Isolated nucleic acid molecules encoding Smad7

Details Isolated nucleic acid molecules encoding Smad7 Isolated nucleic acid molecules encoding Smad7

1998-05-20

2001-06-26

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S243000, C435S320100, C435S325000, C435S410000, C536S023500

Reexamination Certificate

active

06251628

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acids and encoded polypeptides which interact with TGF-&bgr; superfamily receptors and which are negative regulators of signaling by those receptors. The invention also relates to agents which bind the nucleic acids or polypeptides. The invention further relates to methods of using such nucleic acids and polypeptides in the treatment and/or diagnosis of disease.
BACKGROUND OF THE INVENTION
During mammalian embryogenesis and adult tissue homeostasis transforming growth factor &bgr; (TGF-&bgr;) performs pivotal tasks in intercellular communication (Roberts et al., 1993). The cellular effects of this pleiotropic factor are exerted by ligand-induced hetero-oligomerization of two distantly related type I and type II serine/threonine kinase receptors, T&bgr;R-I and T&bgr;R-II, respectively (Lin and Lodish, 1993; Derynck, 1994; Massague and Weis-Garcia, 1996; ten Dijke et al., 1996). The two receptors, which both are required for signaling, act in sequence; T&bgr;R-I is a substrate for the constitutively active T&bgr;R-II kinase (Wrana et al., 1994; Weiser et al., 1995). TGF-&bgr; forms part of a large family of structurally related proteins which include activins and bone morphogenetic proteins (BMPs) that signal in a similar fashion, each employing distinct complexes of type I and type II serine/threonine kinase receptors (Lin and Lodish, 1993; Derynck, 1994; Massague and Weis-Garcia, 1996; ten Dijke et al., 1996).
Genetic studies of TGF-&bgr;-like signalling pathways in Drosophila and
Caenorhabditis elegans
have led to the identification of mothers against dpp (Mad) (Sekelsky et al., 1995) and sma (Savage et al., 1996) genes, respectively. The products of these related genes perform essential functions downstream of TGF-&bgr;-like ligands acting via serine/threonine kinase receptors in these organisms (Wiersdorf et al, 1996; Newfeld et al., 1996; Hoodless et al., 1996). Vertebrate homologs of Mad and sma have been termed Smads (Derynck et al., 1996) or MADR genes (Wrana and Attisano, 1996). Genetic alterations in Smad2 and Smad4/DPC4 have been found in specific tumor subsets, and thus Smads may function as tumor suppressor genes (Hahn et al., 1996; Riggins et al., 1996; Eppert et al., 1996). Smad proteins share two regions of high similarity, termed MH1 and MH2 domains, connected with a variable proline-rich sequence (Massague, 1996; Derynck and Zhang, 1996). The C-terminal part of Smad2, when fused to a heterologous DNA-binding domain, was found to have transcriptional activity (Liu et al., 1996; Meersseman et al., 1997). The intact Smad2 protein when fused to a DNA-binding domain, was latent, but transcriptional activity was unmasked after stimulation with ligand (Liu et al., 1996).
Different Smads specify different responses using functional assays in Xenopus. Whereas, Smad1 induces ventral mesoderm, a BMP-like response, Smad2 induces dorsal mesoderm, an activin/TGF-&bgr;-like response (Graff et al., 1996; Baker and Harland, 1996; Thomsen, 1996). Upon ligand stimulation Smads become phosphorylated on serine and threonine residues; BMP stimulates Smad1 phosphorylation, whereas TGF-&bgr; induces Smad2 and Smad3 phosphorylation (Hoodless et al., 1996; Liu et al., 1996; Eppert et al., 1996; Lechleider et al., 1996; Yingling et al., 1996; Zhang et al., 1996; Macías-Silva et al., 1996; Nakao et al., 1996). Thus certain Smads are pathway specific. Pathway specific Smads include Smad1, Smad2, Smad3 and Smad5.
Smad4 is a common component of TGF-&bgr;, activin and BMP signaling (Lagna et al., 1996; Zhang et al., 1997; de Winter et al., 1997). Smad4 phosphorylation has thus far been reported only after activin stimulation of transfected cells (Lagna et al., 1996). After stimulation with TGF-&bgr; or activin Smad4 interacts with Smad2 or Smad3, and upon BMP challenge a heteromeric complex of Smad4 and Smad1 has been observed (Lagna et al., 1996). Upon ligand stimulation, Smad complexes translocate from the cytoplasm to the nucleus (Hoodless et al., 1996; Liu et al., 1996; Baker and Harland, 1996; Macías-Silva et al., 1996), where they, in combination with DNA-binding proteins, may regulate gene transcription (Chen et al., 1996).
SUMMARY OF THE INVENTION
The invention provides isolated Smad7 nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides encoded by the Smad7 nucleic acids and agents which bind such polypeptides, including antibodies. The foregoing can be used in the diagnosis or treatment of conditions characterized by the expression of a Smad7 nucleic acid or polypeptide, or lack thereof. The invention also provides methods for identifying pharmacological agents usefuil in the diagnosis or treatment of such conditions. Here, we present the identification of Smad7, which opposes pathway specific Smads including Smad1, Smad2 and Smad3 and thus is an inhibitor of the TGF-&bgr; superfamily signalling pathways.
According to one aspect of the invention, an isolated nucleic acid molecule is provided. The molecule hybridizes under stringent conditions to a molecule consisting of the nucleic acid sequence of SEQ ID NOs:3 or 5. The isolated nucleic acid molecule codes for a polypeptide which inhibits TGF-&bgr; superfamily signaling. The invention further embraces nucleic acid lo molecules that differ from the foregoing isolated nucleic acid molecules in codon sequence due to the degeneracy of the genetic code. The invention also embraces complements of the foregoing nucleic acids.
In certain embodiments, the isolated nucleic acid molecule comprises a molecule consisting of the nucleic acid sequence of SEQ ID NO:7 or 8. Preferably, the isolated nucleic acid molecule consists of the nucleic acid sequence of SEQ ID NO:7 or 8, or consists essentially of the nucleic acid sequence of SEQ ID NO:3 or 5.
According to another aspect of the invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule comprises a molecule consisting of a unique fragment of SEQ ID NO:3 between 12 and 1944 nucleotides in length and complements thereof, or a unique fragment of SEQ ID NO:5 between 12 and 1875 nucleotides in length and complements thereof, provided that the isolated nucleic acid molecule excludes sequences consisting only of SEQ ID NO:1 and SEQ ID NO:2. Preferably the isolated nucleic acid molecule excludes molecules consisting solely of nucleotide sequences selected from the group consisting of accession numbers AA061644 (SEQ ID NO: 1), AA022262 (SEQ ID NO:2), AA347307, AA348247, 321995, W78627, W40869, AA033426, AA397050, AA016891, and C85115. In one embodiment, the isolated nucleic acid molecule consists of between 12 and 32 contiguous nucleotides of SEQ ID NO:3, SEQ ID NO:5, or complements of such nucleic acid molecules. In preferred embodiments, the unique fragment is at least 14, 15, 16, 17, 18, 20 or 22 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, or complements thereof.
According to another aspect of the invention, the invention involves expression vectors, and host cells transformed or transfected with such expression vectors, comprising the nucleic acid molecules described above.
According to still other aspects of the invention, transgenic non-human animals are provided. The animals include in certain embodiments the foregoing expression vectors. In certain preferred embodiments, the transgenic non-human animal includes a conditional Smad7 expression vector, such as an expression vector that increases expression of Smad7 in a tissue specific, development stage specific, or inducible manner. In other embodiments, the transgenic non-human animal has reduced expression of Smad7 nucleic acid molecules. In some embodiments, the transgenic non-human animal includes a Smad7 gene disrupted by homologous recombination. The disruption can be homozygous or heterozygous. In other embodiments, the transgenic non

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