WO2006102417A2 - Mechanisms of osteoinduction by lim mineralization protein-1 (lmp-1) - Google Patents
Mechanisms of osteoinduction by lim mineralization protein-1 (lmp-1) Download PDFInfo
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- A61P19/00—Drugs for skeletal disorders
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
Definitions
- rhBMP-2 Food and Drug Administration approved rhBMP-2 for use as a bone graft substitute in interbody spine fusions.
- this technology is not feasible for many patients with bone healing needs due to an unexpectedly high dose required in humans which has resulted in a very high cost (Boden SD, Zdeblick TA, Sandhu HS, and Heim SE. Spine 2000;25:376-81; Acke ⁇ nan SJ, Mafilios MS, and Polly DW, Jr. Spine 2002;27:S94-S99).
- a 15,000-fold higher concentration of BMP-2 is required to induce bone in humans (1.5 mg/mL) than in cell culture (100 ng/mL).
- LMP-I Termed LIM Mineralization Protein
- LMP-I also has considerable potential as a local, regional, or systemic anabolic strategy for increasing bone density in patients with osteoporosis. However, before clinical applications can be seriously considered it will be critical to understand the mode of action of this protein. The present invention addresses this problem.
- Osteoblasts are thought to differentiate from pluripotent mesenchymal stem cells, the maturation of which results in the secretion of an extracellular matrix which can mineralize and form bone.
- the regulation of this complex process involves a group of signaling glycoproteins known as bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF- ⁇ ) superfamily.
- BMPs bone morphogenetic proteins
- TGF- ⁇ transforming growth factor-beta
- LMP-I mesenchymal stem cells
- the present invention relates to combinatorial therapeutic strategies including small molecules and peptide mimics of LIM mineralization proteins, primarily LMP-I, to overcome the dose-related translational barriers for BMP-2 therapeutics.
- LIM mineralization protein (LMP) action in modulating growth factor responsiveness in cells, tissues and organisms.
- LMP LIM mineralization protein
- LMP-I enhances responsiveness to BMP-2 in MSCs.
- LMP-I interacts in vitro with an 85 kDa protein, identified as Smurfl, a key regulator of the degradation of BMP-2 signaling molecules, Smadl and Smad5. It is also demonstrated here that endogenous Smurfl and LMP-I co-immunoprecipitate from cells, suggesting the physiological relevance of the interaction.
- LMP-I phosphorylated Smadl
- P-Smadl phosphorylated Smadl
- BMP-2 phosphorylated Smadl
- LMP-I -induced inhibition of Smurfl WW domain antibody binding to Smurf identified the WW domain as the region of Smurfl that LMP-I binds.
- analysis of LMP-I sequence has identified two potential WW domain interacting motifs within an osteoinductive region of LMP-I. It is also demonstrated that LMP-I increases BMPRlA levels in support of the hypothesis that LMP interrupts the Smurfl/Smad ⁇ mediated degradation of the BMP receptor.
- LMP-I is shown herein to interact with Jabl, an adaptor protein which regulates degradation of the common Smad, Smad4 resulting in increased nuclear Smad4.
- Jabl an adaptor protein which regulates degradation of the common Smad, Smad4 resulting in increased nuclear Smad4.
- the compounds include small proteins and peptides.
- the ability to use a single exposure dose of a recombinant TAT-LMP fusion protein is demonstrated, confirming that continuous LMP-I expression is not required for an effective therapeutic outcome and opens the door for design of an LMP-mimic small compound.
- LMP-I represents a powerful control mechanism over BMP signaling and responsiveness.
- This LMP-I interaction occurs with the Smurf WW2 domain, is dependent on a specific PY motif in LMP-I, and can be mimicked by a small peptide containing only that motif.
- LMP-I competitively binds to Smurf 1, preventing ubiquitin-mediated proteasomal degradation of Smads, contributing to an enhanced cellular responsiveness to BMP -2.
- Such small molecules would be more easily synthesized, stored, and delivered for clinical use to induce bone formation alone or with much lower doses of BMP-2 than are currently required in the clinical setting.
- therapeutics that modulate the effects of LMP-I have the potential to either replace BMP-2 as a strategy to induce bone formation or to serve as a method to enhance the efficacy of rhBMP-2, lowering the dose and cost of its use as an inducer of bone formation.
- Combination therapy The invention relates to treatment of diseases using combination therapy.
- the novel LMP agents described herein may be used in conjunction with BMP agents.
- the present invention provides a method of inducing bone deposition by coadministration of at least one LMP agent and a therapeutically effective dose of at least one BMP agent.
- LMP agents are capable of accelerating bone formation by enhancing the BMP agent's responsiveness.
- the LMP agent accomplishes this by affecting a BMP agent including but not limited to endogenous BMP protein, exogenous BMP protein, exogenous BMP protein fragment, and exogenous BMP protein variant fragment.
- the present invention may therefore be used to decrease the time required to form new bone in the presence of a BMP agent comprising administering at least one LMP agent.
- LMP LIM mineralization protein
- LMP-3 LMP-3 and biologically active fragments thereof. More detailed descriptions, including sequences, can be found in US Patent 6,300,127, pending application USSN 10/951,236, and pending application USSN 09/959,578 filed by Boden et al, the entire teachings of which are incorporated herein by reference.
- LMP-2 is excluded as it is non- osteogenic. Growth factors suitable in the invention include bone morphogenic proteins (BMP) including BMP-2.
- LMP agent includes a functional fragment of an LMP protein, a functional fragment of an LMP protein with a protein transduction domailn (PTD) attached, an LMP protein with a PTD attached, an LMP protein without a PTD attached, a functional fragment of an LMP protein variant, an LMP protein variant with a PTD attached, an LMP protein variant without a PTD attached, an oligonucleotide sequence encoding any of the above, and an LMP gene.
- PTD protein transduction domailn
- BMP agent includes a functional fragment of a BMP protein, a functional fragment of a BMP protein with a PTD attached, a BMP protein, a functional fragment of a BMP protein variant, a BMP protein variant, an endogenous BMP protein, exogenous BMP protein, an exogenous BMP protein fragment, an exogenous BMP protein variant fragment, an oligonucleotide sequence encoding any of the above, and a BMP gene.
- BMP-2 especially rhBMP-2.
- variants molecules derived in sequence from the aforementioned LMP agents or BMP agents in which amino acids have been deleted ("deletion variants"), inserted (“addition variants”), or substituted (“substitution variants”). Molecules having such substitutions, additions, deletions, or any combination thereof are termed individually or collectively "variant(s).” Such variants should, however, maintain at some level (including a reduced level) the relevant activity of the unmodified or "parent” molecule (e.g., an LMP variant possesses the ability to modulate BMP responsiveness or to bind Smurfl).
- parent molecule refers to an unmodified molecule or a variant molecule lacking the particular variation under discussion.
- amino acid sequence variant(s) There are two principal variables in the construction of amino acid sequence variant(s): the location of the mutation site and the nature of the mutation. In designing variant(s), the location of each mutation site and the nature of each mutation will depend on the biochemical characteristic(s) to be modified. Each mutation site can be modified individually or in series, e.g., by (1) deleting the target amino acid residue, (2) inserting one or more amino acid residues adjacent to the located site or (3) substituting first with conservative amino acid choices and, depending upon the results achieved, then with more radical selections.
- An amino acid sequence addition may include insertions of an amino- and/or carboxyl-terminal fusion ranging in length from one residue to one hundred or more residues, as well as internal intra-sequence insertions of single or multiple amino acid residues.
- Internal additions may range generally from about 1 to 20 amino acid residues, preferably from about 1 to 10 amino acid residues, more preferably from about 1 to 5 amino acid residues, and most preferably from about 1 to 3 amino acid residues.
- An example of an amino- or a carboxy-terminus addition includes chimeric proteins comprising the amino-terminal or carboxy-terminal fusion of the parent molecules with all or part of a transduction peptide or other conjugate moiety.
- Amino acid sequence deletions generally range from about 1 to 30 amino acid residues, preferably from about 1 to 20 amino acid residues, more preferably from about 1 to 10 amino acid residues and most preferably from about 1 to 5 contiguous residues.
- a "functional fragment” of a protein is any fragment or portion of a protein which retains the characteristic of interest of the parent protein or peptide.
- biologically active means retaining that characteristic or property in question from the parent molecule.
- the protein or peptide may possess multiple activities such as would be provided by multiple binding sites. These binding sites or domains may be identical or variable and may be in sequence or separated by non-binding site amino acids.
- recombinant proteins, peptides or fusion proteins may be produced.
- the LMP agents or BMP agents of the present invention are conjugated to other proteins or peptides.
- Protein transduction domains (PTDs) and attachment of these to proteins and peptides are contemplated.
- the PTD is the HIV-TAT protein.
- a variant protein or peptide will preferably be substantially homologous to the amino acid of the parent molecule or a portion or a domain of the parent molecule from which it is derived.
- substantially homologous means a degree of homology that is in excess of 80%, preferably in excess of 90%, more preferably in excess of 95% or most preferably even 99%. Homology is determined relative to the smaller peptide or variant and is measured across that domain, site or fragment in the parent from which the variant or peptide is derived.
- the invention also comprises chemically modified derivatives of the parent molecule(s) in which the peptide is linked to a nonproteinaceous moiety (e.g., a polymer) in order to modify its properties.
- a nonproteinaceous moiety e.g., a polymer
- derivatives Such derivatives may be prepared by one skilled in the art given the disclosures herein. Conjugates may be prepared using glycosylated, non-glycosylated or de-glycosylated parent molecule(s) and suitable chemical moieties. Typically non- glycosylated molecules and water-soluble polymers will be used.
- Other derivatives encompassed by the invention include post-translational modifications (e.g., N-linked or
- polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein peptide.
- Water-soluble polymers are desirable because the protein or peptide to which each is attached will not precipitate in an aqueous environment, such as a physiological environment.
- the polymer will be pharmaceutically acceptable for the preparation of a therapeutic product or composition.
- One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/protein conjugate will be used therapeutically and, if so, the therapeutic profile of the protein (e.g., duration of sustained release; resistance to proteolysis; effects, if any, on dosage; biological activity; ease of handling; degree or lack of antigenicity and other known effects of a water-soluble polymer on a therapeutic proteins).
- Variants and/or derivatives may be screened to assess their physical properties in vitro and can be subsequently screened in vivo in the models described herein. It will be appreciated that such variant(s) will demonstrate similar properties to the unmodified molecule, but not necessarily all of the same properties and not necessarily to the same degree as the corresponding parent molecule.
- Oligonucleotide sequences of the present invention include those polymeric nucleic acid sequences which would "code for" the protein or peptide of interest. Those of ordinary skill in the art will appreciate the degeneracy of the genetic code and that variable codons may still produce the same protein on translation.
- oligonucleotide includes nucleic acid sequences which code for the proteins or peptides of the invention or their parent molecules, including but not limited to the LMP and BMP agents and vectors encoding said agents as well as small interfering RNAs (siRNAs) designed to target the genes disclosed herein, especially those involved in BMP and LMP signalling pathways.
- oligonucleotides of the present invention include siRNAs designed to LMP-I, Smurfl, Smurf2 and Jabl.
- Pharmaceutical compositions also provides for pharmaceutical compositions in the form of an osteogenic composition.
- an "osteogenic composition” is a composition comprising a therapeutically effective amount of at least one BMP agent combined with at least one other agent and optionally, at least one pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
- the osteogenic composition comprises a therapeutically effective amount of at least one
- an osteogenic composition comprises at least one Smurf binding agent; and at least one BMP agent; and optionally, at least one pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant, hi the present invention, Smurf binding agents include LMP-I protein, and LMP-I protein fragments, variants or derivatives, and siRNA specific for Smurf mRNA.
- the Smurf is selected from the group consisting of Smad Ubiquitin Regulatory Factor-1 (Smurfl) and
- the osteogenic composition comprises at least one phosphorylated Smad 1 competitive binding agent and at least one BMP agent, hi the present invention, the phosphorylated Smad 1 competitive binding agent includes but is not limited to an LMP-I protein, and LMP-I protein fragment, variant or derivative, and siRNA specific for phosphorylated Smad 1.
- the osteogenic composition comprises at least one phosphorylated Smad 5 competitive binding agent and at least one BMP agent.
- the phosphorylated Smad 5 competitive binding agent includes but is not limited to an LMP-I protein, and LMP-I protein fragment, variant or derivative, and siRNA specific for phosphorylated Smad 5.
- an osteogenic composition comprising at least one phosphorylated Smad 4 competitive binding agent and at least one BMP agent.
- the phosphorylated Smad 4 competitive binding agent includes but is not limited to an LMP-I protein, and LMP-I protein fragment, variant or derivative, siRNA specific for phosphorylated Smad 4.
- compositions may also comprise incorporation of any of the therapeutic molecules or agents into liposomes, microemulsions, micelles or vesicles for controlled delivery over an extended period of time.
- therapeutically effective amount means an amount which provides a therapeutic effect for a specified condition and route of administration. Whether an amount is therapeutically effective may be determined on a stand alone or combinatorial basis.
- an LMP agent is administered in combination with a BMP agent, wherein the dose or amount of BMP agent is subtherapeutic as compared to conventional rhBMP-2 therapy alone.
- a BMP agent at subtherapeutic doses or amounts would not, therefore, be therapeutically effective but when combined with the LMP agents of the present invention would represent a therapeutic dose or amount.
- the pharmaceutical compositions of the present invention may be administered in different ways, i.e., intrathecal injection, subcutaneous, intravenous, intraperitoneal, intramuscular injection, in an implant or combinations thereof.
- the administration of the LMP agents of the present invention may occur before, after or simultaneously with the BMP agent and may be to a single targeted site or separate sites.
- Sites for administration include, but are not limited to an intervertebral space, a facet joint, site of a bone fracture, bones of the mouth, chin and jaw, and an implant site.
- the therapeutic methods of the invention further comprises a co-therapeutic treatment regimen comprising administering a therapeutically effective amount of an LMP agent in combination with a therapeutically effective amount of a BMP agent to treat disease in a patient.
- a co-therapeutic treatment regimen means a treatment regimen wherein two agents are administered simultaneously, in either separate or combined formulations, or sequentially at different times separated by minutes, hours or days, but in some way act together to provide the desired therapeutic response.
- Dosages of the LMP agent, BMP agent or compositions of the present invention may range from InM to 20OnM if delivered as a recombinant fusion protein with a PTD attached or from 0.1 to 100 MOI (multiplicity of infection, i.e. number of infectious viral particles per cell) if delivered by an adenovirus or similar vector.
- MOI multiplicity of infection, i.e. number of infectious viral particles per cell
- doses of one agent may be lowered when the dose of a co-administered agent is raised.
- the dose of the BMP agent administered may be lowered.
- therapeutically effective dose of BMP agent is less than the currently acceptable therapeutically effective amount.
- 1.5mg/mL of bone formed is the therapeutic concentration of rhBMP-2 in primates in vivo with smaller doses effective in cell culture and rodents.
- BMP agent is at least 10-fold less than the dose required in conventional therapy.
- the dose required in conventional therapy can be 20 mg rhBMP-2 per site of 10 cc of bone formation.
- the therapeutically effective dose of BMP is at least 20- fold, 50-fold, 100-fold, 1000-fold, 5000-fold or 10,000-fold less than the dose required in conventional BMP therapy when administered in combination with an LMP agent.
- the therapeutic agents and compositions of the present invention are useful in treating subjects having compromised bone conditions.
- the invention provides for a method of treating a bone disorder using a therapeutically effective amount of an LMP agent in combination with a BMP agent.
- the bone disorder or compromised bone condition may be any disorder characterized by bone loss (osteopenia or osteolysis) or by bone damage or injury.
- Such bone conditions include but are not limited to broken bones, bone defects, bone transplant, bone grafts, bone cancer, joint replacements, joint repair, fusion, facet repair, bone degeneration, dental implants and repair, bone marrow deficits and other conditions associated with bone and boney tissue.
- bone defects include but are not limited to a gap, deformation or a non-union fracture in a bone.
- bone degeneration examples include but are not limited to osteopenia or osteoporosis.
- the bone defect is due to dwarfism.
- the invention is especially useful for joint replacement or repair wherein the joint is vertebral, knee, hip, tarsal, phalangeal, elbow, ankle, sacroiliac or other articulating/non- articulating joint.
- the invention includes a process for engineering bone tissue comprising combining at least one LMP agent and at least one BMP agent with a cell selected from the group consisting of osteogenic cells, pluripotent stem cells, mesenchymal cells, and embryonic stem cells. Also, disclosed is the engineered bone tissue produced by the above process. A method for inducing bone formation in a subject comprising administering the engineered bone tissue of the present invention is contemplated.
- the invention includes a process for engineering bone tissue comprising combining at least one phosphorylated Smad 4 competitive binding agent and at least one BMP agent with a cell selected from the group consisting of osteogenic cells, pluripotent stem cells, mesenchymal cells, and embryonic stem cells. Also included is engineered bone tissue produced by this process. In another aspect, the invention includes a method for inducing bone formation in a subject comprising administering the engineered bone tissue as described in this paragraph.
- Also included in the invention is a method for inducing deposition and maturation of bone in a subject having compromised bone conditions comprising administering to the subject at least one Jab 1 -inhibiting agent and a therapeutically effective dose of at least one BMP agent.
- MSCs Mesenchymal stem cells, (MSCs) at passage 2 are purchased from Cambrex Bio Sciences. Cells are grown at 37 0 C in 5% CO 2 in MSCBM media supplemented with MSCGM Singlequots (Cambrex Bio Sciences), split at confluence, and plated at 3 x 10 4 cells/well in 6-well dishes at passage 4 in these studies. The next day treatments are applied in the presence of 50 uM L-Ascorbic Acid 2-Phosphate and 5 mM ⁇ -glycerol phosphate (Sigma- Aldrich). Medium is changed every 3-4 days with re-application of treatments where appropriate. Cells are transduced for 30 min with adenoviral constructs in 0.5 ml serum free medium. rhBMP-2 will continue to be supplied as a gift from Wyeth
- LMP-I To show that increased levels of LMP-I result in increased levels of phosphorylated Smadl and Smad5, hMSCs are treated with the doses of LMP-I that successfully synergize with BMP-2 to determine the timecourse of increased of phosphorylated Smadl in the nucleus when the agents are applied alone or together.
- Both Smadl and Smad5 are measured.
- Antibody to phosphorylated Smadl is available and is used for Western analysis.
- Antibody to phosphorylated Smad5 is not available so an antibody that detects both phosphorylated and unphosphorylated Smad5 is used; and a phosphoserine antibody on a separate blot is used to determine the phosphorylation state.
- a phosphoserine antibody on a separate blot is used to determine the phosphorylation state.
- comparison of the two blots should allow determination of whether P-Smad5 also increases in the nucleus. Treating cells with the same adenoviral vector carrying GFP cDNA as a control is not expected to have an effect on nuclear levels of P-Smads.
- Increased P-Smad levels are expected to occur rapidly in response to BMP-2, since that involves phosphorylation of existing Smad proteins to activate the intracellular signaling cascade.
- LMP-I cDNA must be transcribed and translated into an intracellular protein, a process requiring several hours.
- a delay in increased nuclear levels of P-Smads is expected in response to LMP-I as compared to the response to BMP-2.
- SDS-PAGE and Westen blotting SDS-PAGE is performed using 10% gels and transferred to nitrocellulose membrane.
- the membrane is blocked with milk protein, incubated with specific antibody, washed with Tris Buffered Saline containing 0.1% Tween 20 (TBST), incubated with anti-rabbit goat IgG-linked to horseradish peroxidase (NEN), and again washed with TBST.
- Chemiluminescent substrates are applied to the membrane and the signal is detected by exposing the membrane to X-ray film 30 seconds.
- RNA extraction RNA is isolated from cells grown in 6-well plates using RNeasy Mini Kits as specified by the manufacturer (Qiagen). Briefly, cells are harvested and disrupted in RLT buffer. The lysate is passed over QiaShredder columns, and the resulting eluate brought to 35% EtOH and passed over RNeasy columns to bind the RNA to the silica-gel membrane. After washing the bound RNA with RWl buffer and then RPE buffer, the
- RNA is eluted from the membrane with water. All RNA samples are DNase treated either using the Qiagen RNase-Free DNase Set during the RNeasy procedure or after final harvest of the RNA using the Ambion DNA-free Kit. After completion of the digestion, 5 ⁇ l of DNase Inactivation Buffer is added, the solution incubated for 2 minutes at RT, and the samples centrifuged for 1 min in a microfuge. The RNA containing supernatant is removed and stored at -7O 0 C.
- the initial RNA prep is digested for 1 h at 37 0 C with the restriction enzyme Rsal to cleave any contaminating plasmid DNA, the RNA reisolated using an RNeasy kit, and DNase treated with the Ambion reagents.
- Each sample consists of RNA isolated from 2 wells of a 6-well plate and at least three samples are isolated for each treatment/time point.
- RNA Two ⁇ g of total RNA is reverse transcribed in a lOO ⁇ l total volume containing 50 mM KCl, 10 mM Tris, pH 8.3, 5.5 mM MgCl 2 , 0.5 mM each dNTPs, 0.125 ⁇ M random hexamer, 40 units RNase Inhibitor, and 125 units MultiScribe (Applied Biosystems). In control samples the RNase inhibitor and MultiScribe are omitted. Samples are incubated for 10 min. at 25 0 C, 30 minutes at 48 0 C, and then 5 min. at 95 0 C to inactivate the enzyme.
- PCR Real-time Reverse Transcription-Polvmerase Chain Reaction
- Real-time PCR is then performed on 5 ⁇ l of the resulting cDNA in a total volume of 25 ⁇ l containing 12.5 ⁇ l of 2X SYBR Green PCR Master Mix (Applied Biosystems), and 0.8 ⁇ M each primer.
- the PCR parameters used are 2 min. at 5O 0 C, 10 min. at 95 0 C, and 45 cycles of 95 0 C for 15 sec. followed by 1 min. at 62 0 C.
- PCR is also performed as described on a 1/800 dilution of the cDNA with 18S primers for normalization of the samples. Relative RNA levels were calculated using the ⁇ ⁇ Ct method (Applied Biosystems).
- the primers listed in Table 1 have been synthesized and successfully measured mRNA levels of gene expression in human MSCs.
- Ad35LMP-l Probe 6FAM-CATCGATGCTCAGCACCCAGTCACC-TAMRA 3 hmrSMAD-1 ACCCTGTCTGAGGAGCGTGTA 4
- siRNA treatment of cells MSCs are transfected with Lipofectamine 2000 (Invitrogen) or Oligofectamine (Invitrogen) transfection reagent and either irrelevant siRNA or specific siRNA sequences (see Table 2). Silencing of the genes and specificity is confirmed by real-time RT-PCR analysis of specific mRNA levels and Western analysis of protein levels.
- siRNA sequences siRNA Sense Sequence (5'-3') SEQ ID NO:
- human MSCs are plated at 3 x 10 4 cells/well in 6- well plates, grown overnight, and treated with an irrelevant siRNA or siRNA specific for Smurfl or Smurf2. Twenty-four hours after siRNA treatment, cells are treated with Ad5F35-LMP-l (0, 5, 10 pfu/cell) or Ad5F35-GFP. Cells are harvested 2, 4, 8, 12, 24, or 48 hr later and both cytoplasmic and nuclear proteins subjected to immunoprecipitation using LMP-I antibody and Western analysis of co-localizing proteins using the WW domain antibody.
- RNA and protein fractions are harvested at the same timepoints and the levels of Smurfl and Smurf2 mRNA measured by real-time RT-PCR using primers specific for Smurfl or Smurf2. Reduction of protein levels by siRNA is confirmed by Western blots using newly acquired antibodies thought to be specific to Smurfl and Smurf2.
- Reduced Smurfl levels are expected to reduce the amount of the WW domain immunoreactive binding protein observed by Western analysis after immunoprecipitation using LMP-I antibody compared with controls that have normal levels of Smurfl .
- reduced Smurf2 levels are expected to have no effect on the amount of WW domain immunoreactive binding protein as we do not expect LMP-I to bind Smurf2.
- human MSCs are plated at 3 x 10 4 cells/well in 6-well plates, grown overnight, and treated with a control irrelevant siRNA or siRNA specific for Smurfl.
- BMP-2 100 ng/ml is applied to some cultures when the siRNA is removed.
- Cells are harvested 4, 8, 12, 24, 36, or 48 hr later and nuclear proteins are analyzed for the presence of P-Smads by Western blot.
- the effectiveness of the siRNA applied to reduce the RNA and protein levels is monitored as described herein.
- Another group of MSCs plated as above are grown in differentiation medium for 21 days and stained with Alizarin Red to assess matrix mineralization.
- siRNA to Smurfl would represent a therapeutic opportunity for matrix mineralization.
- human MSCs are plated at 3 x 10 4 cells/well in 6-well plates, grown overnight, and treated with a control irrelevant siRNA or siRNA specific for Smurfl.
- Cells are harvested 4, 8, 12, 24, 36, or 48 hr later, cytoplasmic proteins are analyzed for the presence of total Smadl and phospho-Smadl by Western blot and ELISA.
- Smurfl are monitored as described herein. Another group of MSCs plated as above are grown in differentiation medium for 21 days and stained with Alizarin Red to assess matrix mineralization. Expected Results: It is expected that treatment will result in increased cytoplasmic Smadl in cells treated with siRNA to Smurfl as this protein should not be targeted for proteasomal degradation in the absence of Smurfl . It has been shown, in other examples herein, that increased cytoplasmic levels of phospho-Smadl in response to overexpression of LMP-I and BMP-2 treatment. This result is believed to have been caused by LMP-I blocking proteasomal degradation of Smadl. Thus, we expect reduction in Smurfl levels to mimic LMP-I in blocking Smurfl targeting of Smadl for degradation.
- Biotin transfer Assay for detection of LMP-I interacting proteins Sulfo-SBED (Pierce), a trifunctional cross-linking agent, contains three functional groups (a photoactivatable aryl azide, a sulfonated N-hydroxy succinimide active ester with a cleavable disulfide group and a biotin moiety) and is widely used to identify interacting proteins (Neely KE, Hassan AH, Brown CE, Howe L, and Workman JL. MoLCeU Biol. 2002;22:1615-25). LMP-I is labeled using this reagent, incubated as bait with nuclear proteins and cross-linked to interacting proteins by UV (365 nm).
- Biotin-containing target proteins are separated using neutravadin beads, detected by Western blotting with neutraviddin-HRP and the signal is developed with chemiluminescent substrate. Corresponding protein bands are in-gel digested with trypsin. Tryptic peptides are recovered, concentrated and their mass profile is analysed by MALDI-TOF at the Emory University Microchemical Facility.
- LMP-It A 223 aa osteoinductive truncated LMP-I variant (missing LIM domains ' ): Although LMP-I is a LIM domain protein, it has been shown that a truncated 223 aa variant of LMP-I which lacks the LIM domains still makes bone in vitro and in vivo (Liu).
- LMP-It truncated LMP-I fusion protein containing a Protein Transduction Domain (PTD) that readily enters cells can be designed.
- PTD Protein Transduction Domain
- RNA samples are analyzed by real-time RT-PCR for mRNA levels of Dlx5, Smad6, and BMP- 2. Similarly secreted, cytoplasmic and nuclear proteins are analyzed by ELISA using commercially available antibodies to Dlx5, Smad 6, and BMP-2. Expected Results: It is expected that LMP-It protein will enhance the osteoinductive responsiveness of MSCs to BMP-2 as seen with overexpression of the full length LMP-I protein. It is also expected that truncated LMP-I will bind to Smurfl and prevent WW domain antibody binding.
- Blots are incubated with Smurf antibody at 1:5000 dilution (Rabbit antibody raised to WW-domain peptide). After washes, blots are incubated with HRP -labeled Anti-rabbit second antibody. The washed blots are then incubated with ECL substrate solution and the membranes are exposed to X-ray film for signal detection.
- Protein A-based immunoprecipitation assay Protein A-agarose beads are incubated with LMP-I antibody, washed 3 times, incubated with nuclear proteins, and washed again to remove unbound protein. Bound proteins are eluted by 2 washes in 0.1 M citric acid, pH 2.7. The eluates are neutralized with Tris base and concentrated by centricon tubes (Ambicon) prior to SDS-PAGE and Western blotting.
- Modified pulse chase assay for nuclear and cytoplasmic analyses of proteins- Nuclear analysis It has been shown herein that phosphorylated Smadl is increased in the nucleus within 4 hrs in response to LMP-I overexpression and is further increased at 8 hours. Possible explanations include: 1) increased gene expression of Smadl; 2) increased phosphorylation of the cytoplasmic pool of unphosphorylated Smadl with subsequent translocation to the nucleus; and 3) reduced degradation of Smadl. The short time required for the P-Smad increase to occur does not rule out increased transcription and translation of Smadl . However, the 4 hr time frame makes it less likely that LMP-I can be transcribed and translated and also cause increased subsequent transcription and translation of Smadl.
- P-Smad5 and result in a reduced rate of degradation compared with P-Smads in cells not overexpressing LMP-I.
- Cytoplasmic analysis Human MSCs are plated at 3 x 10 4 cells/well in 6-well plates, grown overnight and incubated 30 min with Ad5F35-LMP-l (0, 5, 10 pfu/cell), Ad5F35-GFP, or nothing. To determine stability of unphosphorylated Smadl, 35 S- Methionine ( 35 S-Express, NEN) is applied for 30 min on the next day to pulse label the pool of newly synthesized proteins and the cells subsequently washed.
- 35 S- Methionine 35 S-Express, NEN
- Cells are incubated in chase medium containing cold methionine with or without cycloheximide (10 ug/mL) for 2-3 hrs during which cells are harvested at various time points. Cells are harvested in the presence of protease inhibitors and cytoplasmic protein fractions prepared. Radiolabeled total Smadl are immunoprecipitated from the cytoplasmic fraction using specific antibody and analyzed by SDS-PAGE and auto-fluorography. To measure stability of phospho-Smadl, similar experiments are performed in control MSCs or MSCs overexpressing LMP-I, as above.
- Cells are labeled with 32 P-orthophosphate for 2 hr in the presence of the nuclear transport inhibitor, leptomycin B (25uM), to assure that only decay of the labeled phospho-Smad, not the net amount of decay and transport into the nucleus is measured.
- 32 P is removed and cells are treated for 1 hr with BMP-2 (100 ng/ml).
- Cytoplasmic fractions are prepared in the presence of protease and phosphatase inhibitors (to prevent loss of the 32 P label). Radiolabeled 32 P-Smadl are immunoprecipitated from cytoplasmic fractions and analyzed as above. Expected Results: Without wishing to be bound by theory, in the absence of BMP-2, it is expect that unphosphorylated Smadl protein will be synthesized and incorporate 35 S-
- Methionine during the labeling period As overexpressed LMP-I protein is translated it is expected to bind cytoplasmic Smurfl. If the hypothesis is correct, this event should block subsequent degradation of Smadl and result in a reduced rate of degradation of Smadl compared with cells not overexpressing LMP-I . It is expected that this block to be true of both the unphosphorylated and phosphorylated form of Smad.
- MSCs are transfected with Ad5F35-LMP-l or AD5F35-GFP and grown for 24 hr prior to metabolic labeling.
- Cells are washed in methionine-free medium and incubated for 15 min to deplete endogenous methionine.
- Cells are then incubated with the pulse-labeling medium containing [ 35 S]methionine (190 ⁇ Ci/ml) for 30 min. After washing with chase medium (containing 150 mg/L unlabeled methionine).
- chase medium containing 150 mg/L unlabeled methionine
- Cells are incubated in chase medium with or without cycloheximide (10 ug/mL) for 2-3 hr.
- Cells are lysed in buffer containing protease inhibitors (Sigma).
- Smadl is immunoprecipitated from cytoplasmic fractions using specific antibody and analyzed by SDS-PAGE and auto- fluorography.
- [ 32 P] orthophosphate labeling cells are pre-incubated with phosphate-free media for 1 h and exposed to 1 mCi/ml [ 32 P]orthophosphate for 2 h at 37°C.
- Leptomycin B (10ng/ml) or Ratjadone (10ng/ml) (CaI Biochem) is incubated with cells for 30 min to inhibit nuclear translocation of the labeled Smadl.
- Cells are then treated with BMP -2 (100 ng/ml) for 1 hr, lysed in buffer containing protease and phosphatase inhibitors, and the cytoplasmic fractions subjected to immunoprecipitation with phospho-Smadl specific antibody.
- optical density is monitored at specific wavelengths using the BioLumin 960 microtiter plate reader or the SpectraMax M2 microtiter plate reader.
- the assay can be adapted several ways to suit binding partner proteins. When biotin-labeled Smadl is assayed, streptavidin-alkaline phsophatase is used as the secondary reagent. After determining maximum binding between LMP-1/Smurfl and Smadl/Smurfl, mutual competition curves at various concentrations will provide data for Scatchard plot analysis to obtain binding affinity, dissociation constant and number of binding sites.
- Ubiquitin assay for determining extent of ubiquitination of Smadl/5 and/or LMP-I The ELSA assay described above is modified for the ubiquitin assay as follows: Microtiter plates are coated with Smadl/5 or LMP-I antibody and the remaining active sites are blocked by 1% BSA. Smadl/5 or LMP-I are then captured by incubating nuclear proteins in wells. Using specific enzyme/fluorescent-linked Ubiquitin antibody and the appropriate substrate solution, the extent of ubiquitination can be assayed.
- the same assay can be adapted to study inhibitory effect of LMP-I derived peptides on ubiquitination of Smads using an in vitro assay system with purified and commercially available ubiquitination assay reagents (Boston Biochemicals).
- MSCs are grown to confluence, incubated with agents being tested, trypsinized, washed 2x with PBS, suspended to 10- 20M/mL DMEM and 100 uL of the suspension applied to a sterile disc (2 x 5 mm) of bovine collagen. Implants are surgically placed subcutaneously on the chest of 4-6 wk athymic rats (rnuVrnu " ). The animals are euthanized after 4 weeks; the explants removed, fixed in 70% ethanol, and analyzed by radiography and undecalcified histology. Mechanistic Investigations of Ubiquitination 1. Ubiquitination of Smadl and Smad5.
- LMP-I binding to Smurfl is a reduction in the number of ubiquitinated Smadl and Smad5 proteins.
- LMP-I is overexpressed in hMSCs, Smadl or Smad5 are captured on wells coated with the specific Smad antibody, and the level of ubiquitination of the Smads is quantitated by ELISA.
- Human MSCs are plated at 3 x 10 4 cells/well in 6-well plates, grown overnight and treated with Ad5F35-LMP-l (0, 5, 10 pfu/cell), or Ad5F35GFP control plasmid. After 4, 8, 12, 24, 48, and 72 hr, cells are harvested and cytoplasmic and nuclear protein fractions prepared.
- the cell fractions are incubated with antibody to Smadl or Smad5 in a 96 well plate coated with the antibody. After washing to remove nuclear proteins not associated with Smadl or Smad5, fluorescence tagged-ubiquitin antibody is applied and the fluorescence is detected using a Biolumin 960 microtiter plate reader. Use of the fluorescent tagged antibody increases sensitivity and allows quantitation of the low level of ubiquitin expected to be present in the sample. To validate the ELISA results, another aliquot of the cell fractions undergoes immunoprecipitation using anti-Smadl or anti- Smad5 antibody and the precipitated proteins are analyzed by Western blotting using antibody to ubiquitin as well as antibody to Smadl or Smad5.
- siRNA targeting LMP-I are effective at reducing LMP-I levels.
- studies investigating the relationship between LMP-I and ubiquitination of Smads can be performed.
- siRNA to reduce the endogenous LMP-I levels results in increased levels of ubiquitinated Smadl/5 human MSCs are plated at 3 x 10 4 cells/well in 6-well plates, grown overnight, and treated with a control irrelevant siRNA or specific LMP-I siRNA. Cells are harvested 4, 8, 12, 24, 48, or 72 hr later and both cytoplasmic and nuclear fractions prepared.
- the cell fractions are incubated with antibody to Smadl or Smad5 in a 96 well plate coated with the antibody. After washing to remove nuclear proteins not associated with Smadl or Smad5, fluorescence tagged-ubiquitin antibody are applied and the fluorescence detected as in other examples herein.
- This approach allows quantitation of the low level of ubiquitin that is expected to be present in the sample.
- Expected Results It is expected that reduced levels of endogenous LMP-I will increase ubiquitination of both Smadl and Smad5. We would interpret this as being due to greater availability of Smurfl molecules for Smad binding.
- LMP-I prevents Smurfl interaction with Smadl, resulting in decreased ubiquitination of Smadl . Reduced ubiquitination is therefore expected to result in reduced proteasomal degradation of Smadl protein.
- in vitro ubiquitination assays are performed.
- biotin labeled Smadl is prepared by the TNT-coupled reticulocyte lysate system (Promega) and impurities removed by capturing Smadl using neutravidin-linked resin.
- Smadl is mixed with a mixture of purified El and E2 ligases and incubated with the Smad ubiquitin E3 ligase, Smurfl, in the presence or absence of recombinant LMP-I protein.
- the reaction mixture also contains ubiquitin and the creatine kinase-ATP generating system.
- the reaction mixture is analyzed by SDS-PAGE and Western blots using specific antibody to ubiquitin or Strepavidin-HRP (to detect Biotin-Smadl).
- Expected Results It is expected that there will exist a baseline ubiquitination of Smadl by
- Smad 1/5 binding site preventing Smad 1/5 targeting and degradation.
- human MSCs are plated at 3 x 10 4 cells/well in 6- well plates, grown overnight, and Smurfl or control siRNA applied. Cells are harvested 4, 8, 12, 24, 48, or 72 hr later and both cytoplasmic and nuclear fractions prepared. The cell fractions are incubated with antibody to LMP-I in a 96 well plate coated with the antibody. After washing to remove nuclear proteins not associated with LMP-I , fluorescence-tagged ubiquitin antibody is applied and fluorescence detected as above. This approach allows quantitation of the level of ubiquitin in the sample. In other wells, captured LMP-I levels is quantitated by applying fluorescence tagged-LMP-1 antibody.
- Smadl (100 ng) is buffer-exchanged to ubiquitination buffer (50 uM Tris-HCl pH 7.8, 5 mM MgCl 2 , 0.5 mM dithiothreitol (DTT), 2 mM NaF, and 3 ⁇ M okadaic acid). Smadl is then combined with a mixture of purified El and E2 enzymes and incubated with Smurfl (E3 ligase) in the presence or absence of recombinant LMP-I or LMP-2 protein.
- ubiquitination buffer 50 uM Tris-HCl pH 7.8, 5 mM MgCl 2 , 0.5 mM dithiothreitol (DTT), 2 mM NaF, and 3 ⁇ M okadaic acid.
- Smadl is then combined with a mixture of purified El and E2 enzymes and incubated with Smurfl (E3 ligase) in the presence or absence of recomb
- the reaction mixture also contains 2 mM ATP, ubiquitin (150 ⁇ M), ubiquitin aldehyde (5 ⁇ M), and creatine kinase-ATP generating system (Boston Biochem).
- the ubiquitin aldehyde is included to prevent hydrolysis of polyubiquitin chains.
- the reaction mixture (40 ⁇ L) is incubated 4 hr at 37 0 C. Aliquots at various time points are taken for
- I-Smad/Smurfl complexes form in the nucleus and translocate to the plasma membrane where I-Smad binding to the receptor occurs.
- Smurfl ubiquitinates the I-Smad and BMPRlA receptor, targeting them for proteasomal degradation (Ebisawa, T., Fukuchi, M., Murakami, G., Chiba, T., Tanaka, K., Imamura, T., and Miyazono, K., J. Biol. Chem. 2001, 276:12477-12480; Izzi, L. and Attisano, L., Oncogene. 2004, 23:2071-2078).
- Smurfl/I-Smad (Smad ⁇ ) complexes recombinant Smurfl, LMP-I and biotin-Smad ⁇ are purified and tested in an ELSA binding competition assay.
- the assay is described fully herein. Briefly, wells of a 96-well plate are coated with Smurfl protein and preincubated in the presence or absence of different amounts of LMP-I. After washing, biotin-Smad ⁇ is incubated with the Smurfl complexes and binding detected using strepavadin-alkaline phosphatase. Absorbance is monitored at 405 nm using the SpectraMax M2 microplate reader.
- Flag-Smad ⁇ is overexpressed to show that LMP-I blocks the BMP-2-mediated translocation of Smad ⁇ into the cytoplasm. Overexpressed Flag-Smad ⁇ is used rather than endogenous Smad ⁇ because of the specificity of the Flag antibody and cross-reactivity of Smad ⁇ antibodies with other Smads.
- Experimental Design Human MSCs are plated at 3 x 10 4 cells/well in 6-well plates, grown overnight, and transfected with 3 ug empty vector or plasmid containing Flag- Smad ⁇ . After the transfection, cells are allowed to recover for 1 hr prior to transduction with Ad5F35-LMP-l or Ad5F35-GFP (0, 5, 10 pfu/cell).
- BMP-2 (100 ng/mL) is applied to the cells.
- Cells are harvested 1, 2, 4, 8, and 12 hrs after BMP-2 treatment is initiated and cytoplasmic and nuclear fractions prepared. Fractions are subjected to SDS-PAGE and Western analysis using Flag specific antibody.
- cells are harvested and plasma membrane enriched fractions prepared. Fractions are subjected to SDS-PAGE and Western analysis using BMPRlA specific antibody.
- LMP-I is overexpressed due to interruption of Smurfl/Smad ⁇ function. It should be noted that this method may not quantitatively distinguish whether decreased ubiquitination is due to variation in the number of ubiquitin subunits linked to BMPRlA or fewer BMPRlA molecules that are conjugated to ubiquitin chains. Nevertheless, a comparison between treatments can be made, reflecting different amounts of ubiquitinated BMPRlA among various treatments. If ubiquitinated BMPRlA is not detectable it is likely to be caused by rapid degradation of the ubiquitinated receptor.
- the experiment would be performed in the presence of the proteasome inhibitor, lactacystin, to accumulate ubiquitinated BMPRlA for detection in Western blots.
- This method may not be sensitive enough to detect the low levels of ubiquitinated
- Jabl is an adapter protein that targets Smad4 for ubiquitination and regulates the rate of Smad4 degradation, an important role in controlling the cytoplasmic levels of Smad4.
- Smad4 is required for translocation of phospho-Smadl/5 into the nucleus and has been shown to be critical in regulating the responsiveness of cells to BMP-2 (Hata, A., Lagna, G., Massague, J., and
- LMP-I blocks Jabl-induced proteasomal degradation of Smad4.
- the LMP-1/Jabl interaction may either prevent Jabl binding to Smad4 or block the ability of Jabl to serve as an adapter protein that increases targeting of Smad4 for ubiquitination and subsequent proteasomal degradation. This question could be resolved by competition binding studies.
- a recombinant LMP-I fusion protein (TAT-LMP-I) could be used instead of Ad5F35-LMP-l as demonstrated in other studies herein. 4. Enhancement of physiologically relevant markers of BMP-2 responsiveness To investigate the combined effect on BMP-2 responsiveness, a series of experiments are performed to demonstrate the effect of LMP-I overexpression. First, a Smad-responsive luciferase reporter construct (9xGCCG) is first used to determine the effect of LMP-I overexpression on luciferase activity. Next, expression of the BMP-2 regulated gene, Dlx5 is measured. Other studies disclosed herein suggest Dlx5 is important for the synergistic effects of LMP-I and BMP-2 to induce the osteoblast phenotype in MSCs.
- LMP-I overexpression is expected to greatly enhance the reporter construct activity in response to BMP-2.
- a smaller increase in activity is expected with either LMP-I alone or BMP-2 alone. Since LMP-I increases the pool of R- Smads and the Co-Smad, this could result in some increase in activated R-Smad/CoSmad complexes in the nucleus (as suggested by other data herein).
- LMP-I increases the pool of R- Smads and the Co-Smad, this could result in some increase in activated R-Smad/CoSmad complexes in the nucleus (as suggested by other data herein).
- the largest effect observed in enhancing BMP-2 efficacy would be due to the effect of LMP-I to reduce degradation of many important proteins in the BMP-2 signaling cascade.
- LMP-I is further expected to enhance the BMP-2 increase in the Dlx5 gene expression as suggested by results disclosed herein. Therefore, if the results are as expected, one mechanisms by which LMP-I increases responsiveness to BMP-2 may likely involve reduced degradation of BMPRlA, R-Smads, and Co-Smad4.
- Alkaline Phosphatase mRNA levels Alkaline phosphatase activity is an early marker of the osteoblast phenotype. Human MSCs treated with Ad5/35LMP-1 (0, 1, 5, 10 pfu/cell) with and without BMP-2 (100ng/ml) are harvested for RNA at day 8. Cells are washed with PBS once and cell lysates are prepared by sonication.
- RNA is isolated and alkaline phosphatase mRNA was quantified by RT-PCR with the alkaline phosphatase specific primers. Alkaline phosphatase activity is measured using p-nitrophenyl phosphate as substrate where nzyme activity is expressed as p-nitrophenol produced (nmoles/ml). Data is normalized to 18S.
- Osterix is a novel zinc finger-containing transcription factor required for osteoblast differentiation and bone formation.
- Human MSCs are treated with Ad5/35LMP-1 with and without BMP-2 and harvested for RNA at day 8.
- RNA is isolated and osterix mRNA is quantified by RT-PCR with the osterix primers. Data is normalized to 18 S .
- Bacterial Strains and Cloning of cDNAs in bacterial expression vectors All cloning methods are performed according to standard protocols. Escherichia coli XLl blue and BL 21-codon plus (DE3)-RP (Stratagene) hosts are maintained on LB agar plates and grown at 37°C in the presence of ampicillin at 100 mg /L. LMP-I, LMP-It, LMP-2, LMP-3, Smadl and Smad5 cDNAs were cloned into TAT-HA vector. LMP-I mutants were generated using the primers in Table 3.
- the lysate is centrifuged at 10,000 g at 4 0 C and the supernatant applied to Sephacryl S-100/S-200 columns (HiPrep 16 X 60) using AKTA FPLC system with Unicorn 4.0 software (Amersham Pharmacia Biotech) at a flow rate of lml/min. Fractions (2-4 ml) are collected immediately after the void volume (35 ml). Aliquots, from each fraction are assayed by slot blotting, SDS-PAGE and western blotting.
- the fractions, identified by western blots are pooled, dialyzed against 20 mM phosphate buffer, pH 7.5 containing NaCl (50 mM) and imidazole (20 mM) and applied to Ni++ affinity resin (Probond, Invitrogen) previously equilibrated with 4 x 10 ml of buffer.
- Non-specific proteins are washed off the column with 3 x 10 ml of 20 mM phosphate buffer, pH 6.0 containing NaCl (50 mM) and imidazole (20 mM).
- Affinity-bound proteins are eluted using 3 xlO ml washes with 20 mM phosphate buffer, pH 4.0 containing NaCl (50 mM).
- Proteins containing the desired protein are pooled (based on western blot) and then concentrated and de-salted using the centriprep devices (Amicon). Proteins are quantitated using BioRad protein assay reagent. The yield of recombinant protein is routinely 0.5 to 1 mg of pure protein from every 2-liter culture.
- Biotinylation of protein ligands Purified protein ligands are prepared at 10 mg/ml in 50 mM sodium borate buffer, pH 8.5; 0.5 M NaCl.
- Biotinylated ligand (LMP-I, LMP-2, LMP-3, Smadl or Smad5) is mixed with varying concentrations of competing proteins or peptides and incubated in slot blot wells with Smurfl for 90 min. Wells are washed and the blots were blocked with TBST containing 0.5% Tween 20. The blots are then incubated with HRP-labeled avidin for 1 hr. After washes the blots are incubated with ECL substrate solution and the membranes are exposed to X-ray film for signal detection.
- Peptides are synthesized with a protein transduction domain (PTD) at the c-terminal end, (rrqrrtsklmkr, herein incorporated as SEQ ID NO: 32) according to Mi Z, et. al.;MolTl ⁇ er. 2000;2:339- 47.
- hMSCs at passage 4 are seeded at 3 x IQ 4 cells/well in a 6-well plate. The next day, the cells are infected with Ad35LMP-l (1-10 pfu/cell) and incubated with and without BMP-2 (100ng/ml). The medium is replaced every 3-4 days and deposition of mineral observed after 2 weeks. To assess mineralization, cultures are washed with PBS and fixed in a solution of ice-cold 70% ethanol for 2-3 hours. Cultures are rinsed with water and stained for 10 minutes with ImI of 40 mM Alizarin red (pH 4.1). Cultures are rinsed 2-3 times with PBS to reduce non-specific staining, air dried and photographed.
- Chest implants The surgical area is draped using sterile drapes with a hole cut to allow access to the surgical area. Surgery is performed on a draped circulating water heating pad. A 1 cm skin incision is made in 4 locations on the chest, the skin separated from the muscle by blunt dissection and the discs loaded with BMP-2 + cells positioned in separate pockets. Incisions are closed using resorbable sutures. Rats typcially survive 4 weeks after which they are euthanized using canister CO 2 consistent with the recommendation of the AVMA.
- Controls Because of the narrow efficacious dose range for LMP-I it is necessary to test multiple doses of each compound and to include positive control (i.e.TAT-LMP-1) and negative (i.e. cells alone or cells + BMP-2 only) controls. The positive control chosen should not induce bone formation when given at the chosen dose without BMP-2. Each dose or control is tested on multiple sites and there are multiple sites/rat. If compounds are postive, more testing can be performed.
- Anesthesia is adminsitered using 1.5% isoflurane prior to the surgical procedure. Prior to making incisions Bupivicaine 0.1-0.3 ml is given by subcutaneous or intramuscular injection around the surgical site. Buprenorphine (Buprenex) (0.05mg/100g) is injected subcutaneously immediately post-operatively and every 8 hrs for 3 days post-op to relieve pain.
- Buprenorphine Buprenex
- BMP-2 (2.5 ug) that induces bone formation in only 50% of the implants and a lower dose (1 ug) that consistently fails to induce bone formation in the rat ectopic model. These doses are known as the "suboptimal doses.”
- Experimental Design In these studies, multiple doses of each of several peptides are studied. The positive control are MSCs treated with Ad5F35-LMP-l (5 pfu/cell). MSCs (1-2M) are mixed with appropriate doses of peptides in a 100 uL total volume and placed on a collagen disc. The disc is implanted subcutaneously on the chest of athymic rats and explanted after 4 weeks.
- Bone formation is evaluated by palpation, x-ray and semiquantitative scoring of non-decalcified histologic sections (Edwards JT, Diegmann MH, and Scarborough NL. Clin.Orthop. 1998;219-28). It has been previously found that 1-2 million cells transduced with TAT-LMP can induce bone formation in this model, although not consistently. Hence, these studies will attempt to achieve more consistent in vivo bone formation results which will be required for clinical translation. In addition the ability of the successful peptides to enhance the ability of suboptimal doses of BMP-2 to induce ectopic bone formation in this model may also be investigated.
- P-Smad phosphorylated Smad
- R-Smad receptor Smad: I-Smad, inhibitor Smad
- PTD protein transduction domain
- siRNA small interfering RNA
- LMP LIM mineralization protein
- Smurf Smad Ubiquitin Regulatory Factor
- BMP-2 bone morphogenic protein-2
- rhBMP-2 recombinant human bone morphogenic protein-2
- Jabl Jun Activation Domain Binding Protein
- pfu plaque forming units
- MOI multiplicity of infection.
- BMP-2 As used herein, the when referring to treatment of cells, tissues or animals, the terms "BMP-2” and “rhBMP-2” are synonymous.
- Example 1 LMP-I dramatically and svnergisticallv increases the responsiveness of mesenchymal stem cells CMSCs') to BMP-2.
- LMP-I induces bone but produces very small amounts of BMPs.
- LMP-I increases the responsiveness of cells allowing them to respond to lower levels of BMPs with respect to osteoblastice differentiation, was tested.
- MSC cultures transfected with the chimeric Ad5F35 vector overexpressiong LMP-I were treated with either LMP-I (0, 1, 5, 10 pfu/cell) or BMP-2 (100 ng/mL) alone or in combination.
- BMP-2 nor LMP-I alone induce any bone nodule mineralization in human MSCs on day 21, but treating MSCs overexpressing LMP-I (5-10 pfu/cell) with rhBMP-2 (100 ng/mL) induced dramatic bone nodule mineralization as shown by alizarin red staining.
- concurrent exposure to LMP-I enabled an ineffective dose of BMP-2 to facilitate bone formation suggesting that LMP-I increases the BMP signaling pathway activity/sensitivity.
- the chimeric Ad5F35 vector in which the Ad5 fiber protein is replaced with the Ad35 fiber protein, was used to reduce susceptibility to neutralizing antibodies also tranduces human MSCs more effectively than Ad5 vectors.
- LMP-I was also shown to increase the responsiveness of human MSCs to BMP-2 as evidenced by alkaline phosphatase mRNA levels and an increase in enzyme activity.
- Example 2 LMP Variant (LMP-2) does not induce nodule formation in rat calvarial osteoblast cultures.
- rat calvarial osteoblasts do not spontaneously differentiate without exposure to a stimulus such as glucocorticoid (GC).
- GC glucocorticoid
- Cells were transfected with plasmids containing three LMP (hLMP-1; hLMP-2 and hLMP-3) variants and assessed for multilayer mineralized nodule formation 14 days after treatment. Control cells received no treatment and all data were normalized to the control group.
- Overexpression of hLMP- 1 resulted in nodule formation (225 nodules) comparable to that seen with GC (275 nodules).
- hLMP-3 which is a truncated version of LMP-I also induced nodule formation
- Measuring activation of the BMP signaling pathway requires the ability to measure Smadl and Smad5.
- SDS-PAGE separated cytoplasmic and nuclear protein blots were probed with Smad-1 specific antibody.
- a comparison of cytoplasmic and nuclear protein extracts from untreated pleuripotent cells demonstrated that most of the Smadl (54 kDa) was detected in the cytoplasmic fraction.
- P-Smadl phosphorylated Smadl
- SDS-PAGE blots of nuclear proteins were prepared from MSCs overexpressing LMP-I at 4 and 8 hours following infection with Ad5F35-LMP-l. The blots were probed with primary antibody specific to P-Smadl. The binding of primary antibody was detected using the HRP-labeled second antibody after signal development by enhanced chemi-luminescence (ECL).
- ECL enhanced chemi-luminescence
- LMP-I compared to control MSCs not over-expressing LMP-I.
- Human MSCs treated with BMP-2 also have increased levels of cytoplasmic phosphorylated Smadl in the presence of overexpressed LMP-I.
- SDS-PAGE blots of cytoplasmic proteins were prepared from MSCs overexpressing LMP-I at 4 and 8 hours following infection with LMP-I delivered as Ad5F35-LMP-l.
- Cells were treated with rhBMP-2 at lOOng/mL or 200 ng/mL alone or in combination with LMP-I (5 pfu/cell).
- rhBMP-2 treatment at either lOOng/mL or 200 ng/mL resulted in a small increase in cytoplasmic phosphorylated Smadl (64 kDa band).
- MSCs were treated with rhBMP-2 (100 ng/mL) or Ad5F35-LMP-l (5 pfu/cell). Control cells were untreated. After 4 or 8 hrs total RNA was harvested and Smadl mRNA was measured by real time RT-PCR. LMP-I and BMP-2 each increased Smadl mRNA by 4 hours, 5 fold and 6.2 fold, respectively. At 8 hours Smadl mRNA was increased by 4.5 fold and 2 fold for LMP-I and BMP-2 respectively. The data suggest that LMP-I causes increased nuclear levels of P-Smadl via increased expression of Smadl mRNA, which results in increased Smadl protein that can be phosphorylated by the BMP receptor kinase.
- mRNA levels of Dlx5, a gene known to be induced by the BMP-Smads was measured. MSCs were untreated or treated with BMP-2 (100 ng/mL), Ad5F35-LMP-l (5 pfu/cell), or both. After 24 hr total RNA was harvested and Dlx5 mRNA levels measured by real time RT-
- RNA and protein levels of Smad ⁇ , and BMP-2 can also be performed.
- human MSCs are plated at 3 x 10 4 cells/well in 6-well plates, grown overnight, and treated with Ad5F35-LMP-l (0, 5, 10 pfu/cell), BMP-2 (100 ng/ml) or both agents in combination or in series.
- the control treatment would be the vector, Ad5F35-GFP.
- cells are harvested for analysis of total RNA or cytoplasmic and nuclear protein.
- real-time PCR is performed using primers specific for human BMP-2 and Smad ⁇ .
- secreted, cytoplasmic and nuclear proteins can be analyzed by ELISA using commercially available antibodies to the proteins of interest, with alkaline phosphatase conjugated secondary antibodies. Specificity of the antibody can be determined by
- Intracellular BMP-2 is expected to show several immunoreactive bands corresponding to various cleavage steps during processing of pro-BMP -2.
- the processed BMP-2 protein will also likely be found largely in the medium, as it is rapidly secreted.
- Measurement of BMP-2 transcripts can be used for determining the relative effect of
- LMP-I and BMP-2 on BMP-2 gene expression as the majority of the protein measured in the medium from BMP-2 treated cells may be exogenously added as a treatment.
- Example 7 LMP-I is associated with an 85 kPa nuclear protein.
- LMP-I Recombinant LMP-I was labeled with SBED-biotin transfer reagent and incubated as bait with nuclear proteins. Biotin transfer to target proteins was accomplished by photo- activation and decoupling was performed by reduction of bound protein-partners. Enrichment of biotinylated proteins was performed using neutravidin-beads. Biotinylated proteins were separated by SDS-PAGE and detected on Western blots using HRP-labeled neutravidin and ECL. The corresponding coomassie stained bands were excised for tryptic digestion and MALDI-TOF analysis. LMP-I was seen to associate with three protein bands; one 85 IcDa band and two smaller bands.
- Example 8 Peptide mass profile analysis of the 85 kDa protein binding to LMP-I.
- the SDS-PAGE separated LMP-I binding protein bands were in-gel digested by 5% (w/w) trypsin. Molecular mass of tryptic-peptides was obtained by MALDI-TOF and analyzed by Pepldent. The resulting 85 kDa candidate protein matches from the data base were analyzed. Smurfl and its splice-isoform variant showed the best ranking with peptide mass profile. Although the tryptic digestion was done on doublet protein bands, the unique identity was still able to be determined due to identical peptide profiles of both isoforms of Smurfl in tryptic digestion. By comparison the peptide profile obtained did not match the peptide mass profile of Smurf2.
- Example 9 The 85 IcDa protein reacts with Smurfl/2 antibody.
- Example 10 Immunoprecipitation endogenous Smurfl and LMP-I in MSCs.
- Human MSCs were plated at 3 x 10 4 cells/well in 6-well plates, grown overnight, and treated with Ad5F35-LMP-l (0, 5, 10 pfu/cell), or Ad5F35-GFP. After 2, 4, 8, 12, 24, and 48 hrs, cells were harvested and nuclear proteins mixed with LMP-I or Smurfl antibody and subjected to immunoprecipitation. Eluted proteins were separated by SDS-
- Nuclear protein extracts of untreated MSCs were incubated with LMP-I antibody and immunoprecipitated using protein-A beads.
- the immunoprecipitated proteins were concentrated and analysed in Western blots with LMP-I and Smurfl/2 antibody, separately. Both endogenous LMP-I and Smurfl were present in the complex immunoprecipitated with the LMP-I antibody from untreated MSCs. This observation was confirmed by detection of LMP-I when Smurfl antibody was used for the immunoprecipitation.
- Example 11 LMP-I interacts with Smurfl via the Smurfl WW domain.
- LMP-I inhibit binding
- truncated LMP variants also inhibit Smurf WW-domain antibody binding to Smurfl .
- Blots of SDS-PAGE resolved nuclear proteins from untreated MSCs were incubated with the antibody specific for the Smurfl WW- domain.
- LMP-I and recombinant LMP-It were able to prevent the WW-domain antibody from binding to Smurfl.
- Example 12 Characterization of the binding properties of Smurfl, LMP-I, Smadl, and Smad5 using purified proteins to confirm competitive binding of LMP-I.
- Smadl and Smad5 are prepared by bacterial expression and purification. Smads and LMP-I are then fluorescently labeled.
- Sorbent Assay described herein is performed in microtiter plates. Binding curves for evaluating the competitive binding to Smurfl of fluorescently labeled LMP-I with unlabeled Smadl or Smad5 are then obtained. Conversely, the competitive binding of fluorescently labeled Smads with unlabeled LMP-I may also be measured. Fluorescence is monitored using a Biolumin 960 combined fluorescence and absorbance microplate reader. Scatchard analyses, well known in the art, can serve to assess the binding affinity, dissociation constants, number of binding sites, and stoichiometry for each protein involved in the interaction. The results can then predict competition between LMP-I and Smads for binding Smurfl at one or two sites. Competitive binding at two sites might occur, as Smurfl contains two
- LMP-I LMP-I, LMPIt and LMP-3
- LMP-I LMP-I, LMPIt and LMP-3
- AADPPRYTFAPSVSLNKTARPFGAPPPADSAPQQNG SEQ ID NO: 33
- WW interacting site A comprising the sequence ADPPRYTF AP
- WW interacting site B comprising the sequence GAPPP ADSAP; herein referred to as SEQ ID NO: 35
- the WW domain interacting sites are absent in a non-osteogenic LMP variant (LMP-2).
- LMP-2 non-osteogenic LMP variant
- the 45 amino acid osteoinductive region appears to be critical for the bone-forming activing of LMP.
- LMP It and LMP-3 Two LMP variants (LMP It and LMP-3) are truncated at the carboxy-terminus, and do not contain the LIM domains, but do induce bone formation. Thus, it is the 45 amino acid osteoinductive region and not the LIM domains that are required for bone formation (Liu, Y, et. al.; J. Bone Min. Res.,
- two mutant LMP-I proteins that are mutated in either WW interaction site A (Prolines at positions 100 and 101 of the 457 amino acid parent protein being converted to Alanine, herein incorporated as SEQ ID NO: 36; LMP-1 ⁇ WWA) or site B (Prolines at positions 122, 123 and 124 of the 457 amino acid parent protein being converted to Alanine, herein incorporated as SEQ ID NO: 37 ; LMP-1 ⁇ WWB) were prepared.
- the mutations remove proline residues that are required for interaction with the Smurfl WW domain and disrupt the PY motif in each of the two sites.
- Trp VaI Leu Ser lie Asp GIy GIu Asn Ala GIy Ser Leu Thr His lie
- 275 280 285 lie Arg GIy Arg Tyr Leu VaI Ala Leu GIy His Ala Tyr His Pro GIu
- Recombinant Smurfl was ran on SDS-PAGE, transferred to membrane, and incubated with buffer, LMP-I protein, LMP-2 protein (negative control, no osteoinductive region), LMP-1 ⁇ WWA or LMP-1 ⁇ WWB. After washing, Smurfl WW domain antibody was applied and then detected with HRP-labeled secondary antibody. Slot blots of competitive binding assays were analyzed as before and it was found that only WW-domain interaction site B was required for the interaction with Smurfl . The specificity of these interaction sites was confirmed by demonstrating that LMP-I was no longer able to bind to a mutant form of Smurfl which had its WW2 domain deleted (Smurfl ⁇ WW2).
- Example 14 Peptide mimics of LMP-I
- known crystallography data and homology modeling of related homologues similar to the WW-2 domain in Smurfl and the two potential interacting motifs in LMP-I (WW-A and WW-B) as defined by iSPOT were used (Brannetti, B. and Helmer-Citterich, M., Nucleic Acids Res. 2003; 31 :3709-3711).
- the MODELLER program was used to assign structure to the two interacting elements and to model templates.
- the designed Smurfl WW-2 template showed a close (up to 70%) match to the coordinates of crystallographic data available for homologous WW domains from the protein data bank (PDB) (Fiser, A. and SaIi, A., Methods Enzvmol. 2003; 374:461-491).
- DOCKING and SSA Surface Solvant Accessability
- Example 15 Osteoinductive region fusion peptides
- hMSCs were plated at 3 x 10 4 cells/well in 6-well plates, grown overnight, and treated with PTD-LMP-1/OR, BMP-I (100 ng/ml) or both agents.
- the control was PTD- ⁇ Gal.
- cells were harvested and total RNA plus cytoplasmic and nuclear protein fractions prepared.
- the nuclear protein fraction from the untreated sample was analyzed for the ability of purified PTD- LMP-I /OR to compete with Smurfl antibody binding to Smurfl on a Western blot.
- RNA samples were analyzed for the presence of P-Smadl and P-Smad5 by Western blot using appropriate antibody.
- All RNA samples were analyzed by real-time RT-PCR for mRNA levels of Dlx5, Smad6, and BMP-2.
- protein fractions were analyzed by ELISA using commercially available antibodies to Dlx5, Smad6, and BMP-2. The results illustrate that the 36aa peptide has all the ability of full length LMP-I to compete with WW domain antibody binding, to induce increased nuclear levels of P- Smadl and P-Smad5, and to increase expression of BMP/Smad regulated genes.
- Bone marrow cells and leukocytes were transfected with the cDNA of foil length or truncated LMP-I lacking LIM domains.
- Collagen discs containing the engineered cells were implanted subcutaneously into athymic rats. Histology of the implants showed ectopic and orthotopic bone formation.
- New bone trabeculae lined with osteoblasts were seen in the implants containing cells overexpressing truncated LMP-I (LMP-It) and the absence of new bone in implants containing control cells. This demonstrates that genetically engineered cells can form bone in vivo and confirms that the LIM domains of LMP-I are not needed for bone formation.
- Example 17 Bone marrow cells transfected with LMP-I induce bone in the rat model of spine fusion
- Fusion proteins that contain an 1 laa sequence, found in the HIV protein Tat, are known to readily enter cells within minutes (Nagahara H, Vocero-Akbani AM, Snyder EL et al. Nat.Med.1998: 4: 1449-52). This method of protein delivery is superior to adenoviral gene transfer because it avoids immune response issues.
- a FITC labeled LMP-I Tat fusion protein TAT-LMP-I was synthesized and fluroescence monitored for localization in the MSCs when treated at doses of 10, 25 and 10OnM.
- the fusion protein readily entered nucleated blood cells in a dose dependent manner with 2, 25 and 90% of the cells positive for the label, respectively.
- siPvNA small inhibitory RNAs
- siRNA to Smurf were designed and tested for target reduction in TE85 human osteosarcoma cells.
- Cells were treated according the methods taught herein and total RNA was harvested and Smurfl mRNA levels measured by RT-PCR using primers specific for Smurfl.
- Smurf2 mRNA levels were also measured using specific primers and these levels did not change. There was a single product of each primer set which was sequenced to confirm its identity. The results indicate that Smurfl siRNA produced a dose-dependent decrease in Smurfl mRNA levels.
- Osterix is a novel zinc finger-containing transcription factor required for osteoblast differentiation and bone formation.
- Human MSCs were treated with Ad5/35LMP-1 (0, 1,
- Example 23 LMP-I Interacts with Jun Activation Domain Binding Protein (Jabl).
- Jabl Jun Activation Domain Binding Protein
- the yeast-two-hybrid (Y2H) system (Clontech) was used to identify other proteins which could interact with LMP-I. Positive clones were selected based on Y2H screening of a bone marrow library. The sequencing and database matching of 10 putative positive clones identified Jabl as a likely candidate binding partner for LMP-I. It was then determined if this association occurred in cells. Immunoprecipitation of cytoplasmic proteins using LMP-I antibody beads demonstrated that Jabl was found in complexes with LMP-I. Although these data demonstrate that an association between Jabl and LMP-I occurs in cells, they do not establish direct binding of the two proteins.
- Example 24 LMP-I binds to Jabl
- LMP-I LMP-I .
- the bound biotin-LMP-1 was detected using neutravadin-HRP.
- Two bands were present on the blot and demonstrated that LMP-I is capable of binding directly to both Smurfl (85 IdDa) and Jabl (38 IcDa). The identity of these two bands were confirmed by staining with antibody specific to Smurfl and Jabl. These blots provide evidence that LMP-I interacts directly with Jabl supporting the hypothesis that LMP-I may interrupt the binding of Jabl to one of its targets (Smad4).
- Smad4 Smad4
- the sites or protein domains necessary for interaction between LMP-I and Jabl can be determined in the same manner as those for the interaction between LMP-I and Smurfl (see Examples 13 and 14). Once identified, peptides containing those sites or domains may be designed modulate the interaction of the proteins.
- Example 26 LMP-I overexpression increases nuclear Smad4 levels in human MSCs.
- Example 27 Screening of LMP-I mimics To identify small molecules that mimic the effect of LMP-I on induction of bone formation, and have properties that make it more clinically convenient, multiple compounds may be screened which contain or mimic the more important LMP-I interacting domain (B). These compounds are tested for their ability to compete with LMP-I for Smurfl binding.
- recombinant Smurfl is applied to wells of a 96 well plate. After removing excess Smurfl, test compounds (in excess) are pre-incubated with Smurfl followed by incubation of the Smurfl complexes with recombinant biotin-LMP-1. Biotin- LMP-1/Smurfl is detected using Strepavidin-alkaline phosphatase. Absorbance is determined at 405 nm using the SpectraMax M2 microtiter plate reader. Compounds that block Biotin-LMP-1 binding may then be re-screened using appropriate lower amounts of the compound to determine the IC 50 (dose that prevents 50% of the maximum LMP-I binding). Those compounds with the lowest IC 50 are considered the most efficacious and can then be screened for cellular effects. The IC 50 is used to determine the dose of each compound that we will screen.
- Example 28 Screening compounds in vitro
- Candidate compounds that most efficiently inhibit binding of LMP-I to Smurfl in binding competition assays are further evaluated for their ability to mimic LMP-I in cells.
- One appropriate cell line are mesenchymal stem cells (MSCs) and appropriate endpoints include an increase BMPRlA levels and an increase in luciferase production from a Smad- activated reporter construct.
- human MSCs are plated at 3 x 10 4 cells/well in 6-well plates, grown overnight, and treated with candidate compounds at the IC 5O dose and two doses above and below that dose.
- Ad5F35LMP-l and Ad5F35GFP are applied as positive and negative controls. After 1, 2, 4, 8, 12, 24 and 48 hrs, cells are harvested and plasma membrane enriched fractions prepared. Fractions are subjected to SDS-PAGE and Western analysis using BMPRlA specific antibody.
- MSCs plated as above and transfected with a 9xGCCG/Smad-activated luciferase reporter construct are incubated with all compounds (at the successful dose) that increased BMPRlA in the first experiment.
- Example 29 Screening compounds in vivo
- MSCs (2.5 ug) to make bone in more than 50% of the implants.
- the positive control is MSCs treated with TAT-LMP-I (0.625 nM); negative controls include MSCs alone and BMP-2 (2.5 ug) alone.
- MSCs (1-2M) are mixed with appropriate doses of compounds in a 100 uL total volume and placed on a collagen disc. The disc is implanted subcutaneously on the chest of athymic rats and explanted after 4 weeks. Bone formation is evaluated by palpation, x-ray and semi-quantitative scoring of non-decalcified histologic sections (Edwards, J. T., Diegmann, M. H., and Scarborough, N. L., Clin. Orthop., 1998, 219-228).
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US20040034428A1 (en) * | 2000-10-24 | 2004-02-19 | Mckay William F | Spinal fusion methods and devices |
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US20040034428A1 (en) * | 2000-10-24 | 2004-02-19 | Mckay William F | Spinal fusion methods and devices |
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