Cells, plasmids, and antibodies.
hMSCs were cultured under osteoblast differentiation conditions, as instructed by the user’s manuals (Lonza). Primary CalvObs and the C3H10T1/2 cell line (mouse mesenchymal fibroblast-like cell line) were cultured in α-MEM medium (Cellgro) containing 10% FBS, 2 mM l-glutamine, 1% penicillin/streptomycin, 1% HEPES, and 1% nonessential amino acids and differentiated with ascorbic acid and β-glycerophosphate. HEK293 cells (human kidney embryonic cells) and HEK293 FT cells were purchased from ATCC and Invitrogen, respectively. Myc-PDK1 (WT, K111A, and L155E), VP16/CREB, A-CREB, and mouse BMP2-Luc (–150/+165 bp and –2712/+165 bp) cDNAs were gifts from Dan R. Littman (New York University, New York, New York, USA), Angel Barco (Instituto de Neurociencias de Alicante, Alicante, Spain), Charles Vinson (National Cancer Institute, Bethesda, Maryland, USA), and Stephen E. Harris (University of Texas Health Science Center at San Antonio), respectively. Flag-CREB (WT) cDNA was a gift from Marc Montminy (Addgene plasmid no. 22968). Cre recombinase, Myc-PDK1 (WT and K111A), and Myc-RUNX2 cDNAs were PCR amplified and cloned into pHASE/PGK-PURO lentiviral vector. The following antibodies were used: anti-PDK1, anti–phospho-AKT (T308 and S473), anti–phospho–GSK-3β (S9), anti–β-catenin, anti–phospho-AKT substrates (RXXS/T*), anti–phospho-S6 (S235/236), anti–phospho-4EBP1 (T37/T46), anti–phospho-CREB (S133), anti–phospho-SMAD1/5/8, anti–phospho-ERK1/2, anti–phospho-JNK1/2, and anti–phospho-p38 (all from Cell Signaling Technology); anti-HSP90, anti-HA–conjugated agarose, anti-c-Myc–conjugated HRP, and anti-HA–conjugated HRP (all from Santa Cruz Biotechnology Inc.); anti-Flag (M2) and anti-VP16 (both from Sigma-Aldrich); anti-CBP, anti-Tie2, anti-CD45, anti-Ter119, anti-CD105, and anti-Thy1.2 (all from Biolegend); anti-RUNX2 (Calbiochem); and anti-GAPDH (Affinity Bioreagents). As indicated, cells were treated with rhBMP2/7, human IGF-1, human FGF-2, and murine TGF-β (R&D Systems); insulin, forskolin, and isoproterenol (Sigma-Aldrich); and inhibitors for PI3K, PDK1, and AKT (Calbiochem).
Breeding of mouse strains.
mice were intercrossed with the Osx-cre deleter strain (a gift from Andrew McMahon, Harvard University, Boston, Massachusetts, USA) to generate Pdk1osx
mice were gifts from Bjorn Olsen (64
), and Creb+/–
mice and the Dermo1-cre deleter strain were purchased from The Jackson Laboratory. All mice analyzed, including Pdk1osx
, and Cbp+/–
mice, were maintained on the C57BL/6 background.
Skeletal preparation and μCT analysis.
For skeletal preparation, skeletons were prepared for gross analysis using the method of McLeod (65
). Briefly, mice were skinned, eviscerated, and fixed in 95% ethanol. Then, skeletons were stained by Alizarin Red S/Alcian Blue and sequentially cleared in 1% potassium hydroxide.
For μCT analysis, a Scanco Medical μCT 35 system with an isotropic voxel size of 7 μm was used to image the distal femur. Scans were conducted in 70% ethanol and used an X-ray tube potential of 55 kVp, an X-ray intensity of 0.145 mA, and an integration time of 600 ms. From the scans of the distal femur, a region beginning 0.28 millimeters proximal to the growth plate and extending 2.1 millimeters proximally was selected for trabecular bone analysis. A second region 0.6 millimeters in length and centered at the midpoint of the femur was used to calculate diaphyseal parameters. A Gaussian filter, with a support of 0.8 and sigma of 1, was applied to the femur images. In order to distinguish cortical from trabecular bone, a semiautomated contouring approach was used. A contour was drawn by hand a few voxels from the endocortical surface every 20 slices. Contours on slices in this range were interpolated by the software. The region of interest was thresholded using a global threshold that set the bone/marrow cutoff at 438.5 mgHA/cm3
for trabecular bone and 589.4 mgHA/cm3
for cortical bone. Unbiased, 3D microstructural properties of trabecular bone, including bone volume fraction, trabecular thickness, trabecular number, and trabecular separation, were then calculated for the trabecular region of the metaphysis of the distal femur as well as midshaft bone volume fraction and cortical thickness for the diaphysis, using methods based on distance transformation of the binarized images (66
). Scans of the skulls were performed using isotropic voxel sizes of 12 μm and 20 μm. A Gaussian filter, with a support of 0.8 and sigma of 1, was applied to the skull images. A contour was created that encompassed the entire skull, and the volume of interest was thresholded using a global threshold that set the bone/air cutoff at 136.8 mgHA/cm3
. All images presented are representative of the respective genotypes.
Isolation of fetal osteoprogenitor cells.
Fetal osteoprogenitor cells were isolated from E17.5 Pdk1fl/fl
, or Pdk1fl/+dm1
embryos as previously described (30
). Briefly, both hind and fore limbs from E17.5 embryos were digested with type I collagenase (Sigma-Aldrich) and dispase II (Roche) for 40 minutes at 37°C. The cells were stained with antibodies specific to Tie2, CD45, Ter119, Cd105, αν
-integrin, and Thy1.2 after lysing red blood cells. Data were acquired on LSRII and analyzed using Flowjo Flow Cytometry Analysis software.
Inducible PDK1 deletion and osteoblast differentiation analysis.
Primary osteoblast precursors were isolated from calvaria of P2 Pdk1fl/fl neonates by using the digestion solution containing type I collagenase (Sigma-Aldrich) and dispase II (Roche). For inducible deletion of the Pdk1 gene, Pdk1fl/fl osteoblast precursors were transduced with either EGFP-expressing lentivirus (control) or Cre-recombinase–expressing lentivirus (Cre) at the same MOI and cultured in osteoblast differentiation media containing ascorbic acid and β-glycerophosphate. Forty-eight hours after infection, the transduced cells were selected by puromycin.
For osteoblast differentiation assay, ALP activity, extracellular matrix mineralization, and osteoblast marker gene expression were analyzed as previously described (67
). Briefly, for ALP staining, osteoblasts were fixed with 10% neutral buffered formalin and stained with a solution containing Fast Blue and Naphthol (Sigma-Aldrich). Alternatively, osteoblasts were incubated with Alamar Blue solution, washed, and incubated with a phosphatase substrate solution (Sigma-Aldrich), and ALP activity was measured by luminometer (Thermo Electron Co.). For Von Kossa staining of extracellular matrix mineralization, cells were fixed with 10% neutral buffered formalin and stained with a solution containing 2.5% silver nitrate (Sigma-Aldrich). For osteoblast marker gene expression, total mRNAs were purified from osteoblast cultures for use in quantitative RT-PCR reactions that measure the expression level of several genes that are regulated during osteoblast differentiation.
Histology, in situ hybridization, and IHC.
For histological analyses, paraffin sections of bones were produced from E16.5, E18.5, P1, and 4-week-old mice. Limb tissues were dissected and fixed in 4% paraformaldehyde in PBS. They were then decalcified by daily changes of 15% tetrasodium EDTA, until soft and pliable. Tissues were dehydrated by passage through an ethanol series, cleared twice in xylene, embedded in paraffin, and sectioned. For morphological analyses, tissue sections were stained with hematoxylin and eosin.
For in situ hybridization, DIG-labeled probes were prepared to detect osteocalcin and osteopontin mRNA expression using the DIG-labeling Kit (Roche), per the manufacturer’s instructions, and the probes were hybridized with paraffin sections. DIG-labeled probe was then detected by immunostaining with anti-DIG-POD and streptadvidin-HRP (67
For IHC, paraffin tissue sections were dewaxed, and endogenous peroxidase was quenched, as in preparation for in situ hybridization. Sections were blocked with 3% goat serum, 1% BSA, 0.1% Triton X-100 in PBS for 1 hour at room temperature and incubated with antibodies specific for PDK1, phospho-AKT (T308), phospho–GSK-3β (S9), phospho-S6, phospho-CREB (S133), and phospho-SMAD1/5/8 at 4°C overnight. Sections were then treated with TSA-biotin (Perkin Elmer) and streptavidin-HRP, as per manufacturer’s instructions, and HRP visualized with 2,2ι-diaminobenzidine tetrahydrochloride.
Luciferase reporter assay.
Luciferase reporter assay was performed as described previously (67
). Briefly, C3H10T1/2 cells were transfected using Effectene (Qiagen) with the indicated reporter genes and the Renilla
luciferase gene (Promega), together with plasmids of VP16/CREB, Flag-CREB (WT), CBP, or RUNX2. Total DNA concentration in each experiment was maintained by adding the appropriate control vector to the DNA mixture. Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured using the Dual-Luciferase Assay Kit (Promega). Alternatively, primary osteoblast precursors isolated from calvaria of Pdk1fl/fl
neonates were transduced with either EGFP- or Cre-recombinase–expressing lentivirus and cultured in the medium containing puromycin. Puromycin-resistant cells were transfected using Effectene (Qiagen) with various reporter genes and the Renilla
luciferase gene. Six days after culture in differentiation medium, cells were lysed, and luciferase activity was measured using the Dual-Luciferase Assay Kit (Promega).
DNA binding assay.
DNA binding assay was performed as described previously (68
). Briefly, HEK293 cells were transfected with either Flag-CREB or Myc-RUNX2 cDNA, and nuclear proteins were extracted from the cells by using nuclear and cytoplasmic extraction reagents (Thermo Scientific). 150 μg nuclear protein was incubated with biotinylated probe conjugated to streptavidin-agarose (Invitrogen) bound to protein containing the RUNX2-binding (OSE2-binding) site and WT or mutant CREB-binding site (CRE) for 1 hour at 4°C in binding buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.6], 0.1 mM EDTA, 1 mM DTT, 5% glycerol, 1 mg/ml BSA, 20 μg/ml poly dI/dC plus protease inhibitors). The coimmunoprecipitates were washed 3 times in binding buffer, resolved by SDS-PAGE, and immunoblotted for overexpressed Flag-CREB and Myc-RUNX2. The following primer sequences were used: OSE2-WT, GATCCGCTGCCATCACCAACCACAGCA; CRE-WT, CGCGGCCCAGCTAACGCAGAACGTCCGTCCCTCGCCCGGCGAG; and CRE-MUT, CGCGGCCCAGCTAACGCAGAAGCCCCGTCCCTCGCCCGGCGAG.
Treatment of animals with rhBMP2/7.
For treatment of animals with rhBMP2/7, 10 μg rhBMP2/7 (R&D Systems) was dissolved in a total volume of 1 ml, and a dose of 1 μg rhBMP2/7 per day was injected intraperitoneally into pregnant mice on E8.5 through E18.5. Injection of PBS was used as a control.
Where appropriate, 1-way ANOVA was performed, and 2-tailed, unpaired Student’s t tests were used as a post-test for the select biologically relevant comparisons. Otherwise, unpaired Student’s t tests were used directly. A P value equal to or less than 0.05 was considered significant. All data graphed are represented as mean ± SD. Quantification of repeats for all Western blots shown is provided in Supplemental Figure 11.
All animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were handled according to protocols approved by the Harvard University and University of Texas Health Science Center at San Antonio subcommittees on animal care (IACUC).