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Stat1 Controls Postnatal Bone Formation by Regulating Fibroblast Growth Factor Signaling in Osteoblasts

2004; Elsevier BV; Volume: 279; Issue: 26 Linguagem: Inglês

10.1074/jbc.m314323200

1083-351X

Liping Xiao, Takahiro Naganawa, Eneze Obugunde, Gloria Gronowicz, David M. Ornitz, J. Douglas Coffin, Marja M. Hurley,

TGF-β signaling in diseases

Activation of the signal transducers and activators of transcription (STAT) pathway is important in fibroblast growth factor (FGF) modulation of chondrocyte proliferation and endochondral bone formation during embryogenesis. However, it is not known if the FGF/STAT signaling pathway is important for postnatal bone formation. To examine this, we have characterized a novel skeletal phenotype in Stat1-/- mice in which we find a significant increase in bone mineral density, bone mineral content, and other parameters of bone growth. The data show that osteoblasts derived from Stat1-/- mice have decreased expression of cell cycle inhibitor p21WAF/CIP and FGF receptor 3, a known negative regulator of chondrocyte proliferation. Interestingly, Stat1-/- osteoblasts showed increased expression of FGF18 in vivo and increased responsiveness to FGF18 in vitro. These results suggest a mechanism for the regulation of the osteoblast in which Stat1 functions not only to directly regulate the cell cycle but also to modify the repertoire of FGF receptor expression from a potentially inhibitory receptor, FGFR3 to a stimulatory receptor such as FGFR1 or FGFR2. Activation of the signal transducers and activators of transcription (STAT) pathway is important in fibroblast growth factor (FGF) modulation of chondrocyte proliferation and endochondral bone formation during embryogenesis. However, it is not known if the FGF/STAT signaling pathway is important for postnatal bone formation. To examine this, we have characterized a novel skeletal phenotype in Stat1-/- mice in which we find a significant increase in bone mineral density, bone mineral content, and other parameters of bone growth. The data show that osteoblasts derived from Stat1-/- mice have decreased expression of cell cycle inhibitor p21WAF/CIP and FGF receptor 3, a known negative regulator of chondrocyte proliferation. Interestingly, Stat1-/- osteoblasts showed increased expression of FGF18 in vivo and increased responsiveness to FGF18 in vitro. These results suggest a mechanism for the regulation of the osteoblast in which Stat1 functions not only to directly regulate the cell cycle but also to modify the repertoire of FGF receptor expression from a potentially inhibitory receptor, FGFR3 to a stimulatory receptor such as FGFR1 or FGFR2. Cytokines are involved in important biological processes such as inflammation and bone remodeling where they bind to their receptors for association and activation of the Janus (JAK) family of non-receptor protein tyrosine kinases (1Levy J.B. Schindler C. Raz R. Levy D.E. Baron R. Horowitz M.C. Endocrinology. 1996; 137: 1159-1165Crossref PubMed Scopus (43) Google Scholar). Activation of the JAKs mediates tyrosine autophosphorylation, phosphorylation of cytokine receptor subunits, and phosphorylation of a group of cytoplasmic proteins named signal transducers and activators of transcription (STAT, 1The abbreviations used are: STAT, signal transducers and activators of transcription; IL, interleukin; FGF, fibroblast growth factor; BMD, bone mineral density; BMC, bone mineral content; FCS, fetal calf serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BV, bone volume; TV, tissue volume; BS, bone surface; Tb.Th, trabecular thickness; Tb.SP, trabecular separation; Tb.N, trabecular number; ALP, alkaline phosphatase. Ref. 2Kisseleva T.F. Bhattacharya S. Braustein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (925) Google Scholar). Seven mammalian STAT have been identified that play a central role in the biological responses to a number of cytokines including interleukins (ILs) and growth factors to modulate cell proliferation and differentiation (2Kisseleva T.F. Bhattacharya S. Braustein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (925) Google Scholar). Stat1 was originally identified as an essential mediator of the response to interferons (IFNs) (3Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1269) Google Scholar). Stat1 is important in transducing anti-proliferative and growth arrest by inducing expression of cell cycle inhibitors p21WAF/CIP and pro-apoptotic signals by transcriptional activation of genes encoding proteins that directly induce or facilitate the process of cell death, such as caspases, FAS, and TRAIL (4Meraz M.A. White J.M. Sheehan K.C.F. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver-Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1406) Google Scholar, 5Durbin J.E. Hackenmiller R. Simon M.C. Levy D. Cell. 1996; 84: 442-450Abstract Full Text Full Text PDF Scopus (1305) Google Scholar). Activation of STATs results in nuclear translocation where they bind DNA to stimulate transcription. Interleukin-type cytokines play important roles in inflammatory responses in osteoblast function. In osteoblasts, activation of the gp/130/signal transducer, Stat3 and Stat1 by IL-6-type cytokines promoted differentiation and prevented apoptosis in osteoblastic MG-63 cells by increasing p21 (9Bellido T. Victoria Z. Borba C. Roberson P. Manolagas S.C. Endocrinology. 1997; 138: 3666-3676Crossref PubMed Scopus (147) Google Scholar, 10Bellido T. O'Brian C.A. Roberson P. Manolagas S.C. J. Biol. Chem. 1998; 273: 21137-21144Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Studies in mouse fibroblasts lacking Stat3 demonstrated that IL-6 could induce prolonged activation of Stat1 (11Costa-Pereira A.P. Tininini S. Strobl B. Alonzi T. Schlaak J.F. Is'harc H. Gesualdo I. Newman S.J. Kerr I.M. Poli V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8043-8047Crossref PubMed Scopus (218) Google Scholar). Stat1-null mice showed no overt developmental abnormalities and are similar in size to their wild-type littermates (4Meraz M.A. White J.M. Sheehan K.C.F. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver-Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1406) Google Scholar, 5Durbin J.E. Hackenmiller R. Simon M.C. Levy D. Cell. 1996; 84: 442-450Abstract Full Text Full Text PDF Scopus (1305) Google Scholar). However, their immunoregulatory responses to interferon α (IFNα) and interferon γ (IFNγ) are absent. They also have defective Tumor necrosis factor α-induced apoptosis because of low constitutive levels of caspases (7Kumar A. Commaine M. Flickenger T.W. Horvath C.M. Stark G.R Science. 1997; 278: 1630-1632Crossref PubMed Scopus (438) Google Scholar) but have normal responses to growth hormone (GH), epidermal growth factor (EGF), IL-6, IL-10, platelet-derived growth factor (PDGF), and thrombopoietin in non-osseous tissues. These hormones and cytokines have been shown to regulate Stat1 and Stat3 expression in a number of cells and tissues including osteoblasts (6Boyce B.F. Xing L. Jilka R.L. Bellido T. Weinstein R.S. Parfitt A.M. Manolagos S.T. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press, San Diego2002: 151-168Google Scholar), suggesting that Stat1 has a critical role in regulation of bone growth and bone formation. Limited analysis of the long bone development program in Stat1+/+ and Stat1-/- mice was performed by Sahni et al. (12Sahni M. Raz R. Coffin J.D. Levy D. Basilico C. Development. 2001; 128: 2119-2129Crossref PubMed Google Scholar). Histologic studies of E15.5 metatarsals demonstrated expansion of the chondrocyte population in reserve, proliferating, and hypertrophic zones of Stat1-/- mice compared with Stat1+/+ mice. At postnatal day 5, the reserve zone in Stat1-/- femurs was larger than in Stat1+/+ mice. At later stages of development, the differences in the growth plates of Stat1+/+ and Stat1-/- mice became attenuated, but the hypertrophic zone remained larger (12Sahni M. Raz R. Coffin J.D. Levy D. Basilico C. Development. 2001; 128: 2119-2129Crossref PubMed Google Scholar). Fibroblast growth factor ligands (FGFs) and their receptors (FGFRs) are important in both normal bone remodeling and in pathologic disorders of bone (13Hurley M.M. Marie P.J. Florkiewicz R. Bilezikian J. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press Academic Press, San Diego2002: 825-851Google Scholar, 14Smallwood P.M.I. Munoz-Sanjuan P. Tong J.P. Macke S.H. Hendry D.J. Gilbert N.G. Copeland N. Jenkins A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (337) Google Scholar, 15De Luca F. Baror J. TEM. 1999; 10: 61-65Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 16Ornitz D.M. Bioessays. 2000; 22: 108-112Crossref PubMed Scopus (632) Google Scholar, 17Ornitz D.M. Canalis E. Skeletal Growth Factors. Lippincott Williams and Wilkins, 2000: 197-209Google Scholar). FGF signal transduction involves activation of one or more closely related cell surface, high affinity tyrosine kinase receptors (FGFRs, Refs. 1Levy J.B. Schindler C. Raz R. Levy D.E. Baron R. Horowitz M.C. Endocrinology. 1996; 137: 1159-1165Crossref PubMed Scopus (43) Google Scholar, 2Kisseleva T.F. Bhattacharya S. Braustein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (925) Google Scholar, 3Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1269) Google Scholar, 4Meraz M.A. White J.M. Sheehan K.C.F. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver-Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1406) Google Scholar) that are encoded by separate genes (13Hurley M.M. Marie P.J. Florkiewicz R. Bilezikian J. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press Academic Press, San Diego2002: 825-851Google Scholar, 16Ornitz D.M. Bioessays. 2000; 22: 108-112Crossref PubMed Scopus (632) Google Scholar, 18Ornitz D.M. Itoh N. Genome Biol. 2001; 2: 3005.1-3005.12Crossref Google Scholar). Sporadic point mutations in human FGFR1,-2, and -3 genes cause a variety of disorders in skeletal development (17Ornitz D.M. Canalis E. Skeletal Growth Factors. Lippincott Williams and Wilkins, 2000: 197-209Google Scholar, 19Ornitz D.M. Marie P.J. Genes Dev. 2002; 16: 1446-1465Crossref PubMed Scopus (734) Google Scholar). FGFR signaling may result in proliferation, growth inhibition or differentiation depending on the cell type (13Hurley M.M. Marie P.J. Florkiewicz R. Bilezikian J. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press Academic Press, San Diego2002: 825-851Google Scholar, 17Ornitz D.M. Canalis E. Skeletal Growth Factors. Lippincott Williams and Wilkins, 2000: 197-209Google Scholar). Signaling by FGFRs is complex because alternative RNA splicing results in multiple isoforms of each receptor with distinct ligand binding specificities (13Hurley M.M. Marie P.J. Florkiewicz R. Bilezikian J. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press Academic Press, San Diego2002: 825-851Google Scholar, 16Ornitz D.M. Bioessays. 2000; 22: 108-112Crossref PubMed Scopus (632) Google Scholar, 18Ornitz D.M. Itoh N. Genome Biol. 2001; 2: 3005.1-3005.12Crossref Google Scholar). Ligand binding to FGFR results in autophosphorylation, activation and ultimately the induction of transcription regulatory proteins (16Ornitz D.M. Bioessays. 2000; 22: 108-112Crossref PubMed Scopus (632) Google Scholar). Knock-ins of human FGFR3 mutations and FGF2 transgenesis has demonstrated a role for Stat1 in mouse dwarfism models mediated by mutations in FGFR3 (20Li C. Chen L. Iwata T. Kitagawa M. Fu X.Y. Deng C. Hum. Mol. Genet. 1999; 8: 35-44Crossref PubMed Scopus (195) Google Scholar, 21Lievens P.M.-J. Liboi E. J. Biol. Chem. 2003; 278: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 12Sahni M. Raz R. Coffin J.D. Levy D. Basilico C. Development. 2001; 128: 2119-2129Crossref PubMed Google Scholar). Other studies showed that the inhibitory effect of FGF on chondrocyte proliferation requires Stat1 induction of cyclin dependent kinase inhibitor p21WAF/CIP1 (22Sahni M. Davide-Carlo A. Mansukani A. Gertner R. Levy D. Basilico C. Genes Dev. 1999; 13: 1361-1366Crossref PubMed Scopus (323) Google Scholar) to block cell cycle progression from the G1-phase to the S-phase (23Abdollahi A. Lord K.A. Hoffman-Lieberman B. Lieberman D.A. Cell Growth Differ. 1991; 2: 401-407PubMed Google Scholar). Interestingly, bone formation studies in p21-null mice suggest that p21WAF/CIP1 acts as a break on osteoblast proliferation as well as an inhibitor of the osteoblast differentiation program (24Bellosta P. Masramon L. Mansukhani A. Basilico C. J. Bone Miner. Res. 2003; 18: 818-826Crossref PubMed Scopus (43) Google Scholar). Stat1 signaling plays an important role in developmental events involving FGF/FGFR3 endochondral bone formation and chondrocyte differentiation (12Sahni M. Raz R. Coffin J.D. Levy D. Basilico C. Development. 2001; 128: 2119-2129Crossref PubMed Google Scholar, 20Li C. Chen L. Iwata T. Kitagawa M. Fu X.Y. Deng C. Hum. Mol. Genet. 1999; 8: 35-44Crossref PubMed Scopus (195) Google Scholar, 21Lievens P.M.-J. Liboi E. J. Biol. Chem. 2003; 278: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We hypothesized that Stat1 plays an essential role in FGF/FGFR signaling during intramembranous bone formation and osteoblast function in adult mice; and to test this hypothesis we examined the bone phenotype in Stat1-/- adult mice. The result shows that bone mass and bone mineral content were significantly increased in adult Stat1-/- mice. Ex vivo studies revealed increased osteoblast replication and mineralized bone nodule formation in Stat1-/- mice. In addition, calvarial width was significantly increased in Stat1-/- mice compared with Stat1+/+ mice. To gain insight into potential Stat1 mechanisms of action in modulating osteoblast function, we compared expression of FGFRs 1-4 and p21WAF/CIP1 expression in femoral bone sections and osteoblast cultures from Stat1+/+ and Stat1-/- mice. We also compared expression of three FGF proteins (FGF2, FGF9, and FGF18) that are known to modulate FGFR3 expression and function (25Hecht D. Zimmerman N. Bedford M. Avivi A. Yayon A. Growth Factors. 1995; 12: 223-233Crossref PubMed Scopus (101) Google Scholar, 26Ellsworth J.L. Berry J. Bukowski T. Claus J. Feldhaus A. Holderman S. Holdren M.S. Lum K.D. Moore E.E. Raymond F. Ren H. Shea P. Sprecher C. Storey H. Thompson D.L. Waggie K. Yao L. Fernandes R.J. Eyre D.R. Hughes S.D. Osteoarthr. Cart. 2002; 10: 308-320Abstract Full Text PDF PubMed Scopus (160) Google Scholar). There was a marked reduction in the expression of FGFR3 mRNA and protein as well as p21WAF/CIP1 protein in bones from Stat1-/- compared with Stat1+/+ mice. Immunohistochemistry revealed no significant differences in either FGF2 or FGF9 protein expression in bones from Stat1+/+ and Stat1-/- mice. Interestingly, FGF18 labeling in Stat1-/- mice was markedly increased in growth plate hypertrophic chondrocytes, cortical bone endosteal osteoblasts, and trabecular bone surface osteoblasts compared with wild type. Based on these results we propose that FGFR3 signaling via Stat1/p21WAF/CIP1 negatively regulates osteoblast function. Stat1 seem to play a direct role in regulating postnatal bone turnover and responses in differentiated osteoblasts in long bones and also may modulate intramembranous bone formation. Furthermore, the increased expression of FGF18 protein suggests that this ligand may be important in the increased bone mass of Stat1-/- mice. Mice were purchased from Taconic (Germantown, NY) and housed in the transgenic facility in the Center for Laboratory Animal Care at the University of Connecticut Health Center. Mice were sacrificed by CO2 narcosis and cervical dislocation. The University of Connecticut Health Center, Institutional Animal Care and Use Committee approved all animal protocols. Mice were utilized at 13-14 weeks of age. Dual Beam X-ray Absorptiometry—Femoral bones were harvested and stored in 70% ethanol at 4 °C. Bone mineral density (BMD) and bone mineral content (BMC) were measured using a Piximus Mouse 11 densitometer (GE Medical Systems, Madison, WI). Microcomputed Tomography (Micro-CT) Scanning of Trabecular Bone of the Distal Metaphysis of the Femurs of Adult Stat1+/+ and Stat1-/- Mice—The third lumbar vertebrae and metaphyseal cancellous bones of the distal femurs were analyzed by micro-CT system (μCT-20, Scanco Medical, Zurich), as previously reported (27Ruegsegger P. Koller B. Muller R. Calcif. Tissue Int. 1996; 58: 61-75Crossref Scopus (813) Google Scholar). Using two-dimensional data from scanned slices, three-dimensional analysis was performed to calculate morphometric indices including bone volume density (bone volume (BV)/tissue volume (TV)), trabecular thickness (Tb.Th = 2 × BV/bone surface (BS)), trabecular number (Tb.N = (BV/TV)/Tb.Th), and trabecular separation (Tb.SP = (1/Tb.N) - Tb.Th). These parameters were calculated by the parallel plate model of Parfitt et al. (28Parfitt A.M. Mathews C.H. Villanueva A.R. Kleerhofer M. Frame B. Rao D.S. J. Clin. Investig. 1983; 72: 1396-1409Crossref PubMed Scopus (1412) Google Scholar). The Tb.N was defined as the number of intersections between bone and nonbone components per total length of test lines applied to a specimen (29Goulet R.W. Goldstein S.A. Ciarelli M.J. Kuhn J.L. Brown M.B. Feldkamp L.A. J. Biomech. 1994; 27: 375-389Crossref PubMed Scopus (612) Google Scholar). Bone Histomorphometry—For dynamic histomorphometric measurements, Stat1+/+ and Stat1-/- mice at 13 weeks of age were weighed and injected on day 1 with calcein at 1 mg/100 gm body weight, a second injection was administered on day 8, and mice were sacrificed on day 10 postinjection. Femurs were dissected free of tissue at the time of sacrifice. The femurs were dehydrated in increasing concentrations of ethanol, cleared in xylene, and embedded in methyl methacrylate. 5-micron thick longitudinal serial sections were cut on a Reichert-Jung Polycut S microtome (Reichert-Jung) with a D profile knife (Delaware Diamond Knives Corp., Wilmington, DE). Sections were taken from the middle of the femur, where the central vein is located. Unstained sections were evaluated for dynamic parameters. Additional sections were stained with modified Masson trichrome stain (30Narusawa K. Nakamura T. Suzuki K. Matsuaka Y. Lee L. Tanaka H. Seino Y. J. Bone. Miner. Res. 1995; 10: 1853-1864Crossref PubMed Scopus (41) Google Scholar). Osteoblasts were identified as cuboidal cells lining the trabecular bone. Osteoclasts were identified as multinucleated cells on the trabecular bone surface. Histomorphometric measurements were made in a blinded, nonbiased manner using the Bio-Quant computerized image analysis system (R & M Biometrics, Nashville, TN) interfaced with a Nikon E400 microscope (Nikon Inc., Melville, NY). All measurements were confined to the secondary spongiosa and restricted to an area between 400 and 2000 microns distal to the growth plate-metaphyseal junction of the distal femur. The terminology and units used are those recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (31Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. J. Bone Miner. Res. 1987; 2: 595-610Crossref PubMed Scopus (4930) Google Scholar). Mineral apposition rate (MAR μm/day), and bone formation rate (BFR/BS, μm3/μm2/day) were calculated. Trabecular bone volume (BV/TV, %), trabecular thickness (Tb.Th, μm), and trabecular number (Tb.N, mm-1) were measured. For static histomorphometry, calvariae were fixed in 4% paraformaldehyde at 4 °C, decalcified in 15% EDTA, dehydrated in progressive concentrations of ethanol, cleared in xylene, and embedded in paraffin. 5-micron sections were cut and stained for tartrate-resistant acid phosphatase (TRAP) to visualize osteoclasts, and counterstained with hematoxylin (32Bancroft J.D. Enzyme Histochemistry. Churchill Livingstone, Edinburgh, New York1990: 387-389Google Scholar). Histomorphometric analysis was performed in a blinded, nonbiased manner using a computerized image analysis system. Immunohistochemistry—Paraffin-embedded sections of femoral bones from 13-14 week old mice were used. The primary antibodies were a polyclonal anti-p21 (C-19) antibody utilized at (1:50) dilution; a polyclonal anti-FGF9 antibody and a polyclonal anti-FGF18 antibody were utilized at (1:50) dilutions, were all purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA. Anti-FGF2 antibody was utilized at a concentration of 5 μg/ml and was purchased from BD Biosciences, San Diego, CA. A rabbit polyclonal anti FGFR3 C-terminal peptide antibody (33Luke Y. Oh S. Denninger A. Colvin J.S. Vyas A. Tole S. Ornitz D.M. Bansal R. J. Neurosci. Res. 2003; 23: 883-894Google Scholar) was utilized at a 1:500 dilution. Briefly, the sections were blocked (0.3% H202/methanol) for 30 min, followed by blocking in 1:200 normal serum for 30 min, and then primary antibodies were added overnight. The sections were washed 3× for 5 min in phosphate-buffered saline, and then a horseradish peroxidase-conjugated secondary antibody was applied for 30 min. Following 3× 5-min phosphate-buffered saline washes, color was developed with ABC reagent for 30 min. Slides were counterstained with methyl-green for 10 s. Slides were coverslipped, examined, and photographed using bright field microscopy (Nikon Corporation, Shinagawa-ku, Tokyo, Japan). Western Blot Analysis—The expression of Stat1 protein was determined by Western blot. Briefly, protein was extracted using 1× cell lysis buffer (Cell Signaling Technology, Inc., Beverly, MA), and total protein concentration was assayed with BCA protein assay reagent (Pierce). After SDS-polyacrylamide gel electrophoresis on 12% gels, proteins were transferred to Immobilon™ transfer membranes (Millipore). Membranes were blocked overnight in TBS-T containing 5% nonfat dry milk (Bio-Rad). Membranes were incubated with a mouse monoclonal antibody (BD Biosciences, San Diego, CA), for 1 h, washed 1 h with TBS-T, and then incubation with a rabbit anti-mouse secondary antibody (Amersham Biosciences) was performed in TBS-T/1% nonfat milk for 1 h. After incubation with antibodies, membranes were washed 1 h with TBS-T. Western Lighting™ chemiluminescence reagent (PerkinElmer Life Sciences) was used for detection. Band density was quantified by densitometry. Regulation of Cell Growth in Stat1+/+ and Stat1-/- Mice—To assess the effects of knock-out of Stat1 on viable cell number, osteoblastic cells were prepared from calvariae of 14-week-old Stat1+/+ and Stat1-/- mice by sequential digestion with 0.1% collagenase (Worthington Biochemical Co.) and plated in 100-mm dishes in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS, Hyclone, Logan, UT) in a 5% CO2 incubator at 37 °C. At confluence, cells were replated at 5000/cm2 in 96-well dishes in DMEM medium containing either 1 or 10% FCS. After 24, 48, 72, or 96 h, cells were harvested. For the last 1 h of culture, 20 μl per well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Promega) was added, and viable cell number was measured by the MTT assay. Absorbance is directly proportional to the number of proliferating and living cells (34Lucareli E. Sangiorgi L. Maini V. Lattanzi G. Marmiroli S. Reggiani M. Mordenti M. Gobbi G.A. Scrimieri F. Bertoja A.Z. Picci P. Cancer. 2002; 98: 344-351PubMed Google Scholar). Cell replication was also assessed in marrow stromal cells from both genotypes. For these experiments, marrow stromal cells were plated at 2 × 106 in 6-well dishes in α-minimal essential medium (αMEM, Invitrogen) with 10% heat-inactivated FCS for 7 days with media change every 3 days. For labeling studies, [3H]thymidine (10 μCi/well) was added for the last 4 h of the culture to measure cell proliferation. Mouse Bone Marrow Cultures—Mouse bone marrow cells were isolated using a modification of previously published methods (35Montero A. Okada Y. Tomita M. Ito M. Tsurakami H. Nakamura T. Doetschman T. Coffin J.D. Hurley M.M. J. Clin. Investig. 2000; 105: 1085-1093Crossref PubMed Scopus (405) Google Scholar). Tibiae and femurs from 13-14-week-old Stat1+/+ and Stat1-/- mice were dissected free of adhering tissue. Bone ends were removed, and the marrow cavity was flushed with αMEM by slowly injecting medium into one end of the bone using a sterile 25-gauge needle. Marrow cells were collected into tubes washed twice with serum-free αMEM and cultured in αMEM containing 10% heat-inactivated FCS. Cells were plated in 6-multiwell plates (2 × 106) cells/well, and cultures were fed every 3 days by replacing 80% of the medium with fresh medium. Cells for alkaline phosphatase (ALP) staining were harvested on days 7, 14, and 21 of culture. ALP staining was performed with a commercial kit (Sigma). Dishes were scanned and then counterstained for mineral by the von Kossa method as previously described (35Montero A. Okada Y. Tomita M. Ito M. Tsurakami H. Nakamura T. Doetschman T. Coffin J.D. Hurley M.M. J. Clin. Investig. 2000; 105: 1085-1093Crossref PubMed Scopus (405) Google Scholar). mRNA Isolation and Northern Blot Analysis—Total RNA was extracted from cells by the acid guanidinium isothiocyanate extraction and cesium chloride ultracentrifugation methods (36Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63290) Google Scholar, 37Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44969) Google Scholar). For Northern analysis, 20 μg of total RNA was denatured and fractionated on a 0.8% agarose/1.1 m formaldehyde gel, transferred to filters by capillary blotting, and fixed to the filter by UV irradiation. After a 4-h prehybridization, filters were hybridized overnight with a [32P]cDNA probe for the mRNAs of interest. Bands were normalized to glyceraldehyde-3-phosphate dehydrogenase. Northern blots were quantified by autoradiography and densitometry. Statistical Analysis—All results were expressed as means ± S.E. Differences between groups were analyzed using the Student's t test, and differences were considered significant at p values of less than 0.05. DEXA Analysis of Bone Mineral Density and Bone Mineral Content of Femur and Vertebrae of Stat1+/+ and Stat1-/- Mice—The Stat1-/- mice appeared grossly normal with no significant size or weight differences compared with Stat1+/+ mice. BMD and BMC were determined by DEXA analysis of femoral and vertebral bones from 13-week-old mice of both genotypes (Fig. 1). Femoral BMD was significantly increased by 9%, and BMC was increased by 11% in Stat1-/- mice when compared with Stat1+/+ mice. Vertebral BMC was also significantly increased by 16% in Stat1-/- mice compared with Stat1+/+ mice. Microcomputed Tomography Analysis of Bones of Stat1+/+ and Stat1-/- Mice—Bone microarchitecture was examined in adult mice of both genotypes by microcomputed tomography (micro-CT, Ref. 27Ruegsegger P. Koller B. Muller R. Calcif. Tissue Int. 1996; 58: 61-75Crossref Scopus (813) Google Scholar). Three-dimensional images of the third lumbar vertebrae of 13-week-old male Stat1+/+ and Stat1-/- mice are shown in (Fig. 2A). The plate-like structure of the trabecular bone was markedly increased in Stat1-/- mice compared with Stat1+/+ mice. Fig. 2, B and C shows that bone volume/trabecular volume (BV/TV) and trabecular number (Tb.N) were increased by 7 and 11%, respectively. Connective tissue density (2D) was increased by 11% and Tb.SP (2E) was reduced by 14% in Stat1-/- mice compared with Stat1+/+ mice. Trabecular thickness (Tb.Th) was not different between the two genotypes (data not shown). Similar to the observation in vertebrae, micro-CT examination of distal femoral metaphysis revealed that the plate-like structure of the trabecular bone was markedly increased in Stat1-/- mice compared with Stat1+/+ mice (Fig. 3A). The femoral diaphyseal cortical thickness and cortical bone area by micro-CT (Fig. 3B) were also measured. As shown in Fig. 3, C and D there was an 11 and 12% increase in diaphyseal cortical width and cortical bone area, respectively, of femurs of Stat1-/- mice compared with Stat1+/+ mice.Fig. 3Morphologic study by microcomputed tomography scanning of the metapphyseal trabecular and diaphyseal cortical bone of the femurs of adult Stat1+/+ and Stat1-/- mice.A, three-dimensional trabecular bone architecture of femurs from 13-week-old male Stat1+/+ and Stat1-/- mice were analyzed by Micro-CT. Note that the increased plate-like architecture of the trabecular bone and the connecting rods in Stat1-/- mice compared with Stat1+/+. B, note the thickening of the diaphysis of Stat1-/- mice (upper right panel) compared with Stat1+/+. Calculated morphometric indices included Cortical Thickness (C, mm) and Cortical Bone Area (D, mm2). Values are the mean ± S.E. for 7-10 bones/group. *, significantly different from Stat1+/+ group, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Histomorphometry Analysis of Bones of Stat1+/+ and Stat1-/- Mice—To determine whether the increase in bone mass was caused by an increase in bone formation, histomorphometric parameters of bone formation were determined on the left distal femurs of 13-week-old mice. As shown in Fig. 4, A and B BV/TV and Tb.N were increased by 33 and 22%, respectively. Tb.SP (Fig. 4C) was reduced by 27% in Stat