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• W2006062243 abstract "Development and repair of the skeletal system and other organs is highly dependent on precise regulation of bone morphogenetic proteins (BMPs), their receptors, and their intracellular signaling proteins known as Smads. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, control of cellular responsiveness to BMPs is now a critical area that is poorly understood. We determined that LMP-1, a LIM domain protein capable of inducing de novo bone formation, interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads. In the region of LMP responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and can effectively compete with Smad1 and Smad5 for binding. We have shown that small peptides containing this motif can mimic the ability to block Smurf1 from binding Smads. This novel interaction of LMP-1 with the WW2 domain of Smurf1 to block Smad binding results in increased cellular responsiveness to exogenous BMP and demonstrates a novel regulatory mechanism for the BMP signaling pathway. Development and repair of the skeletal system and other organs is highly dependent on precise regulation of bone morphogenetic proteins (BMPs), their receptors, and their intracellular signaling proteins known as Smads. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, control of cellular responsiveness to BMPs is now a critical area that is poorly understood. We determined that LMP-1, a LIM domain protein capable of inducing de novo bone formation, interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads. In the region of LMP responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and can effectively compete with Smad1 and Smad5 for binding. We have shown that small peptides containing this motif can mimic the ability to block Smurf1 from binding Smads. This novel interaction of LMP-1 with the WW2 domain of Smurf1 to block Smad binding results in increased cellular responsiveness to exogenous BMP and demonstrates a novel regulatory mechanism for the BMP signaling pathway. Bone morphogenetic proteins are critical regulators of osteoblast differentiation and bone formation (1Liu Y. Titus L. Barghouthi M. Viggeswarapu M. Hair G. Boden S.D. Bone. 2004; 35: 673-681Crossref PubMed Scopus (27) Google Scholar). BMPs 2The abbreviations used are: BMP, bone morphogenetic protein; TGF, transforming growth factor; MSC, mesenchymal stem cell; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; TBST, Tris-buffered saline containing 0.1% Tween 20; HRP, horseradish peroxidase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; pfu, plaque-forming unit; siRNA, small interfering RNA; STAT, signal transducers and activators of transcription; MAPK, mitogen-activated protein kinase; LMP-1 LIM mineralization protein-1.. were originally identified as molecules that could induce ectopic bone and cartilage formation in rodents (2Wozney J.M. Prog. Growth Factor Res. 1989; 1: 267-280Abstract Full Text PDF PubMed Scopus (317) Google Scholar). Subsequently, various members of the BMP family have been shown to play a role in development of limb bud patterning, kidney, germ cells, nervous system elements, tendons/ligaments, and control of apoptosis (3Ducy P. Karsenty G. Kidney Int. 2000; 57: 2207-2214Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Several members of the BMP family are osteoinductive and able to induce the differentiation of mesenchymal cells into osteoblasts (4Cheng H. Jiang W. Phillips F.M. Haydon R.C. Peng Y. Zhou L. Luu H.H. An N. Breyer B. Vanichakarn P. Szatkowski J.P. Park J.Y. He T.C. J. Bone Joint Surg. Am. 2003; 85: 1544-1552Crossref PubMed Scopus (811) Google Scholar). The various activities of the BMPs are modulated through the control of antagonists, cell surface receptors, intracellular signaling proteins (Smads), and cross-talk with other signaling pathways (5Peng Y. Kang Q. Cheng H. Li X. Sun M.H. Jiang W. Luu H.H. Park J.Y. Haydon R.C. He T.C. J. Cell. Biochem. 2003; 90: 1149-1165Crossref PubMed Scopus (165) Google Scholar, 6von Bubnoff A. Cho K.W. Dev. Biol. 2001; 239: 1-14Crossref PubMed Scopus (342) Google Scholar). Clinically, rhBMP-2 is being used to achieve spine fusion in patients with low back pain and avoids the morbidity of bone harvested from the pelvis (7Goulet J.A. Senunas L.E. DeSilva G.L. Greenfield M.L.V.H. Clin. Orthop. 1997; 339: 76-81Crossref PubMed Scopus (743) Google Scholar, 8Boden S.D. Kang J.D. Sandhu H.S. Heller J.G. Spine. 2002; 27: 2662-2673Crossref PubMed Scopus (599) Google Scholar). Unfortunately, without improving the responsiveness to BMP-2, the high doses (milligrams) required in humans and the resultant cost are prohibitive for routine clinical use (9Martin G.J. Boden S.D. Morone M.A. Moskovitz P.A. J. Spinal Disord. 1999; 12: 179-186PubMed Google Scholar). BMP responsiveness is dependent in part on the availability of intracellular signaling proteins. When activated, the type I BMP receptors phosphorylate intracellular mediators, the Smad proteins (10Hoodless P.A. Haerry T. Abdollah S. Stapleton M. O'Connor M.B. Attisano L. Wrana J.L. Cell. 1996; 85: 489-500Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). The receptor-regulated Smads (R-Smads) are phosphorylated by the type I receptors upon ligand binding (11Lo R.S. Chen Y.G. Shi Y. Pavletich N.P. Massague J. EMBO J. 1998; 17: 996-1005Crossref PubMed Scopus (208) Google Scholar). Smad2 and Smad3 are involved in the TGF-β/activin pathway, whereas Smads 1, 5, and 8 act in response to BMPs (12ten Dijke P. Fu J. Schaap P. Roelen B.A. J. Bone Joint Surg. Am. 2003; 85: 34-38Crossref PubMed Scopus (86) Google Scholar). The phosphorylated R-Smads interact with the single common Smad, Smad4, forming a complex that translocates into the nucleus, associates with DNA, and is responsible for transcriptional regulation of target genes (13Miyazono K. Cytokine Growth Factor Rev. 2000; 11: 15-22Crossref PubMed Scopus (235) Google Scholar). To examine strategies for improving cellular responsiveness to BMPs, we chose to study human mesenchymal stem cell (MSC) and osteoblast differentiation as a model. LMP-1 (LIM mineralization protein-1) was recently cloned and sequenced in our laboratory and is an intracellular protein that induces bone formation in vitro and in vivo (14Boden S.D. Liu Y. Hair G.A. Helms J.A. Hu D. Racine M. Nanes M.S. Titus L. Endocrinology. 1998; 139: 5125-5134Crossref PubMed Scopus (0) Google Scholar, 15Boden S.D. Titus L. Hair G. Liu Y. Viggeswarapu M. Nanes M.S. Baranowski C. Spine. 1998; 23: 2486-2492Crossref PubMed Scopus (209) Google Scholar). LMP-1 overexpression induces BMP-2, -4, -6, and -7 expression in A549 embryonic lung cells and BMPs 4 and 7 in rabbit and human leukocytes during ectopic bone formation (16Minamide A. Boden S.D. Viggeswarapu M. Hair G.A. Oliver C. Titus L. J. Bone Joint Surg. Am. 2003; 85: 1030-1039Crossref PubMed Scopus (69) Google Scholar). The effects of LMP-1 reinforce the action of exogenously applied BMPs, and LMP activity is blocked by noggin, a BMP inhibitor (17Yoon S.T. Park J.S. Kim K.S. Li J. Attallah-Wasif E. Hutton W.C. Boden S.D. Spine. 2004; 29: 2603-2611Crossref PubMed Scopus (132) Google Scholar). In this study we report specific binding between LMP-1 and Smurf1 (Smad ubiquitin regulatory factor 1), an E3 ligase that ubiquitinates many molecules in BMP-2 signaling pathways, causing their degradation by proteosomes (18Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar, 19Izzi L. Attisano L. Oncogene. 2004; 23: 2071-2078Crossref PubMed Scopus (216) Google Scholar). Changes in Smurf1 levels or activity have been shown to affect osteoblast function and bone formation in vitro and in vivo (18Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar, 20Zhao M. Qiao M. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 21Zhao M. Qiao M. Harris S.E. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2004; 279: 12854-12859Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 22Ying S.X. Hussain Z.J. Zhang Y.E. J. Biol. Chem. 2003; 278: 39029-39036Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). We show that LMP-1 competes with Smads 1/5 for Smurf1 binding and propose that LMP-1 interaction with Smurf1 enhances BMP-2 responsiveness by preventing the degradation of key signaling molecules. Bacterial Strains and Cloning of cDNAs in Bacterial Expression Vectors—Escherichia coli XL1 blue and BL 21-codon plus (DE3)-RP (Stratagene) hosts were maintained on LB agar plates and grown at 37 °C in the presence of ampicillin at 100 mg/liter. All of the cloning methods were performed according to standard protocols. LMP-1, LMP-1t, LMP-2, LMP-3, Smad1, and Smad5 cDNAs were cloned into TAT-HA vector. LMP-1 mutants were generated using the following primers: hLMP1Mutant A forward primer, 5′-cgcccccgccgcggacgcagcacggtacacctttgcac-3′; hLMP1Mutant A reverse primer, 5′-gtgcaaaggtgtaccgtgct- gcgtccgcggcgggggcg-3′; hLMP1Mutant B forward primer, 5′-ggcccggccctttggggcggcagcagcagctgacagcgccccgcaac-3′; and hLMP1Mutant B reverse primer, 5′-gttgcggggcgctgtcagctgctgctgccgccccaaagggccgggcc-3′. Smurf1 cDNA was cloned into pTrcHis vector (Invitrogen). For generation of Smurf1ΔWW2 mutant, the following primers were used: hSMURF1WW2 forward primer, 5′-gtgtgaactgtgatgaacttaatcaccagtgccaactc-3′; and hSMURF1WW2 reverse primer, 5′-gagttggcactggtgattaagttcatcacagttcacac-3′. Mutagenesis was performed with a QuikChange site-directed mutagenesis kit (Stratagene). Expression and Purification of Recombinant Proteins—Bacterial cultures were grown at 37 °C until the A600 reached 0.8. Isopropyl β-d-thiogalactopyranoside was added to 200 μm, and the culture was grown for another 8 h. The cells were harvested, and the pellets were suspended in ice-cold lysis buffer (20 mm phosphate buffer, pH 7.0, containing 50 mm Tris-HCl, pH 7.5, and 0.5 m NaCl). The uniform cell suspension was sonicated (Sonicator, model W-385, Heat Systems-Ultrasonics, Inc.) using 4 × 15 s bursts at minimum power output settings in ice with a 2-min interval between each burst. The lysate was centrifuged at 10,000 × g at 4 °C, and the supernatant was applied to Sephacryl S-100/S-200 columns (HiPrep 16 × 60) using an AKTA fast protein liquid chromatography system with Unicorn 4.0 software (Amersham Biosciences) at a flow rate of 1 ml/min. Fractions (2-4 ml) were collected immediately after the void volume (35 ml). Aliquots from each fraction were assayed by slot blotting, SDS-PAGE, and Western blotting. The fractions, identified by Western blots were pooled, dialyzed against 20 mm phosphate buffer, pH 7.5, containing NaCl (50 mm) and imidazole (20 mm), and applied to Ni2+ affinity resin (Probond, Invitrogen) previously equilibrated with 4 × 10 ml of buffer. Nonspecific proteins were washed off the column with 3 × 10 ml of 20 mm phosphate buffer, pH 6.0, containing NaCl (50 mm) and imidazole (20 mm). Affinity-bound proteins were eluted using three 10-ml washes with 20 mm phosphate buffer, pH 4.0, containing NaCl (50 mm). Fractions containing the desired protein were pooled (based on Western blot) and then concentrated and desalted using the centriprep devices (Amicon). The proteins were quantitated using Bio-Rad protein assay reagent. The yield of recombinant protein was routinely 0.5-1 mg of pure protein from every 2-liter culture. Biotinylation of Protein Ligands—Purified protein ligands were prepared at 10 mg/ml in 50 mm sodium borate buffer, pH 8.5, 0.5 m NaCl. Various amounts of sulfo-NHS-biotin (100 mm stock in Me2SO) was mixed with protein ligand to achieve a molar ratio of sulfo-NHS-biotin/protein ligand of 10.0 in a 100-μl reaction volume. After 2 h on ice with occasional shaking, the reaction was terminated with the addition of lysine to a final concentration of 20 mm. The unreacted free biotin was removed by gel filtration, and the concentrated labeled ligand was stored at -20 °C until use. Labeled or unlabeled LMP-1, Smad1, Smurf1, and Smad5 were prepared by using the TnT coupled in vitro transcription/translation system (Promega). The specific activity of biotin incorporation into proteins was normalized by quantitating biotin using the avidin-2-hydroxyazobenzene-4′-carboxylic acid assay (23Green N.M. Adv. Protein Chem. 1975; 29: 85-133Crossref PubMed Scopus (1605) Google Scholar). Cell Culture—MSCs at passage 2 (Cambrex Bio Sciences) were grown at 37 °C in 5% CO2 in MSC basal medium supplemented with Singlequots (Cambrex Bio Sciences), split at confluence, and plated at 3 × 104 cells/well in 6-well dishes at passage 4 in these studies. The next day treatments were applied in the presence of 50 μm l-ascorbic acid 2-phosphate and 5 mm β-glycerol phosphate (Sigma-Aldrich). The medium was changed every 3-4 days with reapplication of treatments where appropriate. The cells were transduced for 30 min with adenoviral constructs in 0.3 ml of serum-free medium. Preparation of Nuclear and Cytoplasmic Protein Fractions—Cell pellets were suspended in buffer A (20 mm HEPES, pH 7.9, 10 mm KCl, 1 mm EGTA, 1 mm EDTA, 0.2% Nonidet P-40, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml protease inhibitor mix (Sigma)), incubated on ice for 10 min, and centrifuged. Supernatants (cytoplasmic fraction) were collected, and nuclear pellets were suspended in high salt buffer B (buffer A plus 600 mm KCl, 20% glycerol), incubated on ice for 30 min, and centrifuged. Supernatants were collected as the nuclear fraction. The protein amounts were determined with Bio-Rad protein assay. SDS-PAGE and Western Blotting—SDS-PAGE was performed using 10% gels and transferred to nitrocellulose membrane. The membrane was 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 (PerkinElmer Life Sciences), and again washed with TBST. Chemiluminescent substrates were applied to the membrane, and the signal was detected by exposure to x-ray film. To demonstrate equal protein loading in each lane, a signal was developed for β-actin in all samples. Biotin Transfer Assay for Detection of LMP-1-interacting Proteins— Sulfo-sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3′-dithiopropionate (Pierce), a trifunctional cross-linking agent was used to label LMP-1. The labeled protein was incubated as a bait with nuclear proteins and cross-linked to interacting proteins by UV (365 nm). Proteins that physically interact with LMP-1 retained the biotin group when suspended in SDS-PAGE reducing buffer. Biotin-containing target proteins were separated using neutravidin beads, detected by Western blotting with neutravidin-HRP, and the signal was developed with chemiluminescent substrate. Corresponding protein bands were in-gel digested with trypsin. Tryptic peptides were recovered and concentrated, and their mass profile was analyzed by MALDI-TOF at the Emory University Microchemical Facility. Smurf1-WW Domain and LMP-1 Interaction Assay (Ligand Blotting)—The proteins were separated by SDS-PAGE and blotted onto nitrocellulose membrane. The protein blots were blocked with 5% milk protein and preincubated with purified LMP-1 or LMP-2 protein (10 μm) or TBST buffer. The blots were incubated with Smurf antibody at 1:5000 dilution (rabbit antibody raised to WW domain peptide; Upstate Biotechnology, Inc, catalog number 07-249). After washes, the blots were incubated with HRP-labeled Anti-rabbit second antibody. The washed blots were then incubated with ECL substrate solution, and the membranes were exposed to x-ray film for signal detection. Slot-Blot Assay—20 μl of purified Smurf1 (50 μg/ml) was blotted onto nitrocellulose in slot blot wells, and the wells were blocked with 0.5% Tween 20 in TBST for 30 min. The biotinylated ligand (LMP-1, LMP-2, LMP-3, Smad1, or Smad5) was mixed with varying concentrations of competing proteins or peptides and incubated in slot blot wells with Smurf1 for 90 min. The wells were washed, and the blots were blocked with TBST containing 0.5% Tween 20. Control wells contained LMP-1 hapten (an antigenic peptide from the c-terminal end of the polypeptide chain) as a competitor peptide. The blots were then incubated with HRP-labeled avidin for 1 h. After washes the blots were incubated with ECL substrate solution, and the membranes were exposed to x-ray film for signal detection. Protein A-based Immunoprecipitation Assay—Protein A-agarose beads were incubated with LMP-1 antibody or Smurf1 antibody (H-60, Santa Cruz catalog number sc25510), washed three times, incubated with nuclear proteins, and washed again to remove unbound protein. The bound proteins were eluted by two washes in 0.1 m citric acid, pH 2.7. The eluates were neutralized with Tris base and concentrated by centricon tubes (Ambicon) prior to SDS-PAGE and Western blotting. Osteogenic Differentiation of hMSCs— hMSCs at passage 4 were seeded at 3 × 104 cells/well in a 6-well plate. The next day, the cells were infected with Ad35LMP-1 (1-10 pfu/cell) and incubated with and without BMP-2 (100 ng/ml). The medium was replaced every 3-4 days, and deposition of mineral was observed after 2 weeks. To assess mineralization, the cultures were washed with phosphate-buffered saline and fixed in a solution of ice-cold 70% ethanol for 2-3 h. The cultures were rinsed with water and stained for 10 min with 1 ml of 40 mm alizarin red (pH 4.1). The cultures were rinsed two or three times with phosphate-buffered saline to reduce nonspecific staining, air-dried, and photographed. RNA Extraction—RNA was isolated from cells grown in 6-well plates using RNeasy mini kits (Qiagen). Briefly, the cells were disrupted in RNeasy lysis buffer (Qiagen) and passed over QiaShredder columns, and the eluate was brought to 35% EtOH and passed over RNeasy columns. The RNA was eluted from the membrane with water. All of the RNA samples were DNase-treated either using the Qiagen RNase-free DNase during the RNeasy procedure or after final harvest of the RNA using the Ambion DNA-free kit. After completion of the digestion, 5 μlofDNase inactivation buffer was added, and the samples were centrifuged for 1 min. The RNA containing supernatant was removed and stored at -70 °C. Each sample consisted of RNA isolated from 2 wells of a 6-well plate. Real Time Reverse Transcription-PCR—Two μg of total RNA was reverse transcribed in a 100-μl total volume containing 50 mm KCl, 10 mm Tris, pH 8.3, 5.5 mm MgCl2, 0.5 mm each dNTPs, 0.125 μm random hexamer, 40 units RNase inhibitor, and 125 units MultiScribe (Applied Biosystems). In control samples the RNase inhibitor and MultiScribe were omitted. The samples were incubated for 10 min at 25 °C, 30 min at 48 °C, and then 5 min at 95 °C to inactivate the enzyme. Real time PCR was then performed on 5 μl of the resulting cDNA in a total volume of 25 μl containing 12.5 μl of 2× SYBR Green PCR Master Mix (Applied Biosystems), and 0.8 μm each primer. The forward primer for osterix was 5′-tcagacgccccgacctt-3′, and the reverse primer was 5′-attggcaagcagtggtctagaga-3′ (PCR conditions: 2 min at 50 °C, 10 min at 95 °C, and 45 cycles of 95 °C for 15 s followed by 1 min at 62 °C). PCR was also performed on a 1:800 dilution of the cDNA with 18 S primers for normalization of the samples. Relative RNA levels were calculated using the ΔΔCt method (Applied Biosystems). siRNA Treatment of Cells—MSCs were transfected with Oligofectamine (Invitrogen) transfection reagent and either irrelevant siRNA or specific siRNA for Smurf1 (5′-ccuugcaaagaaagacuuc-3′). Silencing of the gene and specificity was confirmed by real time reverse transcription-PCR analysis of specific Smurf1 mRNA with forward primer (5′-cccagagaccttaacagtgtgaact-3′) and reverse primer (5′-ttgagttggcactggtgattca-3′). Ubiquitination Reaction in Vitro—Purified Smad1 (100 ng) was buffer-exchanged to ubiquitination buffer (50 μm Tris-HCl pH 7.8, 5 mm MgCl2, 0.5 mm dithiothreitol, 2 mm NaF, and 3 μm okadaic acid). Smad1 was then combined with a mixture of purified E1 and E2 enzymes and incubated with Smurf1 (E3 ligase) in the presence or absence of recombinant LMP-1 or LMP-2 protein. The reaction mixture also contained 2 mm ATP, ubiquitin (150 μm), ubiquitin aldehyde (5 μm), and creatine kinase-ATP generating system (Boston Biochem). The ubiquitin aldehyde was included to prevent hydrolysis of polyubiquitin chains. The reaction mixture (40 μl) was incubated for 4 h at 37°C. Aliquots at various time points were taken for SDS-PAGE and Western blotting using specific antibody for Smad1 and/or ubiquitin. Preparation of Peptides—Peptides were synthesized with a protein transduction domain at the N-terminal end (rrqrrtsklmkr) (24Mi Z. Mai J. Lu X. Robbins P.D. Mol. Ther. 2000; 2: 339-347Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). LMP-1 Interacts with Smurf1—In an effort to understand the mechanism by which LMP-1 enhances osteoblast differentiation and possibly modulates BMP responsiveness, we elucidated proteins that interact with LMP-1 using a biotin transfer assay. Recombinant LMP-1 was labeled with sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3′-dithiopropionate-biotin transfer reagent and incubated with nuclear proteins from MSCs. LMP-1 associated with four protein bands (Fig. 1a). The 85-kDa protein doublet matched the two isoforms of Smurf1 based on peptide mass profile. The other bands were identified as cytoskeletal proteins (tubulin, caldesmon, and meosin) and not likely related to the unique osteoinductive properties of LMP. Binding to cytoskeletal proteins has previously been observed for LMP and other proteins having both PDZ and LIM domains, as does LMP-1 (25Guy P.M. Kenny D.A. Gill G.N. Mol. Biol. Cell. 1999; 10: 1973-1984Crossref PubMed Scopus (91) Google Scholar, 26Vallenius T. Luukko K. Makela T.P. J. Biol. Chem. 2000; 275: 11100-11105Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Identity of the gel purified 85-kDa LMP-1-binding protein was verified by performing a Western blot using antibody that binds Smurf1 (Fig. 1b). Smurf1 is a member of the Hect family of E3 ligases and has been reported to interact with Smad1, Smad5, Runx2, Smad7, TGF-β1, and BMP receptors.(18Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar, 19Izzi L. Attisano L. Oncogene. 2004; 23: 2071-2078Crossref PubMed Scopus (216) Google Scholar, 20Zhao M. Qiao M. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 27Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar, 28Murakami G. Watabe T. Takaoka K. Miyazono K. Imamura T. Mol. Biol. Cell. 2003; 14: 2809-2817Crossref PubMed Scopus (264) Google Scholar) These interactions result in ubiquitination of the targeted protein and subsequent proteasomal degradation. To determine whether the Smurf1/LMP-1 interaction occurs between the endogenous proteins in cells, nuclear protein extracts of MSCs were incubated with an LMP-1-specific antibody, immunoprecipitated, and analyzed in Western blots with LMP-1 and Smurf1 antibody, separately. The expected size bands showing both endogenous LMP-1 (50 kDa) and Smurf1 (85 kDa) are present in the complex immunoprecipitated with the LMP-1 antibody (Fig. 1c). This observation was confirmed by detection of LMP-1 when Smurf1 (H-60) antibody was used for the immunoprecipitation (Fig. 1c). We next determined that LMP-1 protein inhibits Smurf WW domain antibody from binding to Smurf1, suggesting that LMP-1 interacts with Smurf1 via its WW domains, which are also required for interaction with Smads 1/5 (Fig. 1d) (18Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar). Further, we observed that purified nondenatured LMP-1 binds purified nondenatured Smurf1 at the WW domain. LMP-1 substantially reduces binding of the Smurf1 WW domain antibody but has no effect on binding of antibody raised to a different Smurf1 epitope (Fig. 1e). These findings raise the possibility that LMP-1 could increase cellular responsiveness to BMPs by blocking Smurf1 from ubiquitinating Smads 1/5 and thereby increasing the available levels of R-Smads (21Zhao M. Qiao M. Harris S.E. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2004; 279: 12854-12859Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Determination of the Domains of LMP-1 and Smurf1 That Are Involved in Mutual Binding—To better understand the interaction of LMP-1 with Smurf1, we determined the specific region of LMP-1 most likely to interact with Smurf1 by domain and motif analysis. Three bone-inducing LMP variants (LMP-1, LMP1t, and LMP-3) each contain a unique 45-amino acid peptide sequence that includes two putative Smurf1 WW domain interaction sites (A and B) (Fig. 2a) (29Nakayama T. Gardner H. Berg L.K. Christian J.L. Genes Cells. 1998; 3: 387-394Crossref PubMed Scopus (66) Google Scholar). The WW domain-interacting sites are absent in the nonosteogenic LMP variant (LMP-2). Two LMP isoforms (LMP-1t and LMP-3) are truncated at the C 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 (30Liu Y. Hair G.A. Boden S.D. Viggeswarapu M. Titus L. J. Bone Min. Res. 2002; 17: 406-414Crossref PubMed Scopus (38) Google Scholar). We next prepared slot blots with recombinant Smurf1 and used biotin-labeled LMP variants to demonstrate that only the LMP variants with the WW domain interaction sites were able to bind to Smurf1 (Fig. 2b). To determine which of the two WW domain interaction sites were required for the binding of LMP with Smurf1, we prepared two mutant forms of recombinant LMP proteins by substituting alanines for prolines in the putative binding sites. We found that only WW domain interaction site B is required for the interaction with Smurf1 (Fig. 2c). We further demonstrated that LMP-1 was no longer able to bind to a mutant form of Smurf1 that had its WW2 domain deleted, suggesting that the Smurf1 WW2 domain is required for LMP-1 binding (Fig. 2d). To determine whether the LMP/Smurf1 interaction is of sufficient binding affinity to displace the normal binding partners of Smurf1, we studied the competitive binding of LMP-1, Smad1, and Smad5 to Smurf1 and found that the relative binding affinity of LMP-1 for Smurf1 is similar to that of Smad1 and Smad5, suggesting that the LMP-1 interaction with Smurf1 is physiologically relevant (Fig. 2e). LMP-1 Peptides Containing WW Domain-interacting Site B Bind Smurf1—To further confirm the identity of the LMP motif that interacts with Smurf1 and to determine whether the activity of the fulllength LMP protein could be replicated by a small peptide, we synthesized small peptides comprising various portions of the 45-amino acid osteoinductive region containing one, two, or none of the putative LMP WW domain-interacting sites (Fig. 3a). We studied the competitive binding affinity of these LMP peptides and found that the two peptides that contained intact WW domain-interacting Site B were able to compete with full-length LMP-1 (Fig. 3b) and with Smad1 and Smad5 (Fig. 3c) for binding with Smurf1. This result is consistent with the mutational analysis above establishing Site B as the more critical site in LMP. LMP-1 Interaction with Smurf1 Blocks Smurf1-induced Ubiquitination of Smad1 and Smad5 and Increases Cellular Responsiveness to BMP-2—The most relevant physiologic question is whether these mechanisms are active in undifferentiated MSCs, which are the initiating cells in adult osteogenesis. In these cells, we investigated whether the LMP-1 interaction with Smurf1 results in an increased cellular response to BMP-2. We measured the levels of Smad1 that were phosphorylated in response to BMP-2 treatment (100 ng/ml) and found a significantly greater increase in the presence of LMP-1 (Fig. 4a). This response could be observed after 4 or 24 h (data not shown) but was most prominent 8 h after treatment. In a separate experiment we also demonstrated a similar increase in the type 1A BMP receptor in cells treated with LMP-1 and BMP-2 (data not shown). To confirm that this observation could be due to LMP-induced reduction of Smurf1-mediated ubiquitination of Smad1, we performed ubiquitination assays. Less ubiquitinated Smad1 was observed in the presence of LMP-1 (Fig. 4b), demonstrating that LMP-1 can inhibit this critical function of Smurf1. To further suggest that the LMP-1 effects on BMP responsiveness are conveyed by negating the effect of Smurf1, we demonstrated that the expression of small interference RNA to Smurf1 resulted in a similar increase in phosphorylated Smad1 upon BMP-2 exposure as seen with LMP-1 (Fig. 4c). We then investigated whether the LMP-1/Smurf1 interaction that potentiates the initial Smad1 phospho" @default.
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• W2006062243 title "LIM Mineralization Protein-1 Potentiates Bone Morphogenetic Protein Responsiveness via a Novel Interaction with Smurf1 Resulting in Decreased Ubiquitination of Smads" @default.
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