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WO2008105925A2 - Composés et procédés pour moduler les interactions β-caténine/smad-7 - Google Patents

Composés et procédés pour moduler les interactions β-caténine/smad-7 Download PDF

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WO2008105925A2
WO2008105925A2 PCT/US2007/077445 US2007077445W WO2008105925A2 WO 2008105925 A2 WO2008105925 A2 WO 2008105925A2 US 2007077445 W US2007077445 W US 2007077445W WO 2008105925 A2 WO2008105925 A2 WO 2008105925A2
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smad7
catenin
skin
cells
agent
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WO2008105925A3 (fr
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Xiao-Jing Wang
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Oregon Health and Science University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Definitions

  • the present invention relates to the fields of cellular and molecular biology and the treatment of disease. More specifically, the invention provides methods for the identification of agents capable of modulating ⁇ -catenin/Smad-7 interactions, agents so identified, and to therapeutic methods employing such agents .
  • Murine hair follicle morphogenesis starts early in embryogenesis and proceeds through the typical eight stages (Philpott MP and Paus R, 1998) .
  • embryonic day 14.5 E14.5
  • certain epidermal stem cells that reside in the basal layer of the epidermis begin to designate their fate toward hair follicle morphogenesis.
  • some stem cells migrate downward along growing hair germ to form hair progenitor cells (matrix cells) in the hair bulb.
  • the hair progenitor cells will further differentiate into the outer root sheath (ORS) , the inner root sheath (IRS) , and the hair shaft.
  • the hair follicle bulge appears more prominent postnatally and is a niche housing multipotent epidermal stem cells, which are responsible for the self- renewal of hair follicle cells, sebaceous gland cells, and post-injury interfollicular epidermal cells (Fuchs et al., 2004; Watt, 1998; Lavker et al., 2003; Levy et al., 2005) .
  • Hair follicle morphogenesis is tightly controlled by a variety of signal transduction pathways.
  • Wnt signaling is essential for the initiation of hair follicle development (Andl et al., 2002; Huelsken et al., 2001; Gat et al., 1998), whereas hedgehog signaling is required for the down growth of the hair follicle and for sebaceous gland development (St. Jacques et al., 1998; Chiang et al . , 1999; Altaba, 1999; Allen et al., 2003; Mill et al., 2003).
  • Smad7 blocks Smad signaling by inhibiting Smad phosphorylation and by recruiting a Smad ubiquitination-related factor (Smurf) Smurf2 to degrade the type I receptors of transforming growth factor beta (TGF- ⁇ ) and/or Smads (Massague et al . , 2005). Smad7 is often overexpressed under pathological conditions, e.g., in intrinsically aged and photoaged human skin (Quan et al., 2002), and during skin carcinogenesis (He et al., 2001).
  • Smad7 transgene expression has been shown in keratinocytes to inhibit Smad signaling from TGF ⁇ /activin and bone morphogenetic protein (BMP) , which results in multiple developmental defects in stratified epithelia, including decreased hair follicle size (He et al., 2002). These mice die perinatally due to epithelial hyperkeratosis in the upper digestive tract and severe thymic atrophy (He et al., 2002).
  • BMP bone morphogenetic protein
  • compositions and methods which regulate Smad7 activity and function should have efficacy for the treatment of a variety of different disorders .
  • a method for identifying agents which modulate Smad7- ⁇ -catenin complex formation entails incubating cells that express Smad7 in the presence and absence of an agent, and assessing the cells for alterations in Smad7- ⁇ -catenin complex formation which occur in the presence but not in the absence of the test agent.
  • Agents so identified being capable of modulating Smad7- ⁇ -catenin complex formation, thereby regulating signal transduction pathways which include, without limitation, Wnt and TGF- ⁇ biochemical pathways.
  • Another aspect of the invention includes agents identified by the foregoing method.
  • a method for the treatment of cancer comprising increasing ⁇ -catenin degradation via the overexpression of Smad7 encoding nucleic acids in a tumor cell.
  • a Smad7 encoding nucleic acid is delivered to the tumor cell in a vector selected from the group consisting of adenoviral vectors, plasmids, adeno- associated viral vectors, retroviral vectors, hybrid adeno-associated virus vectors, lentivirus vectors, pseudo-typed lentivirus vectors, herpes simplex virus vectors, and vaccinia virus vectors.
  • the Smad7 encoding nucleic acid is delivered to the tumor cell in an antibody studded liposome, wherein the antibody has immuno-specificity for the tumor cell.
  • the methods described above may optionally comprise either prescreening tumor cells to assess TGF-beta receptor expression levels and/or assessing ⁇ -catenin for sequence alterations which result in aberrant ⁇ -catenin degradation.
  • Smad7 proteins or active fragments thereof can be conjugated to biocompatible nanoparticles and delivered to cells.
  • the hair growth is modulated through the Wnt pathway to increase or decrease the ability of hair follicles to form.
  • a method for the treatment of neurodegenerative disorders comprising inhibiting the degradation of ⁇ -catenin via decreasing intracellular levels of Smad7 is disclosed.
  • Smad7 levels are decreased via introduction of siRNA into Smad7 expressing cells.
  • FIG. 1 Generation of gene-switch Smad7 transgenic mice.
  • This inducible transgenic system consists of a transactivator line (GLp65) (Cao et al., 2002a; Lu et al., 2004) and a target line (tata. Smad7 ) .
  • the K5 vector was used to target the GLp65 transactivator to the basal layer of the epidermis and the ORS of the hair follicle, including epidermal stem cells (Arin et al., 2001).
  • the target transgene (tata.Smad7) consists of a GAL4 UAS enhancer upstream of a tata minimal promoter (Cao et al., 2002a; Lu et al., 2004) and cDNA encoding mouse Smad7 with a Flag tag (He et al., 2002).
  • RU486 is used to regulate transgene expression in bigenic mice (GLp65/tata. Smad7) .
  • To induce Smad7 transgene expression in utero, RU486 (dissolved in sesame oil) was subcutaneously injected (100 ⁇ g/kg) daily into pregnant females.
  • RU486 application to P6 skin or daily injection of RU486 (RU) to pregnant females from gestation day 14.5 (E14.5) to Pl. *: p ⁇ 0.01.
  • B Western analysis revealed increased Smad7 protein upon acute (RU 12h) and sustained (RU E14.5-P1) RU486 treatment.
  • C, D In situ hybridization using an antisense probe specific for Smad7 on E15.5 (C) and P ⁇ (E) skins 12 h after RU486 treatment showing that Smad7 transgene was expressed in the epidermis and hair follicles.
  • HF hair follicle.
  • the bar in the first panel of C represents 50 ⁇ m for both micrographs in C.
  • the bar in each panel of D represents 50 ⁇ m.
  • FIG. 3 Smad7 induction perturbed hair follicle development and differentiation.
  • Smad7 expression was induced in a sustained manner beginning either on embryonic day (E) ElO.5 or on E14.5, the effect on bigenic mouse skin from E16.5 throughout Pl was essentially identical. In both cases, histological examination showed that hair follicle morphogenesis was delayed in bigenic skin in comparison with control skin.
  • A H&E staining of El ⁇ .5, E18.5, and Pl skin that was exposed to daily RU486 treatment in utero beginning on E14.5. Arrows point to representative hair follicles in different developmental stages (S).
  • arrows in Smad7 transgenic skin point to the sebaceous glands.
  • arrows in control skin indicate examples of normal staining patterns.
  • arrows in Smad7 transgenic skin point out aberrant staining patterns.
  • Dark staining in Smad7 transgenic follicles represents irregular melanin deposits.
  • PlO control skin hair follicles had each formed a well-differentiated hair shaft.
  • Smad7 transgenic follicles were much shorter and disoriented with prominent sebaceous glands and clustered melanin.
  • FIG. 4 Smad7 induction perturbed hair follicle regeneration but accelerated sebaceous gland morphogenesis. Control (monogenic or wildtype) and gene- switch-Smad7 skins (Smad7) were treated with RU486 as indicated.
  • the left panel is from a control mouse skin with telogen follicles.
  • the bar in the first panel represents 100 ⁇ m for micrographs in B.
  • C Immuno-fluorescence using an antibody against adipophilin. The counterstain is K14. Representative sebocyte clusters and sebaceous glands are pointed out by arrows in control skins and arrows in Smad7 skins. The bar in the first panel represents 75 ⁇ m for all micrographs in C.
  • D qRT-PCR of indian hedgehog (Ihh) expression in control and Smad7 transgenic skin treated with RU486 acutely (12h) or chronically (1Od). *: p ⁇ 0.01.
  • E Immunohistochemistry using an antibody against Ihh. Arrows point to sebaceous glands in P7 control and Smad7 transgenic skin, both of which had been treated with
  • Smad7 transgene expression is induced by topical application of RU486 (RU) .
  • the number of cells positive for pSmad2 nuclear staining in control epidermis was 160 ⁇ 23.5/mm epidermis, but was reduced to 95.0+13.7/mm epidermis (p ⁇ 0.001).
  • the number of cells positive for pSmad2 nuclear staining in control hair follicles was 24.0 ⁇ 3.5/hair follicle, but was reduced to 16.4+2.2/hair follicle after acute Smad7 transgene induction (p ⁇ 0.05).
  • Sustained Smad7 transgene induction for 12 days (d) resulted in a similar reduction in pSmad2 staining in the epidermis and hair follicles.
  • the number of pSmadl/5/8 positive cells in hair follicles did not significantly changed after Smad7 transgene induction.
  • the number of pSmadl/5/8 positive cells was 18.3 ⁇ 2.4/hair follicle in control skin, and 16.0 ⁇ 2.9/hair follicle after acute Smad7 transgene induction (p>0.05).
  • the number of pSmadl/5/8 positive cells was 20.3 ⁇ 3.5/hair follicle in Smad7 skin, in comparison with 22.6 ⁇ 4.6/hair follicle in control skin (p>0.05).
  • a K14 antibody was used as a control. The bar represents 50 ⁇ m for all sections.
  • Lef-1 expression was predominantly expressed in hair buds (arrows) in E15.5 skin (left), which was not significantly altered after sustained Smad7 transgene induction (RU 5d) .
  • Lefl expression was located in hair matrix cells of P6 skin (right) .
  • Acute Smad7 transgene induction (RU 12h) did not significantly affect Lef-1 expression.
  • Prolonged Smad7 transgene induction in adults reduced Lef-1 expression when the hair follicles were degenerated.
  • the bar in the first panel of (C) represents 50 ⁇ m for all sections.
  • FIG. 7 Smad7 transgenic skin exhibited reduction in ⁇ -catenin staining and in ⁇ -catenin-mediated Wnt signaling.
  • A Immunohistochemistry of ⁇ -catenin on P6 control and Smad7 skin. The areas of the epidermis (Epi) and hair follicles (HF) marked by boxes are enlarged to the right of the main panels. Hematoxylin was used as a counterstain. The bar represents 75 ⁇ m for both main panels in A.
  • the bar in the lower panel represents 50 ⁇ m for both sections in B.
  • C Whole mount ⁇ -gal staining on E15.5 TOPGAL+/Smad7- and TOPGAL+/Smad7+ embryos. The embryos were taken after 5 days of in utero RU486 treatment. No difference in staining intensity in the bone and cartilage (open arrows) were observed between the two genotypes. Representative areas from both embryos are enlarged to show positive staining in pelage hair follicles.
  • D Microscopic appearance of the embryonic skin shown in C. The dotted lines denote developing hair germs.
  • FIG. 8 Smad7 transgene expression triggered ⁇ - catenin degradation.
  • A Western analysis of ⁇ -catenin in control and bigenic skin (Smad7) treated with RU486 either acutely (RU 12 h) or chronically (RU 10 d) .
  • E- cadherin is a loading control.
  • B Western analysis of ⁇ -catenin on cultured keratinocytes in the absence and presence of MG132. A K14 antibody was used as a loading control.
  • C Co-immunoprecipitation of Smad7 and ⁇ - catenin in lysates prepared from cultured gene-switch- Smad7 keratinocytes in the presence of MG132.
  • the antibody for Flag which recognizes the Flag-Smad7 transgene, or for ⁇ -catenin, was used for immunoprecipitation and a mouse IgG was used as a negative control in immunoprecipitation.
  • MG132 was used as a positive control for western blotting using an antibody against ⁇ -catenin or Smad7.
  • D Ubiquitin assay for ⁇ -catenin protein. 293T cells were transfected with expression constructs for His-tagged ⁇ - catenin, Flag-Smurf2, Flag-Smurf2C716S, and Flag-Smad7, in combinations as indicated. Western analysis was performed to detect ubiquitinated ⁇ -catenin using an anti-HA antibody (upper) and to detect total levels of ⁇ - catenin in cells using an anti-His antibody (bottom) .
  • E Western analysis for ⁇ -catenin protein. 293T cells were transfected with expression constructs in combinations as indicated. His- ⁇ -catenin level was detected by anti-His antibody, and Smurf2 and Smad7 levels were detected by anti-Flag antibody. A GFP antibody was used as a loading control since a GFP expression vector was included in all transfection experiments .
  • FIG. 9 Low level of Smad7 overexpression did not alter Smad signaling but inhibited Wnt/ ⁇ -catenin signaling.
  • B Immunohistochemistry (pSmad2, upper panels) and double immunofluorescence (pSmadl/5/8, lower panel) show that the numbers of cells positive for nuclear pSmad2 or pSmadl/5/8 in P28 skin were comparable between control and K5.Smad7 skin. Hematoxylin was used as a counterstain for immunohistochemistry. A K14 antibody was used for counterstain with pSmadl/5/8.
  • the bar in the top left panel represents 50 ⁇ m for both immunohistochemistry panels and the bar in the lower left panel represents 50 ⁇ m for both immunofluorescence panels.
  • C ⁇ -gal staining on the anagen phase (P30) hair follicles in TOPGAL skin but was abolished in
  • TOPGAL/K5.Smad7 hair follicles The bar in the left panel represents 50 ⁇ m.
  • FIG. 10 Smurf2 overexpression exacerbated Smad7- mediated hair follicle abnormalities and ⁇ -catenin degradation.
  • A Histology of Pl mouse skin. The bar in the first panel represents 100 ⁇ m for all four sections. Developmental stages (S) of hair follicles in each section are indicated at the arrows.
  • B immunofluorescence (IF) using an adipophilin antibody on Pl mouse skin shows accelerated sebocyte (arrows) differentiation in K5. Smad7 and K5. Smad7/K5. Smurf2 transgenic skin. The counterstain was K14. The bar in the first panel represents 100 ⁇ m for all sections in B.
  • C Gross appearance of P25 littermates.
  • E Western analysis of ⁇ -catenin, Smad7, and Smurf2 protein levels in neonatal transgenic and control skins. A K14 antibody was used as a loading control. Quantified bars with standard deviations were averaged from two samples in each group shown on the western blots.
  • F Co- immunoprecipitation of ⁇ -catenin, Smad7, and Smurf2. Lysates were prepared from cultured keratinocytes treated with MG132 from mice with the indicated genotypes. Immunoprecipitatation was performed using a ⁇ -catenin antibody. Western analyses were performed using an anti- Flag antibody that recognizes both Smad7 and Smurf2 transgenic proteins. A mouse IgG was used as a negative control in immunoprecipitation.
  • FIG. 11 Endogenous Smad7 is involved in ⁇ - catenin degradation.
  • A Histology of 2-month-old (2 mos) and 2-year-old (2 yrs) old mouse skin showing increased sizes and numbers of sebaceous glands and degenerated hair follicles in aged skin. The bar represents 100 ⁇ m for both sections.
  • B Quantitative RT- PCR of Smad7 mRNA in 2 mos and 2 yrs mouse skin. *p ⁇ 0.01.
  • FIG. 1 Smad7 knockdown assay using siRNA specific for Smad7.
  • Western blot (left panel) includes two pairs of mock- and siRNA-transfected wildtype keratinocytes . A K14 antibody was used as a loading control. The intensity of ⁇ - catenin and Smad7 bands in each corresponding lane were further normalized to K14 (right panel) , in which the ⁇ - catenin and Smad7 levels in mock-transfected cells (lane 1 and 3) were both set arbitrarily as 100%.
  • E ⁇ -gal staining for cultured TOPGAL hair follicle cells showing increased ⁇ -GAL positive cell clusters in Smad7 siRNA transfected cells as compared to those in mock- transfected control.
  • Aged skin which often has degenerated hair follicles and enlarged sebaceous glands, over-expresses a TGF ⁇ antagonist, Smad7.
  • Smad7 a TGF ⁇ antagonist
  • transgenic mice that have a Smad7 expression level comparable to aged skin were generated.
  • Biochemical experiments demonstrated that Smad7 overexpression shifts the skin development program from forming hair follicles to sebaceous glands.
  • Smad7 also inhibits Wnt signaling by targeting ⁇ -catenin degradation via recruiting an E3 ligase, Smurf2 to the Smad7/ ⁇ -catenin complex, thereby revealing an alternative ⁇ -catenin degradation pathway.
  • screening methods are provided to identify agents which modulate Smad7- ⁇ -catenin interactions which in turn modulate Wnt signaling.
  • agents should have efficacy for the treatment of a variety of disorders including cancer, neurodegeneration, Alzheimer's, and some of the deleterious side effects associated with aging.
  • Exemplary methods entail the use of cells lines expressing Smad7 and/or whole animal transgenic models wherein Smad7 is over or under expressed.
  • Such transgenic animal models may also express other proteins in the ⁇ -catenin/Wnt signaling cascade, e.g., other Smads and Smurf2.
  • the primary keratinocyte culture system described herein may also be utilized.
  • Smad7 levels For treating cancers with increased ⁇ -catenin signaling, increasing Smad7 levels (either by gene/protein delivery, or via induction with therapeutic agents), should result in enhanced ⁇ -catenin degradation and thereby abrogate Wnt signaling.
  • An exemplary therapeutic agent which is known to increase endogenous Smad7 levels is Halofuginone.
  • agents useful for down- regulating Smad7 expression include, without limitation, siRNA which targets Smad7 RNA.
  • Agents identified using the methods of the invention may be used alone for the treatment of the particular disease in question or may be combined with other agents known to have efficacy for the treatment of the particular disease, (e.g., chemotherapeutic agents if the aberrant ⁇ -catenin signaling disorder is cancer or Aricept® if the disorder is Alzheimer's).
  • agents known to have efficacy for the treatment of the particular disease e.g., chemotherapeutic agents if the aberrant ⁇ -catenin signaling disorder is cancer or Aricept® if the disorder is Alzheimer's.
  • ⁇ -catenin signaling disorder refers to disorders which include, without limitation, cancer, for example, cancer of the colon, brain, lung and skin, neurodegenerative disorders, retinal degradation, bone density defects, and Alzheimer's disease.
  • Wnt signaling refers to a signaling cascade involving the Wnt protein binding to its cell surface receptors resulting in an inhibition of the protein complex that normally degrades ⁇ -catenin. This Wnt-mediated inhibition results in ⁇ -catenin accumulation in the cytoplasm and subsequent nuclear localization where ⁇ -catenin binds with TCF/LEF transcription factors to regulate Wnt target genes. Wnt signaling via ⁇ - catenin transcriptional activation is essential for normal organ development and many physiological functions. However, uncontrolled Wnt signaling can result in hyperproliferation, loss of contact inhibition, and cancer.
  • transgenic animal is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus.
  • the term "transgenic animal” is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule. This molecule may be specifically targeted to defined genetic locus, be randomly integrated within a chromosome, or it may be extra-chromosomally replicating DNA.
  • germ cell line transgenic animal refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring in fact, possess some or all of that alteration or genetic information, then they, too, are transgenic animals.
  • the alteration or genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient.
  • the altered or introduced gene may be expressed differently than the native gene.
  • the altered Smad7 gene generally should not fully encode the same Smad7 protein native to the host animal and its expression product should be altered to a minor or great degree, or absent altogether. However, it is conceivable that a more modestly modified Smad7 gene or one which exhibits inducible expression will fall within the compass of the present invention if it is a specific alteration.
  • the DNA used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof.
  • ES embryonal stem cell
  • ES cells may be obtained from pre-implantation embryos cultured in vitro.
  • Trans- genes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirus-mediated transduction.
  • the resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
  • the introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
  • nucleic acid or a “nucleic acid molecule” as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form.
  • a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5 1 to 3 ' direction.
  • isolated nucleic acid is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated.
  • an "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
  • isolated nucleic acid refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues) .
  • An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • the present invention also includes active portions, fragments, derivatives and functional or non functional mimetics of Smad7 polypeptides or proteins of the invention.
  • An "active portion” of Smad7 polypeptide means a peptide that is less than the full length Smad7 polypeptide, but which retains measurable biological activity.
  • a “fragment” or “portion” of the Smad7 polypeptide means a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to thirteen contiguous amino acids and, most preferably, at least about twenty to thirty or more contiguous amino acids.
  • Smad7 polypeptide sequence Fragments of the Smad7 polypeptide sequence, antigenic determinants, viral antigens or epitopes are useful for eliciting immune responses to a portion of the Smad7 amino acid sequence.
  • a "derivative" of the Smad7 polypeptide or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, and may or may not alter the essential activity of original the Smad7 polypeptide.
  • the Smad7 polypeptide or protein of the invention includes any analogue, fragment, derivative or mutant which is derived from a Smad7 polypeptide and which retains at least one property or other characteristic of the Smad7 polypeptide.
  • Different "variants" of the Smad7 polypeptide exist in nature. These variants may be alleles characterized by differences in the nucleotide sequences of the gene coding for the protein, or may involve different RNA processing or post translational modifications. The skilled person can produce variants having single or multiple amino acid substitutions, deletions, additions or replacements.
  • variants may include inter alia: (a) variants in which one or more amino acids residues are substituted with conservative or non conservative amino acids, (b) variants in which one or more amino acids are added to the Smad7 polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the Smad7 polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the Smad7 polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like.
  • Smad7 polypeptides of the invention include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non conserved positions. In another embodiment, amino acid residues at non conserved positions are substituted with conservative or non conservative residues.
  • the techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques are known to the person having ordinary skill in the art.
  • modulate refers increasing or decreasing.
  • modulate refers to the ability of a compound or test agent to interfere with or strengthen the interaction between interacting protein partners within a complex of the present invention.
  • modulating the Smad7- ⁇ -catenin complex means that an agent or compound inhibits or enhances the binding of these proteins in a complex, and the protein complex may contain other proteins in addition to Smad7 and ⁇ -catenin. Modulating can also refer to increasing or decreasing hair follicle formation.
  • a “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element .
  • tag refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the detection or isolation, to that sequence.
  • a homopolymer nucleic acid sequence or a nucleic acid sequence complementary to a capture oligonucleotide may be added to a primer or probe sequence to facilitate the subsequent isolation of an extension product or hybridized product.
  • histidine residues may be added to either the amino- or carboxy-terminus of a protein to facilitate protein isolation by chelating metal chromatography.
  • amino acid sequences, peptides, proteins or fusion partners representing epitopes or binding determinants reactive with specific antibody molecules or other molecules (e.g., flag epitope, c-myc epitope, transmembrane epitope of the influenza A virus hemaglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S-transferase, and the like) may be added to proteins to facilitate protein isolation by procedures such as affinity or immunoaffinity chromatography.
  • Chemical tag moieties include such molecules as biotin, which may be added to either nucleic acids or proteins and facilitates isolation or detection by interaction with avidin reagents, and the like. Numerous other tag moieties are known to, and can be envisioned by, the trained artisan, and are contemplated to be within the scope of this definition.
  • transform shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion and the like.
  • the introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism.
  • the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid.
  • the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism.
  • the introduced nucleic acid may exist in the recipient cell or host organism only transiently.
  • a “clone” or “clonal cell population” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell or cell population that is capable of stable growth in vitro for many generations.
  • Smad7 and ⁇ -catenin are proteins implicated in the etiology of many diseases and disorders, methods for identifying agents that modulate the Smad7- ⁇ -catenin interaction should result in the generation of efficacious therapeutic agents for the treatment of diseases and disorders involving these proteins.
  • Candidate inhibitor compounds of this interaction may be based on modeling the three-dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics.
  • the Smad7 and ⁇ -catenin polypeptides or fragments employed in drug screening assays may either be free in solution, affixed to a solid support, or within a cell.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptides or fragments, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may determine, for example, formation of complexes between Smad-7- ⁇ -catenin polypeptides or fragments and the agent being tested, or examine the degree to which the formation of a complex between Smad-7 and ⁇ -catenin polypeptides or fragments and a known substrate is interfered with by the agent being tested.
  • Another technique for drug screening provides high- throughput screening for compounds having suitable binding affinity to Smad-7 or ⁇ -catenin polypeptides and is described in detail in Geysen, PCT published application WO 84/03564. Briefly stated, large numbers of different, small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the polypeptides and washed. Bound polypeptides are then detected by methods well known in the art.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo.
  • drugs e.g., Hodgson, (1991) Bio/Technology 9:19-21; U.S. Patent 5,800,998; and U.S. Patent 5,891,628.
  • the peptide compound can then be used as a lead molecule for designing organic molecules or peptide mimetics (Huber et al., Curr. Med. Chem.
  • the compounds designed or selected based on rational drug design or screening can be tested for their ability to modulate (i.e., interfere with or strengthen) the interaction between the interacting partners within a complex of the present invention comprising Smad-7 and ⁇ - catenin.
  • the compounds can also be further tested for their ability to modulate (inhibit or enhance) cellular functions such as carcinogenesis, cell survival, as well as their effectiveness in treating disorders and diseases associated with aberrant ⁇ -catenin signaling.
  • the compounds can be manufactured for further experimental studies to validate them for therapeutic use.
  • the three-dimensional structure of a protein of interest or, for example, of the protein- substrate complex is solved by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches.
  • An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., (1990) Science 249:527-533).
  • peptides e.g., a Smad7 polypeptide
  • alanine scan Wells, (1991) Meth. Enzym. 202:390-411). In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined.
  • Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide. It is also possible to isolate a target-specific antibody, selected by a functional assay, and then to solve its crystal structure. In principle, this approach yields a pharmacophore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original molecule. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides.
  • anti-ids anti-idiotypic antibodies
  • Selected peptides would then act as the pharmacophore.
  • drugs which have, e.g., improved Smad7 polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of the Smad-7 and ⁇ -catenin polypeptide interaction.
  • sufficient amounts of the polypeptide may be made available to perform such analytical studies as x- ray crystallography.
  • the knowledge of the Smad7 and ⁇ -catenin protein sequences will guide those employing computer modeling techniques in place of, or in addition to, x-ray crystallography which permits prediction of structural and functional relationships.
  • compositions useful for treatment in diseases and disorders associated with aberrant ⁇ -catenin signaling such as cancer and hair formation.
  • compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such as a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • administration is preferably in a prophylactically effective amount or a therapeutically effective amount (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • the Smad7 protein, active fragments of Smad7, and agents that modulate Smad7 can be used to modulate the formation of hair follicles.
  • Adenoviral vector systems are of particular utility in the methods of the present invention because they provide several unique features, including, but not limited to: i) the ability to infect all human cells at more than 95% efficiency, making lengthy selection periods unnecessary; ii) the ability to remain episomal and rarely integrate into the human genome; and iii) the generation of replication defective adenoviruses (such as, e.g., the dl7001 adenoviral vector), from which the El gene region (the transforming region) and the E3 gene region (the immune modulatory region) have, for example, been deleted.
  • the high infection efficiency achieved with adenoviral vectors is not generally observed using other gene transfer techniques.
  • the recombinant adenoviruses of the present invention are non-lytic and do not induce apparent phenotypic changes in infected cells.
  • Adenovirus-mediated gene expression in keratinocytes or fibroblasts remains stable in vitro for at least 2 to 6 weeks, depending on the rate of cellular proliferation.
  • gene expression in human skin grafted to SCID mice lasts for at least 2 weeks.
  • an adenoviral vector of the present invention is a type 5 adenovirus having an El deletion, which comprises Smad7 nucleic acid sequences under the control of a cytomegalovirus promoter.
  • an expression construct may further comprise regulatory elements which serve to drive expression in a particular cell or tissue type.
  • regulatory elements are known to those of skill in the art and discussed in depth in Sambrook et al. (1989) and Ausubel et al. (1992).
  • tissue specific regulatory elements in the expression constructs of the present invention provides for at least partial tissue tropism for the expression of Smad7 or functional fragments thereof.
  • adenoviral vectors and methods for producing these vectors have been described in detail in a number of references, patents, and patent applications, including: Mitani and Kubo (2002, Curr Gene Ther. 2 (2) : 135-44) ; Olmsted-Davis et al. (2002, Hum Gene Ther. 13(11) : 1337-47) ; Reynolds et al. (2001, Nat Biotechnol . 19(9) :838-42) ; U.S. Patent Nos.
  • Smad7 proteins or active fragments thereof are based on nanoparticles, such as those described in U.S. Patent Numbers 5,843,509, 6,117,454, and 6,649,192.
  • the gene-switch-Smad7 transgenic mice were generated using the gene-switch transgenic system as previously described (Lu et al., 2004).
  • the activator (GLp65) line was generated previously (Lu et al., 2004).
  • the target line (tata.Smad7) was generated by inserting a full- length mouse Smad7 cDNA with a 5' Flag epitope to a minimal tata promoter with GAL4 binding sites (Lu et al., 2004).
  • the generation of K5.Smad7 mice was described in our previous study (He et al., 2002). The offspring showed the same phenotypes throughout generations. To generate K5.
  • Smurf2 transgenic mice the human Smurf2 cDNA with a Flag epitope (Lin et al., 2000) was inserted into the K5 promoter and microinjected into the pronuclei of mouse embryos obtained from ICR female mice mated to B2D6 male mice.
  • PCR genotyping was performed using primers specific for a fragment between 468 bp and 950 bp in the full-length human Smurf2 cDNA (forward: 5'CTCGGTTGTGTTCGTCTTCTS' , reverse: 5'TGGTAGGTCTGGAGGAGTATS' ) .
  • In situ hybridization for Smad7 and Lefl was performed using digoxigenin-11-dUTP labeled probes as previously described (He et al., 2001; Qiao et al., 2005) .
  • the cDNA fragment was first amplified by reverse transcriptase PCR using primers specific for mouse Smad7 (accession* NM_008543, forward: 5'GCTCACGCACTCGGTGCTCAS', reverse: 5' CCAGGCTCCAGAAGAAGTTG3' ) cDNA sequence and then subcloned into the pGEM-T easy vector (Promega, Madison, WI) .
  • the plasmid was further linearized and purified to create templates for the synthesis of in situ hybridization probes.
  • IF was performed using the same ⁇ -catenin antibody and the sections were treated with 2 mg/ml BSA and 0.1% Triton at 4 0 C overnight (Van Mater et al., 2003), conditions which partially destroy membrane-associated epitopes but increase permeability to the nucleus.
  • Western analysis Western analysis:
  • Protein extraction was performed as previously described (He et al., 2002).
  • the primary antibodies used included Smad7 (He et al., 2002), E-cadherin and ⁇ - catenin (BD Biosciences, San Jose, CA) , Flag (Sigma) and K14 (Fitzgerald) .
  • Gray scale images were obtained and quantified using Odyssey v.1.2 software (LI-COR Biosciences, Lincoln, NE) .
  • Isolation and culture of murine hair follicle cells were performed following an established protocol with minor modifications (Rogers et al., 1987). Briefly, neonatal mouse dermis separated from the epidermis was digested with 0.35% type I collagenase (Worthington Biochemical, Lakewood, NJ) at 37°C for 30 minutes, filtered through a cell strainer (BD Biosciences, San Jose, CA), and then centrifuged at 800 rpm for 5 minutes. The pellet was re- suspended in 1.25 mM Ca 2+ EMEM (Cambrex, Walkersville, MD) and then centrifuged at 300 rpm for 5 minutes.
  • type I collagenase Worthington Biochemical, Lakewood, NJ
  • cell strainer BD Biosciences, San Jose, CA
  • the pellet was re- suspended in 1.25 mM Ca 2+ EMEM (Cambrex, Walkersville, MD) and then centrifuged at 300 rpm for 5 minutes.
  • the pellet was re-suspended in 8 ml 1.25 mM Ca 2+ EMEM/9% Ficoll (1:1) and overlaid on 5 ml 9% Ficoll for centrifugation at 400 rpm.
  • the precipitated hair follicles were then washed in 1.25 mM Ca 2+ EMEM for 3 times and plated in 0.05 mM Ca 2+ EMEM on type I collage- coated plates. The medium was then replaced with 1.25 mM Ca 2+ EMEM 24 hours later.
  • ⁇ -gal staining for whole mount, tissue sections and cells was performed using a ⁇ -gal staining kit (Invitrogen, Calsbad, CA), following the manufacturer's instruction.
  • keratinocytes were cultured as described above. When cells reached subconfluency, RU486 was added at a final concentration of 10 ⁇ 7 M to induce Smad7 overexpression. Twelve hours later, the cells were treated with or without MG132 at a final concentration of 20 ⁇ M for 4 hours prior to harvesting. Subsequently, the cells were lysed in Ix CST cell lysis buffer (Cell Signaling Technology) containing freshly added protease inhibitors that include AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstatin A (Sigma) .
  • Ix CST cell lysis buffer Cell Signaling Technology
  • HEK293T cells were transfected with expression plasmids for His-tagged ⁇ -catenin, Flag-Smurf2, Flag- Smad7, and HA-ubiquitin, as specified in Figure 5D.
  • protein was extracted from transfected cells and subjected to western blotting. His- ⁇ -catenin level was detected by an anti-His antibody (Santa Cruz) , Smurf2 and Smad7 levels were detected by an anti-Flag antibody.
  • Smad7 siRNA oligonucleotides were designed to recognize mouse Smad7 (sense: (SEQ ID NO: 1) 5'GAGGCTGTGTTGCTGTGAAS', (SEQ ID NO: 2) anti-sense:
  • Smad7 transgene induction perturbs embryonic and postnatal hair follicle morphogenesis.
  • the gene-switch-Smad7 mice were generated using the keratin 5 (K5) promoter, in which Smad7 transgene expression can be induced by RU486 in the epidermis and hair follicles (Figure 1) .
  • This inducible system consists of a transactivator line (GLp65) (Lu et al., 2004) and a target line (tata. Smad7) .
  • GLp65 transactivator line
  • target line tata. Smad7
  • Smad7 transgene expression levels were RU486 dose-dependent and correlated with phenotype severity.
  • the data presented here were from the doses of RU486 that induced Smad7 expression levels similar to those when endogenous Smad7 is overexpressed, e.g., in aged skin (Quan et al., 2002) and in cancer lesions (He et al., 2001).
  • Smad7 transgene expression was induced in embryonic skin, beginning on ElO.5, when a single epithelial layer begins transitioning into the stratified epidermis or on E14.5, when hair follicle development is initiated.
  • Skin samples were excised at birth (day one postpartum, Pl) after daily RU486 treatments to verify sustained Smad7 transgene induction.
  • Acute Smad7 transgene induction was assessed by topical RU486 application (20 ⁇ g/mouse) to the skin of P6 mice for 12 hours.
  • Smad7 transgene expression was induced during the telogen to anagen transition of the hair follicles.
  • the dorsal skins of bigenic and monogenic control mice were shaved on postnatal day 52 (P52), when the hair follicles enter into a long period of telogen phase (Paus, 1998) .
  • a depilatory cream, Nair ® was applied to the shaved skin to reinitiate the anagen phase (Paus et al., 1990).
  • the depilated skin was then topically treated with 20 ⁇ g of RU486 3 times/week for 2 weeks.
  • the skin samples at different time points were collected from the RU486 treated area by 3-mm punch biopsy.
  • Smad7 transgene level is sufficient to block Smad signaling
  • phospho- Smad2 (pSmad2) and pSmadl/5/8 were examined in the skin, these proteins represent activation of TGF ⁇ /activin and BMP, respectively.
  • nuclear pSmad2 staining was reduced in the epidermis and hair follicles of Smad7 transgene skin, whereas nuclear staining for pSmadl/5/8 was reduced in Smad7 epidermis but not the hair follicles ( Figure 5) .
  • expression levels of several Smad target genes were reduced in Smad7 transgenic skin ( Figure 6) .
  • Smad7 overexpression resulted in down-regulation of ⁇ - catenin-mediated signaling.
  • ⁇ -catenin was observed in the nucleus of the germ cells of E15.5 control hair follicles ( Figure 7B) and matrix cells of P6 control follicles, but ⁇ -catenin was almost undetectable in these cell populations in Smad7 transgenic follicles ( Figure 7B) .
  • Smad7 bigenic mice were bred with TOPGAL reporter mice in which LacZ gene expression is induced by Wnt/ ⁇ -catenin signaling (DasGupta et al., 2002). Skin samples were taken from E15.5 embryos which had received daily RU486 exposure since ElO.5. Whole mount ⁇ -galactosidase ( ⁇ -GAL) staining on E15.5 embryos yielded numerous blue spots with a well-organized pattern in the trunk skin of TOPGAL+/Smad7- embryos, highlighting the pelage hair follicles ( Figure 7C) .
  • ⁇ -catenin protein levels were examined in cultured control and transgenic keratinocytes treated with and without MG132, an ubiquitin-proteasome inhibitor. Keratinocytes were treated for 12h with 10 "7 M RU486 to induce Smad7 transgene expression (Smad7 transgenic) or with vehicle alone (control) , and then were treated for 4h with or without 20 ⁇ M MG132.
  • the Smad7 protein was examined to determine whether it is physically associated with ⁇ -catenin. Immunoprecipitation with either a Flag antibody, which recognizes the Flag-Smad7 transgene, or the ⁇ -catenin antibody, showed that Smad7 transgenic protein co- precipitated with ⁇ -catenin in the presence of MG132 in cultured keratinocytes ( Figure 8C) , but did not associate with ⁇ -transducin repeat-containing protein ( ⁇ -TrCP), which normally initiates ⁇ -catenin degradation (Latres et al., 1999).
  • ⁇ -TrCP ⁇ -transducin repeat-containing protein
  • a ⁇ -catenin ubiquitination assay was performed to examine whether Smurf2, a ubiquitin E3 ligase that is recruited by Smad7 (Kavsak et al., 2000; Izzi and Attisano, 2004), could induce ⁇ -catenin ubiquitination.
  • This assay involved transfection of a
  • His-tagged ⁇ -catenin His- ⁇ -catenin
  • HEF293T human embryonic kidney cell line
  • additional expression vectors including Flag-tagged Smad7 (Flag-Smad7) , Flag- tagged Smurf2 (Flag-Smurf2) , HA-tagged ubiquitin, and a Smurf2C716A mutant that is a catalytically inactive.
  • Transfected cells were treated with 20 ⁇ M of MG132 for 4 h. His- ⁇ -catenin and its ubiquitinated products were immobilized on Ni-NTA beads, eluted under denaturing conditions, and subjected to western analyses. An antibody specific for HA-ubiquitin was used to detect ubiquitinated ⁇ -catenin. Ubiquitinated ⁇ -catenin was not detected without the addition of exogenous ubiquitin ( Figure 8D, lane 1) , but was detected upon addition of ubiquitin to the lysate ( Figure 8D, lane 2) .
  • Smad7 line was bred with TOPGAL mice, Wnt reporter activity was completely abolished in the anagen hair follicles of TOPGAL/K5. Smad7 skin (Figure 9C).
  • Smad7-induced ⁇ -catenin degradation and subsequent Wnt signaling blockade appeared to be independent of its role in blocking TGF ⁇ /BMP signaling.
  • Smurf2 transgenic mice were generated using the K5 promoter (K5.Smurf2). K5.Smurf2 skin did not show obvious abnormalities in the skin (Figure 10A-10D) or ⁇ -catenin degradation (Figure 10E) .
  • K5.Smurf2 mice were bred with the above K5.Smad7 transgenic line. In neonates, the morphology of K5.Smurf2 skin was indistinguishable from non-transgenic control littermates, i.e., most hair follicles in control and K5.Smurf2 skin are in Stages 3 through 5 ( Figure
  • Smad7 skin exhibit aberrant hair follicle cycling, i.e., a mix of anagen, catagen, and telogen follicles appearing in the same region, and sebaceous glands of similar size among hair follicles at different phases (Figure 10D) .
  • K5. Smad7/Smurf2 littermates exhibited degenerated hair follicles, resulting in papillary cysts and canals ( Figure 10D) .
  • Sebaceous glands were more hypertrophic in K5. Smad7/Smurf2 skin in comparison with those in K5.Smad7 skin ( Figure 10D) .
  • immunoprecipitation assays were performed using an antibody against ⁇ -catenin followed by western analysis using an antibody against the Flag tag fused to the Smad7 and the Smurf2 transgenes.
  • Keratinocytes were isolated from control, K5.Smad7, K5. Smurf2, and K5. Smad7/K5. Smurf2 skins and treated with 20 ⁇ M MG132 for 4 h.
  • the amount of Smad7 that co- precipitated with ⁇ -catenin from lysates of Smad7 transgenic keratinocytes was comparable with the amount from lysates of Smad7/Smurf2 keratinocytes ( Figure 10F) .
  • the Smurf2 protein was co-precipitated with ⁇ -catenin in lysates from Smad7/Smurf2 keratinocytes, but was barely detectable in Smurf2 transgenic keratinocytes, even though these cells contained an amount of Smurf2 protein similar to that in Smad7/Smurf2 transgenic cells ( Figure 10F) .
  • This result indicates that Smurf2 did not bind independently to ⁇ -catenin, but was recruited by Smad7 to the ⁇ -catenin complex. Endogenous Smad7 was involved in ⁇ -catenin degradation and Wnt signaling inhibition.
  • Smad7 siRNA was transfected into hair follicle cells isolated from TOPGAL reporter mice. Similar to that seen in vivo (Figure 7E) , ⁇ -GAL staining was positive in precortex hair follicle cells ( Figure HE) . In mock-transfected TOPGAL follicle cells, -20% of the hair follicles contained ⁇ -GAL positive cells ( Figure HE) . In contrast, in Smad7 siRNA transfected TOPGAL follicle cells, -80% of hair follicles possessed ⁇ -GAL positive cells ( Figure HE) . These data indicate that endogenous Smad7 inhibits Wnt signaling.
  • Smad7 induces ⁇ -catenin degradation independent of its effect on blocking TGF ⁇ /BMP signaling.
  • Smad7-increased ⁇ -catenin degradation contributes to Wnt signaling inhibition in physiological and pathological conditions .
  • ⁇ -catenin protein is expressed at a high level in keratinocytes in both interfollicular epidermis and hair follicles
  • Wnt signaling activity can only be detected in certain cell populations of hair follicles (Kobielak et al., 2003; Lowry et al., 2005; Van Mater et al., 2003), and is crucial for development and differentiation of hair follicles, but not the epidermis (Huelsken et al., 2001).
  • Smad7-initiated ⁇ -catenin degradation Wnt/ ⁇ -catenin signaling in hair follicles was reduced.
  • the degree of ⁇ -catenin degradation correlates with the severity of hair follicle defects. Furthermore, the aged mouse skin, which expresses endogenous Smad7 at a level similar to that in gene-switch-Smad7 skin, also shows significant reduction of ⁇ -catenin protein and histopathological changes strikingly similar to the skin with sustained Smad7 transgene induction. Therefore, under pathological conditions, Smad7-mediated ⁇ -catenin degradation contributes, at least in part, to defects in hair follicle morphogenesis.
  • Endogenous Smad7 is expressed at a very low level in keratinocytes (He et al., 2001), but a marked increase in ⁇ -catenin protein and enhanced Wnt signaling after knocking down endogenous Smad7 in keratinocytes was observed.
  • Accelerated sebaceous gland development induced by Smad7 overexpression represents a shifted balance between ⁇ - catenin/Wnt signaling and hedgehog signaling.
  • Botchkarev, V.A. Li, J., Danielian, P. S . , McMahon, J.A. , Lewis, P.M., Paus, R., and McMahon, A. P. (1998). Curr.

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Abstract

La présente invention concerne des composés et des procédés pour la modulation des interactions β-caténine/Smad-7. De tels composés doivent être efficaces dans le traitement de troubles associés à une signalisation par la β-caténine aberrante.
PCT/US2007/077445 2006-09-01 2007-08-31 Composés et procédés pour moduler les interactions β-caténine/smad-7 Ceased WO2008105925A2 (fr)

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US10350265B2 (en) 2010-09-22 2019-07-16 The Regents Of The University Of Colorado, A Body Corporate Therapeutic applications of Smad7
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