Deficiency of SHP-1 Protein-Tyrosine Phosphatase Activity Results in Heightened Osteoclast Function and Decreased Bone Density
1999; Elsevier BV; Volume: 155; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)65116-4
ISSN1525-2191
AutoresSyuji Umeda, Wesley G. Beamer, Katsumasa Takagi, Makoto Naito, Shin Hayashi, Hiroyuki Yonemitsu, Taolin Yi, Leonard D. Shultz,
Tópico(s)Protein Tyrosine Phosphatases
ResumoMice homozygous for the motheaten (Hcphme) or viable motheaten (Hcphme-v) mutations are deficient in functional SHP-1 protein-tyrosine phosphatase and show severe defects in hematopoiesis. Comparison of femurs from mev/mev mice revealed significant decreases in bone mineral density (0.33 ± 0.03 mg/mm3 for mev/mevversus 0.41 ± 0.01 mg/mm3 for controls) and mineral content (1.97 ± 0.36 mg for mev/mevversus 10.64 ± 0.67 for controls) compared with littermate controls. Viable motheaten mice also showed reduced amounts of trabecular bone and decreased cortical thickness. These bone abnormalities were associated with a 14% increase in numbers of multinucleated osteoclasts and an increase in osteoclast resorption activity. In co-cultures of normal osteoblasts with mutant or control bone marrow cells, numbers of osteoclasts developing from mutant mice were increased compared with littermate control mice. Although mev/mev. osteoclasts develop in the absence of colony-stimulating factor (CSF)-1, nevertheless cultured osteoclasts show increased size in the presence of CSF-1. CSF-1-deficient osteopetrosis (op/op) mutant mice develop severe osteosclerosis. However, doubly homozygous mev/mevop/op mice show an expansion of bone marrow cavities and reduced trabecular bone mass compared with op/op mice. Western blot analysis showed that several proteins that were markedly hyperphosphorylated on tyrosine residues were detected in the motheaten osteoclasts, including a novel 126-kd phosphotyrosine protein. The marked hyperphosphorylation of a 126-kd protein in motheaten osteoclasts suggests that this protein depends on SHP-1 for dephosphorylation. These findings demonstrate that the decreased SHP-1 catalytic activity in me/me and mev/mev mice results in an increased population of activated osteoclasts and consequent reduction in bone density. Mice homozygous for the motheaten (Hcphme) or viable motheaten (Hcphme-v) mutations are deficient in functional SHP-1 protein-tyrosine phosphatase and show severe defects in hematopoiesis. Comparison of femurs from mev/mev mice revealed significant decreases in bone mineral density (0.33 ± 0.03 mg/mm3 for mev/mevversus 0.41 ± 0.01 mg/mm3 for controls) and mineral content (1.97 ± 0.36 mg for mev/mevversus 10.64 ± 0.67 for controls) compared with littermate controls. Viable motheaten mice also showed reduced amounts of trabecular bone and decreased cortical thickness. These bone abnormalities were associated with a 14% increase in numbers of multinucleated osteoclasts and an increase in osteoclast resorption activity. In co-cultures of normal osteoblasts with mutant or control bone marrow cells, numbers of osteoclasts developing from mutant mice were increased compared with littermate control mice. Although mev/mev. osteoclasts develop in the absence of colony-stimulating factor (CSF)-1, nevertheless cultured osteoclasts show increased size in the presence of CSF-1. CSF-1-deficient osteopetrosis (op/op) mutant mice develop severe osteosclerosis. However, doubly homozygous mev/mevop/op mice show an expansion of bone marrow cavities and reduced trabecular bone mass compared with op/op mice. Western blot analysis showed that several proteins that were markedly hyperphosphorylated on tyrosine residues were detected in the motheaten osteoclasts, including a novel 126-kd phosphotyrosine protein. The marked hyperphosphorylation of a 126-kd protein in motheaten osteoclasts suggests that this protein depends on SHP-1 for dephosphorylation. These findings demonstrate that the decreased SHP-1 catalytic activity in me/me and mev/mev mice results in an increased population of activated osteoclasts and consequent reduction in bone density. Osteoporosis is a major public health problem and is characterized by fragility fractures of the skeleton, most notably of the spine, wrist, and hip.1Khosla S Riggs L Melton L Clinical spectrum.in: Riggs L Melton L Osteoporosis: Etiology, Diagnosis and Management. Raven Press, New York1995: 133-160Google Scholar The maintenance of bone mass is a dynamic process requiring a balance between bone resorption and bone formation.2Parfitt AM The cellular basis of bone remodeling: the quantum concept reexamined in light of recent advances in the cell biology of bone.Calcif Tissue Int. 1984; 36: S37-S45Crossref PubMed Scopus (334) Google Scholar This process requires the coordinated regulation of bone-forming cells (osteoblasts. and bone-resorbing cells (osteoclasts). Increased osteoclast activity and/or decreased osteoblast activity may contribute to the development of osteoporosis. Osteoclasts are derived from hematopoietic progenitors and are members of the monocyte/macrophage family, whereas osteoblasts are derived from mesenchymal stem cells.3Walker DG Osteopetrosis cured by temporary parabiosis.Science. 1973; 180: 875Crossref PubMed Scopus (138) Google Scholar, 4Kahn AJ Simmons DJ Investigation of cell lineage in bone using a chimaera of chick and quail embryonic tissue.Nature. 1975; 258: 325-327Crossref PubMed Scopus (169) Google Scholar, 5Ibbotson KJ Roodman GD McManus LM Mundy GR Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cells.J Cell Biol. 1984; 99: 471-480Crossref PubMed Scopus (185) Google Scholar, 6Umeda S Takahashi K Naito M Shultz LD Takagi K Neonatal changes of osteoclasts in osteopetrosis (op/op) mice defective in production of functional macrophage colony-stimulating factor (M-CSF) protein and effects of M-CSF on osteoclast development and differentiation.J Submicrosc Cytol Pathol. 1996; 28: 13-26PubMed Google Scholar The autosomal recessive motheaten mutation (Hcphme) and the less severe allelic viable motheaten mutation (Hcphme-v. cause aberrant splicing of the hematopoietic cell phosphatase (Hcph) gene transcript. The Hcph gene encodes the cytoplasmic protein-tyrosine phosphatase (PTP) Src-homology domain-2 phosphatase 1 (SHP-1) (also known as hematopoietic cell phosphatase, PTP-1C, src homology PTP-1, or PTP nonreceptor type 6).7Shen SH Bastien L Posner BI Chretien P A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.Nature. 1991; 352: 736-739Crossref PubMed Scopus (395) Google Scholar, 8Plutzky J Neel BG Rosenberg RD Isolation of a src homology 2-containing tyrosine phosphatase.Proc Natl Acad Sci USA. 1992; 89: 1123-1127Crossref PubMed Scopus (321) Google Scholar, 9Plutzky J Neel BG Rosenberg RD Eddy RL Byers MG Jani-Sait S Shows TB Chromosomal localization of an SH2-containing tyrosine phosphatase (PTPN6).Genomics. 1992; 13: 869-872Crossref PubMed Scopus (48) Google Scholar, 10Yi TL Cleveland JL Ihle JN Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12–p13.Mol Cell Biol. 1992; 12: 836-846Crossref PubMed Scopus (307) Google Scholar, 11Zhao Z Bouchard P Diltz CD Shen SH Fischer EH Purification and characterization of a protein tyrosine phosphatase containing SH2 domains.J Biol Chem. 1993; 268: 2816-2820Abstract Full Text PDF PubMed Google Scholar The finding that the me and mev mutations disrupt the Hcph. structural gene encoding SHP-1 has illustrated the role of SHP-1 as a negative regulator in many signaling pathways in the hematopoietic and immune systems.12McCulloch J Siminovitch KA Involvement of the protein tyrosine phosphatase PTP1C in cellular physiology, autoimmunity and oncogenesis.Adv Exp Med Biol. 1994; 365: 245-254Crossref PubMed Scopus (5) Google Scholar, 13Ihle JN Cytokine receptor signalling.Nature. 1995; 377: 591-594Crossref PubMed Scopus (1155) Google Scholar SHP-1 is a member of the family of PTPs that contain SH2 domains and is a cytoplasmic PTP expressed primarily in hematopoietic cells.7Shen SH Bastien L Posner BI Chretien P A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.Nature. 1991; 352: 736-739Crossref PubMed Scopus (395) Google Scholar, 8Plutzky J Neel BG Rosenberg RD Isolation of a src homology 2-containing tyrosine phosphatase.Proc Natl Acad Sci USA. 1992; 89: 1123-1127Crossref PubMed Scopus (321) Google Scholar, 10Yi TL Cleveland JL Ihle JN Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12–p13.Mol Cell Biol. 1992; 12: 836-846Crossref PubMed Scopus (307) Google Scholar, 14Matthews RJ Bowne DB Flores E Thomas ML Characterization of hematopoietic intracellular protein tyrosine phosphatases: description of a phosphatase containing an SH2 domain and another enriched in proline-, glutamic acid-, serine-, and threonine-rich sequences.Mol Cell Biol. 1992; 12: 2396-2405Crossref PubMed Scopus (325) Google Scholar, 15Adachi M Fischer EH Ihle J Imai K Jirik F Neel B Pawson T Shen S Thomas M Ullrich A Zhao Z Mammalian SH2-containing protein tyrosine phosphatases.Cell. 1996; 85: 15Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar The me and mev mutations result in a complete or partial loss, respectively, of SHP-1 catalytic activity.16Shultz LD Schweitzer PA Rajan TV Yi T Ihle JN Matthews RJ Thomas ML Beier DR Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene.Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (712) Google Scholar, 17Tsui HW Siminovitch KA de-Souza L Tsui FW Motheaten and viable motheaten mice have mutations in the haematopoietic cell phosphatase gene.Nat Genet. 1993; 4: 124-129Crossref PubMed Scopus (536) Google Scholar These two mutant alleles generate phenotypes that are qualitatively similar but of different severity (Table 1).18Green MC Shultz LD Motheaten, an immunodeficient mutant of the mouse. I: Genetics and pathology.J Hered. 1975; 66: 250-258Crossref PubMed Scopus (156) Google Scholar, 19Shultz LD Pleiotropic effects of deleterious alleles at the "motheaten" locus.Curr Top Microbiol Immunol. 1998; 137: 216-222Google Scholar, 20Shultz LD Coman DR Bailey CL Beamer WG Sidman CL "Viable motheaten," a new allele at the motheaten locus. I. Pathology.Am J Pathol. 1984; 116: 179-192PubMed Google Scholar, 21Shultz LD Hematopoiesis and models of immunodeficiency.Semin Immunol. 1991; 3: 397-408PubMed Google ScholarTable 1Phenotypes of Mutant MiceMutation (gene symbol)Affected geneCharacteristic phenotypeMotheaten (Hcphme)Hematopoietic cell phosphataseComplete loss of SHP-1 catalytic activitySkin lesions appear at 3 to 5 days of ageMean life span of 3 weeksImpaired immunological function:Reduced proliferative response to B cell andT cell mitogensAbsence of cytotoxic T cell responsesSeverely reduced NK cell functionSystemic autoimmunity:Polyclonal B cell activationExpression of multiple autoantibodiesViable motheaten (Hcphme-v)Hematopoietic cell phosphatasePartial activity of SHP-1 (10–20%)Skin lesions appear at 3 to 5 days of ageMean life span of 9 weeksImpaired immunological function, similar to me/me miceSystemic autoimmunity but develops in a chronic fashionOsteopetrosis (Csfmop)Macrophage CSFComplete loss of CSF-1 activityOsteopetrosisAbsence of incisorsMonocyte/macrophage deficiencyOsteoclast deficiency Open table in a new tab Our preliminary experiments showed that me/me and mev/mev mice had fragile bones. Although previous studies demonstrated hematopoietic abnormalities in these mice, little is known about the effect of deleterious alleles at the motheaten mutation on osteoclast development or function. The work described in this paper was carried out to test the hypothesis that SHP-1 protein-tyrosine phosphatase activity is required for the regulation of osteoclastogenesis and osteoclast function. First, we assessed bone density in mev/mev mice by radiography and by peripheral quantitative computed tomography (pQCT). Second, we carried out light microscopic and histochemical studies of bone tissue in me/me and mev/mev mice. Third, we measured the function of osteoclasts by pit formation in dentine slices and examined osteoclast differentiation in co-culture using bone marrow cells from me/me and mev/mev mice. Fourth, to define the potential role and mechanism of SHP-1 in the regulation of osteoclastogenesis and osteoclast function, we localized SHP-1 in osteoclasts from normal and mev/mev mice and analyzed possible SHP-1 substrates in these cells. Colony-stimulating factor 1 (CSF-1 or macrophage colony-stimulating factor (M-CSF)) is known to be essential in the development and differentiation of osteoclasts and certain macrophages. The essential role of CSF-1 in development and differentiation of osteoclasts and certain macrophages is evidenced by the effects of the osteopetrosis mutation (csfmop, hereafter termed op), which causes a total absence of CSF-1 production.22Yoshida H Hayashi S Kunisada T Ogawa M Nishikawa S Okamura H Sudo T Shultz LD Nishikawa S The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene.Nature. 1990; 345: 442-444Crossref PubMed Scopus (1576) Google Scholar The CSF-1 deficiency in op/op. mice causes widespread defects in development of the monocyte/macrophage lineage, including defects in osteoclast development. The failure of osteoclast development and differentiation in osteopetrosis (op/op) mice results in impaired bone resorption and leads to systemic osteopetrosis (Table 1).6Umeda S Takahashi K Naito M Shultz LD Takagi K Neonatal changes of osteoclasts in osteopetrosis (op/op) mice defective in production of functional macrophage colony-stimulating factor (M-CSF) protein and effects of M-CSF on osteoclast development and differentiation.J Submicrosc Cytol Pathol. 1996; 28: 13-26PubMed Google Scholar, 23Marks SC Lane PW Osteopetrosis, a new recessive skeletal mutation on chromosome 12 of the mouse.J Hered. 1976; 67: 1-18Google Scholar, 24Naito M Hayashi S-I Yoshida H Nishikawa S-I Shultz LD Takahashi K Abnormal differentiation of tissue macrophage populations in 'osteopetrosis' (op) mice defective in the production of macrophage colony-stimulating factor.Am J Pathol. 1991; 139: 657-667PubMed Google Scholar, 25Umeda S Takahashi K Shultz LD Naito M Takagi K Effects of macrophage colony-stimulating factor on macrophages and their related cell populations in the osteopetrosis mouse defective in production of functional macrophage colony-stimulating factor protein.Am J Pathol. 1996; 149: 559-574PubMed Google Scholar Immunohistochemical staining and flow cytometry analyses revealed increased numbers of macrophages in the spleen, thymus, lungs, and liver of mev/mev. mice.26Nakayama K Takahashi K Shultz LD Miyakawa K Tomita K Abnormal development and differentiation of macrophages and dendritic cells in viable motheaten mutant mice deficient in haematopoietic cell phosphatase.Int J Exp Pathol. 1997; 78: 245-257Crossref PubMed Scopus (22) Google Scholar It has been reported that macrophages from SHP-1-deficient mice show increased proliferation in response to CSF-1.27Chen HE Chang S Trub T Neel BG Regulation of colony-stimulating factor 1 receptor signaling by the SH2 domain-containing tyrosine phosphatase SHPTP1.Mol Cell Biol. 1996; 16: 3685-3697Crossref PubMed Scopus (181) Google Scholar We also produced mev/mev mice that genetically lacked CSF-1 because of homozygosity for the osteopetrosis mutation to determine the role of CSF-1 in the bone disease observed in mev/mev mice. The doubly homozygous mev/mevop/op mice displayed less severe osteopetrosis than op/op mice. This article shows that SHP-1-deficient mice develop osteoporosis that is due to increased numbers of osteoclasts and heightened osteoclast function. Thus, SHP-1 plays an important role as a negative regulator in osteoclastogenesis and osteoclast function. C57BL/6J-me/me and mev/mev mice were produced in our research colony from matings of +/me or +/mev heterozygotes, respectively. Homozygous me/me and mev/mev mice were identified by their characteristic skin lesions at 3 to 5 days of age. Littermate controls included both heterozygotes (+/me or +/mev) and +/+ mice. The (C57BL/6J ×. B6C3FeJ)F2-op/op mev/mev. mice were produced as follows: (C57BL/6J ×. B6C3FeJ)F1-+/op +/mev doubly heterozygous mice were made from C57BL/6J-+/mev × B6C3FeJ-+/op. matings. The (C57BL/6J × B6C3FeJ)F2-op/op mev/mev mice were produced from matings of (C57BL/6J × B6C3FeJ)F1-+/op +/mev double heterozygotes. Homozygous op/op mev/mev mice were identified by the characteristic (motheaten) skin lesions and characteristic (osteopetrosis) absence of incisors at 10 days of age. The femurs of op/op mev/mev and op/op +/? mice were studied by light microscopy and histochemical analyses. For conventional radiography, mice were anesthetized by intraperitoneal injection with tribromoethanol (0.2 ml/10 g body weight of 1.2% solution).28Papaioannou VE Fox JG Efficacy of tribromoethanol anesthesia in mice.Lab Anim Sci. 1993; 43: 189-192PubMed Google Scholar The mice were radiographed at 45 kV for 20 seconds at a focal distance of 35 cm with a cabinet x-ray system (Hewlett Packard, Wilmington, DE). Isolated femurs from 2-month-old mev/mev and littermate control mice were analyzed by peripheral quantitative computed tomography (pQCT) with a Stratec XCT 960M (Norland Medical Systems, Ft. Atkinson, WI) specifically modified for use on small bone specimens to measure bone mineral and volume as described previously.29Beamer WG Donahue LR Rosen CJ Baylink DJ Genetic variability in adult bone density among inbred strains of mice.Bone. 1996; 18: 397-403Abstract Full Text PDF PubMed Scopus (544) Google Scholar Briefly, isolated femurs from mev/mev and littermate control mice were scanned at 2-mm intervals over their entire lengths, and the unit volume within which mineral was measured was set at 0.1 mm3. The density values were calculated by dividing the mineral content by volume. The femur lengths were divided by body weights. Femurs and tibias from 2-month-old mev/mev and littermate control mice and from 3- to 4-week-old mev/mevop/op, mev/mev +/?, +/? op/op, and +/? +/? mice were fixed and demineralized in Bouin's fixative, processed routinely, and then embedded in paraffin. Sections were cut at 6-μm thickness and stained with hematoxylin and eosin (H&E) for histological examination. For detection of osteoclasts, paraffin sections were processed for the histochemical localization of tartrate-resistant acid phosphatase (TRAP) as described previously.6Umeda S Takahashi K Naito M Shultz LD Takagi K Neonatal changes of osteoclasts in osteopetrosis (op/op) mice defective in production of functional macrophage colony-stimulating factor (M-CSF) protein and effects of M-CSF on osteoclast development and differentiation.J Submicrosc Cytol Pathol. 1996; 28: 13-26PubMed Google Scholar Numbers of TRAP-positive cells per millimeter of bone edge were counted in the distal metaphysis of femurs. The bone surface lengths were measured by using the Quantimet Q600HR system (Leica, Deerfield, IL). In addition, tissue sections in me/me and normal littermate mice were stained by indirect immunoperoxidase using rabbit polyclonal antibody against SHP-1.25Umeda S Takahashi K Shultz LD Naito M Takagi K Effects of macrophage colony-stimulating factor on macrophages and their related cell populations in the osteopetrosis mouse defective in production of functional macrophage colony-stimulating factor protein.Am J Pathol. 1996; 149: 559-574PubMed Google Scholar, 30Jiao H Yang W Berrada K Tabrizi M Shultz L Yi T Macrophages from motheaten and viable motheaten mutant mice show increased proliferative responses to GM-CSF: detection of potential HCP substrates in GM-CSF signal transduction.Exp Hematol. 1997; 25: 592-600PubMed Google Scholar Briefly, the paraffin sections were incubated for 15 minutes in 10% hydrogen peroxide to quench endogenous peroxidase activity. After blocking for 10 minutes with donkey serum, the sections were incubated with rabbit anti-SHP-1 polyclonal antibody. Anti-rabbit immunoglobulin horseradish-peroxidase-linked whole antibody (Amersham, Little Chalfont, UK) was used as secondary antibody. After visualization with 3,3′-diaminobenzidine, the sections were counterstained with hematoxylin and coverslipped. Dentine slices from elephant tusk were the kind gift of Dr. I. Itonaga (Oita, Japan).31Fujikawa Y Shingu M Torisu T Itonaga I Masumi S Bone resorption by tartrate-resistant acid phosphatase-positive multinuclear cells isolated from rheumatoid synovium.Br J Rheumatol. 1996; 35: 213-217Crossref PubMed Scopus (47) Google Scholar, 32Raynal C Delmas PD Chenu C Bone sialoprotein stimulates in vitro bone resorption.Endocrinology. 1996; 137: 2347-2354Crossref PubMed Scopus (65) Google Scholar Dentine slices (4 mm diameter × 0.5 mm) were cut with a low-speed saw. The slices were sterilized by autoclaving and placed in 96-well culture plates. Bone marrow cells were harvested by scraping from the distal metaphysis of femurs and proximal metaphysis of tibias in 1-month-old me/me, mev/mev, and littermate control mice. A suspension (104Kahn AJ Simmons DJ Investigation of cell lineage in bone using a chimaera of chick and quail embryonic tissue.Nature. 1975; 258: 325-327Crossref PubMed Scopus (169) Google Scholar cells) of bone marrow cells from mutant or control mice was added to individual wells of 96-well culture plates. After 48 hours, the dentine slices were sonicated to remove the osteoclasts, and resorption lacunae formed on the slices were detected using a JSM-35C scanning electron microscope (Jeol, Tokyo, Japan). Four to six pits were measured for each animal, and six mice were examined. Pit areas were measured by using NIH Image 1.60 (National Institutes of Health, Bethesda, MD). Osteoblasts were isolated from the calvariae of 3-day-old C57BL/6J-+/+ mice. The calvariae were collected and digested with a solution of phosphate-buffered saline (PBS) containing 0.1. collagenase (Wako Chemical, Dallas, TX) and 0.2% dispase (Boehringer Mannheim, Indianapolis, IN). Isolated cells were cultured until they became confluent in α-minimal essential medium (αMEM) containing 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Grand Island, NY. in 25-cm2 culture flasks at 106 cells/flask. Cells were then detached from the flasks by the addition of 0.05. trypsin in PBS, collected by centrifugation (1500 rpm for 5 minutes), suspended in αMEM containing 10% FBS, and plated in 75-cm2 flasks (Corning Labware and Equipment, Corning, NY. or eight chamber Lab Tek chambers (Nalge Nunc International, Naperville, IL) at 1 × 104 cells/cm2. The me/me, mev/mev, and littermate control mice (8 weeks old) were killed by CO2. asphyxiation, and tibias and femurs were aseptically removed. The bone ends were cut off with scissors, and marrow cavities were flushed with 1 ml of αMEM by using syringes and 27-gauge needles. The bone marrow cells were then filtered through nylon mesh and washed once with αMEM. Bone marrow cells (2.5 × 104/cm2. from the mutant or control mice were cultured with C57BL6J-+/+. osteoblasts in αMEM containing 10% FBS and 10 nmol/L 1,25(OH)2D3 (Calbiochem, San Diego, CA) without or with 1.0 μg/ml human CSF-1 (Cetus, Emeryville, CA) or 10 μg/ml anti-mouse CSF-1 receptor antibody.33Sudo T Nishikawa S Ogawa M Kataoka H Ohno N Izawa A Hayashi S Nishikawa S Functional hierarchy of c-kit and c-fms in intramarrow production of CFU-M.Oncogene. 1995; 11: 2469-2476PubMed Google Scholar Medium was replaced every 3 days. After 8 days, the cells cultured in eight-well Lab Tek chambers were washed twice with PBS and fixed with 10% neutral buffered formalin, permeabilized with 1:1 (v/v) acetone/ethanol for 1 minute, and stained for histochemical localization of TRAP. Numbers of TRAP-positive cells (>2 nuclei/cell) per square millimeter were counted. The cells cultured in 75-cm2 flasks were collected after treating with trypsin and prepared for immunoblotting. Antibodies against SHP-1 have been described previously.30Jiao H Yang W Berrada K Tabrizi M Shultz L Yi T Macrophages from motheaten and viable motheaten mutant mice show increased proliferative responses to GM-CSF: detection of potential HCP substrates in GM-CSF signal transduction.Exp Hematol. 1997; 25: 592-600PubMed Google Scholar Antibodies against phosphotyrosine (anti-ptyr; UBI, Lake Placid, NY) and β-actin (Amersham) were purchased from commercial sources. Immunoprecipitation and Western blotting were performed as described previously.16Shultz LD Schweitzer PA Rajan TV Yi T Ihle JN Matthews RJ Thomas ML Beier DR Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene.Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (712) Google Scholar, 34Tabrizi M Yang W Jiao H DeVries EM Platanias LC Arico M Yi T Reduced Tyk2/SHP-1 interaction and lack of SHP-1 mutation in a kindred of familial hemophagocytic lymphohistiocytosis.Leukemia. 1998; 12: 200-206Crossref PubMed Scopus (15) Google Scholar, 35Yang W Tabrizi M Berrada K Yi T SHP-1 phosphatase C-terminus interacts with novel substrates p32/p30 during erythropoietin and interleukin-3 mitogenic responses.Blood. 1998; 91: 3746-3755Crossref PubMed Google Scholar Briefly, total cell lysates (TCLs) were prepared by lysing the cell samples in cold lysis buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 0.5% sodium deoxycholate, 0.2 mmol/L Na3VO4, 20 mmol/L NaF, 1% Nonidet P-40, 2 mmol/L phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 10% glycerol). The cell lysates were clarified by centrifugation, and Western blotting for SHP-1 protein and phosphotyrosine proteins was carried out as previous described.16Shultz LD Schweitzer PA Rajan TV Yi T Ihle JN Matthews RJ Thomas ML Beier DR Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene.Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (712) Google Scholar For immunoprecipitation experiments, the TCLs were incubated with antibodies at 4°C for 60 minutes. Immune complexes were then collected with protein A/Sepharose beads (Pharmacia, Piscataway, NJ). TCLs and immune complexes were separated by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membrane (Schleicher & Schuell, Keene, NH), probed with specific antibodies, and detected using an enhanced chemiluminescence kit (ECL, Amersham). Data are presented as means ± SEM. Statistical analyses were performed with Stat View software for Macintosh. All data were analyzed first by ANOVA to detect major effects due to genotype. When a significant F ratio was identified, groups were compared using Fisher's protected least significant difference post hoc test. Differences were judged as statistically significant when P < 0.05. Four pairs of mev/mev and littermate control mice at 2 months of age were examined by radiography. The mev/mev mice at 2 months of age were smaller than littermate control mice, and there was no marked disproportion of the limbs and tail. However, absorption of x-rays by bone in mev/mev. mice was markedly decreased, and the cortical bone was thinner compared with littermate control mice (Figure 1). The mid-diaphyseal scans for femurs from littermate control (Figure 2A) and mev/mev (Figure 2B) mice were obtained simultaneously and correspond to the XCT 960M measurements of bone parameters. High-density bone is represented by white and blue-green color; low-density bone and trabecular bone appear yellow and red in these images. The scan from the femur of a littermate control mouse (Figure 2A) shows more higher-density bone than the scan from a mev/mev mouse (Figure 2B). The thickness of cortical bone measured at the mid diaphysis of femurs in mev/mev and normal littermate control mice was 0.20 ± 0.01 mm and 0.25 ± 0.02 mm, respectively (P < 0.02). The body weight and femur parameters (length, density, mineral content, volume, and proportion of femur length to body weight) obtained from 2-month-old mev/mev and littermate control mice are shown in Table 2. There was no effect of sex on femur parameters in mev/mev or in littermate control mice (data not shown). The mineral content and bone volume of mev/mev mice were significantly lower than in littermate control mice (P < 0.001). Bone density of femurs in mev/mev mice were also lower than in littermate control mice (P < 0.02). Finally, the femur lengths were divided by body weight. The femur length/body weight ratios in mev/mev mice were larger than in littermate control mice.Figure 2Decreased levels of high density bone in mev/mev mice, as shown by mid-diaphyseal pQCT tomograms of femurs from a 2-month-old littermate control mouse (A) and mev/mev mouse (B). High-density bone is represented by white and blue-green color, whereas low-density bone is represented by red/yellow color. Littermate control femur (A) shows substantially more high-density cortical bone compared with femur from a mev/mev mouse (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 2Body Weight and Femur Measurements from 2-Month-Old MiceGenotypeBody weight (g)Length (mm)Density (mg/mm3)Mineral (mg)Volume (mm3)Length (mm)/body weight (g)mev/mev (n=11)10.80 ± 0.5212.55 ± 0.180.33 ± 0.031.97 ± 0.365.78 ± 0.841.18 ± 0.05+/? (n = 12)20.28 ± 0.69*P < 0.0001.14.77 ± 0.12*P < 0.0001.0.41 ± 0.01†P < 0.02.10.64 ± 0.67*P < 0.0001.25.60 ± 1.35*P < 0.0001.0.74 ± 0.20*P < 0.0001.Mean ± SEM are presented for groups.* P < 0.0001.† P < 0.02. Open table in a new tab Mean ± SEM are p
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