Role of SMPD3 during Bone Fracture Healing and Regulation of Its Expression

Materials.Recombinant human BMP-2 was purchased from GenScript (Piscataway, NJ), and dorsomorphin (P5499), PD98059 (P215), BI-87G3 (SML0489), SB202190 (S7067), parathyroid hormone-related peptide (PTHrP; SRP4651), and forskolin (F6886) were obtained from Sigma-Aldrich (Saint Louis, MO).

Mice.The generation and genotyping of Smpd3^flox/flox mice were described previously (7). The Osx-Cre mice were obtained from the Jackson Laboratory (stock number 006361). Genotypes were determined by PCR on genomic DNA isolated from tail biopsy specimens. The presence of the “floxed” allele in the gene-targeted mice was confirmed by using the primers 5′-TCAGAGAACGGAGGCTGATC-3′ and 5′-AAGAGACCGTTGGGAATACC-3′ (amplicon sizes, 106 bp [wild type] and 140 bp [“floxed” allele]). The Cre transgene was detected using the primer pair 5′-GCCTGCATTACCGGTCGATGCAACGA-3′ and 5′-GTGGCAGATGGCGCGGCAACACCCATT-3′ (amplicon size, ∼700 bp). In order to detect Cre-mediated deletion, DNA were isolated by using the phenol-chloroform method from tissue collected from the fracture callus of Smpd3^flox/flox and Smpd3^flox/flox; Osx-Cre mice 4 weeks postsurgery. A multiplexed PCR was performed on an equal amount of template DNA using F1 (5′-CATCATTCCCACAGCCTGAA-3′) and R1 (5′-AAGAGACCGTTGGGAATACC-3′) primers (amplicon size, ∼200 bp). The mouse Elastin gene was amplified (amplicon size, ∼500 bp) to be used as a loading control. All animal procedures were reviewed and approved by the McGill Institutional Animal Care and Use Committee following the guidelines of the Canadian Council on Animal Care.

Doxycycline treatment.Drinking water containing 1% dextrose was supplemented with 0.4 mg/ml doxycycline hyclate (Sigma, D9891) to prepare DOX water. A commercially available diet (S38888-DOX Diet; Bio-Serv) containing 200 mg/kg doxycycline was used as the DOX diet. Pregnant female mice were switched to DOX water and diet on day postcoitum (d.p.c.) 10.5, and this regimen was continued until the pups were 7 days old.

Intramedullary nailing surgical procedure.The intramedullary nailing surgery was performed on the right tibia at 2 months of age. The analyses of the fractured bones were performed 2 and 4 weeks postsurgery. The surgical procedures were done as previously described (17). Briefly, the mice were first injected subcutaneously with 0.04 ml of buprenorphine (0.3 mg diluted in 12 ml of normal saline) before the surgery. Mice were then anesthetized using isoflurane gas and oxygen and kept under anesthesia throughout the full length of the procedure. Once mice were anesthetized, they were injected with carprofen (5 μl/g) and normal saline (20 μl/g) subcutaneously. The right leg was then shaved to remove all fur surrounding the tibia. The surgical site was prepped with 2% chlorhexidine and 70% ethanol solution. A 3-mm vertical skin incision was made above the knee, and the patellar ligament was exposed where a vertical medial parapatellar incision (approximately 1 to 1.5 mm in length) was made on the tibial plateau. A 25-gauge (25G) needle was inserted through the incision into the tibial canal and through the needle, the internal wire core guide (Quincke BD 25G 3″ spinal needle, catalog no. 450170; Becton Dickinson, Franklin Lakes, NJ) was inserted. The 25G needle was then removed while keeping the internal wire core guide in the tibial canal. The protruded part of the wire core guide was clipped and bent at 90° at the proximal end and tucked in under the patellar ligament to avoid puncture of the ligament. Using the same skin incision, a mid-tibial shaft osteotomy was made using an extra fine Bonn scissors (catalog no. 14084-08; Fine Science Tools, Vancouver, Canada). The incision was sutured with a single 6-0 vicryl suture (Johnson & Johnson, Skillman, NJ), and 1 to 2 drops of analgesia, lidocaine/bupivacaine, was added to the fracture site. The skin was sutured using three to four horizontal mattress sutures. After the surgery, the mice were placed in their cages on a heated pad and allowed to move around freely. The mice were then given subcutaneous injections of 0.05 ml of buprenorphine and carprofen (5 μl/g) at 24 and 48 h postsurgery. The animals were closely monitored, and any mice showing signs of severe pain, bleeding, or swelling were euthanized. All procedures were performed under aseptic conditions in the procedure room of the animal facility at the Shriners Hospital for Children, Montreal, Quebec, Canada.

Sample collection.The mice were sacrificed using isoflurane and CO₂ at 2 or 4 weeks postsurgery. The tibiae were dissected at the level of the knee and ankle. The intramedullary nail was carefully pulled out using a needle holder. Tibiae were then fixed in buffered formalin overnight and transferred to 70% ethanol for micro-CT and histological analyses.

Micro-CT.The SkyScan 1272 micro-CT machine (SkyScan, Bruker, Belgium) was used to image samples. Tibiae were harvested and imaged in plastic tubes containing 70% ethanol. The scans were performed using the SkyScan 1272 software at a source voltage of 60 kV and a source current of 166 μA. The acquisition parameters were as follows: pixel size, 5.00 μm; rotation step, 0.4; aluminum, 0.5-mm filter; and image size, 2,452 by 1,640 pixels. The generated images were used to reconstruct the three-dimensional image of the scanned bone using the NRecon Reconstruction Software (SkyScan, Bruker, Belgium) using a beam-hardening correction of 15%, a ring artifact correction at 6, smoothing at 2, and a dynamic range of 0.00 to 0.12. The reconstructed images were then rotated using DataViewer and analyzed using CTAn (both from SkyScan).

Skeletal preparation.Skeletal tissues from newborn and adult mice were fixed overnight in 95% ethanol, stained by 0.015% alcian blue dye (Sigma-Aldrich) in a 1:4 solution of glacial acetic acid and absolute ethanol for 24 h, and treated with 2% potassium hydroxide until the soft tissues were dissolved. The mineralized tissues were stained by 0.005% alizarin red (Sigma-Aldrich) solution in 1% potassium hydroxide and clarified in 1% potassium hydroxide–20% glycerol for >2 days.

Histology and histomorphometry.Fractured and nonfractured tibia were fixed overnight in 4% paraformaldehyde–phosphate-buffered saline (PBS), embedded in methyl methacrylate, sectioned (7-μm thickness), and processed for von Kossa and van Gieson (VKVG) staining. Stained bone sections were analyzed for osteoid volume/bone volume (OV/BV) using the Osteomeasure software (Osteometrics, Inc.). The region of interest (ROI) was selected within the fracture healing site. Plastic sections were also decalcified in a Cal-Ex decalcifier (Fisher, Pittsburgh, PA) overnight and processed for alcian blue and van Gieson (AB/VG) staining. AB/VG-stained bone sections were then analyzed for cartilage matrix volume/callus volume using the Osteomeasure software. Images were taken at room temperature using a light microscope (DM200; Leica) with a 20 (numerical aperture of 0.40) or 40 (numerical aperture of 0.65) objective. All histological images were captured using a camera (DP72; Olympus), acquired with DP2-BSW software (XV3.0; Olympus), and processed using Photoshop (Adobe).

Gene expression analysis.Gene expression analysis was performed using a qRT-PCR system (model 7500; Applied Biosystems). Total RNA was extracted from fractured and nonfractured tibia at 13 days postsurgery from 3-month-old WT male mice with TRIzol reagent (Ambion, catalog no. 15596018) and subjected to DNase I (Applied Biosystems, M0303S) treatment. The first-strand cDNA synthesis and qRT-PCR were performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) and SYBR green quantitative PCR master mix (Maxima; Fermentas), respectively. Relative gene expression analysis was performed as described previously (30). The primer sequences for Runx2, Sox9, Osx (Sp7), Col10a1, Smpd3, Alpl, and Col2a1 were the same as those published previously (6, 30). The remaining primer sequences are presented in Table 1.

Immunofluorescence and immunohistochemistry.For immunofluorescence, E15.5 humeri were embedded in paraffin and sectioned (7 μm). Sections were blocked with 5% bovine serum albumin (BSA; Fisher, Pittsburgh, PA) in Tris-buffered saline (TBS)–Triton X-100, followed by incubation with antiosterix (anti-OSX) antibody from Abcam (Cambridge, MA). Localization of the primary antibody was detected with Alexa Fluor 488-conjugated affinity-purified donkey anti-rabbit antibody from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). After three washes with PBS, the nuclei were visualized with Hoechst stain (H33258; Sigma-Aldrich). Images were captured by the EVOS FL cell imaging system (Life Technologies). For immunohistochemistry, plastic sections were blocked with 5% BSA (Fisher) in TBS–Triton X-100, followed by incubation with anti-SMPD3 antibody (Santa Cruz Biotechnology). The samples were further incubated with horseradish peroxidase (HRP)-conjugated goat secondary antibody (1:1,000; Abcam), and then a colorimetric reaction was carried out with 3,3-diaminobenzidine and 0.02% H₂O₂, followed by counterstaining with hematoxylin. Light microscopy images were captured as described above.

DNA constructs.For Smpd3 promoter studies, we used the pSmpd3-luc reporter construct that we generated previously (7). To study the role of SRY binding sites, we deleted a cluster of SRY-binding sites spanning nucleotides –595 to –646 within the 1.9-kb mouse Smpd3 proximal promoter. The modified promoter was then cloned into the SmaI and Sal I sites in the multiple cloning site of the pGL4.10 (luc2) vector (Promega, Madison, WI).

Cell culture.ATDC5 cells were cultured in alpha minimal essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (PAA Laboratories) and 100 U/ml of penicillin-streptomycin at 37°C under 5% CO₂ in a humidified incubator. Cells were treated with 300 ng/ml of BMP-2 for 48 h and then subjected to gene expression analysis. To induce chondrogenic differentiation, cells were cultured in differentiation medium containing ascorbic acid (100 μg/ml) and β-glycerophosphate (5 mM) for 6 days. To inhibit BMP signaling, the cells were treated with 2 μM dorsomorphin for 30 min and washed with PBS, and differentiation medium was added to the culture. The medium was changed in this manner every 2 days and, on the sixth day, the cells were subjected to gene expression analysis.

In order to study the role of MAPK signaling, cells were treated with the indicated inhibitor for 1 h and washed with PBS, and then either inhibitor alone or inhibitor and BMP-2 were added to the medium for a total of 48 h; the cells were then subjected to gene expression analysis. HEK293 cells were cultured in Dulbecco modified Eagle medium (Invitrogen).

To investigate the effect of both BMP-2 and PTHrP on Smpd3 expression, we treated ATDC5 cells with 0.1 μM PTHrP for 24 h and then cotreated the cells with 0.1 μM PTHrP and 300 ng/ml of BMP-2 for another 48 h. The cells were then subjected to gene expression analysis.

To study transcriptional regulation of Smpd3 expression, we transfected ATDC5 cells with 2.5 μg of Mef2c, Sox6, Sox9, or Osx expression vectors using a Lipofectamine 3000 transfection kit (Invitrogen) according to the supplier’s instructions. The cells were then subjected to gene expression analysis after 48 h. The transfection efficiency was determined by transfecting ATDC5 cells with a GFP expression vector (pMax-GFP). The nuclei were visualized with Hoechst stain (Sigma-Aldrich). Images were captured by the EVOS FL cell imaging system at ×20 (Life Technologies). GFP-positive cells were quantified.

Luciferase assay.ATDC5 cells were cotransfected with 500 ng of Smpd3 reporter plasmids or the pIDWT4F-luc reporter plasmid (31) and 50 ng of Renilla luciferase (R-Luc) plasmids using the Lipofectamine 3000 transfection kit (Invitrogen). The pIDWT4F-luc reporter plasmid was used as a control for BMP-2 since the promoter contains four tandem repeats of a BMP-responsive element found in Id1, a direct target of BMP signaling. After 24 h, cells were treated with 300 ng/ml of BMP-2 for 8 or 48 h. HEK293 cells were cotransfected with 500 ng of the plasmid combinations as indicated, as well as 10 ng of R-Luc plasmid. The luciferase activity was measured after 48 h using a Dual-Luciferase reporter assay system (Promega), as described previously (30).

Statistical analysis.All results are shown as means of the standard deviation. Statistical analyses were performed by the Student t test or analysis of variance (Tukey’s multiple-comparison test).