Exported 18-kDa Isoform of Fibroblast Growth Factor-2 Is a Critical Determinant of Bone Mass in Mice
2008; Elsevier BV; Volume: 284; Issue: 5 Linguagem: Inglês
10.1074/jbc.m804900200
ISSN1083-351X
AutoresLiping Xiao, Peng Liu, Xiaofeng Li, Thomas Doetschman, J. Douglas Coffin, Hicham Drissi, Marja M. Hurley,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoThe role of the 18-kDa isoform of fibroblast growth factor-2 (FGF2) in the maintenance of bone mass was examined in Col3.6-18-kDa FGF2-IRES-GFPsaph transgenic (18-kDa TgFGF2) mice in which a 3.6-kb fragment of the type I collagen 5′-regulatory region (Col3.6) drives the expression of only the 18-kDa isoform of FGF2 with green fluorescent protein-sapphire (GFPsaph). Vector only transgenic mice (Col3.6-IRES-GFPsaph, VTg) were also developed as a control, and mice specifically deficient in 18-kDa FGF2 (FGF2lmw-/-) were also examined. Bone mineral density, femoral bone volume, trabecular thickness, and cortical bone area and thickness were significantly increased in 18-kDa TgFGF2 mice compared with VTg. Bone marrow cultures (BMSC) from 18-kDa TgFGF2 mice produced more mineralized nodules than VTg. Increased bone formation was associated with reduced expression of the Wnt antagonist secreted frizzled receptor 1 (sFRP-1). In contrast to 18-kDa TgFGF2 mice, FGF2lmw-/- mice have significantly reduced bone mineral density and fewer mineralized nodules, coincident with increased expression of sFRP-1 in bones and BMSC. Moreover, silencing of sFRP-1 in BMSC from FGF2lmw-/- mice reversed the decrease in β-catenin and Runx2 mRNA. Assay of Wnt/β-catenin-mediated transcription showed increased and decreased TCF-luciferase activity in BMSC from 18-kDa TgFGF2 and FGF2lmw-/- mice, respectively. Collectively, these results demonstrate that the 18-kDa FGF2 isoform is a critical determinant of bone mass in mice by modulation of the Wnt signaling pathway. The role of the 18-kDa isoform of fibroblast growth factor-2 (FGF2) in the maintenance of bone mass was examined in Col3.6-18-kDa FGF2-IRES-GFPsaph transgenic (18-kDa TgFGF2) mice in which a 3.6-kb fragment of the type I collagen 5′-regulatory region (Col3.6) drives the expression of only the 18-kDa isoform of FGF2 with green fluorescent protein-sapphire (GFPsaph). Vector only transgenic mice (Col3.6-IRES-GFPsaph, VTg) were also developed as a control, and mice specifically deficient in 18-kDa FGF2 (FGF2lmw-/-) were also examined. Bone mineral density, femoral bone volume, trabecular thickness, and cortical bone area and thickness were significantly increased in 18-kDa TgFGF2 mice compared with VTg. Bone marrow cultures (BMSC) from 18-kDa TgFGF2 mice produced more mineralized nodules than VTg. Increased bone formation was associated with reduced expression of the Wnt antagonist secreted frizzled receptor 1 (sFRP-1). In contrast to 18-kDa TgFGF2 mice, FGF2lmw-/- mice have significantly reduced bone mineral density and fewer mineralized nodules, coincident with increased expression of sFRP-1 in bones and BMSC. Moreover, silencing of sFRP-1 in BMSC from FGF2lmw-/- mice reversed the decrease in β-catenin and Runx2 mRNA. Assay of Wnt/β-catenin-mediated transcription showed increased and decreased TCF-luciferase activity in BMSC from 18-kDa TgFGF2 and FGF2lmw-/- mice, respectively. Collectively, these results demonstrate that the 18-kDa FGF2 isoform is a critical determinant of bone mass in mice by modulation of the Wnt signaling pathway. A variety of tissues, including bone, produce FGF2 2The abbreviations used are: FGF2, fibroblast growth factor-2; BMD, bone mineral density; BMC, bone mineral content; BV/TV, bone volume/tissue volume; sFRP-1, secreted frizzled receptor 1; β-catenin, TOP-flash; GFP, green fluorescent protein; GFPsaph, with green fluorescent protein-sapphire; BMSC, bone marrow stromal cell; PBS, phosphate-buffered saline; KO, knock-out; VTg, vector only transgenic mice; FZD, frizzled; sFRP, secreted FZD-related protein; FGFR, FGF receptor; WT, wild type; SMI, structure model index; RANKL, receptor activator of the NF-κB ligand; Tb.Th, trabecular thickness; HO, homozygote; ALP, alkaline phosphatase; DXA, dual beam x-ray absorptiometry; siRNA, short interfering RNA. where osteoblasts deposit it in newly forming bone matrix (1Globus R.K. Endocrinology.. 1989; 124: 1539-1547Google Scholar). A single fgf2 gene encodes multiple protein isoforms from alternative translation start sites (2Abraham J.A. Whang J.L. Tumolo A. Mergia A. Friedman J. Gospodarowicz D. Fiddes J.C. EMBO J.. 1986; 5: 2523-2528Google Scholar, 3Florkiewicz R.Z. Sommer A. Proc. Natl. Acad. Sci. U. S. A... 1989; 86: 3981-3987Google Scholar). Humans express four FGF2 isoforms, including three high molecular mass 22-, 23-, and 24-kDa proteins that have nuclear localization sequences and a low molecular mass 18-kDa FGF2 protein that is exported from cells. In rodents, there are two high molecular mass isoforms of 22 and 21 kDa and a low molecular mass 18-kDa FGF2 protein that is exported from cells. Translation of each of the three high molecular mass human FGF2 isoforms (22, 23, and 24 kDa) is initiated from an unconventional CUG translation initiation codon. In contrast, translation of the 18-kDa FGF2 isoform is initiated from a classical AUG codon located downstream of the CUG codons. Thus, multiple isoforms of FGF2 protein can be expressed from a single mRNA as a result of translation at either AUG (18-kDa protein) or CUG (22-, 23-, and 24-kDa proteins) start sites (2Abraham J.A. Whang J.L. Tumolo A. Mergia A. Friedman J. Gospodarowicz D. Fiddes J.C. EMBO J.. 1986; 5: 2523-2528Google Scholar, 3Florkiewicz R.Z. Sommer A. Proc. Natl. Acad. Sci. U. S. A... 1989; 86: 3981-3987Google Scholar, 4Stachowiak M. Moffett J. Joy A. Puchacz E. Florkiewicz R. Stachowiak E. J. Cell Biol... 1994; 127: 203-222Google Scholar). Previous studies showed that constitutive overexpression of all the human FGF2 protein isoforms in transgenic (TgFGF2) mice resulted in chondrodysplasia (5gon Coffin J.D. Florkiewicz R.Z. Neumann J. Mort-Hopkins T. Dorn G.W. II, Lightfoot P. German R. Howles P.N. Kier A. O'Toole B.A. Sasse J. Gonzales A.M. Baird A. Doetschamn T. Mol. Biol. Cell.. 1995; 6: 1861-1873Google Scholar), decreased bone mineral density (BMD), and decreased bone mass (6Sobue T. Naganawa T. Xiao L. Okada Y. Tanaka Y. Ito M. Okimoto N. Nakamura T. Coffin J.D. Hurley M.M. J. Cell. Biochem... 2005; 95: 83-94Google Scholar). We have also reported that overexpression of the 18-kDa FGF2 isoform increased osteoblastic ROS17/2.8 cell proliferation (7Xiao L. Liu P. Sobue T. Lichtler A. Coffin J.D. Hurley M.M. J. Cell. Biochem... 2003; 89: 1291-1301Google Scholar). Counterintuitively, targeted deletion or "knock-out" (KO) of all FGF2 protein isoforms in mice also resulted in decreased bone mass (8Montero A. Okada Y. Tomita M. Ito M. Tsurukami H. Nakamura T. Doetschman T. Coffin J.D. Hurley M.M. J. Clin. Investig... 2000; 105: 1085-1093Google Scholar). The goal of this study was to analyze the role of the exported FGF2 18-kDa protein isoform during in vivo bone formation. Systemic administration of the 18-kDa FGF2 isoform to rats was reported to induce extraskeletal effects such as anemia and glomerular hypertrophy (9Wamsley H.L. Iwaniec U.T. Wronski T.J. Toxicol. Pathol... 2005; 33: 577-583Google Scholar). Therefore, another goal of this study was to analyze whether targeted overexpression of 18-kDa FGF2 in bone modulated development of the extraskeletal side effects. To achieve these goals, we generated transgenic mice expressing the human 18-kDa FGF2 isoform under the control of the Col I 3.6-kb 5′-regulatory region of type 1 collagen, resulting in 18-kDa FGF2 expression in cells of immature and mature osteoblast lineage. In addition, we characterized the bone phenotype of mice with selective deletion of the 18-kDa isoform of FGF2 (10Garmy-Susini B. Delmas E. Gourdy P. Zhou M. Bossard C. Bugler B. Bayard F. Krust A. Prats A.C. Doetschman T. Prats H. Arnal J.F. Circ. Res... 2004; 94: 1301-1309Google Scholar). Recent studies reported a role for modulation of Wnt signaling by fibroblast growth factor receptor-2 (FGFR2) and the ligand fibroblast growth factor-1 (FGF-1) in intramembranous bone formation (11Mansukhani A. Ambrosetti D. Holmes G. Cornivelli L. Basilico C. J. Cell Biol... 2005; 168: 1065-1076Google Scholar). However, possible cross-talk between FGF2 and Wnt signaling during postnatal bone formation has not been investigated, so we examined a putative role of the Wnt pathway in the bone anabolic effect mediated by 18-kDa FGF2. Wnt proteins are a family of secreted glycoproteins that play important roles in many biological processes, including skeletal development (12Church V.L. Francis-West P. Int. J. Dev. Biol... 2002; 46: 927-936Google Scholar) as well as postnatal bone formation (13Patel M.S. Karsenty G. N. Engl. J. Med... 2002; 346: 1572-1574Google Scholar, 14Goltzman D. Nat. Rev. Drug Discovery.. 2002; 1: 784-796Google Scholar, 15Krishnan V. Bryant H.U. MacDougald O.A. J. Clin. Investig... 2006; 116: 1202-1209Google Scholar). Wnt proteins initiate a signaling cascade by binding to a membrane receptor complex composed of the frizzled (FZD) G protein-coupled receptor that is combined with a low density lipoprotein receptor-related protein to activate downstream signaling pathways (15Krishnan V. Bryant H.U. MacDougald O.A. J. Clin. Investig... 2006; 116: 1202-1209Google Scholar). Wnt signaling is controlled by both extracellular and intracellular proteins (15Krishnan V. Bryant H.U. MacDougald O.A. J. Clin. Investig... 2006; 116: 1202-1209Google Scholar). Secreted FZD-related proteins (sFRPs) are extracellular proteins that are able to bind Wnts or FZD receptors thereby attenuating all Wnt-activated pathways. Published results show that sFRP1 is a negative regulator of trabecular bone mass as demonstrated by increased trabecular bone mineral density in sFRP1-/- mice (16Bodine P.V. Zhao W. Kharode Y.P. Bex F.J. Lambert A.J. Goad M.B. Gaur T. Stein G.S. Lian J.B. Komm B.S. Mol. Endocrinol... 2004; 18: 1222-1237Google Scholar). Interestingly, FGF2 and FGFR signaling were shown to modulate heparin-induced sFRP1 accumulation in the HEK293 cell line (17Zhong X. DeSilva T. Lin L. Bodine P. Bhat R.A. Presman E. Pocas J. Stahl M. Kriz R. J. Biol. Chem... 2007; 282: 20523-20533Google Scholar). Because of the similarities between the Sfrp1-/- and the 18-kDa TgFGF2 mice bone phenotypes, we examined sFRP1 mRNA and protein expression in 18-kDa TgFGF2 and VTg mice as well as mice with selective deletion of the 18-kDa FGF2 isoform. We also examined whether differential expression of sFRP-1 altered β-catenin that is important in osteoblast differentiation. These studies demonstrate that transgenic overexpression of the 18-kDa FGF2 protein isoform increased bone mass and that the increase in bone mass is regulated through modulation of the Wnt pathway. Generation and Identification of Mice Expressing 18-kDa Isoform of Human FGF2—To elucidate the role of the endogenous 18-kDa isoform of FGF2 in a bone-specific manner, we generated a construct, called Col3.6-18-kDa FGF2 isoform IRES-GFPsaph. Col3.6-18-kDa FGF2 isoform IRES-GFPsaph was built by replacing a chloramphenicol acetyltransferase fragment in a previously made Col3.6-CAT-IRES-GFPsaph (18Dacic S. Kalajzic I. Visnjic D. Lichtler A.C. Rowe D.W. J. Bone Miner. Res... 2001; 16: 1228-1236Google Scholar) with a human 18-kDa isoform of FGF2 cDNA between AfeI and ScaI sites (Fig. 1A). This expression construct is capable of concurrently overexpressing the 18-kDa isoform of FGF2 (3Florkiewicz R.Z. Sommer A. Proc. Natl. Acad. Sci. U. S. A... 1989; 86: 3981-3987Google Scholar, 19Florkiewicz R.Z. Shibata F. Barankiewicz T. Baird A. Gonzalez A.M. Florkiewicz E. Shah N. Ann. N. Y. Acad. Sci... 1991; 638: 109-126Google Scholar) and GFPsaph from a single bicistronic mRNA. The construct also harbors a neomycin selection gene. Generation of the FGF2 cDNAs was described previously (7Xiao L. Liu P. Sobue T. Lichtler A. Coffin J.D. Hurley M.M. J. Cell. Biochem... 2003; 89: 1291-1301Google Scholar). As control, a Col3.6-IRES/GFP (VTg) construct was also generated. The construct insert was released from Col3.6-IRES/GFP (VTg) or Col3.6-18-kDa FGF2 isoform IRES/GFP (18 kDa) by digestion with AseI and AflII and purified according to standard techniques. Microinjections into the pronucleus of fertilized oocytes were performed at the Gene Targeting and Transgenic Facility at the University of Connecticut Health Center. Founder mice of the F2 (FVBN) strain were mated with wild-type mice to establish individual transgenic lines. Mating heterozygote male with heterozygote female generated homozygote mice. Mice were maintained in a virus- and parasite-free barrier facility under a 12-h light/12-h dark cycle at the Gene Targeting and Transgenic Facility and weaned at 21 days of age onto autoclaved rodent chow. 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. For all the transgenic experiments presented, 4.5-month-old homozygote (HO) male mice were used except where noted. Isoform-specific 18-kDa KO Mice—A variation on the Tag and Exchange strategy was used to introduce a 4-bp change that replaced the methionine translational start site encoding CCATGG NcoI restriction site with an alanine-encoding CTGCAG PstI restriction site. No residual genetic modifications such as antibiotic resistance/marker genes or LoxP recombination recognition sites were present in the targeted allele. Examination of tissues of 18-kDa KO mice revealed only expression of the high molecular mass 21- and 22-kDa FGF2 isoforms compared with their WT littermates that expressed all three mouse FGF isoforms of 18, 21, and 22 kDa. Development of these mice has been previously described in detail (10Garmy-Susini B. Delmas E. Gourdy P. Zhou M. Bossard C. Bugler B. Bayard F. Krust A. Prats A.C. Doetschman T. Prats H. Arnal J.F. Circ. Res... 2004; 94: 1301-1309Google Scholar). To further elucidate the role of the endogenous 18-kDa FGF2 isoform in bone, wild type and 18-kDa homozygote knock-out littermates were used in this study. Reporter Assays—The expression of GFP reporter gene in transfectants was detected under fluorescent microscopy. GFP expression in cell was visualized using an Olympus IX50 inverted system microscope equipped with an IX-FLA inverted reflected light fluorescence (Olympus America, Inc., Melville, NY). A specific excitation wavelength was obtained using filters for GFPsaph (exciter, D395/40; dichroic, 425DCLP; emitter, D510/40 m) and recorded with a SPOT camera (Diagnostic Instrument, Inc., Sterling Heights, MI). Fluorescent images were taken with equal exposure times applied to bones derived from different transgenic constructs. Genotyping—Genomic DNA was extracted from the tails of heterozygote mice using standard techniques. Transgenes were confirmed by PCR. The presence of the GFP gene was detected by PCR using a set of primers from the middle region of the GFP, 5-TCATCTGCACCACCGGCAAGC-3 and 5-AGCAGGACCATGTGATCGCGC-3, which yield a fragment of 525 bp. PCR was performed in a final volume of 50 μl containing 0.2 g of DNA and 1.25 units of TaqDNA polymerase, 1.25 mm 4dNTP mix; 5 μl of amplification buffer, 1.5 μl of 50 mm CaCl2, 10 μl primers, 20-40 pmol of each. PCR was conducted using standard conditions on an GeneAmp PCR System 9700 (Brinkman Instruments Ltd., Westbury, NY) as follows: 30 cycles of 94 °C for 30 s, 62 °C for 30 s, 72 °C for 40 s. Prior to the first cycle, initial denaturation was performed at 94 °C for 3 min, and the last cycle was followed by an extension step of 5 min at 72 °C. The amplification products were evidenced through 1.5% agarose gel electrophoresis. Gels were stained with ethidium bromide and observed under UV light. Dual Beam X-ray Absorptiometry (DXA)—Femurs, tibiae, and vertebrae were harvested and stored in 70% ethanol at 4 °C. BMD and bone mineral content (BMC) were measured using a Piximus Mouse 11 densitometer (GE Medical Systems, Madison, WI). Micro-CT Scanning of Femurs—After measuring the BMD and BMC, the femurs were analyzed by micro-CT system (μCT-20, Scanco Medical, Zurich), as reported previously (20Ruegsegger P. Koller B. Muller R.A. Calcif. Tissue Int... 1996; 58: 24-29Google 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). These parameters were calculated by the parallel plate model of Parfitt et al. (21Parfitt A.M. Mathews C.H. Villanueva A.R. Kleerekoper M. Frame B. Rao D.S. J. Clin. Investig... 1983; 72: 1396-1409Google Scholar). The structure model index (SMI), which reflects the degree of normal plate-like trabeculae versus abnormal rod-like trabeculae, was also determined as described previously (22Hildebrand T. Ruegsegger P. Comput. Methods. Biomech. Biomed. Engin... 1997; 1: 15-23Google Scholar). Tissue Preparation for Histology—For histological analysis, mice expressing VTg and 18-kDa transgenes were sacrificed by CO2 narcosis and cervical dislocation. Following euthanasia, the femurs and other tissue samples were removed and immediately fixed in 4% paraformaldehyde at 4 °C. After processing each sample was embedded in Shandon Cryomatrix (Thermo Electron Corp., Pittsburgh, PA) and completely frozen. In selected cases, femurs were decalcified in 20% EDTA in PBS for 2 days, and paraffin blocks were prepared by standard procedures. Paraffin or frozen samples were cut into 5-μm sections. The sections were stained with von Kossa. For immunohistochemistry staining, sFRP1 (Santa Cruz Biotechnology, Inc.) antibody was used at 1:200 dilution following product protocol. Mouse Bone Marrow Cultures—Bone marrow stromal cells (BMSCs) were isolated using a modification of previously published methods (8Montero A. Okada Y. Tomita M. Ito M. Tsurukami H. Nakamura T. Doetschman T. Coffin J.D. Hurley M.M. J. Clin. Investig... 2000; 105: 1085-1093Google Scholar). Tibiae and femurs from VTg and 18-kDa mice or from WT and FGF2lmw-/- mice were dissected free of adhering tissue. Bone ends were removed, and the marrow cavity was flushed with α-minimum Eagle's medium 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 α-minimum Eagle's medium. BMSCs were plated in 6-multiwell plates (2 × 106 cells/well) in αMEM containing 10% heat-inactivated FCS and on day 3 changed to differentiation medium, and cultures were fed every 3 days by replacing 80% of the medium with fresh medium. BMSCs for ALP or xylenol orange 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 described previously (8Montero A. Okada Y. Tomita M. Ito M. Tsurukami H. Nakamura T. Doetschman T. Coffin J.D. Hurley M.M. J. Clin. Investig... 2000; 105: 1085-1093Google Scholar). mRNA Isolation and Gene Expression—Total RNA was extracted from BMSCs or shaft of flushed long bone by using TRIzol reagent (Invitrogen) according to the manufacturer's instruction. For real time quantitative reverse transcription-PCR analysis, RNA was reverse-transcribed by the Super-Script™ first-strand synthesis system for reverse transcription-PCR (Invitrogen). Quantitative PCR was carried out using the QuantiTect™ SYBR Green PCR kit (Qiagen) on a MyiQ™ instrument (Bio-Rad). β-Actin was used as an internal reference for each sample. Using a formula described previously (23Pfaffl M.W. Nucleic Acids Res... 2001; 29: e45Google Scholar), the relative change in mRNA was normalized against the β-actin mRNA level. RNA Interference—BMSCs were transfected with sFRP-1 siRNA that is able to down-regulate the sFrp1 gene in living cells by introducing a homologous double-stranded RNA. A control siRNA (scrambled sequence) was used as a negative control. The siRNA transfection was performed according to the protocol provided by the manufacturer (Santa Cruz Biotechnology, Inc.). Cells were harvested at 48 h post-transfection and were used for ALP activity assay and/or RNA extraction. The specificity of the silencing was confirmed in three different experiments by real time PCR. Transient Transfections—To determine changes in β-catenin trans-activating activity, bone marrow stromal cells were cultured to 70% confluence in 24-well dishes and transiently transfected with the TOP-flash reporter construct using Lipofectamine reagent and PLUS reagent (25 μl Lipofectamine reagent/160 ng of TOP-flash DNA/80 ng of cytomegalovirus 5′-regulatory region DNA) according to manufacturer's instructions (Invitrogen). Cotransfection with a construct containing the cytomegalovirus 5′-regulatory region driving the β-galactosidase gene (a gift from Dr. Anne M, Delany, University of Connecticut) was used to control for transfection efficiency. Cells were exposed to the Lipofectamine Reagent/PLUS Reagent/DNA mix for 3 h, transferred to regular medium for 48 h, washed twice with PBS, and harvested in a reporter lysis buffer (Promega). Luciferase and β-galactosidase activities were measured using an Optocomp luminometer (MGM Instruments, Hamden, CT). Luciferase activity was corrected for β-galactosidase activity to control for transfection efficiency. Western Blot Analysis—The expression of FGF2 isoform 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-PAGE on 10-20% 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 anti-FGF2 antibody that recognizes all isoforms of FGF2 (BD Biosciences) for 1 h, washed 1 h with TBS-T, and then incubated with a rabbit anti-mouse secondary antibody (Amersham Biosciences) in TBS-T, 1% nonfat milk for 1 h. After incubation with antibodies, membranes were washed 1 h with TBS-T. Western Lightning™ chemiluminescence reagent (PerkinElmer Life Sciences and GE Healthcare) was used for detection. Band density was quantified by densitometry. The membranes were striped and re-probed for sFPR1 (Santa Cruz Biotechnology, Inc.) or β-catenin (BD Biosciences). Biochemistry—Blood was collected from euthanized animals by cardiac puncture. After clotting, the blood was spun, and serum was collected for analysis. Serum phosphate and calcium were measured using the FAST 340 phosphorous reagent SET/calcium reagent SET (Eagle Diagnostics, Desoto, TX). Creatinine was measured using QuantiChrom™ creatinine assay kit (DICT-500) (Sigma). Serum FGF2 level was measured using Quantikine®HS human FGF basic immunoassay kit (R & D Systems) according to the manufacturer's instructions. Indirect Immunofluorescence Staining for sFRP1 in BMSCs—BMSC cells from 2-month-old FGF2lmw-/- and WT littermate mice were plated at 2 × 106 cells/well in 6-well dishes containing coverslips and cultured for 2 weeks on coverslips fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After rinsing twice with PBS, a 1:50 dilution of the rabbit anti-human sFRP1 antibody was added to the cells and then incubated for 1 h at 37 °C. For control studies, primary antibody was replaced with PBS alone. Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma) was then applied to the cells at a dilution of 1:60 for 1 h at 37 °C. After washing, cells were mounted in 50% glycerol in PBS. The cells were examined and photographed with a Zeiss Axiophot fluorescence microscope. 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. For the comparison among multiple groups, analysis of variance was used, and the significant difference was determined by the Bonferroni test (StatView 4.1J Abacus Concepts, Inc., Berkeley, CA). Characterization of 18-kDa TgFGF2 Mice—Fig. 1A shows the maps for the vector only and 18-kDa Fgf2 transgenes that were utilized to develop the vector control (VTg) and 18-kDa TgFGF2 mice. Several founder mice were identified and two viable 18-kDa TgFGF2 lines (18-kDa(305) and 18-kDa(308)) carrying the human 18-kDa FGF2 isoform transgene were established. Analysis was performed on HO 18-kDa TgFGF2 mice compared with HO VTg mice as controls unless noted. Since these mice are on an FVB/N background, HO-breeding pairs were maintained to generate sufficient VTg, 18-kDa(305)TgFGF2, and 18-kDa(308)TgFGF2 mice necessary for experimentation. PCR analysis of genomic DNA sample collected from tail samples demonstrated that both lines of 18-kDa TgFGF2 mice carried the transgene, whereas their non-transgenic littermates lacked the transgene (Fig. 1B). Western blot analysis of protein from tissues of 7-day-old (P7) 18-kDa TgFGF2 mice demonstrated high levels of 18-kDa FGF2 in femurs and calvariae compared with VTg controls. Low levels of FGF2 were observed in liver, but other tissues such as kidney and skin did not overexpress 18-kDa FGF2 (Fig. 1C). There was no difference in serum FGF-2 levels between VTg and 18-kDa TgFGF2 mice (Fig. 1E), confirming bone specificity of FGF-2 overexpression. Expression of the 18-kDa FGF2-GFP transgene in vivo was analyzed in frozen undecalcified sections of femurs from 24-day-old male mice (Fig. 1D). GFP expression was seen in bone-forming osteoblasts and osteocytes, which is consistent with observations in other transgenic animals that have been generated using the col3.6 5′-regulatory region construct (18Dacic S. Kalajzic I. Visnjic D. Lichtler A.C. Rowe D.W. J. Bone Miner. Res... 2001; 16: 1228-1236Google Scholar). Osteoblasts expressing the transgene were present on the surface of trabecular bone and on the endosteal surface and periosteal surface of the metaphyseal cortical bone. GFP expression was stronger in osteoblasts found on trabecular surfaces, endosteum, periosteal (supplemental Fig. 1), and in osteocytes of tibiae from 18-kDa TgFGF2 mice. To determine whether there were extra-skeletal side effects from targeted overexpression of 18-kDa FGF2 in bone, hematocrit, serum creatinine, serum calcium, and phosphorus were measured. There were no differences in any of these parameters between VTg and 18-kDa TgFGF2 mice (data not shown). Histologic examination of hematoxylin and eosin-stained sections of the kidneys showed that targeted overexpression of 18-kDa FGF2 in bone did not cause enlargement, vacuolation and karyomegaly of podocytes in glomeruli, dilatation and cast formation in tubules, thickening of the media in lobular arteries, or hyperplasia of the epithelium of the papilla and collecting ducts (24Mazue G. Newman A.J. Scampini G. Della Torre P. Hard G.C. Iatropoulos M.J. Williams G.M. Bagnasco S.M. Toxicol. Pathol... 1993; 21: 490-501Google Scholar). We assessed body weight and femoral bone lengths in mice in VTg and 18-kDa TgFGF2 mice. Mean body weights were 27.6 ± 0.6 for VTg mice versus 29.4 ± 0.3 and 29.7 ± 0.3 g for 18-kDa(305) TgFGF2 and 18-kDa(308) TgFGF2 mice, respectively (p < 0.05). Femoral bone lengths were 14.37 ± 0.60 for VTg mice versus 14.87 ± 0.26 and 14.68 ± 0.13 mm for 18-kDa(305)TgFGF2 and 18-kDa(308)TgFGF2, respectively. There were no significant differences in bone lengths; therefore, the overexpression of the 18-kDa FGF2 isoform did not cause dwarfism or shortened femurs. DXA Analysis of BMD and BMC in VTg and 18-kDa TgFGF2 Mice—BMD and BMC were determined by DXA analysis of femurs and tibiae from two independent lines of 18-kDa TgFGF2 mice compared with VTg. Tibial BMD (Fig. 2A) was increased by 16 and 12% in 18-kDa(305)TgFGF2 and 18-kDa(308)TgFGF2 mice, respectively (p < 0.05). Tibial BMC (Fig. 2B) was significantly increased by 29 and 18% in 18-kDa(305)TgFGF2 and 18-kDa(308)TgFGF2 mice, respectively (p < 0.05). Femoral BMD (Fig. 2C) was significantly increased by 8 and 6% in 18-kDa(305)TgFGF2 and 18-kDa(308)TgFGF2 mice, respectively (p < 0.05). Femoral BMC (Fig. 2D) was significantly increased by 22 and 12% in 18-kDa(305)TgFGF2 and 18-kDa(308)TgFGF2 mice, respectively (p < 0.05). Micro-CT Analysis of Femurs from VTg and 18-kDa TgFGF2 Mice—To investigate the structural effects of 18-kDa FGF2 isoform overexpression on different bone compartments, bone micro-architecture was examined in adult mice by micro-CT. Three-dimensional images of the femurs from VTg, 18-kDa(305)TgFGF2, and 18-kDa(308)TgFGF2 mice are shown in Fig. 3A. Structural analysis showed that BV/TV was significantly increased by 19% in 18-kDa(305)TgFGF2 and increased by 24% in 18-kDa(308)TgFGF2 mice (Fig. 3B)(p < 0.05). The SMI describes the normal plate-like structure of bone, the closer to 0 the more plate-like the trabecular structure, and the closer to 3 the more abnormal rod-like the trabeculae. As shown in (Fig. 3C), SMI was significantly decreased by 10 and 12% in 18-kDa(305)TgFGF2 and 18-kDa(308)TgFGF2 mice, respectively (p < 0.05). Trabecular thickness (Tb.Th) (Fig. 3D) was significantly increased by 8 and 13% in 18-kDa(305)TgFGF2 and 18-kDa(308)TgFGF2 mice, respectively, compared with VTg mice (p < 0.05). The effect of 18-kDa FGF2 isoform overexpression on femoral cortical bone was also determined by micro-CT. Segmented bone area (Fig. 3E) was significantly increased by 17 and 15% in 18-kDa(305)TgFGF2 and 18-kDa(308)TgFGF2 mice, respectively, compared with VTg mice (p < 0.05). Circumferential cortical thickness (Fig. 3F) was increased by 7% in both 18-kDa(305)TgFGF2 and 18-kDa(308)TgFGF2 mice (p < 0.05). Circumferential periosteal perimeter (Fig. 3G) was increased by 9 and 7% in 18-kDa(305)TgFGF2 and 18-kDa(308)TgFGF2 mice, respectively, compared with VTg mice (p < 0.05). Circumferential endosteal perimeter (Fi
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