cDNA fused with the Gal4 DNA binding domain was used as a bait plasmid to screen a human U-2 OS osteoblast-like cell cDNA library constructed in pACT2 plasmid. After two rounds o f screening, 26 positive clones were obtained. DNA sequence analysis identified one clone as Smadό (Figure 1 ). Smadό a n d Smad7 are immunolocalized in the nucleus of rat epiphyseal plate ( 17), Xenopus embryo (18) and Mink lung epithelial (Mv l Lu) cells (19). The interaction of Smadό with Hoxc-8, a transcription repressor in bone morphogenetic protein signaling pathway , suggests that Smadό may have a novel antagonistic function in the nucleus.
The initial Smadό cDNA clone (SmadόC in Figure 1 ) encodes amino acids 281 to 496 out of a 496 amino acid protein . The interaction between Hoxc-8 and Smadό was further confirmed with a β-gal filter lift assay and quantified by a liquid β-gal assay (Figure 1). When the full length Smadό fused with th e Gal4 transcriptional activation domain was tested in the two- hybrid system, it showed a weaker interaction compared with th e carboxy-terminal domain (SmadόC). Deletion of Smadό amino- terminal domain may change the protein conformation such th at the carboxy-terminal region becomes available to interact with Hoxc-8. The assays of both empty bait vector (pGBT9) with either SmadόC or Smadό full length cDNAs in prey plasmids a s well as empty prey vector (pACT9) with full length Hoxc-8 in bait vector showed very little activity. Compared with the interaction between Smadl and Hoxc-8, the interaction of Smadό with Hoxc- 8 is about 5-fold stronger (Figure 1 ).
EXAMPLE 6
The interaction of Smadό with Hoxc-8 in mammalian cells
To investigate the interaction of Smadό with Hoxc-8 in mammalian cells and the effect of bone morphogenetic protein stimulation on this interaction, COS-1 cells were transiently c o - transfected with expression plasmids for Flag-Smad6, HA-Hoxc-8, and/or constitutively active bone morphogenetic protein type LA receptor ALK3 (Q233D). The cell lysates were immunoprecipitated with anti-Flag antibody and immuno-blotted with anti-HA antibody. The results in Figure 2 demonstrate th at Smadό (lanes 7 and 8) was co-immunoprecipitated with HA- Hoxc-8.
Overexpression of ALK3 (Q233D) did not change th e interaction of Smadό with Hoxc-8 (lane 8), indicating that bone morphogenetic protein stimulation is not required for th e interaction between Smadό and Hoxc-8. Since b one morphogenetic protein induces Smadό mRNA expression ( 20-22 ) , these data suggest that bone morphogenetic protein regulates th e interaction between Smadό and Hoxc-8 at the level of Smadό transcription. The initial Smadό clone only encodes the carboxy- terminal domain, indicating that this region of the protein may b e involved in the interaction with Hoxc-8.
To further investigate this observation, two Flag- tagged Smadό truncated expression plasmids were constructed . As shown in Figure 2, SmadόC exhibits a strong interaction with Hoxc-8 (lanes 4 and 5). In contrast, the Smadό amino-terminal with linker region (SmadόNL) failed to bind to Hoxc-8 in immuno-precipitation assay (Figure 2, lane 6). Smad proteins contain highly conserved carboxy- and amino-terminal domains (referred to as MHl and MH2 domains, respectively). The MH l domain inhibits biological activities of the MH2 domain due t o interactions between these two distal sites (23). Like o ther regulatory Smads, Smadό also contains a conserved MH2 domain and short segments of MHl domain homology (24). Therefore , results herein suggest that the carboxy-terminal domain of Smadό interacts with Hoxc-8, and that the amino-terminus negatively regulates interaction between the two proteins.
EXAMPLE 7
The effect of Hox -8/Smadό on Hoxc-8 DNA binding activity
Next, the effect of the interaction between Hoxc-8 an d Smadό on Hoxc-8 DNA binding activity was examined. Gel shift assays were performed with purified GST-Smadό and GST-Hoxc-8 fusion proteins and using osteopontin Hoxc-8 DNA binding element as a probe.
As expected, Hoxc-8 protein binds to the DNA probe , which is inhibited by Smadl (Figure 3A, lanes 5 and 6). Smadό alone did not bind to the DNA element (lane 4). Interestingly, incubation of both Hoxc-8 and Smadό proteins yields a distinct shifted band with a molecular weight higher than Hoxc-8 binding alone, indicating that Hoxc-8 and Smadό bind to the DNA element cooperatively (lane 7). More importantly, the formation of th e Smadό and Hoxc-8 complex blocked the interaction of Hoxc- 8 with Smadl (lane 8). Yeast two-hybrid assays already demonstrated that the interaction between Hoxc-8 and Smadό is much stronger than that between Hoxc-8 and Smadl (Figure 1). The formation of the complex between Hoxc-8 an d
Smadό on the DNA element was confirmed by the fact that a n anti-Smadό polyclonal antibody inhibited the development of th e retarded band (lanes 9 and 10). Smad4, also interacting with Hoxc-8, was examined for the same purpose in gel shift assays (Figure 3B). The complex of Smadό and Hoxc-8, however, did n o t block the interaction of Smad4 with Hoxc-8 completely (Figure 3B, lanes 7 and 9). In fact, it has been shown that Smadό inhibits Smadl phosphorylation and prevents its translocation into nucleus (5,7), whereas Smad4, a common partner for all regulatory Smads, can only be passively translocated into nucleus by forming hetero-oligomers with regulatory Smads ( 25 ) . Therefore, the preference of Smadό inhibition for Smad l - mediated gene transactivation suggests the importance of Smadό antagonistic function for BMP signaling pathway. EXAMPLE 8
Inhibition of S ad l /Hoxc-8-activated gene transcription by t h e
Smad6/Hoxc-8 complex To investigate whether the Smad6/Hoxc-8 complex inhibits the interaction of Smadl with Hoxc-8 in activating gene transcription, a model was used ( 13). The interaction domains within the amino-terminal 87 amino acid residues of Smadl w ere mapped to interact with Hoxc-8. Overexpression of cDNAs encoding the Hoxc-8 interaction domains of Smadl linked to a nuclear localization signal (SmadlB) effectively activated osteopontin gene transcription. Stable expression of these S mad l fragments in 2T3 osteoblast precursor cells stimulated endogenous osteoblast differentiation-related gene expres sion and mineralized bone matrix formation. When the BMP-inducible construct (Hox-pGL3) was co-transfected into MvlLu cells with the SmadlB expression plasmid, luciferase activity was stimulated in a dose-dependent manner (Figure 4A). _ This model provides an ideal assay to examine the Smadό antagonistic function in th e nucleus directly. Because SmadlB mimics BMP-induced gene transcription without involving BMP receptor phosphorylation and interaction with Smad (67), this assay avoids Smadό inhibitory function in the cytoplasm.
Hox-pGL3 construct was co-transfected in MvlLu cells with Hoxc-8 or Smadό expression plasmid, or both. As shown in Figure 4B, overexpression of Hoxc-8 or Smadό alone inhibited SmadlB-induced transcription activity. In addition to th e interaction between Smadό and Hoxc-8 in the nucleus, Smadό binds to BMP type I receptor to block phosphorylation of o ther regulatory Smads. Smadό has also been shown to interacted with phosphorylated Smadl, inhibiting Smadl translocated into nucleus. Most importantly, co-transfection of both Hoxc-8 and Smadό plasmids completely abolished the SmadlB-induced luciferase activity. To validate this observation, MvlLu cells were transfected with a mutated construct (mHox-pGL3) in which the core nucleotides of the Hoxc-8 binding site were mutated from TAAT to GCCG. As expected, transfection of the mutant construct completely abolished SmadlB-induced reporter activity and eliminated Smad6/Hoxc-8 complex-mediated inhibition (Figure 4C). These results demonstrate for the first time that Smadό has an antagonistic function towards BMP signaling in the nucleus in addition to its interaction with BMP type I receptor and Smadl in the cytoplasm. The following references were cited herein:
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Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the s ame extent as if each individual publication was specifically an d individually indicated to be incorporated by reference.
One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects an d obtain the ends and advantages mentioned, as well as tho se objects, ends and advantages inherent herein. The pre sent examples, along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope o f the invention. Changes therein and other uses will occur to tho se skilled in the art which are encompassed within the spirit of th e invention as defined by the scope of the claims .

Claims

WHAT IS CLAIMED IS:
1 . A method of regulating bone formation in a n individual, comprising the step of: ( a ) administering a composition that alters th e binding activity of Smadό protein in said individual, wherein a n increase in Smadό protein results in an increase in S madό/Hoxc- 8 complexes which maintains transcriptional repression of genes involved in bone formation, wherein a decrease in Smadό protein binding activity results in a decrease in S madό/Hoxc- 8 complexes, which relieves transcriptional repression of genes involved in bone formation, thereby regulating bone formation i n said individual.
2 . The method of claim 1, wherein said composition is selected from the group consisting of a trans gene encoding Smadό, an antisense molecule directed towards Smadό , an antibody directed towards Smadό 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 b on e morphogenetic protein signaling in an animal, comprising th e step of:
( a) administering a composition that alters th e binding activity of available Smadό protein in a cell in said individual, wherein an increase in available Smadό 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 Smadό protein binding activity results in a decrease in Smadό/Hoxc-8 complexes which relieves transcriptional repression of genes involved in b on e 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 Smadό, an antisense molecule directed towards Smadό, an antibody directed towards Smadό 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 th e steps of: ( a ) combining Smadό proteins and Hoxc-8 proteins in the presence and absence of a compound; and
( b ) detecting complex formation between said
Smadό proteins and said Hoxc-8 proteins, wherein a lack o f complex formation between said Smadό 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 Smadό/Hoxc-8 protein complex formation is by mean s 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 th e 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 th e absence of said compound is indicative of a compound th at disrupts transcriptional repression of said gene.
10. The method of claim 9, wherein said DNA binding by said Smadό/Hoxc-8 protein complex is determined b y means selected from the group consisting of a gel-shift assay, a competitive binding assay, pull-down assay and immunoprecipitation assay.
1 1 . A method of screening for a compound that disrupts transcriptional repression of a gene, comprising th e steps of: ( a ) combining a Smadό/Hoxc-8 protein complex with a gene in the presence and absence of a compound, wherein said gene comprises a Hox DNA binding element; and
( b ) assaying for transcription of said gene, wherein an increase in the level of transcription in the presence of said compound relative to the level of transcription in the absence o f said compound is indicative of a compound that disrupts transcriptional repression of said gene.
1 2. The method of claim 11 , wherein said transcription is assayed by means selected from the group consisting of a Northern blot, a Western blot, an enzymatic assay and a chemiluminescent assay.
1 3 . The method of claim 11, wherein said gene is a reporter gene.
1 4. The method of claim 13, wherein said reporter gene is selected from the group consisting of β-galactosidase, luciferase, secreted alkaline phosphotase and CAT assay.
1 5 . A method of regulating expression of gene th at binds Hoxc-8, wherein binding by Hoxc-8 results i n transcriptional repression of said gene, comprising the step of: altering the amount of Smadό protein, wherein a n increase in said Smadό protein binding activity results in a n increase in Smadό/Hoxc-8 protein complexes, wherein a n increase in said Smadό/Hoxc-8 protein complexes maintains said transcriptional repression of said gene, wherein a decrease in s aid Smadό protein binding activity results in a decrease in Smadό/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.
1 6. The method of claim 15, wherein said gene i s selected from the group consisting of osteopontin, osteoprotegrin, OPGLand RANK.
1 7. The method of claim 15, wherein said Smadό protein is increased by means selected from the group consisting of overexpression of a Smadό gene and upregulation of a Smadό gene .
1 8. The method of claim 15, wherein said Smadό protein is decreased by means selected from the group consisting of antisense hybridization to Smadό RNA, antibody binding to a Smadό protein and mutagenesis of a gene encoding Smadό.
1 9. The method of claim 15, further comprising th e step of: increasing the amount of Smadl protein, wherein said
Smadl 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 Smadό, wherein in the presence of Smad l , said inhibition of Smadό removes transcriptional repression of a gene encoding osteopontin, thereby inducing transcription of said gene .
PCT/US2000/040563 1999-08-04 2000-08-03 The interaction of smad6 with hox proteins and uses thereof WO2001011013A2 (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1998056913A1 (en) * 1997-06-13 1998-12-17 Ludwig Institute For Cancer Research Smad6 and uses thereof
WO1999050296A1 (en) * 1998-03-27 1999-10-07 Eli Lilly And Company Treatment and prevention of vascular disease

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Publication number Priority date Publication date Assignee Title
WO1998056913A1 (en) * 1997-06-13 1998-12-17 Ludwig Institute For Cancer Research Smad6 and uses thereof
WO1999050296A1 (en) * 1998-03-27 1999-10-07 Eli Lilly And Company Treatment and prevention of vascular disease

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