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• W2023150899 abstract "Increasingly a number of proteins important in the regulation of bone osteoclast development have been shown primarily influence osteoclastogenesis under conditions of physiologic or pathologic stress. Why basal osteoclastogenesis is normal and how these proteins regulate stress osteoclastogenic responses, as opposed to basal osteoclastogenesis, is unclear. LIM proteins of the Ajuba/Zyxin family localize to cellular sites of cell adhesion where they contribute to the regulation of cell adhesion and migration, translocate into the nucleus where they can affect cell fate, but are also found in the cytoplasm where their function is largely unknown. We show that one member of this LIM protein family, Limd1, is uniquely up-regulated during osteoclast differentiation and interacts with Traf6, a critical cytosolic regulator of RANK-L-regulated osteoclast development. Limd1 positively affects the capacity of Traf6 to activate AP-1, and Limd1–/– osteoclast precursor cells are defective in the activation of AP-1 and thus induction of NFAT2. Limd1–/– mice, although having normal basal bone osteoclast numbers and bone density, are resistant to physiological and pathologic osteoclastogenic stimuli. These results implicate Limd1 as a potentially important regulator of osteoclast development under conditions of stress. Increasingly a number of proteins important in the regulation of bone osteoclast development have been shown primarily influence osteoclastogenesis under conditions of physiologic or pathologic stress. Why basal osteoclastogenesis is normal and how these proteins regulate stress osteoclastogenic responses, as opposed to basal osteoclastogenesis, is unclear. LIM proteins of the Ajuba/Zyxin family localize to cellular sites of cell adhesion where they contribute to the regulation of cell adhesion and migration, translocate into the nucleus where they can affect cell fate, but are also found in the cytoplasm where their function is largely unknown. We show that one member of this LIM protein family, Limd1, is uniquely up-regulated during osteoclast differentiation and interacts with Traf6, a critical cytosolic regulator of RANK-L-regulated osteoclast development. Limd1 positively affects the capacity of Traf6 to activate AP-1, and Limd1–/– osteoclast precursor cells are defective in the activation of AP-1 and thus induction of NFAT2. Limd1–/– mice, although having normal basal bone osteoclast numbers and bone density, are resistant to physiological and pathologic osteoclastogenic stimuli. These results implicate Limd1 as a potentially important regulator of osteoclast development under conditions of stress. Vertebrate bone development and homeostasis requires the exquisite balance between the bone resorptive capacity of osteoclasts and bone forming capacity of osteoblasts. Perturbation of this equilibrium can have pathologic consequences. For example, pathologic bone loss, as occurs in osteoporosis, rheumatoid arthritis, Paget disease of the bone, and tumor metastasis to bone represents enhanced net osteoclastic activity. Alternatively, defective osteoclastogenesis can lead to increased bone mass or osteopetrosis. These pathologies can result from enhanced or inhibited osteoclast development, altered osteoclast function without change in number of osteoclasts, or both.The extent of bone resorption is directly related to the control of osteoclast differentiation. Osteoclasts derive from bone marrow-derived macrophages (BMDM) 3The abbreviations used are: BMDM, bone marrow-derived macrophage; TRAF, TNF receptor-associated factor; NFAT, nuclear factor of activated T cells; PTH, parathyroid hormone; aPKC, atypical protein kinase C; FHL2, four and one-half LIM domains 2; M-CSF, macrophage colony-stimulating factor; RANK-L, receptor activator of NF-κB ligand; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; PKC, protein kinase D; GST, glutathione S-transferase; TRAP, tartrate resistant acid phosphatase; preOC, prefusion osteoclast precursors; OC, osteoclast; EMSA, electrophoretic mobility shift assay; WT, wild type. 3The abbreviations used are: BMDM, bone marrow-derived macrophage; TRAF, TNF receptor-associated factor; NFAT, nuclear factor of activated T cells; PTH, parathyroid hormone; aPKC, atypical protein kinase C; FHL2, four and one-half LIM domains 2; M-CSF, macrophage colony-stimulating factor; RANK-L, receptor activator of NF-κB ligand; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; PKC, protein kinase D; GST, glutathione S-transferase; TRAP, tartrate resistant acid phosphatase; preOC, prefusion osteoclast precursors; OC, osteoclast; EMSA, electrophoretic mobility shift assay; WT, wild type. under the influence of macrophage colony stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANK-L), and incompletely understood co-stimulatory factors acting through immunoreceptor tyrosine-based activation motif-containing receptors to give rise to large, motile, multinucleated, terminally differentiated osteoclasts (1Boyle W.J. Simonet W.S. Lacey D.L. Nature. 2003; 423: 337-342Crossref PubMed Scopus (4806) Google Scholar, 2Koga T. Inui M. Inoue K. Kim S. Suematsu A. Kobayashi E. Iwata T. Ohnishi H. Matozaki T. Kodama T. Taniguchi T. Takayanagi H. Takai T. Nature. 2004; 428: 758-763Crossref PubMed Scopus (675) Google Scholar, 3Reddy S.V. Crit. Rev. Eukaryotic Gene Expression. 2004; 14: 255-270Crossref PubMed Scopus (56) Google Scholar). Other RANK-L-related inflammatory cytokines, such as TNFα and interleukin-1β, also influence osteoclastogenesis and function, either independently or in synergy with RANK-L (4Kwan Tat S. Padrines M. Theoleyre S. Heymann D. Fortun Y. Cytokine Growth Factor Rev. 2004; 15: 49-60Crossref PubMed Scopus (704) Google Scholar). Whereas M-CSF is thought to largely provide a survival/proliferative signal to macrophage precursor cells, RANK-L signals are critical for osteoclast differentiation (1Boyle W.J. Simonet W.S. Lacey D.L. Nature. 2003; 423: 337-342Crossref PubMed Scopus (4806) Google Scholar).The cellular receptor for RANK-L, RANK, is a member of the TNFR superfamily that includes the interleukin-1 receptor and Toll-like receptors (5Dunne A. O'Neill L.A. Sci. STKE. 2003; 2003: re3Crossref PubMed Scopus (510) Google Scholar). Like other TNFRs, RANK recruits adapter proteins after ligand-induced multimerization. A central family of such adapters is the TNF receptor-associated factors or TRAFs. RANK binds multiple TRAFs but only Traf6 has been shown to be critical for osteoclast development and function (6Lomaga M.A. Yeh W. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. van der Heiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z. Penninger J. Paige C. Lacey D. Dunstan C. Boyle W. Goeddel D.V. Mak T.W. Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1068) Google Scholar, 7Naito A. Azuma S. Tanaka S. Miyazaki T. Takaki S. Takatsu K. Nakao K. Nakamura K. Katsuki M. Yamamoto T. Inoue J. Genes Cells. 1999; 4: 353-362Crossref PubMed Scopus (538) Google Scholar). In the absence of Traf6 or in the presence of inhibitory peptides osteoclast differentiation is blocked (7Naito A. Azuma S. Tanaka S. Miyazaki T. Takaki S. Takatsu K. Nakao K. Nakamura K. Katsuki M. Yamamoto T. Inoue J. Genes Cells. 1999; 4: 353-362Crossref PubMed Scopus (538) Google Scholar, 8Ye H. Arron J.R. Lamothe B. Cirilli M. Kobayashi T. Shevde N.K. Segal D. Dzivenu O.K. Vologodskaia M. Yim M. Du K. Singh S. Pike J.W. Darnay B.G. Choi Y. Wu H. Nature. 2002; 418: 443-447Crossref PubMed Scopus (522) Google Scholar). TRAFs share a common C-terminal TRAF domain that serves to localize TRAFs to their target proteins and results in oligomerization of the N-terminal effector domain leading to the activation of IKK, NF-κB, the MAPKs (particularly JNK and p38), and AP-1 (9Arch R.H. Gedrich R.W. Thompson C.B. Genes Dev. 1998; 12: 2821-2830Crossref PubMed Scopus (511) Google Scholar, 10Baud V. Liu Z.G. Bennett B. Suzuki N. Xia Y. Karin M. Genes Dev. 1999; 13: 1297-1308Crossref PubMed Scopus (405) Google Scholar, 11Bradley J.R. Pober J.S. Oncogene. 2001; 20: 6482-6491Crossref PubMed Scopus (512) Google Scholar). Recently, Traf6 was also found to form a complex with the atypical PKC-interacting adapter protein p62 in osteoclasts (aPKC-p62-Traf6 complex), and this complex was shown to be important for the activation of NF-κB by RANK-L (12Duran A. Serrano M. Leiyges M. Flores J.M. Picard S. Brown J.P. Diaz-Meco M.T. Dev. Cell. 2004; 6: 303-309Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The importance of p62 to bone physiology is evident as mutations in p62 have been identified in a group of patients with 5q35-linked Paget disease (13Laurin N. Brown J.P. Morissette J. Raymond V. Am. J. Hum. Genet. 2002; 70: 1582-1588Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar), and deletion of the p62 gene in mice results in normal basal bone structure but inhibited osteoclastogenic response to parathyroid hormone (PTH) challenge (12Duran A. Serrano M. Leiyges M. Flores J.M. Picard S. Brown J.P. Diaz-Meco M.T. Dev. Cell. 2004; 6: 303-309Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar).The NF-κB and AP-1 transcriptional complexes are particularly critical for osteoclast development (14Wagner E.F. Matsuo K. Ann. Rheum. Dis. 2003; 62: ii83-ii85PubMed Google Scholar, 15Franzoso G. Carlson L. Xing L. Poljak L. Shores E.W. Brown K.D. Leonardi A. Tran T. Boyce B.F. Siebenlist U. Genes Dev. 1997; 11: 3482-3496Crossref PubMed Scopus (861) Google Scholar, 16Iotsova V. Caamano J. Loy J. Yang Y. Lewin A. Bravo R. Nat. Med. 1997; 3: 1285-1289Crossref PubMed Scopus (873) Google Scholar). AP-1 synergizes with the nuclear factor of activated T cells, NFAT1 (or NFATc2), and transcription factor to induce transcription of NFAT2 (or NFATc1) (17Ikeda F. Nishimura R. Matsubara T. Tanaka S. Inoue J. Reddy S.V. Hata K. Yamashita K. Hiraga T. Watanabe T. Kukita T. Yoshioka K. Rao A. Yoneda T. J. Clin. Investig. 2004; 114: 475-484Crossref PubMed Scopus (396) Google Scholar, 18Macian F. Lopez-Rodriguez C. Rao A. Oncogene. 2001; 20: 2476-2489Crossref PubMed Scopus (610) Google Scholar). NFAT2 expression is a critical cell fate determinant for osteoclast development (19Takayanagi H. Kim S. Koga T. Nishina H. Isshiki M. Yoshida H. Saiura A. Isobe M. Yokochi T. Inoue J. Wagner E.F. Mak T.W. Kodama T. Taniguchi T. Dev. Cell. 2002; 3: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1946) Google Scholar). Whereas there is abundant genetic evidence demonstrating the importance of AP-1 and NF-κB component proteins in the regulation of osteoclastogenesis (for review see Ref. 14Wagner E.F. Matsuo K. Ann. Rheum. Dis. 2003; 62: ii83-ii85PubMed Google Scholar), it is still not completely understood how RANK-L-induced Traf6 activation leads to the activation of these transcriptional complexes.LIM domains are unique protein-protein interacting modules found in multiple proteins throughout all cellular compartments. The Ajuba/Zyxin family are cytosolic, complex LIM proteins that associate with cellular cytoskeletal components particularly at sites of cell adhesion where they can regulate cell-cell adhesion and migration (20Marie H. Pratt S.J. Betson M. Epple H. Kittler J.T. Meek L. Moss S.J. Troyanovsky S. Attwell D. Longmore G.D. Braga V.M. J. Biol. Chem. 2003; 278: 1220-1228Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 21Kadrmas J.L. Beckerle M.C. Nat. Rev. Mol. Cell. Biol. 2004; 5: 920-931Crossref PubMed Scopus (563) Google Scholar, 22Pratt S.J. Epple H. Ward M. Feng Y. Braga V.M. Longmore G.D. J. Cell Biol. 2005; 168: 813-824Crossref PubMed Scopus (91) Google Scholar), shuttle from sites of cell adhesion into the nucleus where they have the potential to affect cell fate (21Kadrmas J.L. Beckerle M.C. Nat. Rev. Mol. Cell. Biol. 2004; 5: 920-931Crossref PubMed Scopus (563) Google Scholar, 23Nix D.A. Beckerle M.C. J. Cell Biol. 1997; 138: 1139-1147Crossref PubMed Scopus (196) Google Scholar, 24Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (114) Google Scholar), and are also found in the cytoplasm, free of cytoskeletal association, where their function is incompletely understood.Recently one member, Ajuba, was found to interact with the p62 adapter protein and affect interleukin-1-induced NF-κB activation in epithelia (25Feng Y. Longmore G.D. Mol. Cell. Biol. 2005; 25: 4010-4022Crossref PubMed Scopus (70) Google Scholar). Within this family Ajuba is most closely related to Limd1. LIMD1 was originally identified as a gene present at chromosome locus 3p21 in humans (26Kiss H. Kedra D. Yang Y. Kost-Alimova M. Kiss C. O'Brien K.P. Fransson I. Klein G. Imreh S. Dumanski J.P. Hum. Genet. 1999; 105: 552-559Crossref PubMed Scopus (54) Google Scholar), which in human twin studies has been a locus implicated as containing gene(s) important for the regulation of bone mineral density (27Wilson S.G. Reed P.W. Bansal A. Chiano M. Lindersson M. Langdown M. Prince R.L. Thompson D. Thompson E. Bailey M. Kleyn P.W. Sambrook P. Shi M.M. Spector T.D. Am. J. Hum. Genet. 2003; 72: 144-155Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). We found that the expression of Limd1 is uniquely regulated during osteoclast differentiation, interacts with Traf6, and affects the ability of Traf6 to activate AP-1. Limd1–/– mice, although having normal basal bone osteoclast numbers and bone density, are resistant to physiological and pathologic osteoclastogenic stimuli. We discuss the potential role of Limd1 as a positive regulator of Traf6 activity, only during states of osteoclastogenic stress.EXPERIMENTAL PROCEDURESAntibodies and Protein Purification—Mouse Limd1 cDNA was PCR amplified from a mouse kidney cDNA library, sequenced, and subcloned. The full-length protein and the N-terminal PreLIM region were cloned into pBacPAK9 containing a His6-FLAG tag (HF-PreLIM), baculovirus generated, and proteins purified from infected Sf9 cells using Talon metal affinity resin (Clontech). Polyclonal rabbit antiserum was raised against the purified PreLIM peptide and partially purified by mixing with purified HF-PreLIM peptide charged to polyvinylidene difluoride membrane. Ajuba antiserum has previously been described (20Marie H. Pratt S.J. Betson M. Epple H. Kittler J.T. Meek L. Moss S.J. Troyanovsky S. Attwell D. Longmore G.D. Braga V.M. J. Biol. Chem. 2003; 278: 1220-1228Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 28Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar). Mouse monoclonal antibodies against p62 were from BD Transduction Laboratories (San Diego, CA). Traf6 mouse monoclonal, and NFAT2, c-Jun, c-Fos, and Traf6 rabbit polyclonal antibodies were from Santa Cruz (Santa Cruz, CA). M2AG (mouse monoclonal anti-FLAG antibody immobilized on agarose), horseradish peroxidase-conjugated monoclonal anti-FLAG, horseradish peroxidase-conjugated monoclonal anti-Myc, horseradish peroxidase-conjugated monoclonal anti-HA antibodies were all from Sigma. Phospho-ERK, ERK1, phospho-JNK, JNK1, phosphor-p38, p38, and phospho-c-Jun rabbit polyclonal antibodies were all from Cell Signaling (Beverly, MA). Nucleophosmin antibodies were from J. Weber (Washington University). p65 RelA antiserum was from Oncogene Research Products (San Diego, CA).In Vivo Bone Analyses—Bone mineral density (BMD) was determined in male mice, 6–7 weeks of age, by dual-energy x-ray absorptiometry using a PIXCImus2 scanner (Lunar Corp., Madison, WI). Seven-week-old female mice were injected subcutaneously every 6 h for 3 days with 10 μg of hPTH peptide-(1–34) (Bachem, King of Prussia, PA). Animals were sacrificed 24 h after the last PTH injection. GST and GST-RANK-L were prepared, as described (29McHugh K.P. Hodivala-Dilke K. Zheng M.H. Namba N. Lam J. Novack D. Feng X. Ross F.P. Hynes R.O. Teitelbaum S.L. J. Clin. Investig. 2000; 105: 433-440Crossref PubMed Scopus (572) Google Scholar). Eight to 10-week-old mice were subcutaneously injected daily for 7 days at the base of the skull with 100 μg of GST or GST-RANK-L, then sacrificed. In both instances following sacrifice calvariae were isolated, fixed overnight in 10% neutral buffered formalin, and decalcified in 14% EDTA for 4–5 days. Following sample dehydration, paraffin-embedded sections were prepared and stained for TRAP with a hematoxylin counterstain. Histomorphometric analysis of osteoclast number per millimeter of trabecular surface and percent surface covered by osteoclasts were measured and analyzed using Osteomeasure (OsteoMetrics, Atlanta, GA) in a blinded fashion. Three to four calvaria slices per mouse were analyzed and statistics performed with the unpaired Student's t test. K/BxN serum (200 μl, intraperitoneally) was injected into 8-week-old mice on days 1 and 4. On day 7 clinical evidence for ankle inflammation was assessed and ankle swelling measured. Mice were sacrificed and ankles and feet prepared for histological analysis. Serum was also obtained on day 7 and levels of type I collagen C-terminal fragments determined, as described by manufacturer (Nordic Biosciences, Denmark).Bone Marrow Macrophage Isolation and Osteoclast Differentiation—Primary BMDM were extracted from femora and tibia of 6–8-week-old mice and cultured overnight in α-10 medium (lipopolysaccharide-free α-minimal essential medium containing 10% inactivated fetal bovine serum). Nonadherent cells were collected by centrifugation and re-plated in fresh α-10 medium containing 1/10 volume of CMG 14–12 culture supernatant (which was equivalent to 130 ng/ml of recombinant M-CSF) for 4 days. Fresh media and M-CSF were supplemented every other day. For osteoclast differentiation cells were washed in phosphate-buffered saline, lifted, and reseeded at 1.5 × 106 cells/10-cm dish, in osteoclast differentiation medium (α-10 medium containing 10 ng/ml M-CSF and 100 ng/ml of recombinant RANK-L). Media was changed every other day. TRAP+ mononuclear prefusion osteoclast precursors (preOCs) are present after 3 days of differentiation, whereas TRAP+ mature, terminally differentiated, multinucleated osteoclast cells (OCs) are produced after 5 or 6 days in culture. TRAP staining was performed as described by the manufacturer (Sigma).Osteoclast Bone Matrix Resorption Assay—BMDM were plated on synthetic calcium matrices (BD Biosciences), and allowed to differentiate in the presence of 100 ng/ml RANK-L and 10 ng/ml MM-CSF for 10 days. Cells were removed with a bleach solution and calcium matrix washed 3 times with water. Resorption pits were analyzed by phase-contrast microscopy and the total area of bone resorption was determined using the Metamorph program (Molecular Devices).Retroviral Production and Macrophage Transduction—Full-length mouse Limd1 was cloned into the pMX-IRES-Bsr retrovirus vector (30Onishi M. Nosaka T. Misawa K. Mui A.L. Gorman D. McMahon M. Miyajima A. Kitamura T. Mol. Cell. Biol. 1998; 18: 3871-3879Crossref PubMed Scopus (346) Google Scholar), and transiently transfected into Plat-E packaging cells (31Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1338) Google Scholar) using FuGENE 6 transfection reagent (Roche). Virus was collected 48 h after transfection. BMDMs were infected with virus for 24 h in the presence of M-CSF and 4 μg/ml Polybrene (Sigma). Cells were then selected in the presence of M-CSF and 1 μg/ml blasticidin (Calbiochem) for 3 days.Subcellular Fractionation—Subconfluent cultures of BMDMs, preOCs, or OCs were starved of serum, M-CSF, and RANK-L in α-minimal essential medium for 6 h, then stimulated with RANK-L (100 ng/ml). Cell fractionation (nuclear, cytosolic, and total cell lysates) was performed as described (25Feng Y. Longmore G.D. Mol. Cell. Biol. 2005; 25: 4010-4022Crossref PubMed Scopus (70) Google Scholar). Protein concentration was determined with Bio-Rad protein assay kit. For Western blots, 5 μg of nuclear extract or 25 μg of cytosolic or total cell lysate were loaded in each lane.Immunoprecipitation and Western Blots—For immunoprecipitation cells were harvested, washed with cold phosphate-buffered saline, and lysed with IP buffer (20 mm HEPES, pH 7.5, 120 mm NaCl, 5 mm NaF, 1 mm sodium orthovanadate, 0.5 mm EDTA, 1 mm dithiothreitol, 5% glycerol, 0.1% Nonidet P-40, and protease inhibitor mixture from Sigma). Extracts were clarified by centrifugation at 15,000 × g for 15 min. For each IP, cell extract proteins were mixed with primary antibody or preimmune serum on ice for 2 h, and then incubated with 25 μl of protein AG/slurry (1:1, v/v) overnight with gentle rotation at 4 °C. The immunoprecipitates were washed five times with IP buffer, and boiled in SDS-loading buffer. After SDS-PAGE, under reducing conditions, products were transferred to nitrocellulose ECL membrane and subjected to Western blot analysis with ECL detection reagent (Amersham Biosciences).Electrophoretic Mobility Shift Assay (EMSA)—DNA oligos were labeled with biotin at the 5′ ends during synthesis, annealed, and purified as described (25Feng Y. Longmore G.D. Mol. Cell. Biol. 2005; 25: 4010-4022Crossref PubMed Scopus (70) Google Scholar). 3 μg of nuclear extract were mixed with EMSA binding buffer containing 10 mm HEPES, pH 7.5, 1.5 mm MgCl2, 50 mm KC1, 2.5% glycerol, 1 mm dithiothreitol, 0.5 mm EDTA, 0.2 μg of poly(dI-dC), 0.2 nm NF-κB or AP-1 binding site to a final volume of 20 μl and kept at room temperature for 30 min. The mixture was then subjected to 6% polyacrylamide gel electrophoresis. Biotin complex signals were developed with the Pierce kit following the manufacturer's instruction. Oligos used were: NF-κB, 5′-biotin-AAGTTGAGGGGACTTTCCCAGGCT-3′ and 5′-biotin-AGCCTGGGAAAGTCCCCTCAACTT-3′ and AP-1, 5′-biotin-ACGCTTGATGACTCAGCCGGAAT-3′ and 5′-biotin-ATTCCGGCTGAGTCATCAAGC-3′.Luciferase Assay—HEK293T cells (6 × 104 cells/well) were transfected with pAP-1-luciferase (0.025 μg/well, Stratagene), pTK-Renilla luciferase (0.025 μg/well, Promega), pcDNA3-Flag-Limd1 isoforms (0.1 μg/well), and pcDNA3-Flag-Traf6 (0.1 μg/well). Total plasmid amount was balanced to 0.25 μg/well with pcDNA3-Stop-Limd1 (stop codon after the ATG start site) or pcDNA3, as needed. Forty-eight hours post-transfection cells were lysed in 100 μl of lysis buffer (Promega) and firefly and Renilla luciferase activity was determined using substrates from Promega and a luminometer.RESULTSLimd1 Associates with Traf6—LIMD1 was found to interact with the atypical PKC interacting adapter protein p62/sequestosome in a yeast two-hybrid protein-protein interactive screen (25Feng Y. Longmore G.D. Mol. Cell. Biol. 2005; 25: 4010-4022Crossref PubMed Scopus (70) Google Scholar). Because the LIMD1 gene localizes to a chromosomal locus, 3p21 (26Kiss H. Kedra D. Yang Y. Kost-Alimova M. Kiss C. O'Brien K.P. Fransson I. Klein G. Imreh S. Dumanski J.P. Hum. Genet. 1999; 105: 552-559Crossref PubMed Scopus (54) Google Scholar), a region implicated in the regulation of bone density in humans (27Wilson S.G. Reed P.W. Bansal A. Chiano M. Lindersson M. Langdown M. Prince R.L. Thompson D. Thompson E. Bailey M. Kleyn P.W. Sambrook P. Shi M.M. Spector T.D. Am. J. Hum. Genet. 2003; 72: 144-155Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and p62 appears to be important for bone osteoclast development (12Duran A. Serrano M. Leiyges M. Flores J.M. Picard S. Brown J.P. Diaz-Meco M.T. Dev. Cell. 2004; 6: 303-309Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), we asked whether LIMD1 might contribute to osteoclast development.The expression pattern of Limd1, and related LIM proteins, during osteoclast differentiation was determined. BMDM were isolated from wild type adult mice, expanded in M-CSF, and then induced to differentiate into osteoclasts by adding RANK-L. At each of 6 days in cultures containing M-CSF and RANK-L, total cell protein extracts were prepared and the level of Limd1 protein determined by quantitative Western blot. BMDM were found to express low levels of Limd1 protein (Fig. 1), but during RANK-L-induced osteoclast differentiation Limd1 protein levels significantly increased (Fig. 1). This occurred, in part, at the level of transcriptional regulation as reverse transcriptase-PCR analysis revealed an increase in Limd1 mRNA during osteoclast differentiation (data not shown). The induced expression of Limd1 protein during osteoclast differentiation was similar to previously reported induced expression of Traf6 (12Duran A. Serrano M. Leiyges M. Flores J.M. Picard S. Brown J.P. Diaz-Meco M.T. Dev. Cell. 2004; 6: 303-309Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), an important cytosolic regulator of osteoclast development (Fig. 1). Ajuba, a closely related LIM protein abundant in epithelia (20Marie H. Pratt S.J. Betson M. Epple H. Kittler J.T. Meek L. Moss S.J. Troyanovsky S. Attwell D. Longmore G.D. Braga V.M. J. Biol. Chem. 2003; 278: 1220-1228Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) that also interacts with p62 (25Feng Y. Longmore G.D. Mol. Cell. Biol. 2005; 25: 4010-4022Crossref PubMed Scopus (70) Google Scholar), was present in BMDM, at a low level, but in contrast to Limd1 its level did not change during osteoclast differentiation (Fig. 1). Likewise, the level of other related LIM proteins: Wtip, Zyxin, and Lpp, did not fluctuate significantly during osteoclast differentiation (data not shown). These results indicated that of the Ajuba/Zyxin LIM protein family Limd1 protein expression was uniquely up-regulated during osteoclast differentiation, and thus, might contribute to osteoclast development and bone homeostasis.When bone osteoclast progenitors are stimulated with RANK-L, p62 assembles into a multiprotein complex containing Traf6 and atypical protein kinase C (aPKC) (12Duran A. Serrano M. Leiyges M. Flores J.M. Picard S. Brown J.P. Diaz-Meco M.T. Dev. Cell. 2004; 6: 303-309Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Because Traf6 has been shown to be a critical regulator of RANK-L-induced osteoclast development and function (6Lomaga M.A. Yeh W. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. van der Heiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z. Penninger J. Paige C. Lacey D. Dunstan C. Boyle W. Goeddel D.V. Mak T.W. Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1068) Google Scholar, 7Naito A. Azuma S. Tanaka S. Miyazaki T. Takaki S. Takatsu K. Nakao K. Nakamura K. Katsuki M. Yamamoto T. Inoue J. Genes Cells. 1999; 4: 353-362Crossref PubMed Scopus (538) Google Scholar), we asked whether Limd1 associates with Traf6. HEK293T cells were co-transfected with epitope-tagged plasmids expressing Limd1 and Traf6, Limd1 was immunoprecipitated, and bound products Western blotted for the presence of Traf6. Limd1 was found to associate with Traf6 (Fig. 2A). Mapping studies identified the C-terminal LIM region as directing the interaction of Limd1 with Traf6 (Fig. 2A). The N-terminal PreLIM region of Limd1 did not interact with Traf6 (Fig. 2A). The association of Limd1 with Traf6 was specific, as related LIM proteins LPP and Zyxin did not co-immunoprecipitate with Traf6 (Fig. 2B), nor did Traf2 interact with Limd1 (data not shown).FIGURE 2Limd1 interacts with Traf6. A, a representation of the modular organization of Limd1. The C-terminal LIM region is comprised of three LIM domains, whereas the PreLIM region is the N-terminal end of the protein. HEK293T cells were transiently co-transfected with myc-Traf6, full-length FLAG-Limd1, FLAG-PreLIM region, or FLAG-LIM region as indicated. Limd1 isoforms were immunoprecipitated (anti-FLAG) and bound products Western blotted for the presence of Traf6 (anti-myc) and Limd1 (anti-FLAG). Input controls are shown on the right panels. B, HEK293 cells were transiently co-transfected with FLAG-Traf6, myc-tagged Lpp, Zyxin, or Limd1. Traf6 was immunoprecipitated (anti-FLAG) and bound products Western blotted for the presence of bound LIM protein (anti-myc) and Traf6 (anti-FLAG). Input controls are shown in the third and fourth lanes of each panel. C, Limd1+/+ or Limd1–/– BMDM were differentiated to form preOCs, cells were lysed, and endogenous Limd1 immunoprecipitated. Bound products were Western blotted for the presence of Traf6, p62, PKCζ, and Limd1. Input controls are shown in the third and fourth lanes. D, aliquots of purified His-FLAG-tagged Limd1 (HF-LIMD1), purified GST, and GST-Traf6 were run on SDS-PAGE and the gel Coomassie stained (lower panel). Control GST (lane 1) or GST-Traf6 (lane 2) proteins were mixed with HF-Limd1. Glutathione-agarose beads were added, pelleted, washed, and bound products incubated with Prescission enzyme to cleave Traf6 from GST. The final reaction was then run on SDS-PAGE and Western blotted for Limd1 and Traf6. Input controls are shown in the third and fourth lanes. In the second lane both cleaved Traf6 and uncleaved GST-Traf6 are present. E, p62 does not affect the amount of Traf6 associated with Limd1. As in A and B HEK293T cells were transfected with the indicated plasmids, Limd1 was immunoprecipitated and bound products Western blotted for the presence and amount of Traf6, p62, and Limd1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To establish that Limd1 and Traf6 interact in physiologically relevant primary cells expressing endogenous levels of each protein" @default.
• W2023150899 created "2016-06-24" @default.
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• W2023150899 date "2007-01-01" @default.
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• W2023150899 title "The LIM Protein, LIMD1, Regulates AP-1 Activation through an Interaction with TRAF6 to Influence Osteoclast Development" @default.
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• W2023150899 doi "https://doi.org/10.1074/jbc.m607399200" @default.
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