WO2013119893A1 - Dosages permettant d'identifier des composés modulant la fonction des ostéoblastes et la formation osseuse - Google Patents

Dosages permettant d'identifier des composés modulant la fonction des ostéoblastes et la formation osseuse Download PDF

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WO2013119893A1
WO2013119893A1 PCT/US2013/025258 US2013025258W WO2013119893A1 WO 2013119893 A1 WO2013119893 A1 WO 2013119893A1 US 2013025258 W US2013025258 W US 2013025258W WO 2013119893 A1 WO2013119893 A1 WO 2013119893A1
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shn3
erk
phosphorylation
protein
compound
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Laurie H. Glimcher
Jae-Hyuck SHIM
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Cornell 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Definitions

  • Bone mass reflects the balance between production of bone by osteoblasts and resorption of bone by osteoclasts, and disturbance of this balance results in bone pathology such as osteoporosis.
  • osteoblasts have a limited ability to directly perceive the thickness of the bone they overlay, they rely on the ability to integrate extracellular cues to appropriately adjust the rate of bone formation.
  • extracellular cue the WNT/p-catenin pathway
  • WNT/p-catenin pathway is well established as a positive regulator of osteoblast differentiation, appearing to divert early progenitors away from a chondrocyte fate into becoming osteoblasts.
  • a subset of ligands of the WNT family bind to the seven-pass transmembrane receptor Frizzled (FZD) and the single-pass low- density lipoprotein receptor-related protein 5 or 6 (LRP5/6).
  • FZD seven-pass transmembrane receptor Frizzled
  • LRP5/6 low- density lipoprotein receptor-related protein 5 or 6
  • Mitogen Activated Protein Kinase (MAPK) pathways are essential for skeletal development and the maintenance of bone mass by mediating the response to a wide range of extracellular ligands relevant to osteoblast activity such as BMPs, WNTs, PTH, TNF, insulin, IGF, and FGFs.
  • the Schnurri family of large zinc finger proteins consists of three mammalian
  • SHN1 homologues
  • SHN2 homologues
  • SHN3 SHN3 proteins are critical regulators of postnatal bone mass.
  • Shn2 'A mice display a mildly reduced bone turnover due to reduced activity of both osteoblasts and osteoclasts.
  • screening assays based on elucidation of biological mechanisms involved in osteoblast differentiation, in particular those involving the critical regulator protein Shn3 would be very useful and are still needed to facilitate identification of compounds that may be useful in the treatment of various skeletal disorders.
  • the instant invention is based, at least in part, on the discovery of the mechanism by which SHN3 regulates bone mass, finding that SHN3 controls osteoblast function via regulating WNT/LRP5 -induced ERK activation.
  • SHN3 dampens ERK kinase activity thereby preventing excessive activation of downstream substrates in response to WNT stimulation.
  • These pathways operate in vivo as evidenced by recapitulation of the Shn3 ⁇ ' ⁇ high bone mass phenotype in mice bearing a Shn3 allele with a mutation of the ERK interaction domain, and by observation of a genetic interaction between null alleles of Shn3 and the WNT co-receptor Lrp5.
  • inducible knockdown of Shn3 in adult mice increases bone mass, suggesting that compounds blocking SHN3 expression or activity would be attractive therapeutic agents for the treatment of osteoporosis.
  • This invention provides screening assays based on elucidation of biological mechanisms involving the zinc finger protein Schnurri-3 (Shn3) that are important in regulating osteoblast differentiation.
  • Shn3 is a tissue-specific regulator of nuclear ERK activity in osteoblasts.
  • ERK or "extracellular- signal related kinase”
  • MAPK or "mitogen activated protein kinase” super-family member.
  • Sequestration of ERK within the nucleus by Shn3 establishes an activation threshold within the osteoblast by limiting the ability of this MAPK to phosphorylate specific substrates. Mutations in Shn3 that abolish its interaction with ERK result in aberrant nuclear ERK activation resulting in elevated osteoblast activity. Moreover, the presence of a high-bone mass phenotype in mice bearing this same mutation in the ERK docking site of Shn3 provides in vivo confirmation that Shn3 controls bone formation through localized regulation of ERK activity.
  • the invention provides a method of identifying compounds useful in modulating bone formation and mineralization comprising, a) providing an indicator composition comprising Shn3 and ERK, or biologically active portions thereof; b) contacting the indicator composition with each member of a library of test compounds; c) selecting from the library of test compounds a compound of interest that modulates the interaction of Shn3 and ERK, or biologically active portions thereof, to thereby identify a compound useful in modulating bone formation and mineralization.
  • the indicator composition comprises, or consists essentially of, or consists of, (i) a BAS region of Shn3; or (ii) amino acids 850-900 of Shn3; or (iii) a D domain of Shn3; or (iv) a D domain of Shn3 comprising an amino acid sequence PPKKKRLRL (SEQ ID NO: 1); or (v) a peptide comprising an amino acid sequence PPKKKRLRL (SEQ ID NO: 1).
  • the indicator composition comprises, or consists essentially of, or consists of, a CD domain of ERK.
  • the ERK is ERK2 MAPK (e.g., human ERK2 MAPK.)
  • the ability of the compound of interest to modulate the interaction of Shn3 and ERK can be measured, for example, by determining an indicator of osteoblast differentiation.
  • indicators of osteoblast differentiation include formation of mineralized matrix, expression of one or more osteoblast marker genes and activation of RUNX2.
  • Activation of RUNX2 can be determined using, for example, a reporter gene comprising one or more (e.g. , tandem repeating) RUNX-binding elements.
  • two or more indicators of osteoblast differentiation are deteremined as a measure of the interaction of Shn3 and ERK.
  • the compound identified in the method is useful in inhibiting bone formation and/or mineralization.
  • a compound is selected that enhances or stimulates the interaction of Shn3 with ERK.
  • the compound identified in the method is useful in stimulating bone formation and/or mineralization.
  • a compound is selected that represses or inhibits the interaction of Shn3 with ERK.
  • the invention also demonstrates that interaction of Shn3 with ERK leads to inhibition of phosphorylation of ERK substrates. Accordingly, in another aspect, the invention provides a method of identifying a compound as a modulator of the functional activity of Shn3/ERK interaction, the method comprising a) providing an indicator composition comprising Shn3, ERK and an ERK substrate; b) contacting the indicator composition with a test compound; c) determining level of phosphorylation of the ERK substrate in the presence of the test compound; and d) identifying the compound as a modulator of functional activity of Shn3/ERK when the level of phosphorylation of the ERK substrate in the presence of the test compound is different than the level of phosphorylation of the ERK substrate in the absence of the test compound.
  • the ERK is ERK2.
  • Non-limiting examples of ERK substrates include RUNX2 and ELK1.
  • the invention also demonstrates that interaction of Shn3 with ERK leads to
  • the invention provides a method of identifying a compound as a modulator of phosphorylation of Shn3 by ERK, the method comprising a) providing an indicator composition comprising Shn3 and ERK; b) contacting the indicator composition with a test compound; c) determining level of phosphorylation of Shn3 in the presence of the test compound; and d) identifying the compound as a modulator of phosphorylation of Shn3 by ERK when the level of phosphorylation of Shn3 in the presence of the test compound is different than the level of phosphorylation of Shn3 in the absence of the test compound.
  • the ERK is ERK2.
  • the level of phosphorylation of Shn3 is determined at one or more amino acid positions selected from the group consisting of S811, S812, T851, S911 and S913.
  • Figure 1A is a schematic diagram showing the truncated Shn3 mutants.
  • Figure IB shows the results of experiments examining the subcellular localization of Shn3.
  • Immortalized CalvObs were fractionated into cystolic (CYT) and nuclear (NUC) fractions and immunoblotted with anti-Shn3 antibody.
  • HDAC1 and HSP90 were used as markers for nuclear and cytosolic fractions, respectively (left).
  • Localization of C-terminally GFP-tagged Shn3 mutants (AC and ⁇ ) was analyzed in HeLa cells by immunofluorescence (right).
  • Figure 1C shows the results of mineralization experiments in which human MSCs were in infected by vector or Shn3 mutants expressing lentivirus, cultured under OBD conditions for 21 days and analyzed for Von Kossa staining for mineralization activity.
  • Figure ID shows the evolutionarily conserved domain in Shn3 contains D domain (810-933 aa).
  • the asterisks (*) denote phosphorylation sites.
  • Figure IE shows the results of mineralization experiments performed as described in Figure 1C.
  • Figure IF shows the results of osteoblast gene expression experiments in which RNA levels of the indicated genes were analyzed by quantitative PCR on human MSCs infected by vector or Shn3 mutants expressing lentivirus.
  • Osx Osterix
  • Bsp bone sialoprotein
  • Ocn osteocalcin. Values are mean + SD.
  • Figure 2A is a schematic diagram showing amino acid sequences in the D domains of ERK substrates, which are important for regulation of osteoblast function.
  • Figure 2B shows the results o experiments in which HEK293 cells were transfected with HA- ERK2 together with wt or BAS-deletion Shn3 expression constructs. Cells were lysed, immunoprecipitated with anti-HA antibody-conjugated agarose, and immunoblotted with anti- Shn3 antibody.
  • Figure 2C shows the results of experiments in which immortalized Shn3 +/+ and Shn3 ⁇ A CalvObs were lysed, immunoprecipitated by anti-ERKl/2 antibody and protein A-agarose, and immunoblotted by anti-Shn3 antibody.
  • Figure 2D shows the results of experiments in which immortalized wt CalvObs were stimulated with FGF2 (10 ng/ml) for 30 minutes, nuclear proteins were fractionated, immunoprecipitated by either rabbit IgG or anti-phospho-ERKl/2 antibody and protein A-agarose, and immunoblotted by anti-Shn3 antibody. Input indicates increased phosphorylation of nuclear ERK1/2 in response to FGF2 stimulation.
  • FIGS 2E and 2F show the results of experiments in which hMSCs were infected with control lentivirus or lentivirus expressing the various Shn3 point-mutants.
  • Cells were cultured under OBD conditions for 21 days, and analyzed for Von Kossa staining for mineralization activity (E) or RNA was harvested and gene expression for Osx, BSP and Ocn was determined by qRT-PCR (F).
  • Figures 2G and 2H show the results of interaction experiments in which HA-ERK2 protein was incubated with recombinant GST or GST-BAS protein and immunoprecipitated with glutathione- agarose, and immunoblotted with anti-HA antibody. Input indicates loading controls of HA- ERK2, GST, or GST-BAS protein.
  • Figure 21 shows the results of interaction experiments in which HA-ERK2 protein was incubated with recombinant GST or GST-BAS (WT) protein along with different concentrations of ERK peptide inhibitor (PI) and immunoprecipitated with glutathione-agarose, and immunoblotted with anti-HA antibody. Input indicates loading controls of HA-ERK2 proteins.
  • Figures 2J and 2K show results from examination of femurs from p 15 Mekl ⁇ ;Mek2 +/+ (WT) and Mek /fl ;Mek- A ; Osx (DKO) mice. Femurs were analyzed by ⁇ CT. Displayed are three- dimensional reconstructions of trabecular bone and midshaft cortical bone (Figure 2J), and BV/TV, trabecular number, trabecular thickness, and cortical thickness parametes (Figure 2K). Values are mean + SD. indicates a significant difference by the Student's t-test, K0.0005.
  • Figure 2L shows the results of examination of gene expression in pl5 Mekl ⁇ ;Mek2 +/+ (WT) and Mekl fl/fl ;Mek A ; Osx (DKO) mice.
  • Total RNAs were extracted from calvaria of mice and analyzed by quantitative PCR. The value of each sample is indicated with a circle and the average value of each group indicated with a red line.
  • Genes examined are alkaline phosphatase (Tnalp), osteocalcin (Osn), osterix (Osx) and collagen la (Coll).
  • Figure 3A shows the results of ERK kinase assays in which rERK2 protein was incubated with rHis-Shn3 proteins along with either GST- ELK 1 or Myc-Runx2 protein and ERK kinase activity was analyzed by in vitro kinase assay.
  • FIG. 3B shows the results of experiments in which immortalized Shn3 +/+ and Shn3 ⁇ A CalvObs were stimulated with PMA for 30 minutes and then immunoprecipitated with anti-ERKl/2 antibody. The immunoprecipitates were mixed with recombinant ELK1 protein and ERK kinase activity was analyzed by in vitro kinase assay.
  • Figure 3C shows the results of experiments in which rERK2 or rCDKl protein was incubated with GST or GST-BAS (WT, KA, or KR) protein and Shn3 phosphorylation was analyzed by in vitro kinase assay.
  • Figure 3E shows the results of experiments in which rERK2 protein was incubated with GST- BAS (WT) protein along with different concentrations of ERK peptide inhibitor (PI) and Shn3 phosphorylation was analyzed by in vitro kinase assay.
  • WT GST- BAS
  • PI ERK peptide inhibitor
  • Figure 3F is a schematic diagram showing phosphorylation sites on Shn3 (636-930 aa) (Top) and their Alanine- substitution mutations (Bottom). Red: ERK2 phosphorylation sites; Blue: serum phosphorylation sites.
  • Figure 3G shows the results of experiments in which rERK2 protein was incubated with rHis- Shn3 (WT, KA or ml23) protein in the absence or presence of Myc-Runx2 protein and ERK kinase activity (top) or Shn3 phosphorylation (bottom) was analyzed by in vitro kinase assay.
  • Figure 3H shows the results of experiments in which rERK2 protein was incubated with rHis- Shn3 (WT or ml23) protein and immunoprecipitated with anti-His antibody-conjugated agarose, and immunoblotted with anti-HA antibody. Input indicates loading controls of HA-ERK2 proteins.
  • Figure 31 shows the results of experiments in which human MSCs infected by vector or Shn3 mutants expressing lenti virus were cultured under OBD conditions for 21 days and analyzed for Von Kossa staining for mineralization activity.
  • Figure 3J is a schema depicting the regulatory mechanism of Shn3 and ERK MAPK in osteoblasts.
  • Figures 4A-J show the results of experiments analyzing the in vivo function of the three lysines within the D domain of Shn3 protein in bone formation.
  • Figure 4A shows the results of experiments in which primary Shn3 and Shn3 BMSCs were lysed and immunoblotted with anti-Shn3 antibody.
  • Figure 4B shows a histological analysis of the proximal femur of Shn3 and Shn3 mice. "*" indicates a significant difference by the Student's t-test, t ⁇ 0.05. indicates K0.005.
  • FIGS 4D, 4E, 4F, 4G and 4H show results of experiments in which Shn3 and Shn3
  • BMSCs were cultured under OBD conditions and then mineralization was analyzed by Von Kossa staining (Figure 4D) or, alternatively, total RNA was extracted for RT-PCR analysis ( Figures 4E, 4F, 4G and 4H). Values are mean + SD ( Figures 4E, 4F, 4G and 4H).
  • Figures 41 and 4J show the results of experiments in which femurs from 8 week old Shn3 +/+ ,
  • Figure 5 shows the subcellular localization of various Shn3 mutants.
  • Figure 5A C3H10T1/2 cells were transiently transfected with various Shn3 constructs, fractionated into cytosolic (CYT) and nuclear (NUC) fractions, and immunoblotted with anti-Shn3 antibody. HDACl was used as a marker for nuclear fraction.
  • Figure 5B C3H10T1/2 cells were transiently transfected with Shn3-NT constructs, fractionated into cytosolic (CYT) and nuclear (NUC) fractions, and immunoblotted with anti-Shn3 antibody. HDACl was used as a marker for nuclear fraction (left).
  • FIG. 6 shows the effects of various Shn3 mutants on osteoblast differentiation.
  • FIG. 6A Human MSCs were infected by vector or Shn3 mutants expressing lentivirus and cultured under OBD conditions for 14 days. RNA levels of the indicated genes were analyzed by quantitative PCR on osterix (Osx), bone sialoprotein (Bsp), and osetocalcin (Ocn). Values are mean + SD.
  • Figure 6B Shn3 A CalvObs were infected by vector or Shn3 mutants expressing lentivirus, cultured under OBD conditions for 21 days, and analyzed for Von Kossa staining for mineralization activity (left). Alternatively, RNA levels of the indicated genes were analyzed by quantitative PCR at 14 day OBD culture (right).
  • FIG. 6C Human MSCs were infected by vector or Shn3 mutants expressing lentivirus, and effects of Shn3 mutants on osteoblast differentiation (OBD) were analyzed by mineralization activity and expression of osteoblast marker genes.
  • Figure 6D Effect of Shn3-KR mutant (mutation of three lysines to arginines) on Runx2 activity.
  • C3H10T1/2 cells were transiently transfected with various Shn3 constructs, OSE2 luciferase reporter, and Renilla along with either vector or Runx2. Results are expressed as relative luciferase activity normalized by Renilla control. Values are mean + SD.
  • Figure 7 shows the effects of various Shn3 mutants on transcriptional activities of Runx2, NF- KB, or AP- 1.
  • Figure 7A C3H10T1/2 cells were transiently transfected with various Shn3 constructs, OSE2 luciferase reporter, and Renilla along with either vector or Runx2. Results are expressed as relative luciferase activity normalized by Renilla control. Values are mean + SD.
  • Figure 7B HEK293 cells were transiently transfected with various Shn3 constructs along with PB-II luciferase reporter and Renilla. 24 hours after transfection, cells were incubated with TNFa (10 ng/ml) for 24 hours.
  • Results are expressed as relative luciferase activity normalized by Renilla control. Values are mean + SD.
  • Figure 7C Jurkat T cells were transiently transfected with various Shn3 constructs along with AP-1 luciferase reporter and Renilla. 24 hours after transfection, cells were incubated with PMA (20 ng/ml) and ionomycin (1 ⁇ ) for 24 hours. Results are expressed as relative luciferase activity normalized by Renilla control. Values are mean + SD.
  • Figure 8 shows how ERK1 and 2 MAPKs interact with nuclear Shn3.
  • Figure 8 A Schematic diagram showing Myc-tagged Zn/B or BAS domain expressing constructs, which are enforced to be localized in nucleus by adding three tandem nuclear localization sequence (3x NLS).
  • Figure 8B C3H10T1/2 cells were transiently transfected with Shn3 constructs, fractionated into cytosolic (CYT) and nuclear (NUC) fractions, and immunoblotted with anti-Myc antibody (right).
  • Myc-tagged ZnB construct containing 3xNLS was transiently transfected into HEK293 cells along with T7-ERK1 or HA-ERK2. Cells were lysed, immunoprecipiated by either anti-T7 antibody or anti-HA antibody and protein A agarose, and immunoblotted with the indicated antibodies.
  • Figure 9 shows a characterization of Mekl ⁇ ;Mek2 ⁇ / ⁇ ;osx mice.
  • Figure 9A Alizarin Red/Alcian Blue-stained skeletal preps of whole bodies, calvaria, hyoid bones, clavicles, fore limbs, and hind limbs in various mice at P15. Arrows indicate the rib fractures.
  • Figure 10 shows how ERK MAPK regulates Shn3 activity through Shn3 phosphorylation.
  • FIG. 10A Recombinant ERK2 protein was incubated with recombinant His-Shn3 s (636-930 aa) protein along with either Myc-Runx2 or GST-Smad3 protein (top).
  • recombinant p38cc or JNK1 MAPK was incubated with recombinant His-Shn3 s (636-930 aa) protein along with either GST-ATF2 or GST-cJUN protein (middle and bottom).
  • MAPK kinase activity was analyzed by in vitro kinase assay.
  • HEK293 cells were transiently transfected with HA-ERK2 and Myc-RUNX2 in the presence or absence of the indicated Shn3 constructs. Cells were lysed, immunoprecipiated by anti-HA antibody and protein A agarose, and immunoblotted with the indicated antibodies.
  • Figure IOC HA-ERK2 (D319N) protein was incubated with either GST, GST-BAS or GST-ELK 1 protein and ERK2-induced
  • Figure 11 shows a characterization of Shn3 mice.
  • Figure 11 A Schematic diagram showing three lysine knock-in mutations on Shn3 gene.
  • Figure 11B Shn3 transcript levels were analyzed by quantitative PCR on primary Shn3 +/+ and Shn3 KI/KI CalvObs.
  • Figure 11C TRAP stain of the trabecular bone below the growth plate of the tibia in 8 week old Shn3 and Shn3 mice. TRAP-positive osteoclasts stain a magenta color.
  • Figure 11D Fasting CTX levels were
  • Figure 12 shows the results of expermints demonstrating selective function of Shn3 on ERK substrates.
  • Figure 12A Myc- or Flag-tagged Shn3 (Full length or ABAS) was transiently transfected into HEK293 cells along with HA-, Flag-, or Myc-tagged various proteins. Cells were lysed, immunoprecipiated by the indicated antibodies and protein A agarose, and immunoblotted with the indicated antibodies. N.D.; non-determined.
  • Flag-tagged JNK1 or 2 and p38cc or ⁇ proteins were incubated with recombinant GST-BAS protein, immunoprecipiated by glutathione-agarose, and immunoblotted with anti-Flag antibody.
  • FIG. 12B C3H10T1/2 cells were infected with vector or Shn3 (WT or KA) expressing lentivirus, lysed, and immunoblotted with the indicated antibodies. Expression of Shn3 proteins was analyzed by immunoblotting with anti-Shn3 antibody.
  • Figure 13 shows the in vivo function of the three lysine motif of SHN3 in bone formation and shows that SHN3 inhibits ERK MAPK activity in osteoblasts via the D-domain three lysine motif.
  • Shn3 ⁇ and Shn3 ⁇ ' BMSCs were cultured in differentiation medium and
  • FIG. 14 demonstrates that SHN3 inhibits the WNT-mediated ⁇ -catenin pathway via ERK regulation.
  • C3H10T1/2 cells were infected by vector or SHN3 expressing lentivirus and transfected with top-flash Luc and Renilla along with either a xWNT8/Fz5 fusion protein, a constitutively active mutant of LRP5 (LRP5-CA), or a xWNT8/Fz5 fusion protein plus LRP5.
  • Results are expressed as relative luciferase activity normalized to Renilla control. Values are mean + SD ( Figure 14A.) Primary Shn3 +/+ and Shn3 'A COBs were transfected with top- flash Luc along with Renilla.
  • Luciferase activity was analyzed 6 days after culture in differentiation medium. Results were normalized to a Renilla control. Values are mean + SD ( Figure 14B.) Primary Mek /fl ;Mek2 'A COBs were infected by vector (Mekl/2 +/+ ) or Cre recombinase (Mekl/2 'A ) expressing lentivirus and cultured in differentiation medium and lysed and immunoblotted with the indicated antibodies (Figure 14C.) Primary Mekl ⁇ I ;Mek2 ⁇ / ⁇ COBs were infected by vector (Mekl/2 +/+ ) or Cre recombinase (Mekl/2 ⁇ A ) expressing lentivirus and transfected with top-flash Luc and Renilla.
  • Luciferase activity was analyzed 6 days after culture in differentiation medium. Results were normalized to a Renilla control. Values are mean + SD ( Figure 14D.) Femurs from 8 week old Shn3 , Lrp5 ⁇ Shn3 ⁇ and Lrp5 ⁇ ⁇ ;Shn3 ⁇ ' mice were analyzed by ⁇ . Displayed are three-dimensional reconstructions of proximal femur ( Figure 14E), and BV/TV, trabecular number, trabecular thickness, and cortical thickness parameters. Values are mean + SD ( Figure 14F).
  • Figure 15 shows that SHN3 regulates GSK3 activity and ⁇ -catenin level in osteoblasts.
  • HEK293 cells were transfected with a xWNT8/Fz5 fusion protein along with LRP5, lysed and immunoprecipiated by anti-ERKl/2 antibody and protein A agarose. The immunoprecipitates were incubated with GST-ELK1 in the absence or presence of recombinant His-SHN3 (WT, KA) and ERK kinase activity was analyzed by in vitro kinase assay.
  • Figure 16 depicts inducible knock-down of SHN3 increases bone mass in adult mice.
  • 8 week old Shn3WT and Shn3KD mice were fed with doxycycline-containing chow for 6 weeks and total RNA was extracted from long bones for RT-PCR analysis (Figure 16A.) Values are mean + SD.
  • 8 week old Shn3WT and Shn3KD mice were fed with either PBS- or doxycycline-containing chow for 6 weeks.
  • Femurs from 14 week old mice were analyzed by ⁇ CT.
  • Values for bone volume fraction (BV/TV) and cortical thickness (C.Th) are displayed ( Figure 16B.)
  • Values are mean + SD.
  • "*" indicates a significant difference by the Student's t-test, t ⁇ 0.05.
  • Figure 17 dpicts genetic interaction analysis of SHN3 with RUNX2 in bone. ⁇ CT analysis of the femurs from 10 week old Shn3+/+, Runx2+/-, Runx2+/-;Shn3-/-, and Shn3-/- mice. Displayed are three-dimensional reconstructions of trabecular bone (Figure 17A), and values for bone volume fraction (BV/TV), trabecular number (Tb. N), and trabecular thickness (Tb. Th). Values are mean + SD ( Figure 17B).
  • Figure 18 depicts effects of various SHN3 mutants on osteoblast differentiation.
  • Human MSCs were infected by vector or SHN3 mutants expressing lentivirus, and effects of SHN3 mutants on osteoblast differentiation were analyzed by Alkaline phosphatase (ALP) activity ( Figure 18 A) and nodule numbers ( Figure 18B).
  • Human MSCs were infected with lentiviruses containing vector or constructs encoding SHN3 mutants, and the effects of SHN3 mutants on osteoblast differentiation were analyzed by expression of osteoblast marker genes using RT-PCR (Figure 18C.)
  • Figure 19 depicts data showing that he BAS domain is not required to form a regulatory SHN3- WWP1-RUNX2 protein complex.
  • HEK293 cells were transfected with Myc-SHN3 along with either GFP-WWP1 or Xpress- RUNX2. Cells were lysed, immunoprecipitated by anti-Myc antibody conjugated agarose, and immunoblotted with the indicated antibodies ( Figures 19A-B).
  • HEK293 cells transfected with Flag-RUNX2 and His-Ub along with Myc-WWPl in the absence or presence of SHN3 (WT, ABAS). After 48 hour transfection, cells were treated with MG132 for 6 hours and lysed, immunoprecipitated by Ni-NTA beads and immunoblotted with the indicated antibodies (Figure 19C.)
  • FIG. 20 decicts Shn3 activities in vitro and in vivo.
  • Shn3 transcript levels were analyzed by RT-PCR on primary Shn3 +/+ and Shn3 KI/KI BMSCs (Figure 20A.)
  • HEK293 cells were transfected with Flag-p38a, Flag-JNKl, or Flag-ERK2 along with SHN3 (WT).
  • WT SHN3
  • Cells were lysed, immunoprecipitated by anti-SHN3 antibody and protein A agarose, and immunoblotted with the indicated antibodies (Figure 20B.)
  • Data are as in Figure 4J. Values are mean + SD ( Figure 20C).
  • Figure 21 depicts data showing that SHN3 regulates ERK MAPK pathway downstream of WNT.
  • C3H10T1/2 cells were infected by vector or SHN3 expressing lentivirus and after puromycin selection cells were stimulated with FGF18, BMP2, or TGFP for 6 hours.
  • RNA levels of the indicated genes were analyzed by RT-PCR ( Figure 21A.)
  • RNA levels of ⁇ -catenin and HPRT genes were analyzed by RT-PCR on primary Shn3+/+ an Shn3-/- COBs (top).
  • Meklfl/fl;Mek2-/- COBs were infected by vector (Mekl/2+/+) or Cre recombinase (Mekl/2-/-) expressing lentivirus and RNA levels of ⁇ -catenin and HPRT genes were analyzed by RT-PCR (bottom) ( Figure 21B).
  • C3H10T1/2 cells were stimulated with mWNT3a (200 ng/ml) for the indicated time points.
  • the skeleton is a complex organ system that serves a critical role in numerous
  • the ability of the skeleton to facilitate these various functions requires the existing bone elements to be in a dynamic state known as bone remodeling.
  • the initial resorptive phase of the remodeling process is driven by large multinucleated osteoclasts that degrade the existing bone matrix through an enzymatic and acidic process (Edwards, J.R. and Mundy, G.R. (2011) Nat Rev Rheumatol 7:235.)
  • a complex milieu of growth factors and cytokines that are released into the microenvironment following the resorptive phase cue the differentiation and localization of osteoblasts to the area of bone resorption where these mesenchymal-derived cells secrete a matrix rich in Type I collagen (Karsenty, G. and Wagner, E.F.
  • osteoblasts and its progenitor cells interpret the various extracellular cues to ensure that the differentiation and function of these cells is regulated in both a spatial and temporal manner is only beginning to be understood. Furthermore, a number of signaling pathways important in osteoblast biology share a number of signaling components. Thus, understanding how the genetic program that drives osteoblast differentiation is regulated in response to the various microenvironmental stimuli may provide not only a greater
  • Shn3 a member of the Schnurri family of zinc finger proteins, has been identified as an essential regulator of adult bone formation (Jones, D. C. et al. (2006) Science 312: 1223). Mice lacking Shn3 display an osteosclerotic phenotype with profoundly increased bone mass due to augmented osteoblast activity, thereby establishing Shn3 as a negative regulator of osteoblast activity. However, while a critical role for Shn3 in controlling bone formation has been established, the molecular mechanism through which Shn3 controls osteoblast activity has not been established.
  • the present invention is based, at least in part, on the finding that Shn3 modulates bone formation and mineralization by interacting with ERK in the nucleus of osteoblasts and acting as a downregulator of ERK activity, thereby leading to inhibition of osteoblast differentiation and bone formation.
  • the present invention is also based, at least in part, on the discovery that Shn3 and ERK specifically interact through a binding interaction between the D-domain of Shn3, located in the central BAS region of the protein, and the CD domain of ERK.
  • the present invention is also based, at least in part, on the discovery that interaction of Shn3 and ERK leads to phosphorylation of Shn3 by ERK, which enhances Shn3's repressive ability, and leads to inhibition of phosphorylation of other ERK substrates involved in osteoblast regulation.
  • Shn3 and “Schnurri-3” are used interchangeably and refer to a protein member of the human immunodeficiency virus type 1 enhancer-binding protein family. Members of this protein family contain multiple zinc finger and acid-rich (ZAS) domains and serine- threonine rich regions. This protein is able to regulate nuclear factor kappaB-mediated transcription in target genes as an adaptor protein. This protein also binds the recombination signal sequence that flanks the V, D, and J regions of immunoglobulin and T-cell receptors. Alternate splicing results in both coding and non-coding transcript variants. "Shn3" or
  • Shn3 or “Schnurri-3” is also known in the art as FLJ16752, Human immunodeficiency virus type I enhancer-binding protein 3 (HIVEP3), Kappa-B and V(D)J recombination signal sequences-binding protein, Kappa-binding protein 1, KBP1, KBP-1, KIAA1555, Transcription factor HIVEP3, ZAS3, Zinc finger protein ZAS3,and ZNF40C.
  • the Shn3 protein is also referred to in the art as "KRC.”
  • the terms "Shn3 activity”, “Shn3 biological activity” or “activity of an Shn3 polypeptide” includes modulation of bone growth, modulation of bone mineralization, modulation of osteoclastogenesis, modulation of osteoblast versus osteoclast activity, e.g., in bone formation and/or remodeling of bone, modulation of osteocalcin gene transcription, degradation of Runx2, e.g., modulation of Runx2 protein levels, ubiquitination of Runx2, modulation of the phosphorylation of Runx2, modulation of the expression of RSK2, degradation of RSK2, e.g., modulation of RSK2 protein levels, ubiquitination of RSK2, modulation of the phosphorylation of RSK2, modulation of the expression of BSP, ColI(cc)l, OCN, Osterix, RANKL, and ATF4, modulation of ATF4 protein levels, and/or modulation of the
  • modulate include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).
  • bone formation and mineralization refers to the cellular activity of osteoblasts to synthesize the collagenous precursors of bone extracellular matrix, regulate mineralization of the matrix to form bone, as well as their function in bone remodeling and reformation, e.g., bone mass is maintained by a balance between the activity of osteoblasts that form bone and the osteoclasts that break it down.
  • the mineralization of bone occurs by deposition of carbonated hydroxyapetite crystals in an extracellular matrix consisting of type I collagen and a variety of non-collagenous proteins.
  • osteoblast includes bone-forming cells that are derived from mesenchymal osteoprognitor cells and forms an osseous matrix in which it becomes enclosed as an osteocyte.
  • a mature osteoblast is one capable of forming bone extracellular matrix in vivo, and can be identified in vitro by its capacity to form mineralized nodules which reflect the generation of extracellular matrix.
  • An immature osteoblast is not capable of forming mineralized nodules in vitro.
  • ERK refers to "extracellular- signal-regulated kinases", which are members of the MAPK (mitogen-activated protein kinases) family and which have been established as major participants in the regulation of cell growth and differentiation.
  • ERKs include ERK1 and ERK2.
  • the nucleotide sequence and amino acid sequence of human ERK1 is described in, for example, GenBank Accession No. gi:232066.
  • the nucleotide sequence and amino acid sequence of murine ERK1 is described in, for example, GenBank Accession No. gi:52001483.
  • the nucleotide sequence and amino acid sequence of human ERK2 is described in, for example, GenBank Accession No. gi: 119554.
  • the nucleotide sequence and amino acid sequence of murine ERK2 is described in, for example, GenBank Accession No. gi:52001076.
  • ERK substrate refers to compounds (e.g., proteins) that are phosphorylated by ERK, non-limiting examples of which include Runx2, ELK1 and Shn3.
  • ELK1 is a member of the TCF family of Ets domain proteins that has been shown to be phosphorylated at multiple serine/threonine residues upon activation of ERK, as described in, for example, Cruzalegui, F.H. et al. (1999) Oncogene 18:7948-7957.
  • Runx2 is one of three mammalian homologues of the Drosophila transcription factors, Runt and Lozenge (Daga, A., et al. (1996) Genes Dev. 10: 1194-1205). Runx2 is also expressed in T lymphocytes and cooperates with oncogenes c-myc, p53, and Piml to accelerate T-cell lymphoma development in mice (Blyth, K., et al. (2001) Oncogene 20:295-302). Runx2 expression also plays a key role in osteoblast differentiation and skeletal formation.
  • Runx2 regulates expression of several other genes that are activated during osteoblast differentiation, including alkaline phosphatase, type I collagen, osteopontin, and osteoprotegerin ligand. These genes also contain Runx2-binding sites in their promoters. These observations suggest that Runx2 is an essential transcription factor for osteoblast differentiation. This hypothesis is strongly supported by the absence of bone formation in mouse embryos in which the cbfal gene was inactivated. Furthermore, cleidocranial dysplasia, a human disorder in which some bones are not fully developed, has been associated with mutations in a cbfal allele. In addition to its role in osteoblast differentiation, Runx2 has been implicated in the regulation of bone matrix deposition by differentiated osteoblasts.
  • Runx2 is regulated by factors that influence osteoblast differentiation. Accordingly, BMPs can activate, while Smad2 and glucocorticoids can inhibit, Runx2 expression. In addition, Runx2 can bind to an OSE2 element in its own promoter, suggesting the existence of an autoregulatory feedback mechanism of transcriptional regulation during osteoblast differentiation.
  • the nucleotide sequence and amino acid sequence of human Runx2 is described in, for example, GenBank Accession No. gi: 10863884.
  • the nucleotide sequence and amino acid sequence of murine Runx2 is described in, for example, GenBank Accession No. gi:20806529.
  • an "osteoblast marker gene” refers to a gene whose level of expression is regulated by Shn3, non-limiting examples of which include, for example, BSP, ColI(cc)l, OCN, RANKL, Osterix, RSK2, and/or ATF4.
  • BSP bone sialoprotein
  • the nucleotide sequence and amino acid sequence of human BSP is described in, for example, GenBank Accession No. gi:38146097.
  • the nucleotide sequence and amino acid sequence of murine BSP is described in, for example, GenBank Accession No. gi:6678112.
  • OCN also referred to as osteocalcin and bone gamma-carboxyglutamic acid (Gla) protein (BGLAP, or BGP)
  • BGLAP bone gamma-carboxyglutamic acid
  • BGP bone gamma-carboxyglutamic acid
  • gi: 158517828 The nucleotide sequence and amino acid sequence of murine OCN, is described in, for example, GenBank Accession No gi:83816951.
  • ATF4 also called “CREB2”, and “Osterix”, also called “SP7”
  • CREB2 CREB2
  • S7 stereosterix
  • bZIP protein family CREB2
  • C2H2-type zinc-finger protein family respectively, that are key regulators of bone matrix biosynthesis during remodeling of bone, e.g., during bone formation and mineralization (see, for example, Yang, X., et al. (2004). Cell 117, 387-398;
  • BSP, ColI(.alpha.)l, ATF4, and Osterix are specific markers of bone formation and development.
  • the nucleotide sequence and amino acid sequence of human ATF4 is described in, for example, GenBank Accession No. gi:33469975 and gi:33469973.
  • the nucleotide sequence and amino acid sequence of murine ATF4 is described in, for example, GenBank Accession No. gi:6753127.
  • the nucleotide sequence and amino acid sequence of human SP7 is described in, for example, GenBank Accession No.
  • gi:22902135 The nucleotide sequence and amino acid sequence of murine SP7, is described in, for example, GenBank Accession No gi: 18485517.
  • Type I collagen (cc)l (“ColI(cc)l”), is a collagenous bone matrix protein.
  • the nucleotide sequence and amino acid sequence of human ColI(.alpha.)l is described in, for example, GenBank Accession No. gi: 14719826.
  • the nucleotide sequence and amino acid sequence of murine ColI(cc)l is described in, for example, GenBank Accession No. gi:34328107.
  • the amino acid sequence of human RANKL is known and can be found in, for example, GenBank accession number GL4507595 or
  • GI: 14790152 The nucleotide sequence of human RANKL can be found in, for example, GenBank accession number GL4507595 or GI: 14790152.
  • the nucleotide and amino acid sequence of mouse RANKL may be found in, for example, GenBank accession number gi: 6755833.
  • Rsk2 also referred to as Ribosomal Protein S6 Kinase, 90-KD, 3; RPS6KA3, is a member of the RSK (ribosomal S6 kinase) family of growth factor-regulated serine/threonine kinases, known also as p90(rsk).
  • RSK2 ribosomal S6 kinase family of growth factor-regulated serine/threonine kinases, known also as p90(rsk).
  • the highly conserved feature of all members of the RSK family is the presence of 2 nonidentical kinase catalytic domains.
  • RSK2 is required for osteoblast differentiation and function.
  • ATF4 is a critical substrate of RSK2 that is required for the timely onset of osteoblast differentiation, for terminal differentiation of osteoblasts, and for osteoblast- specific gene expression. Additionally, RSK2 and ATF4 posttranscriptionally regulate the synthesis of type I collagen.
  • nucleotide sequence and amino acid sequence of human RSK2 is described in, for example, GenBank Accession No. gi:56243494.
  • nucleotide sequence and amino acid sequence of murine Rsk2 is described in, for example, GenBank Accession No gi:22507356.
  • the term "interact" as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a yeast two hybrid assay or
  • the term interact is also meant to include “binding" interactions between molecules. Interactions may be protein-protein or protein-nucleic acid in nature.
  • the term "contacting" is intended to include incubating the compound and the cell together in vitro (e.g., adding the compound to cells in culture) or administering the compound to a subject such that the compound and cells of the subject are contacted in vivo.
  • the term "contacting” is not intended to include exposure of cells to an Shn3 modulator that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).
  • the term "compound of interest” or “test compound” includes a compound that has not previously been identified as, or recognized to be, a modulator of Shn3 activity and/or expression and/or a modulator of cell growth, survival, differentiation and/or migration.
  • library of test compounds is intended to refer to a panel comprising a multiplicity of test compounds.
  • the term "indicator composition” refers to a composition that includes a protein of interest (e.g., Shn3 and/or ERK, or a molecule in a signal transduction pathway involving Shn3 and/or ERK), for example, a cell that naturally expresses the protein, a cell that has been engineered to express the protein by introducing an expression vector encoding the protein into the cell, or a cell free composition that contains the protein (e.g., purified naturally- occurring protein or recombinantly-engineered protein).
  • a protein of interest e.g., Shn3 and/or ERK, or a molecule in a signal transduction pathway involving Shn3 and/or ERK
  • a cell that naturally expresses the protein e.g., a cell that has been engineered to express the protein by introducing an expression vector encoding the protein into the cell, or a cell free composition that contains the protein (e.g., purified naturally- occurring protein or recombin
  • “Shn3” or “Schnurri-3” is a protein member of the human immunodeficiency virus type 1 enhancer-binding protein family. Members of this protein family contain multiple ZAS domains that are composed of C2H2-zinc finger, acid-rich region, and serine-threonine rich region. This protein is able to regulate nuclear factor kappaB-mediated transcription in target genes as an adaptor protein. This protein also binds the recombination signal sequence that flanks the V, D, and J regions of immunoglobulin and T-cell receptors. Alternate splicing results in both coding and non-coding transcript variants. "Shn3” or “Schnurri-3” has been shown to be is an essential regulator of adult bone formation.
  • Shn3 (Schnurri-3) has been shown to be is an essential regulator of adult bone formation. Mice lacking Shn3 display adult-onset osteosclerosis with increased bone mass due to augmented osteoblast activity. Shn3 was found to control protein levels of Runx2, the principal transcriptional regulator of osteoblast differentiation, by promoting its degradation through recruitment of the E3 ubiquitin ligase WWP1 to Runx2. By this means, Runx2-mediated extracellular matrix mineralization was antagonized, revealing an essential role for Shn3 as a central regulator of postnatal bone mass.
  • Exemplary Shn3 activities include modulation of bone growth, modulation of bone mineralization, modulation of osteoclastogenesis, modulation of osteoblast versus osteoclast activity, e.g., in bone formation and/or remodeling of bone, modulation of osteocalcin gene transcription, degradation of Runx2, e.g., modulation of Runx2 protein levels, ubiquitination of Runx2, modulation of the phosphorylation of Runx2, modulation of the expression of RSK2, degradation of RSK2, e.g., modulation of RSK2 protein levels, ubiquitination of RSK2, modulation of the phosphorylation of RSK2, modulation of the expression of BSP, ColI(cc) l, OCN, Osterix, RANKL, and ATF4, modulation of ATF4 protein levels, and/or modulation of the phosphorylation of ATF4.
  • Shn3 Exemplary amino acid and nucleotide sequences of Shn3 (Schnurri-3) are given in PCT/US2006/014295, incorporated herein by reference.
  • Murine and human Shn3 (Schnurri-3) are also set forth below for reference. Protein domains and/or motifs of interest are delineated.
  • TITLE Dampening of death pathways by schnurri-2 is essential for T-cell
  • the large zinc finger protein ZAS3 is a critical modulator of
  • REMARK GeneRIF ZAS3 was a crucial molecule in osteoclast differentiation.
  • TITLE A high-resolution anatomical atlas of the transcriptome in the mouse embryo
  • REMARK GeneRIF report that mice bearing parallel null mutations in the adapter proteins Schnurri2 (Shn2) and Schnurri3 (Shn3) exhibit defects in patterning of the axial skeleton during embryogenesis .
  • KRC dominant-negative KRC enhances NFkappaB-dependent transactivation and JNK phosphorylation and consequently, apoptosis and cytokine gene expression
  • V(D)J recombination signal sequences contains composite DNA-protein interaction domains and belongs to a new family of large transcriptional proteins
  • This RefSeq record includes a subset of the publications that are available for this gene. Please see the Gene record to access additional publications.
  • Region 844..865 /region_name "Acidic 2"
  • HIVEP3 isoform b [Homo sapiens] .
  • Eukaryota Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi ; Mammalia; Eutheria; Euarchontoglires ; Primates; Haplorrhini;
  • REFERENCE 1 (residues 1 to 2405)
  • TITLE ZAS3 promotes TNFalpha-induced apoptosis by blocking
  • REMARK GeneRIF The inhibitory effect of ZAS3 on NF-kappa B activity is mediated by neither direct association with N-kappa B nor disrupting nuclear localization of NFvarkappaB.
  • REFERENCE 2 (residues 1 to 2405)
  • TITLE ZAS3 accentuates transforming growth factor beta signaling in
  • REMARK GeneRIF An interaction between ZAS3 and Smad proteins enhances transforming growth factor beta signaling.
  • REFERENCE 3 (residues 1 to 2405)
  • TITLE ZAS3 represses NFkappaB-dependent transcription by direct
  • REFERENCE 4 (residues 1 to 2405)
  • REMARK GeneRIF Single nucleotide polymorphisms strongly support HIVEP3 as a candidate for PARK10 in Parkinson disease.
  • TITLE Foxp3 interacts with nuclear factor of activated T cells and
  • REFERENCE 7 (residues 1 to 2405)
  • TITLE Schnurri-3 (KRC) interacts with c-Jun to regulate the IL-2 gene in
  • REMARK GeneRIF Overexpression of KRC in transformed and primary T cells leads to increased IL-2 production.
  • REFERENCE 8 (residues 1 to 2405)
  • REFERENCE 9 (residues 1 to 2405)
  • TITLE Structure of the human zinc finger protein HIVEP3 molecular cloning, expression, exon-intron structure, and comparison with paralogous genes HIVEP1 and HIVEP2
  • REFERENCE 10 (residues 1 to 2405)
  • TITLE Two genes encode factors with NF-kappa B- and H2TFl-like
  • REVIEWED REFSEQ This record has been curated by NCBI staff, The reference sequence was derived from BC152563.1, AC119676. 2 , AL445933.32 and AI082114.1.
  • This RefSeq record includes a subset of the publications that are available for this gene. Please see the Gene record to access additional publications.
  • This gene encodes a member of the human immunodeficiency virus type 1 enhancer-binding protein family.
  • Members of this protein family contain multiple zinc finger and acid-rich (ZAS ) domains and serine-threonine rich regions.
  • ZAS zinc finger and acid-rich
  • This protein acts as a transcription factor and is able to regulate nuclear factor kappaB-mediated transcription by binding the kappaB motif in target genes.
  • This protein also binds the recombination signal sequence that flanks the V, D, and J regions of immunoglobulin and T-cell receptors. Alternate splicing results in both coding and non-coding transcript variants, [provided by RefSeq, Sep 2011] .
  • Transcript Variant This variant (2) lacks an alternate exon in the 5' UTR and uses an alternate splice site in the 3' coding region, compared to variant 1.
  • the resulting isoform (b) is shorter than isoform a.
  • the RefSeq transcript and protein were derived from genomic sequence to make the sequence consistent with the reference genome assembly.
  • the genomic coordinates used for the transcript record were based on alignments.
  • /region_name No DNA binding activity or transactivation activity, but complete prevention of TRAF-dependent NF-Kappa-B activation, associates with TRAF2 and JUN (By similarity) "
  • /gene_synonym "KBP-l; KBP1; KRC; Schnurri-3; SHN3 ; ZAS3; ZNF40C"
  • the invention provides a method of identifying compounds useful in modulating bone formation and mineralization comprising, a) providing an indicator composition comprising Shn3 and ERK, or biologically active portions thereof; b) contacting the indicator composition with each member of a library of test compounds; c) selecting from the library of test compounds a compound of interest that modulates the interaction of Shn3 and ERK, or biologically active portions thereof, to thereby identify a compound useful in modulating bone formation and mineralization.
  • a compound is selected that is useful in inhibiting bone formation and mineralization. In another embodiment, a compound is selected that is useful in stimulating bone formation and mineralization.
  • a test compound that stimulates the interaction between Shn3 and ERK is identified based upon its ability to increase the degree of interaction between Shn3 and ERK as compared to the degree of interaction in the absence of the test compound and such a compound would be expected to increase the down-regulatory activity of Shn3 in osteoblast function and thereby inhibit osteoblast differentiation and/or bone formation.
  • a test compound that inhibits the interaction between Shn3 and ERK is identified based upon its ability to decrease the degree of interaction between Shn3 and ERK as compared to the degree of interaction in the absence of the compound and such a compound would be expected to decrease the down-regulatory activity of Shn3 in osteoblast function and thereby stimulate osteoblast differentiation and/or bone formation.
  • Shn3 and ERK As described in detail in Example 1, the portions of Shn3 and ERK that mediate their binding interaction have been mapped in detail.
  • full-length Shn3 and/or ERK can be used, or for one or both of the proteins, a "biologically active portion thereof can be used, which in this instance refers to a portion of Shn3 that retains the ability to bind to ERK and/or a portion of ERK that retains the ability to bind to Shn3.
  • full-length Shn3 and ERK are used, a full-length Shn3 protein is used and a biologically active portion of ERK is used, a full-length ERK protein is used and a biologically active portion of Shn3 is used or biologically active portion of both Shn3 and ERK are used.
  • the biologically active portion of Shn3 is a BAS region, i.e., the indicator composition comprises a BAS region of Shn3.
  • the biologically active portion of Shn3 comprises (or consists essentially of or consists of) amino acids 850-900 of Shn3, i.e., the indicator composition comprises (or consists essentially of or consists of) amino acids 850-900 of Shn3.
  • the biologically active portion of Shn3 comprises (or consists essentially of or consists of) a D domain of Shn3, i.e., the indicator composition comprises (or consists essentially of or consists of) a D domain of Shn3.
  • the biologically active portion of Shn3 comprises (or consists essentially of or consists of) an amino acid sequence PPKKKRLRL (SEQ ID NO: 1), i.e., the indicator composition comprises (or consists essentially of or consists of) an amino acid sequence
  • PPKKKRLRL (SEQ ID NO: 1).
  • the biologically active portion of ERK is a CD domain
  • the indicator composition comprises (or consists essentially of or consists of) a CD domain of ERK.
  • the ERK is ERK2.
  • test compound to modulate the interaction of Shn3 and ERK (or biologically active portions thereof) can be assessed directly using an in vitro binding assay, wherein the read-out that is determined is the amount of binding between Shn3 and ERK in the presence of the test compound as compared to the amount of binding between Shn3 and ERK in the absence of the test compound.
  • a test compound can be selected that either increases the amount of binding between Shn3 or ERK or decreases the amount of binding between Shn3 and ERK, as compared to the amount of binding in the absence of the test compound.
  • Suitable types of in vitro binding assays for assessing the interaction of Shn3 and ERK in the presence or absence of a test compound are well known in the art, and described in the Examples, and the components can be prepared using standard recombinant DNA techniques.
  • a recombinant GST-Shn3 fusion protein or fusion protein comprising a biologically active portion of Shn3, such as GST-BAS or GST-D-domain
  • HA-ERK2 fusion protein or fusion protein comprising a biologically active portion of ERK2, such as HA-CD-domain
  • the ability of a test compound to modulate the interaction of Shn3 and ERK can be assessed indirectly, for example using a cell-based assay, wherein the read-out is measured by determining an indicator of osteoblast differentiation.
  • an indicator of osteoblast differentiation For example, osteoblast progenitor cells deficient in Shn3 and ERK can be transfected with suitable expression vector constructs, along with appropriate controls, and the effect of test compounds on osteoblast differentiation of the cells can be assessed.
  • suitable examples of indicators of osteoblast differentiation include formation of mineralized matrix, expression of one or more osteoblast marker genes and/or activation of RUNX2. The effect of the test compound on one or more indicators of osteoblast differentiation can be compared to the indicator(s) of osteoblast differentiation in the absence of the test compound.
  • a test compound can be selected that either increases the amount of binding between Shn3 or ERK or decreases the amount of binding between Shn3 and ERK, as compared to the amount of binding in the absence of the test compound.
  • Suitable types of cell based assays for assessing the interaction of Shn3 and ERK in the presence or absence of a test compound are well known in the art, and described in the Examples, and the components can be prepared using standard recombinant DNA techniques.
  • skeletal architecture can be assayed by digital radiography of, trabeculation (i.e., the anastomosing bony spicules in cancerous bone which form a meshwork of intercommunicating spaces that are filled with bone marrow) can be determined by three-dimensional .mu.-QCT imaging, and by analyses of bone cross-sections.
  • trabecular number, trabecular thickness, trabecular spacing, bone volume per tissue volume (BV/TV), and bone mineral density (BMD) can also be determined by .mu.-QCT imaging.
  • Mineralized bone and non-mineralized cartilage formation can be determined by histochemical analyses, such as by alizarin red/alcian blue staining.
  • the total osteoblast surface which a reliable indicator of osteoblast population, can be measured, as can osteoid thickness, i.e., the thickness of bone that has not undergone calcification.
  • Sections of bone can also be analyzed by staining with Von Kossa and Toluidine Blue for analysis of in vivo bone formation and serum levels of, for example, Trabp5b and deoxypyridinoline can be determined as an indication of bone formation.
  • the ex vivo culturing of osteoblast precursors and immature osteoblasts can also be performed to determine if cells possess the capacity to form mineralized nodules, which reflects the generation of extracellular matrix, i.e., the mineralized matrix of bone. Furthermore, these cultures can be assayed for their proliferative ability, e.g., by cell counting, and can be stained for the presence of various markers of bone formation, such as for example, alkaline phosphatase.
  • a mesenchymal stem cell may be used in an assay for bond formation.
  • a pluripotent cell capable to forming an osteoblast i.e., a mesenchymal stem cells (e.g., a primary cell or a cell line, can be contacted with a compound of interest and the differentiation of the pluripotent cell into an osteoblast can be visually assessed.
  • the differentiation of the pluripotent cell into an osteoblast can also be assessed by assaying the level of cellular alkaline phosphatase using a colorimetric assay.
  • total cell number is normalized to the level of cellular alkaline phosphatase by staining the cells with, for example, Alamar blue.
  • the mineralization of such cultured, differentiated cells can be determined by, for example calcin staining and/or von Kossa staining human) may be plated for culture on day 0. On day 1, cells may be differentiated. Also on day 1, test compounds may be added to the cultures. Differentiation may be analyzed (e.g., on day 4-10) using an alkaline phosphatase assay and cell viability may be measured using alamar blue. Extracellular matrix formation may also be measured, e.g., on day 21.
  • screening assays are provided that are useful in identifying compounds that modulate this functional activity, to thereby identify compounds that modulate osteoblast differentiation, osteoblast function, osteoblast activity, bone formation and/or bone mineralization.
  • the invention provides a method of identifying a compound as a modulator of the functional activity of Shn3/ERK interaction, the method comprising a) providing an indicator composition comprising Shn3, ERK and an ERK substrate; b) contacting the indicator composition with a test compound; c) determining level of phosphorylation of the ERK substrate in the presence of the test compound; and d) identifying the compound as a modulator of functional activity of Shn3/ERK when the level of phosphorylation of the ERK substrate in the presence of the test compound is different than the level of phosphorylation of the ERK substrate in the absence of the test compound.
  • the ERK is ERK2.
  • ERK substrates suitable for use in the above described screening assay include RUNX2 and ELK1.
  • test compound to modulate the phosphorylation of ERK substrates, in the indicator composition comprising Shn3, ERK and an ERK substrate, can be assessed using an in vitro kinase assay, wherein the read-out that is determined is the amount of phosphorylation of the ERK substrate in the presence of the test compound as compared to the amount of phosphorylation of the ERK substrate in the absence of the test compound.
  • a test compound can be selected that either increases the amount of ERK substrate phosphorylation or decreases the amount of ERK substrate phosphorylation, as compared to the amount of phosphorylation in the absence of the test compound.
  • Suitable types of in vitro kinase assays for assessing the phosphorylation of ERK substrate(s) in an indicator composition comprising Shn3, ERK and an ERK substrate, in the presence or absence of a test compound are well known in the art, and described in the Examples, and the components can be prepared using standard recombinant DNA techniques.
  • the invention provides a method of identifying a compound as an modulator of phosphorylation of Shn3 by ERK, the method comprising a) providing an indicator composition comprising Shn3 and ERK; b) contacting the indicator composition with a test compound; c) determining level of phosphorylation of Shn3 in the presence of the test compound; and d) identifying the compound as a modulator of
  • the ERK is ERK2.
  • the level of phosphorylation of Shn3 is determined at one or more amino acid positions selected from the group consisting of S811, S812, T851. S911 and S913.
  • test compound to modulate the phosphorylation of Shn3, in the indicator composition comprising Shn3 and ERK, can be assessed using an in vitro kinase assay, wherein the read-out that is determined is the amount of phosphorylation of Shn3 in the presence of the test compound as compared to the amount of phosphorylation of Shn3 in the absence of the test compound.
  • a test compound can be selected that either increases the amount of Shn3
  • Suitable types of in vitro kinase assays for assessing the phosphorylation of Shn3 in an indicator composition comprising Shn3 and ERK, in the presence or absence of a test compound, are well known in the art, and described in the Examples, and the components can be prepared using standard recombinant DNA techniques.
  • the indicator composition can be a cell that expresses the Shn3 protein and an ERK protein, for example, a cell that naturally expresses or, more preferably, a cell that has been engineered to express the proteins by introducing into the cell an expression vector encoding the proteins.
  • the cell is a mammalian cell, e.g., a mouse cell and/or a human cell.
  • the cell is derived from an adult.
  • the cell is a T cell.
  • the cell is a B cell.
  • the cell is an osteoblast.
  • the osteoblast is a primary calvarial osteoblast.
  • the cell is a mature osteoblast. In another embodiment, the cell is a mesenchymal stem cell.
  • cells for use in the screening assays of the invention are primary cells, e.g., isolated cells cultured in vitro that have not been immortalized.
  • cells for use in the screening assays of the invention are immortalized cells, i.e., cells from a cell line.
  • the cell line is the C3H10T1/2 cell line.
  • the cell line is the MC3T3-E1 cell line.
  • the cell line is the 293T cell line.
  • the indicator composition can be a cell-free composition that includes the proteins (e.g., a cell extract or a composition that includes e.g., either purified natural or recombinant protein).
  • the cell based and/or cell free assays are performed in a high-throughput manner.
  • the assays are performed using a 96-well format.
  • the assays of the invention are performed using a 192- well format.
  • the assays of the invention are performed using a 384-well format.
  • the assays of the invention are semi- automated, e.g., a portion of the assay is performed in an automated manner, e.g., the addition of various reagents.
  • the assays of the invention are fully automated, e.g., the addition of all reagents to the assay and the capture of assay results are automated.
  • the assays of the invention generally involve contacting an assay composition with a test compound or a compound of interest or a library of compounds for a predetermined amount of time or at a predetermined time of growth (either in vitro or in vivo) and assaying for the effect of the compound on a particular read-out.
  • an assay composition is contacted with a compound of interest or a library of compounds for the duration of the assay.
  • an assay composition is contacted with a compound of interest or a library of compounds for a period of time less than the entire assay time period.
  • cells may be cultured for a period of days or weeks and may be contacted with a compound following, for example, 14 days in culture.
  • cells are contacted with a compound of interest for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.
  • assay compositions of the invention are contacted with a compound for a predetermined time period, the compound is removed, and the assay composition is maintained in the absence of the compound for a predetermined period prior to assaying for a particular read-out.
  • the compounds of the invention may be assayed at concentrations suitable to the assay and readily determined by one of skill in the art.
  • assay compositions are contacted with millimolar concentrations of compounds.
  • assay compositions are contacted with micromolar concentrations of compounds. In another embodiment, assay compositions are contacted with nanomolar concentrations of compounds.
  • an Shn3 chimeric or fusion protein is used and/or an ERK chimeric or fusion protein is used.
  • an Shn3 or ERK “chimeric protein” or “fusion protein” comprises an Shn3 or ERK polypeptide operatively linked to a non-Shn3 or ERK polypeptide.
  • Shn3 polypeptide refers to a polypeptide having an amino acid sequence corresponding to Shn3 polypeptide
  • a non- Shn3 polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the Shn3 protein, e.g., a protein which is different from the Shn3 protein and which is derived from the same or a different organism.
  • an "ERK polypeptide” refers to a polypeptide having an amino acid sequence corresponding to ERK polypeptide
  • a “non-ERK polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the ERK protein, e.g., a protein which is different from the ERK protein and which is derived from the same or a different organism.
  • the Shn3 or ERK polypeptide can correspond to all or a portion of an Shn3 or ERK protein.
  • an Shn3 or ERK fusion protein comprises at least one biologically active portion of an Shn3 or ERK protein, e.g., an Shn3 BAS domain or D-domain or an ERK CD-domain.
  • an Shn3 BAS domain or D-domain or an ERK CD-domain e.g., an Shn3 BAS domain or D-domain or an ERK CD-domain.
  • the term "operatively linked" is intended to indicate that the Shn3 or ERK polypeptide and the non- Shn3 or ERK polypeptide are fused in-frame to each other.
  • the non-Shn3 or ERK polypeptide can be fused to the N-terminus or C-terminus of the Shn3 or ERK polypeptide.
  • the fusion protein is a GST-Shn3 or GST-ERK member fusion protein in which the Shn3 or ERK member sequences are fused to the C-terminus of the GST sequences.
  • the fusion protein is an Shn3-HA or ERK-HA fusion protein in which the Shn3 or ERK member nucleotide sequence is inserted in a vector such as pCEP4-HA vector (Herrscher, R. F. et al. (1995) Genes Dev. 9:3067-3082) such that the Shn3 or ERK member sequences are fused in frame to an influenza haemagglutinin epitope tag.
  • pCEP4-HA vector Herrscher, R. F. et al. (1995) Genes Dev. 9:3067-3082
  • Such fusion proteins can facilitate the purification of a recombinant Shn3 or ERK member.
  • Fusion proteins and peptides produced by recombinant techniques may be secreted and isolated from a mixture of cells and medium containing the protein or peptide. Alternatively, the protein or peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • a cell culture typically includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. Protein and peptides can be isolated from cell culture media, host cells, or both using techniques known in the art for purifying proteins and peptides. Techniques for transfecting host cells and purifying proteins and peptides are known in the art.
  • an Shn3 or ERK fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide, an His epitope tag, or an HA epitope tag).
  • An Shn3 or ERK encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the Shn3 or ERK protein.
  • test compound includes any reagent or test agent which is employed in the assays of the invention and assayed for its ability to influence the expression and/or activity of Shn3, ERK or a molecule in a signal transduction pathway involving Shn3 and ERK. More than one compound, e.g., a plurality of compounds, can be tested at the same time for their ability to modulate the expression and/or activity of, e.g., Shn3 in a screening assay.
  • screening assay preferably refers to assays which test the ability of a plurality of compounds to influence the readout of choice rather than to tests which test the ability of one compound to influence a readout.
  • the subject assays identify compounds not previously known to have the effect that is being screened for.
  • high throughput screening can be used to assay for the activity of a compound.
  • the compounds to be tested can be derived from libraries (i.e., are members of a library of compounds). While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds, such as benzodiazepines (Bunin et al. (1992). J. Am. Chem. Soc.
  • the compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12: 145).
  • Other exemplary methods for the synthesis of molecular libraries can be found in the art, for example in: Erb et al. (1994). Proc. Natl. Acad. Sci. USA 91: 11422-; Horwell et al. (1996) Immunopharmacology 33:68-; and in Gallop et al. (1994); J. Med. Chem. 37: 1233.
  • the combinatorial polypeptides are produced from a cDNA library.
  • Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries.
  • Candidate/test compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam, K. S. et al. (1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature 354:84-86) and
  • combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopep tides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang, Z. et al. (1993) Cell 72:767-778); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab').sub.2, Fab expression library fragments, and epitope-binding fragments of antibodies); 4) small organic and inorganic molecules (e.g., molecules obtained from D- and/or L-configuration amino acids); 2) phosphopep tides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang, Z. et al. (1993) Cell 72:767-778); 3) antibodies (e.g.,
  • KRC mutant forms of KRC (e.g., dominant negative mutant forms of the molecule).
  • enzymes e.g., endoribonucleases, hydrolases, nucleases, proteases, synthatases, isomerases, polymerases, kinases, phosphatases, oxido- reductases and ATPases
  • mutant forms of KRC e.g., dominant negative mutant forms of the molecule.
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • biological libraries are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12: 145).
  • Biotechniques 13:412-421 or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89: 1865-1869) or phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).
  • Compounds identified in the subject screening assays can be used in methods of modulating one or more of the biological responses regulated by Shn3. It will be understood that it may be desirable to formulate such compound(s) as pharmaceutical compositions prior to contacting them with cells.
  • test compound that directly or indirectly modulates, e.g., Shn3 or ERK expression or activity, or a molecule in a signal transduction pathway involving Shn3 and ERK, by one of the variety of methods described hereinbefore
  • the selected test compound can then be further evaluated for its effect on cells, for example by contacting the compound of interest with cells either in vivo (e.g., by administering the compound of interest to a subject) or ex vivo (e.g., by isolating cells from the subject and contacting the isolated cells with the compound of interest or, alternatively, by contacting the compound of interest with a cell line) and determining the effect of the compound of interest on the cells, as compared to an appropriate control (such as untreated cells or cells treated with a control compound, or carrier, that does not modulate the biological response).
  • an appropriate control such as untreated cells or cells treated with a control compound, or carrier, that does not modulate the biological response.
  • kits for carrying out the screening assays of the invention can include an indicator composition comprising Shn3, or a biologically active portion thereof, ERK, or a biologically active portion thereof, means for measuring a readout (e.g., protein interaction, substrate phosphorylation) and instructions for using the kit to identify modulators of biological effects of Shn3 and/or ERK.
  • an indicator composition comprising Shn3, or a biologically active portion thereof, ERK, or a biologically active portion thereof, means for measuring a readout (e.g., protein interaction, substrate phosphorylation) and instructions for using the kit to identify modulators of biological effects of Shn3 and/or ERK.
  • a readout e.g., protein interaction, substrate phosphorylation
  • Oligonucleotide Synthesis (M. J. Gait ed., 1984); MuUis et al. U.S. Pat. No. 4,683, 195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.
  • SHN3, HIVEP3 Schnurri-3 (SHN3, HIVEP3)-deficient mice display increased bone formation, but harnessing this observation for therapeutic benefit requires an improved understanding of how SHN3 functions in osteoblasts.
  • SHN3 as a novel dampener of ERK activity downstream of WNT signaling in osteoblasts.
  • a D-domain motif within SHN3 mediates the interaction with and inhibition of ERK activity and osteoblast differentiation, and knockin of a mutation in Shn3 that abolishes this interaction results in aberrant ERK activation and consequent osteoblast hyperactivity in vivo.
  • mice lacking SHN3 display an osteosclerotic phenotype with profoundly increased lamellar bone mass due to augmented osteoblast activity.
  • SHN3 controls protein levels of RUNX2, the master regulator of osteoblast differentiation, by promoting its proteasome-dependent degradation via WWP1 E3 ubiqutin ligase-mediated ubiquitination.
  • Wwpl 'A mice were examined and found to have no obvious alteration in bone mass (unpublished data).
  • the osteosclerotic bone phenotype in Shn3 ' mice was unable to be reversed by haploinsufficency of Runxl (Supplementary Fig. 1), however this may reflect that bone mass in long bones is less sensitive to reductions in Runxl gene dosage than the calvarium or clavicle.
  • Shn3 is a relatively large protein (>260KD) that contains few conserved structural motifs.
  • Mammalian Schnurri proteins contain two highly conserved ZAS domains that consist of a separated pair of C 2 H 2 zinc-fingers followed by an acidic and serine amino acid-rich sequence (Wu, L.C. (2002) Gene Expr. 10: 137).
  • a BAS domain which contains a basic amino acid-rich region followed by an acidic and serine amino acid-rich sequence is also found in each of the mammalian Schnurri proteins (see Figure 1A). Results from previous studies suggest that this family of proteins function as scaffolds that facilitate the formation of multimeric complexes that regulate certain signaling pathways and gene expression.
  • a series of Shn3 mutants were generated and their functional activity assessed.
  • SHN3-WT a fragment of SHN3 consisting of the first 1186 amino acids of SHN3
  • lenti viral-mediated transduction of human mesenchymal stem cells with the N-terminal portion of Shn3 consisting of the first 1186 amino acids (referred to as Shn3-NT) blocks the differentiation of these cells into osteoblasts as determined by the reduced formation of mineralized matrix and inhibited expression of osteoblast marker genes.
  • Shn3-NT also retains the ability to repress osteoblast differentiation when both the N-terminal and C-terminal ZAS domains are deleted. Deletion of the central BAS domain renders Shn3 incapable of repressing osteoblast differentiation of hMSCs.
  • the D-domain has been shown to be important for a MAPK interaction in many transcription factors.
  • this D-domain is observed in several key regulators of osteoblasts whose activities are controlled by ERK-phosphorylation (Ge, C. et al. (2009) J. Biol. Chem. 284:32533; Anjum, R. et al. (2008) Nat. Rev. Mol. Cell. Biol. 9:747) (see also Figure 2A). Therefore, we tested if MAPKs can bind to Shn3 through this D-domain. As shown in Figure 2B ERK could interact with full length Shn3 but not mutant Shn3 protein lacking this D-domain.
  • Shn3 expression constructs containing point mutations at various residues using alanine mutagenesis in which two prolines (PA; NP_001121186.1: p.[P884A; P885A]), three lysines (KA; p.[K886A;K887A;K888A]), two arginines (RA; p.[R889A; R891A]), or two leucines (LA; p.[L890A;L892A]) were substituted with alanines (see Figure 2E, left).
  • Lysine to arginine mutation has been shown to alter the post-translational modification of the protein but not protein-protein interaction due to sharing similar electronic and structural amino acid residues. Therefore, we tested if the lysine to arginine substitution in the BAS region of Shn3 (referred to as KR-BAS) was not altering to the ability of Shn3 to interact with ERK and inhibit RUNX2 activity and osteoblast differentiation (see Figure 2G and Figure 6D-E). Unlike the KA-BAS mutant, the KR-BAS mutant was able to bind to ERK2 protein, and to inhibit both RUNX2 activity and osteoblast differentiation, consistent with the model that these three lysines function by regulating the interaction of SHN3 with ERK2.
  • CD domain of MAPKs has been reported to be crucial for interaction with D-domain motifs of its target substrates (T. Tanoue et al. (2000) Nat. Cell. Biol. 2: 110; Liu, S. et al. (2006) Proc. Natl. Acad. Sci. USA 103:5326; Akella, R. et al. (2008) Biochim. Biophys. Acta 1784:48).
  • D319N an ERK2 protein containing CD domain mutation
  • GST-BAS protein interacts strongly with WT-ERK2 while its interaction with D319N ERK2 mutant is ablated.
  • interaction of GST-BAS with WT ERK2 is decreased in a dose dependent manner by adding ERK peptide inhibitor (ERK PI, described in Keleman, B.R. et al. (2002) J. Biol. Chem.
  • Example 1 The results in Example 1 suggest that the interaction between Shn3 and ERK is important for controlling osteoblast function. Therefore, to better understand how this interaction influences bone biology, we first sought to better understand the importance of the ERK MAPK pathway in osteoblast function. For this, we generated compound mutant mice with a global deletion of MEK2 and a specific deletion of MEK1 in osteoblast (referred to as Mekl osx Mek2 - " /- " ) that abolishes ERK activation within the osteoblast. Analysis of bone formation and remodeling revealed that compared to control mice, the Mekl osx"Mek2 - ⁇ /'- ⁇ mice display a short stature and severe osteopenia, along with rib fractures and die around d20.
  • HA-ERK2 and MYC-RUNX2 were transfected into HEK293 cells along with full-length Shn3. Immunoprecipitation analysis revealed that ERK2 could still bind to RunX2 even in the presence of Shn3 ( Figure 10B).
  • Shn3 aj+ ;Mekl iiJ+ ;Mek2 +A prxl-cre (Shn3/Mekl/2-DHet) triple heterozygous mice ( Figure 13C).
  • Shn3-Het mice display increased bone mass in the trabecular and cortical compartments of the appendicular skeleton, and Mekl/2-Het mice display near normal bone mass.
  • the triple heterozygous mice (Shn3/Mekl/2-DHet) display a reversal of the high bone mass phenotype imparted by Shn3 haploinsufficency.
  • Shn3 proteins were immunoprecipitated from immortalized Shn3 ⁇ A COB lysates that were previously infected with Flag-tagged Shn3-NT expressing lentivirus. Analysis of Shn3 via mass spectrometry revealed four phosphorylation sites at serine, namely amino acid positions S811, S812, S911 and S913.
  • phosphorylation sites contributes to Shn3 function to regulate osteoblast function and that no single site has a dominant effect.
  • Shn3 functions as a nuclear suppressor of the ERK MAPK in osteoblasts. Once ERK is phosphorylated, the active ERK translocates to the nucleus. In turn, phosphorylated and activated Shn3 represses the ERK activation in the nucleus thus providing a finely tuned mechanism to regulate osteoblast activity (schematized in Figure 3J).
  • Shn3 Kl/Kl mice mice bearing a knock-in allele of the KA mutant into the endogenous Shn3 locus (hereafter referred to as Shn3 Kl/Kl mice) (see Figure 11 A).
  • Shn3 Kl/Kl mice were born healthy at expected Mendelian ratios and do not display any gross developmental abnormalities.
  • analysis of WT and Shn3 mice revealed comparable levels of Shn3 transcript or protein levels (see Figure 4A and Figure 1 IB).
  • Shn3 Kl/Kl osteoblasts exhibited an increased mineralization and expression of Bsp and Ocn genes, but transcript levels of Osx and Runx2 were comparable between WT and Shn3 Kl/Kl osteoblasts (see Figures 4D, 4E, 4F, 4G and 4H). These results demonstrate that these three lysines (within the D domain) are critical for Shn3's ability to regulate osteoblast-mediated bone formation, both in vitro (see Example ID) and in vivo.
  • Shn3 acts to establish an activation-threshold within osteoblasts that arises in part through Shn3's localized regulation of ERK.
  • the ability of Shn3 to bind to ERK dampens this kinase activity and thereby prevents it from over-activating downstream substrates in response to high level of stimulation as a feedback loop.
  • ERK MAPK activity in osteoblasts deficient for Shn3 or expressing mutant Shn3 that is unable to bind ERK would be sustained when compared to WT osteoblasts.
  • ⁇ -catenin is a positive regulator of osteoblast differentiation, and limb-specific deletion of ⁇ - catenin results in a complete loss of bony structure in mouse embryos.
  • ⁇ -catenin is constitutively phosphorylated by ⁇ 8 ⁇ 3 ⁇ , which results in ⁇ - catenin ubiquitination and proteasome-dependent degradation. 4
  • This GSK3 ⁇ -mediated suppression of ⁇ -catenin is relieved by ERK-induced phosphorylation of ⁇ 8 ⁇ 3 ⁇ Thr43.
  • ERK may additionally contribute to ⁇ 8 ⁇ 3 ⁇ suppression by phosphorylating p90RSK, which in turn can promote ⁇ 8 ⁇ 3 ⁇ suppression via phosphorylation of Ser9. 40
  • phosphorylation levels of ⁇ 8 ⁇ 3 ⁇ (S9) and p90RSK were analyzed in COBs lacking MEK1 and MEK2 (Mekl/2 'A ).
  • Phosphorylation levels of ⁇ 8 ⁇ 3 ⁇ (S9) and p90RSK were significantly decreased in Mekl/2 'A COBs when compared with WT COBs ( Figure 14E). This was accompanied by a decrease in ⁇ - catenin levels and
  • SHN3 is able to suppress ERK activity and WNT-mediated ⁇ -catenin transcriptional activity.
  • C3H10T1/2 cells infected by vector or SHN3 expressing lentivirus were stimulated with various ligands relevant to osteoblast function, including FGF18, BMP2, TGF- ⁇ and a canonical WNT activator (xWNT8/Fz5 and LRP5) ( Figures 14A and 21A).
  • Shn3 KD mice 8 week old Shn3 KD and littermate control mice were fed either with PBS- or doxycycline-chow for 6 weeks and bone RNAs were extracted from femurs to measure Shn3 knockdown efficiency (Figure 16A). After 6 weeks of induction
  • Shn3 KD mice displayed a 30% reduction in Shn3 mRNA levels in bone relative to doxycycline- treated WT mice. Remarkably, this modest reduction of Shn3 transcripts was sufficient to enhance bone mass up to 25% in trabecular and cortical bones ( Figure 16B). Likewise, histomorphometric analysis revealed that Shn3 knockdown results in an increase of osteoblast numbers and activity on the bone surface ( Figure 16C). Therefore, even modest levels of reduction of SHN3 activity in adult mice results in enhanced bone formation. These results imply that compounds designed to block SHN3 expression or activity are attractive therapeutic agents for the treatment of osteoporosis.
  • Osteoblasts overlying a bone surface have no direct means to sense the thickness of the bone beneath them. Thus, they rely on the integration of extrinsic signals to modulate their activity to maintain consistent architecture throughout a given bone.
  • SHN3 appears to function as a rheostat that acts to interpret and transmit signals governing bone production.
  • Shn3 accomplishes this by dampening ERK activity in the context of WNT stimulation in osteoblasts ( Figure 16D).
  • SHN3 acts distal to the activation of ERK itself to prevent the phosphorylation of ERK substrates such as p90RSK and GSK3 .
  • SHN3 may regulate other pathways in addition to WNT signaling, possibly including the growth factor-mediated signaling pathways such as FGF2, FGF18, EGF, IGF1, BMP2, or TGFP that are able to activate ERK in osteoblasts.
  • the phenotype of Shn3 KI/KI is less severe than that of Shn3 - ⁇ /- " mice leaves open the possibility that SHN3 works via other mechanisms in addition to those described here ( Figures 41 and 20C).
  • this corresponds at a structural level to the binding of other possible SHN3 effectors such as WWPl outside of the BAS domain ( Figure 19A).
  • the ability of SHN3 to regulate osteoblast/osteoclast crosstalk likely maps outside of the BAS domain based on the absence of basal alterations in osteoclasts in Shn3 mice, though testing in a provocative model may be useful for further deconvolution of this point.
  • SOST Sclerostin
  • ⁇ -catenin appears to have diverse functions at various stages of osteoblast differentiation. At the level of very early osteoblasts or osteoblast progenitors, ⁇ -catenin serves to promote osteoblast differentiation at the expense of chondrocytic differentiation. (Hill, et al. (2005). Dev. Cell 8:727-738; Day, et al. (2005). Dev. Cell 8:739-750) In mature osteoblasts, ⁇ -catenin acts to suppress the production of signals that promote osteoclast differentiation. (Glass, et al. (2005). Dev.
  • SHN3 can be assigned to the category of regulatory MAPK scaffold proteins including JNK- interacting proteins (JIPs).
  • JIPs JNK- interacting proteins
  • this large (2283 aa) adaptor protein, SHN3, binds to many key regulators of osteoblast differentiation in a selective manner. Interestingly, these interactions do not require the Ddomain in the BAS domain (Table 1).
  • hMSCs Human mesenchymal stem cells
  • hMSCs Human mesenchymal stem cells
  • Primary calvarial osteoblasts (CalvOb), C3H10T1/2 cell line (mouse mesenchymal fibroblast-like cell line), immortalized calvarial osteoblasts (CalvObs or COBs), and bone marrow stromal cells (BMSC) were cultured in cc-MEM medium (Cellgro) containing 10% FBS, 2mM L-glutamine, 1% penicillin/streptomycin, 1% HEPES, and 1% nonessential amino acids and differentiated with ascorbic acid and ⁇ -glycerophosphate.
  • Primary CalvObs were differentiated underosteoblastt differentiation (OBD) conditions for 14 days and immortalized by transfecting with SV40 large T Ag using Amaxa nucleofector (Lonza).
  • OBD underosteoblastt differentiation
  • HEK293 cells human kidney embryonic cells
  • HEK293 FT cells were purchased from ATCC and Invitrogen, respectively. Plasmids were HA-ERK2 (wt, L198A, L232A, Y261A, D319N; Addgene plasmids), T7-ERK1 (Addgene plasmid), and HA-RSK2, HA-GSK3 , BIM, Flag-CREB (Addgene plasmid), HA-ELK1 (Addgene plasmid), Flag-Schnurri-2, Flag-TWISTl and 2, Flag-JNKl, 2, and 3 (Addgene plasmids), Flag-p38cc and ⁇ (Addgene plasmids), HA- CYCLIN Dl (Addgene plasmid), Flag-TAZ (Addgene plasmid), HA-ATF4, Flag-p65 and p50, Flag-PPARy (Addgene plasmid), Flag
  • Antibodies used were anti-phospho-BIM (S69), anti-phospho-GSK3 (S9), anti-phospho-MNKl (T197/202), anti-phospho-ERKl/2, anti-phospho-JNKl/2, anti-phospho- p38, anti-p38 (Cell Signaling); anti-JNKl/2, anti-HSP90, anti-GFP, anti-HA conjugated agarose, anti-c-Myc conjugated HRP and anti-HA conjugated HRP (Santa Cruz); anti-Xpress
  • cDNAs were PCR- amplified and cloned into pEF-Nuc mammalian expression vector (Invitrogen), pMyc-CMV mammalian expression vector (Clontech), pHASE/PGK-PURO lentiviral vector, or
  • pLenti6/capTEV-CT-DEST lentiviral vector (Invitrogen).
  • FL full length (1-2283 aa), AC; C- terminal deletion (1-2011 aa), ⁇ ; BAS deletion ( ⁇ 844-928 aa), ⁇ / ⁇ ; ZnF and BAS deletion ( ⁇ 635-928 aa), AZsl; ZAS1 deletion ( ⁇ 184-327 aa), AZs2; ZAS2 deletion ( ⁇ 1719-1902 aa), AZsl/2; ZAS1 and 2 deletion ( ⁇ 184-327 and ⁇ 1719-1902 aa), AZsl/2/B; ZAS1 and 2 and BAS deletion ( ⁇ 184-327, ⁇ 1719-1902, and ⁇ 844-928 aa), NT-WT; N-terminal fragments (1-1186 aa, 1-1084 aa, 1-900 aa, 1-850 aa, and 1-817 aa), ⁇ - ⁇ ; N-terminal fragment/ BAS
  • ZAS1, ZAS2, BAS, and ZnF and BAS domains were PCR-amplified and cloned into pEGFP-C2 mammalian expression vector (Clontech) or pEF-Nuc mammalian expression vector containing 3x nuclear localization sequences (Invitrogen). All amino acid numbering is relative to the reference sequence NP_001121186.1.
  • the TetR/O system uses a reverse tet iraws-repressor protein that responds to doxycycline (DOX).
  • DOX doxycycline
  • TetR protein recognizes the sequence of a tet-inducible promoter (TetO) and blocks gene transcription.
  • DOX bound TetRprotein is unable to bind to the TetO sequence, resulting in gene transcription.
  • shRNA of mouse Shn3 (5'-CCGG-CCTGCTCTCAAGTAGTTTGTA- CTCGAG- TACAAACTACTTGAGAGCAGG-TTTTTG-3') is cloned into the transgenic vector placing the transgene downstream of the TetO.
  • the transgenic vector is placed into the Rosa26 locus and transmitted through the germline of chimeric mice. Treatment of these transgenic mice with DOX prevents the TetR protein to the TetO sequence, resulting in transactivation of mouse Shn3 shRNA.
  • mice were skinned, eviscerated and fixed in 95% ethanol. Then skeletons were stained by Alizarin Red S/Alcian Blue and sequentially cleared in 1% potassium hydroxide.
  • paraffin sections of bones were produced from 8 week old mice. Limb tissues were dissected and fixed in 4% paraformaldehyde (PFA) in PBS. They were then decalcified by daily changes of 15% tetrasodium EDTA until soft and pliable. Tissues were dehydrated by passage through an ethanol series, cleared twice in xylene, embedded in paraffin, and sectioned. For morphological analyses tissue sections were stained with haematoxylin and eosin.
  • PFA paraformaldehyde
  • ⁇ CT analysis a Scanco Medical ⁇ CT 35 system with an isotropic voxel size of 7 and 20 ⁇ resolution was used to image the distal femur and skull, respectively. Scans were conducted in 70% ethanol and used an X-ray tube potential of 55 kVp, an X-ray intensity of 0.145 mA and an integration time of 600 ms.
  • femoral bone mass a region of trabecular bone 2.1mm wide was contoured, starting 280 microns from the proximal end of the distal femoral growth plate. Femoral trabecular bone was thresholded at 211 permille. Femoral cortical bone was thresholded at 350 permille.
  • Calvarium was thresholded at 260 permille.
  • a Gaussian noise filter optimized for murine bone was applied to reduce noise in the thresholded 2-diemensional image.
  • Three-dimensional reconstructions were created by stacking the thresholded, 2- dimensional images from the contoured region (greenblatt).
  • BMSCs bone marrow stromal cells
  • OBD osteoblast differentiation
  • Total mRNAs were purified from osteoblast cultures for use in quantitative RT-PCR reactions that measure the expression level of several genes that are regulated during osteoblast differentiation, including alkaline phosphatase (Alp), oxterix (Osx), osteocalcin (Ocn), collagen la (Coll), Runx2, and bone sialoprotein (Bsp).
  • Alpha alkaline phosphatase
  • Osx oxterix
  • Ostcalcin osteocalcin
  • Coll Coll
  • Runx2 bone sialoprotein
  • C3H10T1/2 or HEK293 cells grown on 12-well plates were transiently transfected using Effectene (QIAGEN) with the Runx2- (6xOSE2-luc) or NF- ⁇ - (PBII-luc) responsive reporter gene and the Renilla luciferase vector (Promega) together with plasmids expressing various Shn3 mutants in the absence or presence of Runx2.
  • HEK293 cells were stimulated with TNFcc (lOng/ml) for 24 hours 1 day after transfection with PBII-luc and the Renilla reporter genes along with various Shn3 mutants.
  • Jurkat T cells were transiently transfected using Amaxa nucleofector (Lonza) with 6xOSE-luc or APl-luc and the Renilla reporter genes along with the plasmids expressing various Shn3 mutants in the absence or presence of Runx2.
  • Amaxa nucleofector Lixza
  • APl-luc a nucleofector for expressing various Shn3 mutants in the absence or presence of Runx2.
  • PMA 20 ng/ml
  • ionomycin (1 ⁇ ) for 24 hours 1 day after transfection with API -responsive reporter gene (2xAPl-luc) and the Renilla reporter genes along with various Shn3 mutants.
  • C3H10T1/2 cells grown on 10 cm petri-dish were infected by vector or SHN3 expressing lentivirus and after puromycin selection, cells were transfected using Effectene (QIAGEN) with the ⁇ -catenin responsive reporter gene (Top-flash luc) and the Renilla luciferase vector (Promega) in the absence or presence of a WNT/Frizzed fusion protein (xWNT8/Fz5), a constitutively active mutant of LRP5 (LRP5-CA), or a
  • HEK293 cells were lysed in TNT lysis buffer (10 mM Tris, 50 mM NaCl, 5 mM EDTA, 2 mM NaF, 30 mM sodium
  • pyrophosphate 100 mM Na 3 V0 4 , 0.5 mM PMSF, 1 ⁇ g/ml leupeptin, and 5 ⁇ g/ml aprotinin, 1 % Triton X-100).
  • Cell lysates were incubated with Flag-, HA-, or Myc-conjugated agarose beads, fractionated by SDS-PAGE, and transferred to Immunobilon-P membranes (Millipore). Protein levels were normalized by immunoblotting with anti-GAPDH or HSP90 antibody.
  • Membranes were blocked in TTBS buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 % Tween-20) with 4 % Skim Milk and then incubated with the indicated antibodies. Finally, membranes were washed and incubated with HRP-conjugated secondary antibodies and developed with ECL (Thermo scientific). For endogenous interaction of Shn3 with ERK1/2, immortalized Shn3 +/+ and Shn3 A CalvObs were lysed and incubated with IgG control or anti-ERKl/2 antibody along with protein A-agarose beads for immunoprecipitation.
  • HEK293 cells were transfected with a construct expressing a xWNT8/Fz5 fusion protein and LRP5 and lysed and immunoprecipitated with anti ERKl/2 antibody and protein A-agarose. The immunoprecipitates were incubated with GST- ELKl in the absence or presence of recombinant His-SHN3.
  • kinase buffer (20 mM HEPES, pH 7.5, 20 mM MgCl 2 , 1 mM EDTA, 2 mM NaF, 2 mM-glycerophosphate, 1 mM DTT, 10 ⁇ ATP) containing either GST, GST-ELKl, GST-BAS, GST-Zn/B, GST-Smad3, Myc-Runx2, Shn3 L (50-930 aa), or Shn3 s (630-930 aa) and 10 ⁇ of [ ⁇ 32 ⁇ ] ⁇ (Perkin Elmer).

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Abstract

La présente invention concerne les interactions spécifiques intervenant entre la protéine Shn3 et la kinase ERK dans le noyau des ostéoblastes et le rôle essentiel desdites interactions dans la régulation de la différentiation et de la fonction des ostéoblastes et, donc, dans la régulation de la formation osseuse. Nous avons démontré que la protéine Shn3 est l'un des principaux agents de régulation de la fonction des ostéoblastes par son rôle de régulateur à la baisse, spécifique d'un tissu, de l'activité de la kinase ERK. L'invention concerne également des procédés d'identification de modulateurs de la formation osseuse agissant par modulation des interactions intervenant entre la protéine Shn3 et la kinase ERK, par modulation de la phosphorylation des substrats de ERK ou par modulation de la phosphorylation de la protéine Shn3 par la kinase ERK.
PCT/US2013/025258 2012-02-08 2013-02-08 Dosages permettant d'identifier des composés modulant la fonction des ostéoblastes et la formation osseuse WO2013119893A1 (fr)

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US9745589B2 (en) 2010-01-14 2017-08-29 Cornell University Methods for modulating skeletal remodeling and patterning by modulating SHN2 activity, SHN3 activity, or SHN2 and SHN3 activity in combination

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9745589B2 (en) 2010-01-14 2017-08-29 Cornell University Methods for modulating skeletal remodeling and patterning by modulating SHN2 activity, SHN3 activity, or SHN2 and SHN3 activity in combination

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