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EP1058729A1 - Elements fixateurs de la proteine smad et leurs applications - Google Patents

Elements fixateurs de la proteine smad et leurs applications

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Publication number
EP1058729A1
EP1058729A1 EP99911099A EP99911099A EP1058729A1 EP 1058729 A1 EP1058729 A1 EP 1058729A1 EP 99911099 A EP99911099 A EP 99911099A EP 99911099 A EP99911099 A EP 99911099A EP 1058729 A1 EP1058729 A1 EP 1058729A1
Authority
EP
European Patent Office
Prior art keywords
smad
nucleic acid
binding
binding element
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99911099A
Other languages
German (de)
English (en)
Inventor
Luigi J. C. Jonk
Susumo Itoh
Carl-Henrik Heldin
Peter Ten Dijke
Wiebe Kruijer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ludwig Institute for Cancer Research Ltd
Ludwig Cancer Research
Original Assignee
Ludwig Institute for Cancer Research Ltd
Ludwig Cancer Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ludwig Institute for Cancer Research Ltd, Ludwig Cancer Research filed Critical Ludwig Institute for Cancer Research Ltd
Publication of EP1058729A1 publication Critical patent/EP1058729A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • JunB The product of the JunB gene is a member of the AP-1 family of transcription factors that activate transcription by binding to TPA response elements (TRE) within the promoter of target genes.
  • AP-1 components are immediate early gene products whose expression is rapidly induced byva variety of extra-cellular stimuli.
  • JunB differs in biological properties from its homologs and appears to be a negative regulator of AP-1 function. This functional difference is due to a small number of amino acid changes between its DNA binding and dimerization motifs and the corresponding c-Jun sequences as well as to differences in phosphorylation status in response to mitogenic stimulation.
  • the action of JunB as a negative regulator of TRE response elements corresponds with its induction by negative regulators of cell growth including TGF ⁇ and related factors.
  • junB is among other known genes, e.g. cyclin dependent inhibitors (CDI) pi 5 and p21, and the plasminogen activator inhibitor (PAI-1) gene, that are known to be transcriptionally induced in response to TGF ⁇ .
  • CDI cyclin dependent inhibitors
  • PAI-1 plasminogen activator inhibitor
  • TGF ⁇ initiates its action by binding to transmembrane receptors that possess serine/threonine kinase activity.
  • Receptor activation involves binding of TGF ⁇ to the type II receptor (T ⁇ R-II) which then recruits the type I receptor (T ⁇ R-I) in a tetrameric complex consisting of two ligand-bound type II and two type I receptors. This is followed by phosphorylation of the type I receptor by the type II receptor and phosphorylated type I receptor then further activates signal transmission leading to activation of gene transcription.
  • Smads which play an important role in TGF ⁇ receptor-downstream signal transduction. At present at least nine family members have been identified among vertebrates. Smads are 40-62 kDa proteins with N- and C-terminal homology domains (MH1 and MH2) connected by a proline-rich linker. Recent evidence suggests that in their inactive states the MH1 and MH2 domains interact and after - 2 - phosphorylation by receptor, Smad molecules undergo a conformational change that leads to heteromeric Smad complexes and translocation to the nucleus.
  • Smadl, Smad5 and presumably Smad9 associate with and are phosphorylated by BMP -mediated type I receptor activation, while Smad2 and Smad3 are phosphorylated after activation of TGF ⁇ R-I and activin type IB receptor (ActR-IB)-mediated activation, following phosphorylation, which occurs at a conserved SSXS motif at the extreme carboxy-terminus.
  • ActR-IB activin type IB receptor
  • Smad ⁇ and Smad 7 are inhibitory Smads that have been shown to block Smad mediated signal transduction in a dominant-negative manner by stable receptor association.
  • Smad2 and Smad4 together with Fast 1 , a winged-helix DNA binding protein, form activin response factor (ARF) that bind to the activin response element of the Mix-2 promoter (Chen et al., Nature 383:691- 696, 1996; Chen et al., Nature 389:85-89, 1997).
  • ARF activin response factor
  • Smad3/4 overexpression transactivates the PAI- 1 and p3TP-lux promoters, which has been attributed to potentiation of AP 1 -dependent transcriptional activation.
  • Drosophila Mad domain binds a GC rich sequence in the Drosophila quadrant enhancer.
  • the invention provides isolated nucleic acids including one or more Smad binding elements (SBEs), and Smad proteins and fragments thereof which bind to Smad binding elements. As described herein, binding of Smad proteins to SBEs results in transactivation of downstream nucleic acid sequences.
  • SBEs Smad binding elements
  • the invention also provides TGF ⁇ response elements which confer TGF ⁇ superfamily responsiveness to genes which have such elements in transcriptional control regions; the SBEs surprisingly confer responsiveness via Smad binding to TGF ⁇ , activin, and bone morphogenetic protein factors.
  • the TGF- ⁇ superfamily members are - 3 - well known to those of ordinary skill in the art and include TGF- ⁇ s, activins, bone morphogenetic proteins (BMPs), Vgl, Mullerian inhibitory substance (MIS) and growth/differentiation factors (GDFs). Also provided are methods for using the Smad binding element in methods of screening for compounds which bind the Smad binding element and in methods for modulating the binding of Smad proteins to Smad binding elements to modulate TGF ⁇ signal transduction. Further, the identification of a mammalian Smad binding sequence permits identification of the SBE binding determinants of Smad proteins by mutagenesis or crystallography.
  • the isolated nucleic acid molecule includes at least two of the Smad binding elements, and preferably includes four or more of the Smad binding elements.
  • the Smad binding elements are arranged as one or more inverted repeats.
  • the Smad binding elements are arranged in a non-natural arrangement, such as one which is not found in a naturally occurring nucleic acid known as of the effective filing date of this application, including arrangements which vary the sequence, spacing, inversion, flanking sequences, multiplicity and/or distance from promoter sequences of the Smad binding elements.
  • the Smad binding elements include the nucleotide sequence CAGACAGTCTGTCTG (SEQ ID NO: 10).
  • the Smad binding element included in the isolated nucleic acid molecule is a fragment of the junB gene upstream region, wherein the nucleotide sequence of the nucleic acid molecule which flanks the Smad binding element is not derived ⁇ x v ⁇ JunB.
  • Preferred fragments of the junB gene upstream region include nucleotides -2813 through -2792, nucleotides -2908 through -2788, nucleotides -2908 through -2611, and nucleotides -3004 through -1534.
  • the fragment of the junB gene upstream region is less than 50 nucleotides in length, preferably - 4 - including at least two Smad binding elements which more preferably are arranged as one or more inverted repeats.
  • the foregoing isolated nucleic acid molecules are double stranded.
  • isolated polypeptides are provided which are fragments of Smad proteins which bind to Smad binding elements.
  • such polypeptides include the Smad MH1 domain.
  • Preferred isolated polypeptides include fragments of Smad proteins consisting of amino acids 1-279 of Smad3 or amino acids 1-321 of Smad4.
  • the foregoing isolated polypeptides are fusion proteins.
  • SBE binding complexes of the foregoing polypeptides also are provided.
  • methods of screening for compounds which bind to a Smad binding element include the steps of contacting a nucleic acid comprising a Smad binding element with a test compound, and determining the binding of the test compound to the Smad binding element.
  • the methods also include the steps of contacting a mutant Smad binding element with the test compound, determining the binding of the test compound to the mutant Smad binding element, and comparing the binding of the test compound to the Smad binding element and the mutant Smad binding element as a measure of the specific binding of the test compound to the Smad binding element.
  • the mutant Smad binding element does not bind a Smad protein.
  • methods for identifying compounds that compete for binding of a Smad binding element with a Smad protein include the steps of providing a nucleic acid comprising a Smad binding element, forming a complex comprising the nucleic acid and a Smad protein which binds the Smad binding element, contacting the complex with a test compound, and determining the binding of the Smad protein - 5 - to the nucleic acid as a measure of the displacement of the Smad protein by the test compound.
  • the invention provides methods of screening for compounds that modulate (i.e., increase or decrease) transcription mediated by Smad binding elements.
  • the methods include providing a transcription system comprising a Smad protein which binds a Smad binding element and a nucleic acid molecule including a Smad binding element, contacting the transcription system with a test compound, and determining the transcription of the nucleic acid molecule as a measure of transcriptional modulation by the test compound.
  • Increased transcription of the nucleic acid molecule in the presence of the test compound indicates that the test compound increases transcription mediated by the Smad binding element and decreased transcription indicates that the test compound decreases transcription mediated by the Smad binding element.
  • the transcription system is a cell, preferably one which includes a Smad binding element reporter vector which is stably contained within the cell, such as a stably transfected cell line.
  • the Smad protein which binds Smad binding element is Smad 1, Smad2, Smad3, Smad3 ⁇ MH2, Smad4, Smad4 ⁇ MH2, Smad5, a Smadl/Smad4 complex, a Smad2/Smad4 complex, a Smad3/Smad4 complex, or a
  • Such methods further include the steps of contacting the transcription system with a first ligand which induces transcription mediated by Smad binding elements, wherein the transcription system is responsive to the ligand.
  • a reduction in ligand induced transcription in the presence of the test compound indicates that the test compound interferes with the ligand induced transcription mediated by Smad binding elements and an increase in ligand induced transcription in the presence of the test compound indicates that the test compound enhances the ligand induced transcription mediated by Smad binding elements.
  • the Smad binding elements described herein have the unexpected property of conferring transcriptional responsiveness to multiple TGF ⁇ superfamily ligands, including at least TGF ⁇ , activin and BMPs.
  • the methods described above can be used to - 6 - screen for and distinguish between compounds which selectively modulate transcription induced by a single TGF ⁇ superfamily ligand (or other ligand which modulates transcription through SBEs) and compounds which modulate transcription induced by more than one such ligand.
  • the foregoing procedure is repeated at least once in the presence of a second ligand which induces transcription mediated by Smad binding elements.
  • a reduction or increase in transcription induced by the first ligand but not the second ligand indicates that the test compound selectively modulates the transcription mediated by Smad binding elements induced by the first ligand.
  • a reduction or increase in transcription induced by the first ligand and the second ligand indicates that the test compound generally modulates the transcription mediated by Smad binding elements as induced by the first and the second ligands.
  • the method can be repeated with several ligands in parallel assays to select for general and selective modulators of transcription.
  • Preferred ligands include TGF ⁇ , activin, OP-1, BMP-2, and GDF5.
  • the methods include administering to a subject in need of such treatment an agent which decreases the level of binding of a Smad protein to an endogenous Smad binding element in an amount effective to reduce the abnormally elevated level of Smad-mediated transcription.
  • the agent is an isolated nucleic acid molecule including a SBE, preferably which includes the nucleotide sequence CAGACA, an antisense nucleic acid which binds to a nucleic acid molecule encoding the Smad protein which binds a Smad binding element, or an antibody which binds to the Smad protein which binds a Smad binding element, the Smad binding element itself, or a complex of the Smad protein and the Smad binding element.
  • methods for modulating in a cell transcription of genes which include a Smad binding element in a transcription control region.
  • the methods include contacting the cell with an effective amount of an agent which increases in the cell the level of a Smad protein which binds a Smad binding element or a fragment of the Smad protein which binds a Smad binding element.
  • the agent is an expression vector which encodes the Smad protein which binds a Smad binding element or a fragment of the Smad protein which binds a Smad binding element.
  • the agent is the Smad protein which binds a Smad binding element or a fragment of the Smad protein which binds a Smad binding element.
  • Preferred Smad proteins in these methods include Smad3, Smad4 and fragments thereof.
  • methods for modulating in a cell transcription of genes comprising a Smad binding element in a transcription control region.
  • the methods include contacting the cell with an effective amount of an agent which modulates binding of a Smad protein to a Smad binding element, or which itself binds to a Smad binding element, wherein the agent modulates transcription.
  • Agents may be identified in accordance with the methods disclosed herein
  • methods of identifying a Smad protein - 8 - having altered binding to a Smad binding element nucleic acid sequence include contacting a sample containing a Smad protein, the nonmutated form of which binds a Smad binding element in a first amount, with an isolated nucleic acid molecule comprising the Smad binding element, determining a second amount of binding of the Smad binding element by the Smad protein, and comparing the first amount and the second amount of binding of the Smad binding element. A difference between the first amount and the second amount indicates that the sample contains a mutant Smad protein with altered nucleic acid binding characteristics.
  • the Smad binding element includes the nucleotide sequence CAGACA.
  • the Smad protein preferably is prepared by mutagenesis, more preferably by site-directed mutagenesis.
  • nucleic acid molecules which include a Smad binding element, including antisense nucleic acids, in the manufacture of a medicament is provided.
  • the invention also embraces functional variants and equivalents of all of the molecules described above.
  • Fig. 1 shows that Smad3 and Smad4 activate the JunB promoter through a region that binds a TGF ⁇ -induced protein.
  • the JunB promoter is activated by Smad3 and Smad4 between nucleotides -3004 and -1534.
  • the Smad3 and Smad4-responsive region within the JunB promoter is located in a 297 bp (nt -2908/-2611) fragment.
  • Fig. 2 shows the identification of a 22 bp fragment containing a 7 bp inverted repeat as a Smad-responsive element in the JunB promoter.
  • Fig. 4 shows that the SBE is a responsive element for TGF ⁇ superfamily.
  • the JunB promoter is activated by different pathway-specific Smad proteins.
  • Pathway-specific Smads differentially activate the SBE.
  • SBE is activated by TGF ⁇ , activin and osteogenic protein- 1 (OP-1).
  • OP-1 osteogenic protein- 1
  • D Activation of SBE is dependent on Smad4 protein.
  • the invention provides nucleic acid molecules which include a Smad binding element.
  • the minimal Smad binding element is the hexanucleotide CAGACA.
  • One or more nucleotides can be added to one or both ends of the SBE without destroying the Smad binding characteristics of the nucleotide sequence.
  • one nucleotide can be added to the 3' end of the hexanucleotide to form the heptanucleotide sequence CAGACAG, which is shown below to be a SBE.
  • the invention includes SBEs formed by the addition of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 75, and more nucleotides to one or both ends of the hexanucleotide CAGACA.
  • certain fragments of the junB transcriptional control region were found to effectively confer Smad transactivation upon a reporter construct.
  • the Smad transactivation properties of the isolated junB transcriptional control region were not previously recognized, and were unexpected. These fragments range in size from 22 nucleotides to 1471 nucleotides and more.
  • the nucleotide sequence of the nucleic acid which flanks the Smad binding element is not derived from junB. In other embodiments where the sequence flanking the SBE is derived from junB, it is preferred that the fragment of the junB gene upstream region flanking region is less than 50 nucleotides in length in total.
  • the Smad binding element in the junB gene was found to be a multimer of the hexanucleotide CAGACA, arranged as an inverted repeat (CAGACAGTCTGTCTG). As - 10 - described below, this inverted repeat multimer confers TGF ⁇ responsiveness of transcription when the inverted repeat is included in a transcriptional control region.
  • the junB SBE inverted repeat is thus a TGF ⁇ response element. Additional experiments described herein demonstrate that other multimeric SBEs (including 2x and 4x direct repeat multimers) also provide TGF ⁇ response element activity. Thus SBEs arranged as multimers are preferred when TGF ⁇ response element activity is desired. SBE multimers are also preferred when it is desired to have Smad binding activity, as multimers have greater numbers of Smad binding sites per molecule.
  • the number of SBEs in a multimer is not limited in the broadest embodiments of the invention. However, in certain embodiments of the invention it may preferred that the number of SBEs in a multimeric configuration is less than 50, more preferably less than 25, and still more preferably less than 10, and yet still more preferably less than 5. Limitation on the number of SBEs in a multimer may be of importance in those multimeric configurations which favor formation of stem-loop structures, such as inverted repeats. One of ordinary skill in the art can readily prepare and test a variety of multimeric configurations in accordance with the methods described herein.
  • the Smad binding elements can be arranged in non-natural arrangements or configurations, such as an arrangement or configuration which is not found in a naturally occurring nucleic acid known as of the effective filing date of this application.
  • Non-natural arrangements include arrangements which vary the sequence, spacing, inversion, flanking sequences, multiplicity and/or distance from promoter sequences of the Smad binding elements from those found in natural arrangements.
  • Sequence means the nucleotide sequence of the Smad binding element itself.
  • Spacing means the number of nucleotides between individual SBEs of a direct or inverted repeat, or the number of nucleotides between one SBE (or repeat) and a 5' or 3' SBE or repeat.
  • the SBE repeat set forth in SEQ ID NO: 10 contains a single nucleotide between the halves of the repeat, and thus has a spacing of one nucleotide.
  • the number of nucleotides between the SBE repeat of SEQ ID NO: 10 in the JunB transcriptional control region and another SBE in that control region would be the other use of the term spacing with respect to SBEs.
  • “Inversion” means the linear relation of SBE repeats to each other.
  • the SBE repeat set forth in SEQ ID NO: 10 is an inverted repeat; direct repeats are set forth in other sequences described herein. Thus, one can vary the inversion of SBE repeats, for example, by altering a direct repeat to an inverted repeat, and vice versa.
  • “Flanking sequences” are the nucleotide sequences 5' and 3' of a natural SBE.
  • “Multiplicity” means the number of - 11 -
  • SBEs in a transcriptional control region or other nucleic acid, such as an oligonucleotide are referred to as “Distance from promoter sequences”
  • Distance from promoter sequences means the number of nucleotides between a particular Smad binding element and a promoter sequence.
  • Smad binding element sequences described above having a wild-type nucleotide sequence are preferred, but by no means the only functional SBE sequences.
  • certain variant SBEs having one or more mutations in the SBE nucleotide sequence were "functional variant SBEs"; that is, the variant SBEs retained the ability to bind Smad proteins, as shown by direct binding and competition experiments.
  • functional variant SBEs which differ in one or more nucleotides from the wild-type SBE sequences provided above are also embraced by the invention.
  • Exemplary functional variant SBEs which bind Smads include gAGACA (SEQ ID NO:3), gAGACAG (SEQ ID NO:4), CtGACA (SEQ ID NO:5), CtGACAG (SEQ ID NO:6), CAGACt (SEQ ID NO:7), CAGACtG (SEQ ID NO:8) and CAGACAc (SEQ ID NO:9).
  • the mutation in each variant SBE is indicated by a lower case letter.
  • Non-functional variant SBEs in which nucleotides in the central GAC sequence of the CAGACA SBE were mutated (non-functional variant SBEs") were found to have substantially decreased Smad binding characteristics as determined by a loss of the ability to compete for binding to Smad proteins with a wild type SBE in an electrophoretic mobility shift assay.
  • non-functional variant SBEs are useful as negative controls in the assays and methods described herein.
  • variant SBE As exemplified below, one of ordinary skill in the art can readily test a variant SBE to determine if retains the Smad binding property of wild type SBEs, i.e., that the variant SBE is a functional variant SBE.
  • Additional variant SBEs can be made by standard methods in the art, including chemical synthesis of random nucleotide sequences, site directed mutagenesis, random mutagenesis, etc. The skilled artisan also will be familiar with methods of selecting additional SBE sequences by computer database searches using standard software packages which select sequences based on nucleotide homology. Preferably such a search is conducted on sequences derived from transcriptional control regions of genes.
  • isolated means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; - 12 -
  • PCR polymerase chain reaction
  • nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art.
  • a nucleotide sequence contained in a vector in which 5' and 3 ' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not.
  • An isolated nucleic acid may be substantially purified, but need not be.
  • the Smad binding characteristics of a putative SBE can be determined experiments in which a Smad protein or complex of Smad proteins is contacted with the putative SBE under conditions which permit binding of the Smad protein to the SBE.
  • the binding experiments can assay Smad-SBE binding directly, or indirectly, such as in competition experiments wherein the putative SBE is used as a competitor nucleic acid for a known SBE- Smad binding pair.
  • These conditions are generally known to one of ordinary skill in the art of protein-nucleic acid binding and can be performed using routine experimentation. Certain preferred binding conditions are set forth in greater detail in the Examples below.
  • Smad proteins bound in vitro by SBEs include Smad4 and derivatives of Smad3 and Smad4 in which the MH2 domain is deleted (Smad3 ⁇ MH2 and Smad4 ⁇ MH2).
  • Smad3 ⁇ MH2 and Smad4 ⁇ MH2 In cells, it is believed that other Smad proteins, including Smadl, Smad2, Smad3, and Smad5 bind to SBEs. It is also believed that complexes of the foregoing Smad proteins with Smad4 bind to SBEs in cells. As shown below, cells transfected with expression vectors encoding Smad proteins and Smad complexes exhibit increased transactivation of SBE-containing reporter constructs. The Smad binding elements are useful for binding Smad proteins.
  • SBEs can be used to isolate Smad proteins and complexes thereof, as well as to detect Smad proteins and complexes thereof.
  • Smad binding elements as a TGF ⁇ superfamily response element, are also useful for regulating transcription of genes.
  • SBEs When placed in a transcription control region, SBEs confer transcriptional responsiveness to ligands of the TGF ⁇ superfamily in transcriptional systems having the appropriate ligand receptor and signal transduction apparatus. Receptors and signal transduction factors for TGF ⁇ superfamily members are known in the art.
  • a detectable gene e.g., a reporter gene having a detectable nucleic acid or protein gene product
  • a detectable gene e.g., a reporter gene having a detectable nucleic acid or protein gene product
  • Smad binding elements can further be used in nucleic acid hybridizations to identify related sequences and identify additional gene transcriptional control regions which include SBEs.
  • Smad binding element sequences can be incorporated into primers useful in polymerase chain reaction assays of SBEs in transcriptional control regions.
  • Conditions, reagents and so forth for hybridization and polymerase chain reaction assays are well known in the art and it is within the skill of the person of ordinary skill in the art to modify such conditions to achieve the appropriate stringency of probe or primer hybridization to selectively isolate or identify SBE sequences.
  • Examples of such conditions, reagents etc. are provided in compilations of molecular biology methods, including, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a Smad polypeptide which binds to a Smad binding element, e.g., Smad3, Smad4 and derivatives thereof. Nucleic acid molecules which bind to SBEs and block binding of proteins thereto are also contemplated. This is desirable in virtually any medical condition wherein a reduction of Smad protein binding to a Smad binding element is desirable, e.g., to modulate TGF- ⁇ superfamily transcriptional response or reduce Smad transactivation of SBEs by reducing the amount of Smad.
  • the antisense oligonucleotides also are useful in screening assays for identifying compounds which bind to or regulate binding to a Smad binding element, or which regulate TGF- ⁇ superfamily transcriptional response mediated by a Smad binding element.
  • antisense oligonucleotide or “antisense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA.
  • the antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript.
  • the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the - 14 - particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon known Smad gene sequences, or upon allelic or homologous genomic and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.
  • antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides (Wagner et al., Nature Biotechnol. 14:840-844, 1996). Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.
  • oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3'-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs.
  • the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439-457, 1994) and at which proteins are not expected to bind.
  • the nucleic acids which contain a Smad binding element of the invention, or the antisense oligonucleotides of the invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester intemucleoside linkage.
  • These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.
  • the nucleic acids of the invention also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from binding proteins or hybridizing to their target nucleic acids but which enhance their stability or targeting or which otherwise enhance their effectiveness. - 15 -
  • modified oligonucleotide as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic intemucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.
  • a synthetic intemucleoside linkage i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide
  • a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.
  • Preferred synthetic intemucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
  • modified oligonucleotide also encompasses oligonucleotides with a covalently modified base and/or sugar.
  • modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified oligonucleotides may include a 2'-O-alkylated ribose group.
  • modified oligonucleotides may include sugars such as arabinose instead of ribose.
  • compositions containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding Smad polypeptides which bind a Smad binding element, optionally together with a carrier.
  • Antisense oligonucleotides may be administered as part of a pharmaceutical composition.
  • a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.
  • the compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The characteristics of the carrier will depend on the route of administration.
  • Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of DNA although RNA vectors are also available.
  • Vectors include, but are not limited to, plasmids, phagemids and - 16 - virus genomes.
  • a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., luciferase, ⁇ -galactosidase and alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
  • Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • a coding sequence and regulatory sequences are said to be "operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences.
  • two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' - 17 - non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribed regulatory sequences especially will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences may also include, in addition to one or more SBE sequences, other enhancer sequences or upstream activator sequences as desired.
  • the vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • RNA heterologous DNA
  • the transcriptional elements include one or more Smad binding elements sufficient to confer Smad transactivation or TGF ⁇ superfamily ligand responsiveness.
  • Preferred systems for mRNA expression in mammalian cells are those such as pRc/CMV (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences.
  • pRc/CMV available from Invitrogen, Carlsbad, CA
  • CMV human cytomegalovirus
  • suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr vims (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element.
  • kits which allow the artisan to test compounds for their ability to bind a Smad binding element, to increase or reduce Smad binding to a Smad binding element, or to modulate transcription of nucleic acids which are controlled by a Smad binding element.
  • kits include at least the previously discussed Smad binding element nucleic acid sequences. Other components may be added, as desired, as long as the previously mentioned sequences, which are required, are included.
  • the invention also provides isolated polypeptides and complexes of polypeptides which bind a Smad binding element.
  • the polypeptides include Smad proteins such as Smadl, Smad2, Smad3, Smad4 and Smad5, as well as Smad derivatives such as Smad proteins having a truncated C-terminus, e.g. a complete or partial deletion of the MH2 domain, such as the Smad3 ⁇ MH2 and Smad4 ⁇ MH2 proteins described below.
  • Smadl/Smad4 complex a Smad2/Smad4 complex
  • Smad3/Smad4 complex a Smad3/Smad4 complex
  • Smad5/Smad4 complex each component of which can be a wild-type Smad protein or a derivative thereof.
  • Such polypeptides are useful, for example, in in vitro assays of SBE- regulated transcription.
  • Such protein also are useful in therapeutic methods for modulating binding to a Smad binding element, or for modulating transcription regulated by a Smad binding element.
  • the proteins also can be used in screening methods for identifying compounds which bind to a Smad binding element, or which modulate transcription regulated by a Smad binding element.
  • a "variant" of a Smad polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a Smad polypeptide. Modifications which create a Smad variant can be made to a Smad polypeptide 1) to reduce or eliminate an activity of a Smad polypeptide, such as binding to a SBE; 2) to enhance a property of a Smad polypeptide, such as protein stability in an expression system or the stability of protein-protein binding or such as binding to a SBE; or 3) to provide a novel activity or property to a Smad polypeptide, such as addition of an antigenic epitope or addition of a detectable moiety.
  • Modifications to a Smad polypeptide are typically made to the nucleic acid which encodes the Smad polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and additions of - 19 - amino acids or non-amino acid moieties. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the various Smad amino acid sequences. In general, variants include Smad polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity.
  • cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages.
  • certain amino acids can be changed to enhance expression of a Smad polypeptide by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present).
  • Mutations of a nucleic acid which encode a Smad polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the variant polypeptide. Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities such as binding to a Smad binding element to determine which mutation provides a variant polypeptide with the desired properties.
  • variants or to non-variant Smad polypeptides
  • which are silent as to the amino acid sequence of the polypeptide but which provide preferred codons for translation in a particular host.
  • the preferred codons for translation of a nucleic acid in, e.g., E. coli are well known to those of ordinary skill in the art.
  • Still other mutations can be made to the noncoding sequences of a Smad gene or cDNA clone to enhance expression of the polypeptide.
  • variants of Smad polypeptides can be tested by cloning the gene encoding the variant Smad polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the variant Smad polypeptide, and testing for a functional capability of the Smad polypeptides as disclosed herein.
  • the variant Smad polypeptide can be tested for inhibition of TGF ⁇ (and/or activin or BMP) signalling activity as disclosed in the Examples, or for inhibition of Smad transactivation from SBEs or for SBE binding as is also disclosed herein. Preparation of other variant polypeptides may favor testing of other activities, as will be known to one of ordinary skill in the art.
  • conservative amino acid substitutions may be - 20 - made in Smad polypeptides to provide functionally equivalent variants of the foregoing polypeptides, i.e, variants which retain the functional capabilities of the Smad polypeptides.
  • a "conservative amino acid substitution” refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J.
  • Smad polypeptides include Smads having a deletion of the MH2 domain, such as Smad4 ⁇ MH2.
  • Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • amino acid sequence of Smad polypeptides typically are made by alteration of the nucleic acid encoding Smad polypeptides.
  • substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488- 492, 1985), or by chemical synthesis of a gene encoding a Smad polypeptide.
  • the polypeptide may be purified from cells which naturally produce the polypeptide by chromatographic means or immunological recognition.
  • an expression vector may be introduced into cells to cause production of the - 21 - polypeptide.
  • mRNA transcripts may be microinjected or otherwise introduced into cells to cause production of the encoded polypeptide.
  • Translation of mRNA in cell-free extracts such as the reticulocyte lysate system also may be used to produce polypeptide.
  • Those skilled in the art also can readily follow known methods for isolating Smad polypeptides. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.
  • the invention also makes it possible isolate Smad protein complexes such as Smadl/Smad4, Smad2/Smad4, Smad3/Smad4 and Smad5/Smad4 by the binding of such proteins to a SBE as disclosed herein.
  • the identification of this binding also permits one of skill in the art to block the binding of Smad proteins to endogenous Smad binding elements (e.g. in gene control regions of endogenous genes).
  • binding of such proteins can be affected by introducing into a biological system (e.g., a cell) in which the proteins bind an oligonucleotide including a Smad binding element in an amount sufficient to reduce or even block the binding of Smad proteins and endogenous SBEs.
  • Smad binding to SBEs also enables one of skill in the art to identify Smad amino acid sequences which bind to such SBEs and prepare modified proteins, using standard recombinant DNA techniques, which can bind to SBEs. For example, when one desires to place a certain gene under the control of a particular transcription factor, one can prepare a fusion polypeptide of the transcription factor protein and the Smad SBE binding site. Inclusion of SBEs in the transcriptional control region of the desired gene will then confer control of the transcription of that gene by the modified transcription factor.
  • the invention also provides, in certain embodiments, "dominant negative" Smad polypeptides which have mutated SBE binding sites.
  • a dominant negative polypeptide interacts with the cellular machinery (such as another Smad protein in a Smad/Smad complex), thereby displacing an active protein from its interaction with the cellular machinery or competing with the active protein, thereby reducing the effect of the active protein.
  • the end result of the expression of a dominant negative polypeptide in a cell is a reduction in function of active proteins.
  • One of ordinary skill in the art can assess the potential for a dominant negative variant of a protein, and using standard mutagenesis techniques to create one or more dominant negative variant polypeptides.
  • Smad polypeptides which bind SBEs one of ordinary skill in the art can modify the sequence of a Smad polypeptide by site-specific mutagenesis, scanning mutagenesis, partial gene - 22 - deletion or t ncation, and the like. See, e.g., U.S. Patent No.
  • the invention also involves the use of agents such as polypeptides which bind to Smad polypeptides and/or to complexes of Smad polypeptides and/or to SBEs to modulate SBE- mediated activities.
  • agents such as polypeptides which bind to Smad polypeptides and/or to complexes of Smad polypeptides and/or to SBEs to modulate SBE- mediated activities.
  • the invention therefore, embraces peptide binding agents which, for example, can be antibodies or fragments of antibodies having the ability to selectively bind to Smad polypeptides.
  • Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology.
  • Agents also include antisense nucleic acid (described above) which are useful for reducing the expression of Smad proteins, thereby reducing SBE- mediated activities.
  • an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F(ab') 2 fragment
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • the present invention also provides for the use of F(ab') 2 , Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab') 2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes the use of so-called single chain antibodies.
  • the invention involves the use of polypeptides of numerous size and type that bind specifically to Smad polypeptides, and complexes of Smad polypeptides.
  • polypeptides may be derived also from sources other than antibody technology.
  • polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries.
  • Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties (e.g., peptidomimetics).
  • peptoids and non-peptide synthetic moieties e.g., peptidomimetics
  • Phage display can be particularly effective in identifying binding peptides useful according to the invention. Briefly, one prepares a phage library (using e.g. ml 3, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures.
  • the inserts may represent, for example, a completely degenerate or biased array.
  • DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides.
  • the minimal linear portion of the sequence that binds to the SBE or Smad polypeptide can be determined.
  • Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the Smad polypeptides.
  • the Smad binding element nucleotide sequences can be used to screen peptide libraries, including phage display libraries, to identify and select molecules which bind to SBEs.
  • Other non-peptide compounds, including libraries of such compounds can also be screened for SBE binding activity.
  • Such molecules can be used, as described, for screening assays, for purification protocols, for interfering directly with the functioning of SBEs and for other purposes that will be apparent to those of ordinary skill in the art.
  • a Smad binding element, or a multimers thereof, also can be used to isolate their native binding partners, including, e.g., the Smad proteins and Smad protein complexes. Isolation of such binding partners may be performed according to well-known methods. For example, isolated nucleic acids including one or more Smad binding elements can be attached to a substrate (e.g., chromatographic media, such as polystyrene beads, or a filter), and then a solution suspected of containing the Smad binding element binding proteins may be applied to the substrate. If a protein or protein complex which can bind a Smad binding element is present in the solution, then it will bind to the substrate-bound Smad binding element. The protein or protein complex then may be isolated. Other molecules which bind Smad binding elements, may be isolated by similar methods without undue experimentation.
  • the invention further provides methods for modulating (reducing or increasing) TGF ⁇ superfamily signal transduction in a cell.
  • Such methods are useful in vitro for altering the TGF- ⁇ - 25 - signal transduction, for example, in testing compounds for potential to block abnormally elevated TGF- ⁇ signal transduction or increase deficient TGF- ⁇ signal transduction.
  • In vivo such methods are useful for modulating growth, e.g., to treat cancer and fibrosis.
  • Increasing TGF- ⁇ signal transduction in a cell by, e.g., introducing a reporter gene under the control of one or more Smad binding elements (TGF ⁇ response elements) in the cell, can be used to provide a model system for testing the effects of putative inhibitors of TGF- ⁇ signal transduction.
  • TGF- ⁇ signal transduction can be measured by a variety of ways known to one of ordinary skill in the art, such as the reporter systems described in the Examples.
  • Various modulators of Smad-SBE binding activity can be screened for effects on TGF- ⁇ signal transduction using the methods disclosed herein. The skilled artisan can first determine the modulation of a SBE activity, such as TGF- ⁇ signalling activity, and then apply such a modulator to a target cell or subject and assess the effect on the target cell or subject.
  • cells in culture can be contacted with Smad-SBE binding modulators and the increase or decrease of growth or focus formation of the cells can be determined according to standard procedures.
  • Smad-SBE binding activity modulators can be assessed for their effects on other TGF- ⁇ signal transduction downstream effects by similar methods in many cell types.
  • TGF- ⁇ superfamily activity in certain conditions characterized by abnormally elevated TGF- ⁇ superfamily activity.
  • conditions involving abnormally elevated TGF- ⁇ superfamily activity include ossification of the posterior longitudinal ligament (Yonemori et al., Am. J. Pathol.
  • ossification of the ligament flavum (Hayashi et al., Bone 21 :23-30, 1997); liver fibrosis including cirrhosis and veno-occlusive disease; kidney fibrosis including glomerulonephritis, diabetic nephropathy, allograft rejection and HIV nephropathy; lung fibrosis including idiopathic fibrosis and autoimmune fibrosis; skin fibrosis including systemic sclerosis, keloids, hypertrophic bum scars and eosinophilia-myalgia syndrome; arterial fibrosis including vascular restenosis and atherosclerosis; central nervous system fibrosis including intraocular fibrosis; and other fibrotic diseases including rheumatoid arthritis and nasal polyposis. (see, e.g., Border and Noble, N. Engl. J. Med. 331 :1286-1292, 1994).
  • Smad-SBE binding or transcriptional effects thereof is administered to treat the condition, which amount can be determined by one of ordinary skill in the art by routine experimentation.
  • an SBE-containing nucleic acid can be administered and the progress of the fibrosis or ossification monitored using standard medical diagnostic methods.
  • An amount of an SBE-containing nucleic acid, or an antagonist of Smad-SBE binding which reduces the progression of the fibrosis or ossification, or even halts the progression of the fibrosis or ossification is an effective amount.
  • the person of ordinary skill in the art will be familiar with such methods.
  • Smad-SBE binding antagonists include antibodies to Smad proteins, nucleic acids including SBE sequences and antisense Smad nucleic acids described above.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the preparations of the invention are administered in effective amounts.
  • An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, produces the desired response.
  • the desired response is inhibiting the progression of the cancer.
  • the desired response is inhibiting the progression of the ossification.
  • the desired response is inhibiting the progression of the fibrosis. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods or can be monitored according to diagnostic methods of the invention discussed herein.
  • the invention also contemplates gene therapy.
  • the procedure for performing ex vivo gene therapy is outlined in U.S. Patent 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents. In general, it involves introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene, and returning the genetically engineered cell(s) to the subject.
  • the functional copy of the gene is under operable control of regulatory elements which permit expression of the gene in the genetically engineered cell(s).
  • a functional gene need not be expressed; the gene therapy can be the product of the administration of nucleic acids, especially double stranded nucleic acids, which contain one or preferably more than one SBEs and thereby can act by binding endogenous SBE binding factors.
  • nucleic acids especially double stranded nucleic acids, which contain one or preferably more than one SBEs and thereby can act by binding endogenous SBE binding factors.
  • Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654.
  • In vivo gene therapy using vectors such as adenovirus, retroviruses, herpes vims, and targeted liposomes, as well as naked DNA, also is contemplated according to the invention.
  • the invention further provides efficient methods of identifying pharmacological agents or lead compounds for agents active at the level of SBE-Smad binding or a Smad binding element modulatable cellular function.
  • such functions include TGF ⁇ superfamily signal - 28 - transduction, Smad transactivation and formation of a SBE-Smad complex.
  • the screening methods involve assaying for compounds which interfere with one of these activities. Such methods are adaptable to automated, high throughput screening of compounds.
  • An exemplary cell-based assay involves transfecting a cell with a nucleic acid encoding a Smad polypeptide, such as Smad4, and a nucleic acid encoding a test peptide which potentially modulates Smad-SBE binding.
  • the cell also contains a reporter gene operably linked to a gene expression regulatory region containing one or more Smad binding elements. A change in the activation of reporter gene transcription occurs when the test peptide modulates Smad-SBE binding such that the Smad-mediate transcription of the reporter gene is modulated.
  • Agents which modulate Smad transactivation of the reporter gene are then detected through a change in the expression of reporter gene.
  • Methods for determining changes in the expression of a reporter gene are known in the art and exemplified below.
  • assays in which non-nucleic acid test compounds are added to the cell are provided.
  • the foregoing cell-based assays can be used to screen for compounds which modulate TGF ⁇ superfamily signal transduction activity, in the presence of TGF ⁇ superfamily ligands or in the absence of TGF ⁇ superfamily ligand. Addition of ligand to the cell based assays will permit identification and selection of compounds which reduce TGF ⁇ superfamily ligand stimulated signal transduction.
  • the cell based assays permit the selection of compounds - 29 - which modulate TGF ⁇ superfamily signal transduction at any stage in the signal transduction pathway upstream of SBE-mediated transcriptional induction, including ligand binding, activation of receptors independent of ligand, prevention of Smad nuclear translocation, modulation of receptor-Smad interaction, upregulation of inhibitory Smad expression, etc.
  • the SBE (e.g. as a reporter construct) is responsive to several members of the TGF ⁇ superfamily, including TGF ⁇ , activin and OP-1.
  • the assays can be carried out in parallel by stimulating with one, two, or more TGF ⁇ superfamily ligands in individual assays, thereby permitting the identification of compounds which modulate the signal transduction induced by one or more of the ligands.
  • the compound is specific for that member of the TGF ⁇ superfamily.
  • the compound has a broader spectrum of activity, e.g., is a more general or less specific modulator of TGF ⁇ superfamily signal transduction.
  • Recombinantly produced Smad polypeptides include chimeric proteins comprising a fusion of a Smad protein with another polypeptide, e.g., a polypeptide capable of providing or enhancing protein-protein binding, sequence specific nucleic acid binding (such as GAL4), enhancing stability of the Smad polypeptide under assay conditions, or providing a detectable moiety, such as green fluorescent protein or Flag epitope as provided in the examples below.
  • one of these concentrations serves as - 30 - a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection.
  • Candidate agents encompass numerous chemical classes, although typically they are organic compounds.
  • the candidate pharmacological agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents.
  • reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components.
  • reagents that improve the efficiency of the assay such as protease, inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used. - 31 -
  • the mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the Smad polypeptide or complex thereof specifically binds a Smad binding element, a multimer thereof or analog thereof.
  • the conditions permit transactivation of a reporter gene under the control of the Smad binding element.
  • the order of addition of components, incubation temperature, time of incubation, and other perimeters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4°C and 40 °C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours.
  • a separation step is often used to separate bound from unbound components.
  • the separation step may be accomplished in a variety of ways. Conveniently, at least one of the components is immobilized on a solid substrate, from which the unbound components may be easily separated.
  • the solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc.
  • the substrate preferably is chosen to maximum signal to noise ratios, primarily to minimize background binding, as well as for ease of separation and cost.
  • Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent.
  • the separation step preferably includes multiple rinses or washes.
  • the solid substrate is a microtiter plate
  • the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc.
  • the solid substrate is a magnetic bead
  • the beads may be washed one or more times with a washing solution and isolated using a magnet.
  • Smad-SBE binding modulator pharmacological agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving Smad proteins, e.g., TGF- ⁇ superfamily ligand induced signal transduction.
  • Novel Smad- and SBE- specific binding agents include Smad- and SBE-specific antibodies and other natural intracellular binding agents identified with assays such as two hybrid screens, and non-natural intracellular binding agents identified in screens of chemical libraries, phage-display libraries and the like.
  • Such techniques include transfection of nucleic acid-CaPO 4 precipitates, transfection of nucleic acids associated with DEAE, transfection with a retrovims (or other vims) including the nucleic acid of interest, liposome mediated transfection, electroporation and the like.
  • a vehicle used for delivering a nucleic acid of the invention into a cell e.g., a retrovims, or other vims; a liposome
  • a molecule such as an antibody - 33 - specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle.
  • proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.
  • proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo intemalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.
  • Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids.
  • a deletion series was constmcted by restriction digestion of the pJBl plasmid with EcoRI-PinAI ( ⁇ JB2), Sad (pJB3), SacI-BssHII (pJB4), or SacI-SacII (pJB5) and subsequent self-ligation.
  • a minimal promoter construct (pGL3ti) was made from pGL3-basic by inserting oligonucleotides carrying the Adenovims Major Late promoter TATA box (AdTu: gatctGGGGCTATAAAAGGGGGTAGGGGgagct; SEQ ID NO:l 1 and AdThcCCCCTACCCCCTTTTATAGCCCCa; SEQ ID NO: 12) and the mouse Terminal deoxynucleotidyl Transferase gene Initiator sequence (TiuxGCCCTCATTCTGGAGACAg; SEQ ID NO:13 and Til: gatccTGTCTCCAGAATGAGGGCgagct; SEQ ID NO:14) in the Bglll site.
  • AdTu AdTu: gatctGGGGCTATAAAAGGGGGTAGGGGgagct; SEQ ID NO:l 1 and AdThcCCCCTACCCCCTTTTATAGCCCCa; SEQ ID NO: 12
  • a deletion series (pJBl 1-17) was constmcted as follows: an Asp718-BseAI (-3004/1561) fragment from pJB3 was inserted into the Asp718-XmaCl sites from pGL3ti (pJBl 1), a T4 DNA polymerase blunted Sad-BamHI fragment from pJB3 was inserted in the Smal site of pGL3ti (pJB12), a BamHI-Bglll fragment from pJBl 1 was inserted into the Bglll site of pGL3ti (pJB13), pJB12 was restriction digested with Asp718-PvuII or PvuII-Bglll and subsequently blunted and self ligated (pJB14 and pJB15 respectively), pJB15 was restriction digested with - 34 -
  • pJB17 Asp718-MluNI and subsequently blunted and self ligated (pJB17), and pJBl 1 was digested with MluNI and Bglll and subsequently blunted and self ligated (pJB16).
  • pJB15 was used as template for PCR reactions with the Til oligonucleotide and the upper strands of the following oligonucleotides:
  • PCR products were phosphorylated, restriction digested with Sad and ligated into a Asp718 digested and blunted, Sad digested pGL3ti vector.
  • the internal deletion series was constmcted as follows: a Pstl/PvuII fragment (-2762/-2611) from pJB12 was cloned into the Pstl and EcoRV sites of pBluescript SKII- (pSK-PsPv).
  • pJB15 was used as template for PCR reactions with the upper strand of oligonucleotide A and the lower strands of oligonucleotides B, C, D and G.
  • PCR products were cloned into the Smal site of pSK-PsPv. Also double stranded oligonucleotides A and D were ligated into pSK-PsPv.
  • the resulting plasmids were restriction digested with Xbal and Sail and the fusion fragments were inserted into Nhel-Xhol opened pGL3ti, pGL3ti-PsPv and pGL3ti-AfPv was constmcted by digestion of pJB15 with Asp718-Psl or Asp718-AfII, blunting and self ligation.
  • Reporter constructs carrying multimerized versions of Smad Binding Element I V5 were created by ligating 4 oligonucleotides into the Bglll site of ⁇ GL3ti (inv5u:gatccTTTCTCAGACAGTCTGTCTGCa; SEQ ID NO:25 and inv51: gAAAGAGTCTGTCAGACAGACGtctag; SEQ ID NO:26).
  • pGL3ti(DIR)4 was created by inserting one 4xCAGACA-WT oligonucleotide (upper strand: - 35 - gtaccCAGACAGTCAGACAGTCAGACAGTCAGACAGTc; SEQ ID NO:27, lower strand: gGTCTGTCAGTCTGTCAGTCTGTCAGTCTGTCAgagct; SEQ ID NO:28) into Asp718-XhoI opened pGL3ti.
  • pGL3ti was generated by the insertion of adenovims major late promoter in pGL3 vector (Promega, Madison, WI).
  • pGL3ti-(CAGACAGACTGTCTG) 2 was constmcted using the double strand oligonucleotides and cloned into pGL3ti. All constmcts were verified by sequencing.
  • Smad expression plasmids were constmcted as follows: pSG5-XMADl and pSG5- XMAD2 were created by inserting EcoRI fragments from pSP64TEN-DOTl and -2 into EcoRI opened pSG5; pSG5-hSmad3f was created by inserting a blunted BamHI-Sall fragment from pRK5-hSmad3f into blunted BamHI opened pSG5, and pSG5-hSmad4 was created by inserting a blunted BamHI-EcoRI fragment from pDPC-wt3 into blunted EcoRI-BamHI opened pSG5.
  • GST-Smadl ⁇ MH2 (residues 1-269), GST-Smad2 ⁇ MH2 (residues 1-272), GST-Smad3 ⁇ MH2 (residues 1-229), GST-Smad4 ⁇ MH2 (residues 1-321), and GST-Smad5 ⁇ MH2 (residues 1-269) contained MH1 plus linker regions of Smadl, Smad2, Smad3, Smad4 and Smad5, respectively.
  • GST-Smad2 was also constructed using pGEX4T-l vector. GST-Smadl, GST-Smad3 and GST-Smad4 were generously provided by Dr. R. Derynck.
  • P19EC embryonal carcinoma cells were maintained in a 1 :1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium supplemented with 7.5% fetal bovine serum (Integro, Zaandam, The Netherlands).
  • DMEM Dulbecco's modified Eagle's medium
  • Ham's F12 medium supplemented with 7.5% fetal bovine serum
  • NIH3T3 embryonic fibroblast cells were maintained in DMEM supplemented with 10% Newborn Calf Serum (Life Technologies, Gaithersburg, MD).
  • HaCaT human keratinocytes, HepG2 human hepatocellular carcinoma cells, MDA-MB468 breast cancer cells and MvlLu mink lung epithelial cells were grown in DMEM medium supplemented with 10% FCS.
  • TGF ⁇ l R&D Systems, Minneapolis, MN and Abingdon, UK
  • Activin or OP-1 OP-1 at 10, 20 or 100 ng/ml respectively.
  • Luciferase activities were measured using the Luciferase Assay System following lysis in lx Reporter Lysis Buffer (Promega).
  • ⁇ -galactosidase activity was quantified in 100 mM Na 2 HPO 4 /NaH 2 PO 4 , 1 raM MgCl 2 , 100 mM 2-mercapto-ethanol and 0.67 mg/ml o- Nitrophenyl-Galactopyranoside, pH 7.3.
  • GST fusion protein expression was induced in logarithmically growing cultures of E coli, TGI, by the addition of isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and growing the bacteria at 30 °C for an additional 5 h.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • Bacteria suspended in 50 ml of cold phosphate-buffered saline (PBS) were sonicated, mixed with 1% Triton X-100 and centrifuged at 12,000g for 10 min.
  • an extract containing GST fusion protein was mixed with 0.25% glutathione-Sepharose 4B beads (Pharmacia) and incubated at 4°C under the constant agitation for 12 h.
  • the beads were washed four times with PBS containing 1% Triton X-100, and then eluted three times with 50 mM Tris (pH 8.0) including 10 mM glutathione. After the dialysis of eluates with PBS containing 2 mM DTT and 0.5 mM PMSF, the sample was stored at -70°C until used.
  • WT tcgagCAGACAGTCAGACAGTc; SEQ IDNO:29 cGTCTGTCTGTCTGTCAgagct; SEQ IDNO:30
  • Ml tcgaggAGACAGTgAGACAGTc; SEQ IDNO:31 ccTCTGTCTcTCTGTCAgagct; SEQ IDNO:32 - 37 -
  • M2 tcgagCtGACAGTCtGACAGTc; SEQ ID NO:33 cGaCTGTCTGaCTGTCAgagct; SEQ ID NO:34
  • M4 tcgagCAGtCAGTCAGtCAGTc; SEQ ID NO:37 cGTCaGTCTGTCaGTCAgagct; SEQ ID NO:38
  • M5 tcgagCAGAgAGTCAGAgAGTc;SEQIDNO:39 cGTCTcTCTGTCTcTCAgagct; SEQ ID NO:40
  • M6 tcgagCAGACtGTCAGACtGTc; SEQ ID NO:41 cGTCTGaCTGTCTGaCAgagct; SEQ ID NO:42
  • M7 tcgagCAGACAcTCAGACAcTc; SEQ ID NO:43 cGTCTGTgTGTCTGTgAgagct; SEQ ID NO:44
  • Binding reactions contained 4 microgram nuclear extract, 100 mM KC1, 0.2 mM EDTA,
  • the binding reaction using GST fusion proteins was the following procedure: GST fusion proteins were mixed with the binding buffer consisting of 20% glycerol, 20 mM Hepes (pH 7.9), 30 mM KC1, 4 mM MgCl 2 , 0.1 mM EDTA, 0.8 mM sodium phosphate, 4 mM spermidine, 0.3 ⁇ g/ ⁇ l poly (dLdC) (Pharmacia) and 0.25 nM 32 P-labeled probe at the final - 38 - volume of 20 ⁇ l. If necessary, 500-fold molar excess of the cold competitor was added in the reaction mixture. The mixture was incubated at 25 °C for 1 h and ran on a 5% non-denatured polyacrylamide gel with 0.5X TBE.
  • JunB gene is a direct target for transcription activation in response to activation of signal transduction by TGF ⁇ (de Groot and Kruijer, Biochem. Biophys. Res. Comm. 168:1074-1081, 1990).
  • TGF ⁇ signal transduction by TGF ⁇
  • a JunB-luciferase fusion gene was constructed, containing the JunB TATA box and transcriptional start site, the complete 5' untranslated region as well as approximately 6.4kb of promoter upstream sequences (pJBl, Fig. 1 A).
  • a nested set of restriction fragments was derived from this region and cloned in front of a heterologous minimal promoter fused to the luciferase gene.
  • Co-transfection of P19EC cells with these constmcts and Smad3 and Smad4 expression plasmids allowed localization of a Smad responsive region to between nucleotides -2908 and -2611 (Fig. IB). After 40 hours, cells were lysed to measure luciferase activity. Luciferase activity was normalized using ⁇ -galactosidase activity. Data are shown with the mean ⁇ SD of triplicates.
  • TGF ⁇ treated HaCaT and MvlLu cells contained an induced DNA binding activity that migrated with a - 39 - lower mobility than that of a constitutively expressed binding entity.
  • a minimal Smad-responsive region was defined next (Fig. 2).
  • a nested series of deletion fragments derived from the 297 bp region were generated by PCR using oligonucleotides A, B, C, D, and G and a downstream primer or restriction digestion with Aflll or Pstl.
  • a series of internal deletion fragments were made by PCR and fused to the Pstl-PvuII (PsPv) fragment.
  • nt -2788 and -2616 The region between nt -2788 and -2616 is not responsive to overexpression of Smad3 and Smad4. However, it appeared to be required for transactivation of upstream located sequences by Smad3 and Smad4. Extending this region with sequences located just upstream restored transactivation by Smad 3 and -4 (D-PvuII), while further incorporation of 5' sequences appeared to have a slight repressive effect except when sequences unique to the A oligo were included. This analysis was complemented by a series of deletions of sequences located between nt -2908 and -2762. Exclusion of sequences upstream from nt -2792 abrogated transactivation by Smad3 and Smad4. This analysis therefore defines the 22 basepair region between -2813 and -2792 as that minimally required for transactivation by Smad3 and Smad4.
  • Example 2 Smad3 and Smad4 bind an inverted CAGACA repeat
  • the location of the Smad binding element on the 297 bp Smad responsive region was more specifically determined by detailed binding competition analysis.
  • a 120 bp subfragment (-2908/-2788) was found to carry the binding site for the TGF ⁇ induced complex.
  • An end labeled restriction fragment derived from the 120 bp Smad-responsive region was incubated with - 40 - nuclear extracts from mouse NIH-3T3 embryonic fibroblasts, human HaCaT keratinocytes, and mink MvlLu lung epithelial cells treated with or without 10 mg/ml TGF ⁇ for 1 h. Protein binding was visualized as above. A TGF ⁇ -induced complex was observed.
  • This fragment (-2908/-2788) contains the minimal region required for transactivation by overexpressed Smad3 and Smad4 (-2813 to -2792).
  • Smad 1-5 were produced as GST fusion proteins in E. coli and analyzed for their ability to bind the 120 bp MluNI/Aflll subfragment from the 297 bp region.
  • the purified proteins were incubated with the labeled probe and complexes were resolved on a 5% non-denaturing polyacrylamide gel. Binding of full length fusion protein was only observed for GST-Smad4. However, C-terminally tmncated Smad3 and Smad4 strongly interacted with the probe, while binding of C-terminally truncated Smadl, Smad2, and Smad5 to the 120 bp fragment was below the threshold of detection.
  • GST-Smad4 readily complexed with the probe while interaction with GST-Smad3 was not detectable.
  • the CAGACA repeat is a TGF ⁇ response element
  • the minimal region required for transactivation of the 297 bp cis-acting region by overexpressed Smad3 and Smad4 is by itself not sufficient to render a heterologous promoter activatable by TGF ⁇ or Smad3 and Smad4. It therefore was reasoned that efficient activation by Smad proteins requires multimerization of CAGACA repeat containing region.
  • the JunB SBE (INV5) and a direct repeat version thereof (DIR) were multimerized and cloned in pGL3ti. These reporter plasmids with or without Smad expression plasmids were transfected into HepG2 cells. The cells were treated overnight with or without 10 ng/ml TGF ⁇ .
  • Smad7 has been isolated and identified as a negative regulator of TGF ⁇ superfamily receptor mediated signaling (Nakao et al, Nature 389:631-635, 1997). Smad7 acts - 43 - by binding to the docking site for receptor-activated Smads thereby preventing phosphorylation and thus signaling.
  • the (INV)4 reporter plasmid was cotransfected into HepG2 cells along with increasing amounts of a Smad7 expressing plasmid (Fig. 3B). The transfected cells were treated with TGF ⁇ .
  • Example 5 The CAGACA repeat is a response element for other members of the TGF ⁇ superfamily.
  • TGF ⁇ Besides TGF ⁇ , other members of the TGF ⁇ superfamily such as activin and OP-1 have been shown to induce JunB mRNA expression.
  • Co-transfection of the Smad3 -responsive (-3000/-2333) reporter with Smad4 and a receptor-activatable Smad protein (Smadl, Smad2, or Smad3) expressing plasmids showed that each receptor-activatable Smad induced reporter activity when co-transfected with Smad4 expressing plasmid (Fig. 4A).
  • P19EC cells were transfected with the pJB12 plasmid with or without Smadl, Smad2, Smad3, and/or Smad4 expression plasmids as in Example 1.
  • TGF ⁇ preferentially activated the CAGACA repeat ((INV)4) through Smad3 over Smad2 Fig. 4B.
  • HepG2 cells were transfected with the pGL3ti-(INV)4 plasmid and with the combinations of Smad2, Smad3, and/or Smad4 expression plasmids shown. The cells were incubated for 16 h with or without 10 ng/ml TGF ⁇ . Normalized luciferase activities are shown as the mean ⁇ SD of triplicates. Co-transfection of Smad2+Smad4 did not enhance TGF ⁇ mediated induction of the reporter constmct while Smad3+Smad4 superactivated ligand induced reported activity.
  • the JunB gene is activated by TGF ⁇ , activin and BMP2. Besides Smad3, Smadl and Smad2 also activated the JunB promoter when co-transfected with Smad4, indicating that overexpression of Smads mimics the ligand mediated activation of the endogenous promoter. - 44 -
  • Smad4-negative MDA-MB468 cells were transfected with the CAGACA reporter plasmid with or without the Smad4 expression plasmid. Following transfection cells were incubated for 16 h with or without 10 ng/ml TGF ⁇ , 20 ng/ml activin or 100 ng/ml OP-1 (Fig. 4D). Normalized luciferase activities are expressed as the mean ⁇ SD of triplicates. In the absence of Smad4, the reporter constmct was virtually unresponsive to any of the ligands. Co-transfection of Smad4 resulted in a slight activation of the reporter.

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Abstract

Cette invention a trait à des acides nucléiques isolés comprenant un ou plusieurs éléments fixateurs de la protéine Smad (SBE) ainsi que des protéines Smad et leurs fragments se liant à ces éléments fixateurs de la protéine Smad. Elle concerne également des éléments de réponse apportés au facteur de croissance transformant bêta (TGFβ) qui confèrent à la super-famille TGFβ une sensibilité à des gènes possédant ces éléments dans des régions de commande transcriptionnelle. L'invention porte, en outre, sur des méthodes d'utilisation de l'élément fixateur de la protéine Smad dans des techniques de criblage de composés liant ledit élément et dans des techniques de modulation de la fixation de protéines Smad auxdits éléments fixateurs, par exemple, pour moduler la transduction de signal de TGFβ. De surcroît, l'identification d'une séquence, d'origine mammalienne, de fixation de la protéine Smad permet d'identifier des déterminants de fixation de SBE de protéines SMAD.
EP99911099A 1998-03-03 1999-03-03 Elements fixateurs de la proteine smad et leurs applications Withdrawn EP1058729A1 (fr)

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