Conditional Inactivation of Cxcr4 in Osteoprecursors Reduces Postnatal Bone Formation Due to Impaired Osteoblast Development

Cystine (C)-X-C motif chemokine receptor 4 (CXCR4), the primary receptor for stromal derived factor-1 (SDF-1), is involved in bone morphogenic protein 2 (BMP2)-induced osteogenic differentiation of mesenchymal progenitors. To target the in vivo function of CXCR4 in bone and explore the underlying mechanisms, we conditionally inactivated CXCR4 in osteoprecursors by crossing osterix (Osx)-Cre mice with floxed CXCR4 mice (CXCR4) to generate knockouts with CXCR4 deletion driven by the Osx promoter (Osx::CXCR4). The Cremediated excision of CXCR4 occurred exclusively in bone of Osx::CXCR4 mice. When compared to littermate controls, Osx::CXCR4 mice developed smaller osteopenic skeletons, evidenced by reduced trabecular and cortical bone mass, lower bone mineral density, and a slower mineral apposition rate. In addition, Osx::CXCR4 mice displayed chondrocyte disorganization in the epiphyseal growth plate, associated with decreased proliferation and collagen matrix syntheses. Moreover, mature osteoblast-related expression of type I collagen α1 (Col1α1) and osteocalcin (OCN) was reduced in bone of Osx::CXCR4 mice versus controls, suggesting that CXCR4deficiency results in arrested osteoblast progression. Primary cultures for osteoblastic cells derived from Osx::CXCR4 mice also showed decreased proliferation and impaired osteoblast differentiation in response to BMP2 or BMP6 stimulation, and suppressed activation of intracellular BMP receptor-regulated Smads (RSmads) and Erk1/2 was identified in CXCR4deficient cells and bone tissues. These findings provide the first in vivo evidence that CXCR4 functions in postnatal bone development via regulating osteoblast development in cooperation with BMP signaling. Thus, CXCR4 acts as an endogenous signaling component necessary for bone formation. Introduction Cystine (C)-X-C motif chemokine receptor 4 (CXCR4) is the primary transmembrane receptor for signaling chemokine stromal derived factor 1 (SDF-1, also named CXCL12 or pre B cell stimulating factor (PBSF)) (1-4). Both CXCR4 and SDF-1 are highly conserved in various mammalian cell types, and have broad functions in cell proliferation, morphogenesis and migration. The CXCR4 and SDF-1 mice die in utero or perinatally due to multiple defects in developing brain, heart, vasculature, intestine and hematopoietic tissues (5-7). Mutations at the carboxyl terminus of CXCR4 gene also lead to the WHIM syndrome in humans, a complex immunodeficient disease associated with neutropenia and defective B cell development (8). The binding of SDF-1 to CXCR4 induces cytoskeleton rearrangement and integrin activation, and eventually results in the migration of CXCR4expressing cells towards high gradients of SDF-1 (9-16). This SDF-1/CXCR4-mediated chemotaxis is involved in a variety of physiological and pathological events including the blood homeostasis (9), cellular inflammatory and immune response (10), bone remodeling (11), homing of stem/progenitors to bone marrow reservoir (17), tumor metastasis to bone or other organs containing high levels of SDF-1 (18), and cell recruitment in injured tissues (15, 19-24). 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M111.250985 The latest version is at JBC Papers in Press. Published on June 2, 2011 as Manuscript M111.250985 Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from Previous studies suggested the expression of CXCR4 and SDF-1 in bone; however, their direct function as well as underlying mechanisms remained poorly defined. This is probably due to the absence of demonstrable reports about skeletal abnormalities in patients with CXCR4 mutations or mice lacking CXCR4 or SDF-1. In fact, accumulating evidence has suggested the intimate association of the SDF-1/CXCR4 pathway with progenitor cells that have potentials to become bone-producing osteoblasts or form bone. Expression of SDF-1 and CXCR4 is found in both mesenchymal cell cultures (15, 25-27) and bone sections (28-32), with greater levels in less differentiated cells or immature osteoblasts relative to mature osteoblasts and osteocytes. High levels of SDF-1 are also present in regions of perichondrium of embryonic bones and periosteum of injured bones, where osteoprogenitors arise and differentiate (15, 28-32). In addition, retrovirus-mediated over expression of SDF-1 in human MSCs was found to enhance the MSCs’ induction of ectopic bone formation in nude mice (27). In mesenchymal cultures, such as the human and mouse bone marrow-derived stromal cells (33), C2C12 and ST2 cells (25), we demonstrated that blocking of the SDF-1/CXCR4 signal axis inhibits the differentiation of these cells towards the osteoblastic lineage in response to bone morphogenic protein 2 (BMP2) stimulation. This reveals the direct involvement of the SDF1/CXCR4 pathway in osteogenic differentiation in vitro. Moreover, recent evidence showing the effect of CXCR4 and SDF-1 on enhancing chondrocyte hypertrophy at the chondro-osseous junction of long bone further implicates the involvement of SDF-1 signaling in endochondral ossification (34). In light of these findings, we hypothesized that SDF-1 signaling functions in bone formation via affecting osteoblast development. To address the direct role of SDF-1 signaling in bone formation in vivo, in this study, we used the Cre/loxP genetic approach to conditionally remove CXCR4 from osteoblastic lineage precursors under the control of an osterix (Osx) promoter. Osx (35) is a zinc-finger domaincontaining transcription factor, downstream of Runx2, mainly expressed by osteoprecursors during early phases of osteogenic differentiation. Osx is considered one of the “master” regulators for bone formation, because Osx mice (35), like Runx2 (36, 37), develop no bone in their skeletons. Via this Osx-controlled conditional deletion of CXCR4, we demonstrated the requirement for CXCR4 in bone formation of the mouse skeleton. Moreover, we identified the function of CXCR4 in regulating osteoblast activities, and the interaction of CXCR4 with BMP signaling in this process. Experimental Procedures Antibodies and reagents: Anti-CXCR4 and anti-Osx antibodies were purchased from eBioscience (San Diego, CA) and Abcam (Cambridge, MA), respectively. Antibodies for type X collagen α1 (Col10α1), ProCol1α1, and proliferating cell nuclear antigen (PCNA) were purchased from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Anti-osteocalcin (OCN) was purchased from Millipore (Billerica, MA). Antibodies for Smad1/5/8, phosphorylated Smad1/5, total Erk, phosphorylated Erk1/2, and βTubulin were obtained from Cell Signaling (Danvers, MA). Anti-phosphorylated Smad2 was purchased from Rockland Immunochemicals (Gilbertsville, PA). Antibodies for SDF-1, Col2α1, isotype-matched control antibodies and secondary goat, sheep or rabbit IgGs were from Santa Cruz (Santa Cruz, CA). Recombinant human (rh) BMP2 and BMP6 proteins were purchased from R&D Systems (Minneapolis, MN). General chemicals were from Sigma (St. Louis, MO). All cell culture media and supplements were from Gibco (Invitrogen, Carlsbad, CA). Animals and genotyping: All animal experiments were carried out following review and approval by IACUC at The Hospital for Special Surgery. CXCR4 mice (3840), in which CXCR4 gene is flanked by loxP sequences, were provided by Dr. Yong-Rui Zou (Columbia University, New York, NY). Osx-Cre mice (41, 42), in which the expression of a tetracycline (Tet)-off regulatable GFP/Cre fusion protein is transcriptionally controlled by an Osx promoter, were purchased from The Jackson Laboratory (Bar Harbor, Maine). These two strains were crossed and maintained on a C57BL/6J background. We first generated double heterozygous mice for Cre and floxed CXCR4 2 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from (Osx-Cre;CXCR4), which were then bred to CXCR4 mice via a back-mating strategy to generate excised floxed CXCR4 homozygous (Osx::CXCR4, used as conditional knockouts), along with heterozygous for floxed CXCR4 (OsxCre;CXCR4) and Cre-null mice (CXCR4 and CXCR4, used as littermate controls throughout the study). These mice were born at the expected Mendelian frequency. As additional controls, Osx::CXCR4 mice were fed with 200μg/ml doxycycline (Tet analog, Sigma) in drinking water, which prevents the Osx promoter from driving Cre expression, as suggested by the manufacturer. For genotyping, genomic DNA was isolated from tail tips using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA). PCR reactions were performed using PCR Master Mix (Fermentas, Glen Burnie, MI) with primer sequences for Cre transgene and floxed CXCR4 gene relative to wild type CXCR4 as shown in Table 1. X-ray and micro-computed tomography (μCT) analyses: For skeletal analysis, mice were sacrificed by CO2 inhalation, and x-rays (40Kv, 4mAS, SID130, Faxitron, Lincolnshire, IL) were obtained in both lateral and anterior-posterior views to assess the spinal length and the femoral/tibia length, respectively, in the mouse skeleton. μCT (Scanco Medical Micro CT 35) was performed on both calvarias and tibias of mice. Entire calvarias and tibias were scanned (isotropic resolution of 15-20μm) to obtain grayscale images, which were Gaussian-filtered and globally thresholded (15.2% of maximum gray value) to form binarized images for morphological analyses. Quantitative volumetric analyses of cortical and trabecular microstructures were performed on entire parietal calvarias and on a region of 200 micro tomographic slices (2.1mm) at the proximal tibia with an isotropic resolution of 6μm. These analyses measured cortical and trabecular bone volume fraction, thickness, and bone mineral density, as well as trabecular number, separation and connectivity density. Double fluorochrome labeling and mineral apposition rate (MAR): To measure bone mineralization in vivo, mice were subcutaneously and sequentially injected with xylenol orange (orange red) and tetracycline (green) at 20μg/g (body weight) each in a 4-day time interval. One day after the second injection, tibias were collected and fixed in 80% ethanol, and then subjected to polymethyl methacrylate (PMMA) embedding and sectioned to 8-10μm. Histomorphometry analysis using OsteoII software (Bioquant, Nashville, TN) was performed on tibia sections to measure the distance between two fluorochrome-labeled mineralization fronts at the midshaft of tibia. Periosteal MAR was calculated by dividing the measured distance with the time interval. Histology, immunohistochemistry, and histomorphometry: For phenotypic analysis, tibias including knee joints or calvarias were fixed in sodium phosphate-buffered 4% paraformaldehyde at 4C for 2-4 days, decalcified in 5% EDTA, and transferred to 70% ethanol until paraffin-wax embedding. These tissues were sectioned to 4-5μm and subjected to alcian blue staining; and PMMAsections were subjected to von Kossa staining following the standard histology protocols. To reveal patterning changes in cartilage and bone of the entire skeleton, E18.5 whole embryos of mice were sequentially stained with alcian blue (for cartilage) and alizarin red (for bone) as described in previous studies (43). In short, after fixation with 95% ethanol for 48-72 hours, embryos were stained with 0.03% (w/v) alcian blue in 80% ethanol and 20% acetic acid solution for 1-3 days, followed by 0.03% (w/v) alizarin red in 1% KOH solution for another 12-24 hours, and then maintained in a solution of 2% KOH:glycerol (20:80) until analysis. For immunohistochemistry, deparaffinized sections were treated with antigen retrieval in heated 10mM citrate buffer (pH6.0), and then incubated with the primary antibody at 4C overnight. On the second day, sections were incubated with appropriate secondary antibodies for 30-60 minutes at room temperature, and developed with DAB or TMB as the chromogen, following the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). After histology or immunohistochemistry, histomorphometric analysis (Bioquant) was performed on tissue sections to quantify the number of positively stained cells per region of 3 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from interest or per bone surface and the fraction of mineralized bone, following the manufacturer’s protocols. RT-PCR, real time PCR, and Western blotting: Total RNA of mouse tissues was extracted using the RNeasy Extraction Kit (Qiagen), and the equal amount of 1μg of total RNA per sample was reverse-transcripted (First strand RT kit, Fermentas). Regular PCR or quantitative real time PCR (qPCR) was performed using PCR Master Mix (Fermentas) or SYBR Green Supermix (BioRad, Hercules, CA) with primer sequences listed in Table 1. The level of target gene expression was normalized to the level of the housekeeping gene, GAPDH (25, 33). Western blotting was performed as previously described (25, 33). In short, whole cell lysates were assayed for the amount of total cellular protein (Pierce bicinchoninic acid assay, Thermo Scientific, Rockford, IL). Equal loading of 20μg aliquots of total protein from each sample were fractionated on 12% Bis–Tris gels, following the manufacturer’s protocol (Invitrogen, Carlsbad, CA). After incubating with appropriate antibodies, immunoreactive bands were visualized with enhanced chemiluminescence (ECL) detection reagents (Amersham Biosciences, Piscataway, NJ). The intensities of bands were measured using gel image analysis software (ImageJ_1.32, NIH). Primary calvaria culture, proliferation, and differentiation assay: Calvarias were dissected from newborn (1-3 days after birth) mice, rinsed with PBS, and digested in freshly made 1mg/ml Collagenase/Dispase mixture (Roche, South San Francisco, CA) in αMEM medium at 37C for 20 minutes and repeated three times. After digestion, supernatants were combined and centrifuged to pellet cells. Cells were then cultured in αMEM supplemented with 10% FBS and 1% penicillin/streptomycin (all from Gibco). Medium was replaced every 2-3 days. Proliferation of calvaria cells was measured by evaluating cell cycle progression using bromodeoxyuridine (BrdU) incorporation assays (Chemicon, Billerica, MA) as the manufacturer describes. To induce osteoblast differentiation, calvaria cells were stimulated with rhBMP2 or rhBMP6 at 100ng/ml for 4 and 7 days. ALP activity and OCN synthesis were evaluated in these cells as markers for immature and mature osteoblasts, respectively. As previously described (25, 33), ALP activity was measured in cell lysates using a colorimetric assay (Sigma), and was normalized to the level of total cellular protein. OCN synthesis was measured in culture medium, using a commercially available ELISA kit (Biomedical Technologies, Stoughton, MA). Serum OCN and tartrate-resistant acid phosphatase (TRAP) assays: OCN levels in mouse serum were also measured using ELISA kits from Biomedical Technologies. Serum TRAP, a marker of bone resorption, was measured using the ELISA-based mouse TRAP assay kit from Immunodiagnostic Systems (Fountain Hills, AZ). Statistical analysis: Experiments were repeated two to three times independently, with five to six samples included in each experimental group. All quantitative measurements were reported as the mean ± standard deviation (SD) calculated using SigmaPlot8 or Instat statistical software. Differences between two experimental groups were compared by One Way ANOVA or Student’s t-test, with significance accepted at p<0.05.